EP4658689A2 - Phosphomimetic mutants, phosphospecific antibodies, and uses thereof - Google Patents

Phosphomimetic mutants, phosphospecific antibodies, and uses thereof

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Publication number
EP4658689A2
EP4658689A2 EP24751139.7A EP24751139A EP4658689A2 EP 4658689 A2 EP4658689 A2 EP 4658689A2 EP 24751139 A EP24751139 A EP 24751139A EP 4658689 A2 EP4658689 A2 EP 4658689A2
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EP
European Patent Office
Prior art keywords
acid sequence
cse
amino acid
nucleic acid
antibody
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EP24751139.7A
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German (de)
French (fr)
Inventor
Christopher G. Kevil
Gopi K. Kolluru
Xinggui SHEN
Shaufil ALAM
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Louisiana State University
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Louisiana State University
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Publication of EP4658689A2 publication Critical patent/EP4658689A2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/13—Transferases (2.) transferring sulfur containing groups (2.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y208/00—Transferases transferring sulfur-containing groups (2.8)
    • C12Y208/01—Sulfurtransferases (2.8.1)
    • C12Y208/01002—3-Mercaptopyruvate sulfurtransferase (2.8.1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y404/00—Carbon-sulfur lyases (4.4)
    • C12Y404/01—Carbon-sulfur lyases (4.4.1)
    • C12Y404/01001—Cystathionine gamma-lyase (4.4.1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/14—Post-translational modifications [PTMs] in chemical analysis of biological material phosphorylation

Definitions

  • This invention is directed to CSE phosphomimetic mutants, phosphospecific antibodies against CSE, and methods of use thereof.
  • Hydrogen sulfide synthesis and metabolism is an important participant in cardiovascular health and function.
  • Hydrogen sulfide can be generated by mammalian cells via a variety of enzymes, including cystathionine -synthase (CBS), cystathionine y-lyase (CSE), and 3 -mercaptopyruvate sulfur-transferase (MPST). Cystathionine y-lyase (CSE).
  • An aspect of the invention is directed to an isolated antibody or fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR).
  • HCVR heavy chain variable region
  • LCVR light chain variable region
  • the HCVR comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is SGGIS, the amino acid sequence of HCDR2 is SISTSGNTYYASWAKG, the amino acid sequence ofHCDR3 is ALAGI, the amino acid sequence of LCDR1 is QASQSVSKNNRLA, the amino acid sequence of LCDR2 is SASTLAS, and the amino acid sequence of LCDR3 is LGGYDCKSADCYI; the amino acid sequence of HCDR1 is SYGMI, the amino acid sequence of HCDR2 is AISSSGNTYYAKWAKG, the amino acid sequence of HCDR3 is NHYGSGDI, the amino acid sequence of LCDR1 is QSSQSVYDANRLA, the amino acid sequence of LCDR2 is GASTLDS, and the amino acid sequence of LCDR3 is QGYYSGY
  • CDRs complementar
  • the antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the amino acid sequence of the LCVR is AQVLTQTPSSVSAAVGSTVTINCQASQSVSKNNRLAWFQQKPGQPPKGLIYSASTLASGVSS RFKGSGSGTQFTLTISDVQCDDAATYYCLGGYDCKSADCYIFGGGTEVVVK, and the amino acid sequence of the HCVR is
  • HCVR DVVMTQTPASVSEPVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYLASNMASGVPSRF SGSGYGTEFTLTISGVQCEDAATYYCQCTYYHSSTSSTVGGAFGGGTEVVVK, and the amino acid sequence of the HCVR is
  • HCVR AQVLTQTPSSVSAAVGGTVTINCQSSKSVGNNNRLSWYQQKPGQPPKQLIYGASTLASGVP SRFSGSGSGTQFTLTISDVQCDDAATYYCLGAYSSSSDNSFGGGTEVVVK, and the amino acid sequence of the HCVR is
  • HCVR AQVLTQTPSSVSAAVGGTVTINCQASQSVYSNNYLSWFQQKPGQPPKLLIYYASSLASGVPS RFSGSGTRFTLTISDVQCDDAAAYYCLGSYDCRAADCMAFGGGTEVVVR, and the amino acid sequence of the HCVR is
  • the antibody binds to cystathionine gamma lyase (CSE).
  • CSE cystathionine gamma lyase
  • the antibody binds CSE phosphorylated at amino acid position serine 346 according to the sequence in Table 10.
  • the antibody comprises a wildtype Fc or a modified Fc.
  • the antibody fragment comprises an Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody.
  • the antibody further comprises a detectable moiety.
  • aspects of the invention are further directed to a nucleic acid encoding the antibody as described herein.
  • the nucleic acid sequence encoding LCVR is GCCCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAAGCACAGTCAC CATCAATTGCCAGGCCAGTCAGAGTGTTTCTAAGAACAACCGCTTAGCCTGGTTTCAGC AGAAACCAGGGCAGCCTCCCAAAGGCCTGATCTATTCTGCATCCACTCTGGCATCTGGG GTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGA CGTGCAGTGTGACGATGCTGCCACTTACTATTGTCTAGGCGGTTATGATTGTAAAAGTG CTGATTGTTATATTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCA CCTGCACAGTCTCTGGAATCGACCTCAG
  • nucleic acid sequence encoding LCVR is GATGTTGTGATGACCCAGACTCCAGCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCAC CATCAAGTGCCAGGCCAGTCAGAGCATTAGTAGCTACTTAGCCTGGTATCAACAGAAAC CAGGGCAGCC
  • nucleic acid sequence encoding LCVR is GCCCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCAC CATCAATTGCCAGTCCAGTAAGAGTGTTGGTAATAACAACCGCTTATCCTGGTATCAGC AGAAACCAGGGCAGCCT
  • nucleic acid sequence encoding LCVR is GCCCAAGTGCTGACCCAGACTGCATCCCGTGTCTGCGGCTGTTGGAGGCACAGTCAC CATCAATTGCCAGGCCAGTCAGAGTGTTGCTAATAACAACCGCTTATCCTGGTTTCAGC AGAAACCAGGGCAGCCTCC
  • CGTGCAATGTGACGATGCTGCCGCTTACTACTGTCTAGGCAGTTATGATTGTAGGGCTG CTGATTGTATGGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAGA or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCA CCTGCGCAGTCTCTGGATTCTCCGTCAGTGGTAGTGCAGTGAACTGGGTCCGCCAGGCT CCAGGGGAGGGGCTGGAATGGATCGGGACAATTAGTAAGAATGGTAACACATACTACG CGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTG AGAATGACCACTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCCGGGCGCAATCC TGATACTAGTGGTGGTTTGGCCTTGTGGGGCCAAGGCACCCTGGTCACCGTCCTCA, or a degen
  • Embodiments further comprise a vector encoding the nucleic acid as described herein, and a cell comprising the vector as described herein.
  • compositions comprising the antibody as described herein, the nucleic acid as described herein, or the cell as described herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • aspects of the invention are drawn towards a method of detecting phosphorylated CSE in a subject in need thereof.
  • the method comprises contacting a biological sample with the antibody as described herein, wherein the antibody specifically binds to phosphorylated CSE.
  • phosphorylated CSE is phosphorylated at amino acid position serine 346.
  • the phosphorylated CSE is phophosphorylated at amino acid position 346.
  • aspects of the invention are drawn towards a synthetic nucleic acid encoding a CSE mutant, wherein the CSE mutant mimicks phosphorylation at Serine 346, Threonine 355, or both.
  • the synthetic nucleic acid is an mRNA molecule.
  • compositions comprising a nucleic acid as described herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • aspects of the invention are drawn towards a method of treating tissue injury in a subject.
  • the method comprises administering to a subject a nucleic acid encoding a CSE mutant, wherein the CSE mutant mimicks phosphosporylation at Serine 346, Threonine 355, or both.
  • the nucleic acid increases cellular hydropersulfide, polysulfide, or both.
  • the nucleic acid comprises a synthetic nucleic acid expression construct, wherein the expression construct comprises a promoter operatively linked to the nucleic acid encoding a CSE mutant.
  • aspects of the invention are drawn towards a method of increasing cellular hydropersulfide, polysulfide, or both.
  • the method comprises administering to a subject a nucleic acid encoding a CSE mutant, wherein the CSE mutant mimicks phosphosporylation at Serine 346, Threonine 355, or both.
  • Figure 1 shows changes in sulfide metabolite levels under hypoxia and mass spec analyses depicting CSE (cystathionine gamma lyase) phosphorylation.
  • Panel A shows mouse aortic endothelial cells (MAECs) exposed to either normoxia (21% oxygen) or hypoxic (1% oxygen) conditions for 30min and analyzed for various sulfide metabolites, including free, acid-labile sulfide and bound sulfane sulfur (that includes persulfide and polysulfides) using MBB/HPLC method developed in our lab.
  • a significant increase in bound sulfane sulfur was observed under hypoxia compared to normoxia.
  • Panel B illustrates per- and polysulfides quantified using the fluorescent probe Sulfane Specific Probe 4 (SSP4) in MAECs treated with either normoxia (21% oxygen) or hypoxia (1% oxygen) for 30min.
  • MAECs treated under hypoxia showed a significant 50% increase in per-polysulfide production compared to normoxia.
  • Panel C illustrates that MAECs show a significant increase in cystathionine (a classical CSE substrate) consumption revealing an increase in CSE enzyme activity under hypoxia compared to normoxia.
  • Panel D shows MAECs transfected with either mock, CSE, CBS (cystathionine beta synthase), MPST (3 -mercaptopyruvate sulfurtransferase), CARS-1 (cysteinyl-tRNA synthetase 1) or CARS-2 (cysteinyl-tRNA synthetase 2) siRNAs to reduce respective enzyme expression and then respectively were probed with SSP4 under hypoxic conditions.
  • siCSE siRNA showed significant reduction in polysulfide generation compared to mock siRNA, demonstrating a dominant CSE role in hypoxia poly sulfide generation.
  • siRNA to CARS1 and CARS2 also showed significant reduction in SSP4 compared to mock sima; however, they were significantly higher than siCSE revealing a lesser role of these enzymes in hypoxic per- polysulfide generation.
  • Panel E shows LC/MS HCD fragmentation spectrum of trypsin digested human CSE that was purified from normoxic versus hypoxic treated cells. An over two-fold significant increase in CSE amino acid fragment 334-364 (LFTLAESLGGFESLAELPAIMTHASVLKNDR) phosphorylation was observed, which were identified by Protein Discoverer 2.5.
  • HCD spectra of native Human CSE [334-364] peptide (i) HCD spectra of singly phosphorylated Human CSE [334-364] at Ser346. (iii) HCD spectra of singly phosphorylated Human CSE [334-364] at T355.
  • Panel F shows various human control, WT, or CSE phosphonegative alanine mutant constructs of S346A or T355A were transfected into HEK293 cells and cystathionine consumption measured.
  • Panel G shows HEK293 cells transfected with either Control, WT, CSE glutamic acid (E) phospho-mimetics, S346E or T355E.
  • Figure 2 shows hydrogen sulfide (SF7 fluorophore) or per-polysulfide (SSP4 fluorophore) signal in HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-negative mutant S346A or T355A or S346A/T355A combined under normoxia. H2S levels quantified with fluorescent probe SF7 showed no significant difference. Panels A and B show the ability of cysteine as a substrate, and panels C and D show cystine as a substrate.
  • Figure 3 shows hydrogen sulfide (SF7 fluorophore) or per-polysulfide (SSP4 fluorophore) signal in HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-mimetics mutant S346E or T355E or S346E/T355E combined under normoxia. H2S levels quantified with fluorescent probe SF7 showed no significant difference. Panels A and B show the ability of cysteine as a substrate, and panels C and D show cystine as a substrate.
  • Figure 4 shows hydrogen sulfide (SF7 fluorophore) or per-polysulfide (SSP4 fluorophore) signal in HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-negative mutant S346A or T355A or S346A/T355A combined under hypoxia (1% oxygen) for 30 minutes. H2S levels quantified with fluorescent probe SF7 showed no significant difference. Panels A and B show the ability of cysteine as a substrate, and panels C and D show cystine as a substrate.
  • Figure 5 shows hydrogen sulfide (SF7 fluorophore) or per-polysulfide (SSP4 fluorophore) signal in HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-mimetics mutant S346E or T355E or S346E/T355E combined under hypoxia (1% oxygen). H2S levels quantified with fluorescent probe SF7 showed no significant difference. Panels A and B show the ability of cysteine as a substrate, and panels C and D show cystine as a substrate.
  • Figure 6 shows the effect of cellular hypoxia or tissue ischemia on phosphorylation of CSE and its regulation in vitro and in vivo.
  • Panel A shows SSP4 polysulfide levels were significantly reduced upon inhibition of AMPK (AMPK-I; Dorsomorphin) compared to control (mock).
  • Panel B illustrates representative western blots of p-AMPK and GAPDH from MAECs treated under normoxia or hypoxia for 30 minutes with a graph representing the quantification shown below. Phospho- AMPK (pAMPK) was significantly increased under hypoxic conditions.
  • Panel C shows western blots of pCSE346 and GAPDH from MAECs treated under normoxia or hypoxia.
  • FIG. 1 Graphic representation of the densitometry quantification depicted below shows a significant increase in pCSE346 protein expression under 30 minutes of hypoxia.
  • Panel D illustrates representative blots of pCSE346 and GAPDH from MAECs treated under normoxia, hypoxia, or hypoxia+ AMPK-I.
  • Graphic representation of the densitometry quantification shows a significant increase in pCSE346 protein expression under hypoxia, which is significantly reduced with AMPK-I.
  • pAMPK and pCSE346 were next examined in muscle tissue from mice subject to the femoral artery ligation (FAL) model that establishes tissue ischemia.
  • FAL femoral artery ligation
  • Western blots were performed from non-ischemic (NI) and ischemic (I) skeletal muscle (SkM) tissues collected from day 4 post femoral artery ligation. Graphs represent densitometry quantification of phospho-molecules compared to GAPDH loading control.
  • Panel E shows representative western blots of pAMPK and GAPDH blots with pAMPK significantly increased in ischemic SkM tissues.
  • Figure 7 shows schematic depicting embodiments of the invention.
  • FIG 8 shows CSE Sulfide metabolite formation.
  • CSE uses cysteine or cystathionine as substrates to make H2S but can also use cystine (CysSSCys) to generate cysteine persulfide (CysSSH).
  • CysSSCys cystine persulfide
  • H2S can form HS* radical leading to hydropersulfide or polysulfide.
  • Figure 9 shows endothelial specific CSE genetic deficiency.
  • Panel A shows creation of CSE tmlc conditional mutant allele from the European Mutant Mouse Archive (EMMA).
  • Tmla mice were bred with Flp recombinase mice creating the tmlc allele and subsequently bred with VE- Cad Cre- driver mice to create the VE-Cad cre/CTHA/A.
  • Panel B shows the PCR genotyping of the CTHA/A mutant allele.
  • Panel C shows representative images of gastrocnemius muscle tissue from control and VE-Cad/CTHA/A mice co-stained with CSE, CD31 and DAPI shows significant reduction in CSE expression.
  • Panel D and E shows significant reduction in CSE mRNA in skeletal muscle and aorta respectively.
  • Panel F and G represents reduction in Total sulfide levels of plasma and skeletal muscle tissues in ecCSEKO compared to the control. *p ⁇ 0.01 ecCSEKO vs control.
  • FIG. 10 shows endothelial CSE and ischemic vascular remodeling.
  • Panel A reports a decrease in blood flow in CSEKO and ecCSEKO compared to WT.
  • Panel B shows representative heatmap images of blood flow in WT and ecCSEKO at pre, post and at day7 after ligation respectively.
  • Panel C and D shows representative images and quantification of skeletal muscle tissues showing reduced vascular angiogenic index and arteriole density in ecCSEKO ischemic tissue. *p ⁇ 0.01 ecCSEKO vs WT.
  • FIG 11 shows hypoxia CSE and sulfide metabolism responses.
  • Panel A shows 30 min of hypoxia does not change CSE protein levels, but CSE enzyme activity was increased >2 fold.
  • Panel B reports a significant decrease in acid labile sulfide and increase in bound sulfane sulfur. *p ⁇ 0.01 hypoxia vs control.
  • Figure 12 shows Per/polysulfide effects on MAEC proliferation and permeability.
  • Panel A shows increases in BrdU incorporation due to per and polysulfide treatment.
  • Panel B shows the effect of per/polysulfides on albumin permeability across MAEC monolayers. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 001 vs vehicle.
  • FIG. 13 shows H2S increases hypoxic HUVEC NO formation.
  • Panels A & B show the amount of NO produced in response to increasing amounts of H2S in normoxic and hypoxic HUVEC, respectively.
  • Panel C illustrates the amount of NO that was produced from normoxic and hypoxic HUVEC with various sulfide levels. Treatment with NOS inhibitor L-NAME did not, but the XO inhibitor febuxostat did blunt sulfide dependent NO formation.
  • Figure 14 shows CSE expression and activity in mouse spleen or bone marrow monocytes upon femoral artery ligation.
  • Panels A & B report qRT-PCR CSE mRNA in monocytes from different tissue niches.
  • Panels C & D demonstrate the monocyte CSE activity in WT control and (pCSEKO.
  • Figure 15 shows myeloid CSE and ischemic vascular remodeling.
  • Panel A reports a significant decrease in blood flow in CSEKO and (pCSEKO compared to WT.
  • Panel B shows a representative heatmap images of blood flow in WT control and (pCSEKO at pre, post and at day7 after ligation respectively.
  • Panel C and D shows representative images and corresponding quantification of skeletal muscle tissues showing reduced vascular angiogenic index and arteriole density in (pCSEKO compared to WT. *p ⁇ 0.01 (pCSEKO vs WT.
  • FIG. 16 shows CSE deficiency inhibits myeloid recruitment in ischemic tissues.
  • Representative images of ischemic skeletal muscle tissues of A. Control B. (pCSEKO C. VE-Cad Control and D. ecCSEKO, co-stained with DAPI (blue) and MAC-2 (green) reports a significant decrease in MAC-2 staining.
  • Corresponding quantification is shown in panels E and F. *p ⁇ 0.01 WT vs cell specific CSE KO.
  • Figure 17 shows identification of CSE phosphorylation by mass spectrometry.
  • Panels A and B illustrates peptide motifs of human CSE under normoxic and hypoxic conditions identifying phospho-S346, T355, and S358.
  • Panel C illustrates CSE activity via cystathionine consumption in response to normoxia vs hypoxia (*p ⁇ 0.01).
  • Panel D reports sulfide metabolite levels between cells under normoxic vs hypoxic treatments with a significant increase in bound sulfide (*p ⁇ 0.01).
  • Figure 18 shows inhibition of regulators of CSE affects activity under hypoxia. A significant reduction in CSE activity was observed with inhibition of AMPK, PKC and ATR. *p ⁇ 0.01 inhibitors vs control.
  • Figure 19 shows models of in situ vascular remodeling.
  • Panel A shows SPY angiographic image showing tissue perfusion and collateral arterioles in mouse hindlimbs.
  • Panel B shows microfil vascular casting image of ischemic gracillis arterioles at day 7 following FAL.
  • Figure 20 shows LC-MS/MS detection of different sulfide metabolites.
  • Panels A, B and C illustrates MS of Sdibimane for H2S, SS-dibimane for H2S2 and SSSdibimane for H2S3, respectively. This LC-MS/MS method allows accurate detection of multiple sulfide species.
  • Figure 21 shows increased Ml and reduced M2 macrophage signature in CSE KO and (pCSEKO mice. Ischemic gastrocnemius muscle tissue was harvested 5 days post FAL in WT, CSEKO, (pCSEKO and ecCSEKO mice for Ml vs M2 marker measurement. *p ⁇ 0.01 control vs (pCSEKO.
  • Figure 22 shows specific site mutations of CSE phospho-amino acids for dominant negative or constitutively active analysis. Mutations of Ser346, Thr355, Ser358, or various combinations will be made to analyze effects on CSE enzyme activity and sulfide metabolite formations.
  • Figure 23 shows measurement of PLP by HPLC analysis. Analytical PLP measurement is performed by HPLC achieving nanomole detection sensitivity. Panel A illustrates PLP standard curve; Panel B reports PLP bioavailability in mouse plasma with or without spiked 20 nM PLP. **p ⁇ 0.01 versus plasma alone.
  • Figure 24 shows hypoxic per-polysulfide formation and CSE phosphorylation.
  • Panel a shows MAECs exposed to either normoxia (21% oxygen) or hypoxic (1% oxygen) conditions for 30min and analyzed for various sulfide metabolites, including free, acid-labile sulfide and bound sulfane sulfur (including persulfide and polysulfide) using MBB/HPLC method.
  • Panel b Per- polysulfides levels in MAECs treated with either normoxia or hypoxia for 30min using the fluorescent probe SSP4.
  • Figure 25 shows phospho-negative mutants reduces per-polysulfide levels under hypoxia.
  • HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-negative mutant S346A or T355A under hypoxia (1% oxygen) for 30 minutes and probed for per-polysulfide (SSP4 fluorophore) or hydrogen sulfide (SF7 fluorophore) signal.
  • Figure 26 shows phospho-mimetics induces per-polysulfide levels under hypoxia.
  • HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-mimetic mutants S346E or T355E under hypoxia (1% oxygen) for 30 minutes and probed for per-polysulfide (SSP4 fluorophore) or hydrogen sulfide (SF7 fluorophore) signal.
  • Figure 27 shows molecular dynamics simulations of cystathionine gamma lyase.
  • Molecular dynamics simulations of 350 ns were performed with linear constraint solver (LINCS) constraints for all bonds for a. WT CSE, and b. CSE with phospho sites S346 (purple), and T355 (gold).
  • Molecular dynamics simulations Phosphorylation of residues S346 and T355 modeled in Pymol using the PyTMs plugin. All were performed using GROMACS 2019 software with the GROMOS 54A7 force field and SPC216 water model. Frames were recorded every 2 ps.
  • LINCS linear constraint solver
  • Panel c The 300 ns simulation showing extensive intra- and inter-molecular contacts induced by phosphorylation of 346. Each p346 monomer were color coded belongs to and kept the scheme as shown panel A to illustrate which contacts are intra vs inter-molecular contacts. The backbone RMSD was monitored over the production run of each protein to ensure the stability and convergence of the simulated trajectories.
  • p346 leads to a new interaction between monomer A (p346) and B (K260, R257), monomer B (p346, H217, E345) and monomer A (K260, R257), monomer C (p346, T336) and monomer A (K48), and monomer D (p346, E345, E381) and monomer C (K260).
  • Panel d Intra- and inter-molecular electrostatic interactions formed by phosphorylation of T355.
  • Panel F HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-negative mutants S346A or T355A with or without PAG under hypoxia (1% oxygen) for 30 minutes and probed for per-polysulfide (SSP4 fluorophore). All the data are averaged from triplicates from each experiment with at least n 5. ****P ⁇ 0.0001; ***P ⁇ 0.0002; *P ⁇ 0.01.
  • Figure 28 shows hypoxia induces per-polysulfide via AMPK-mediated phosphorylation of CSE.
  • Panel a Representative blots of MAECs treated under hypoxia for 0, 5, 15, 30, 60 and 90 min probed for pCSES346, total CSE, pAMPK, AMPK and GAPDH.
  • Panel b Quantitation of pCSES346 and p-AMPK protein levels, respectively from western blots in Panel A.
  • Panel c Representative blots of MAECs treated with mock or AMPK inhibitor (AMPK-I), Dorsomorphin under hypoxia for 30min followed by per-polysulfide (SSP4 fluorophore) signal.
  • AMPK-I mock or AMPK inhibitor
  • SSP4 fluorophore per-polysulfide
  • Panel d Quantitation of pCSES346 and pAMPK protein levels from western blots in Panel c.
  • Panel f Representative blots from HUVECs treated under Normoxia or hypoxia or hypoxia+ AMPK-I analyzed for protein levels of pCSES346, total CSE, pAMPK, AMPK and GAPDH.
  • Panel h HUVECs treated under Normoxia or hypoxia or hypoxia+ AMPK-I for 30min followed by per-polysulfide (SSP4 fluorophore) signal. All the data are averaged from triplicates from each experiment with at least n 5. ****P ⁇ 0.0001; ***P ⁇ 0.0002; *P ⁇ 0.01.
  • Figure 29 shows AMPK inhibition reduces CSE phosphorylation, per-polysulfide and ischemic blood flow.
  • Panel a Representative blots of ischemic gastrocnemius muscle tissues from mice subject to the femoral artery ligation (FAL) Ohrs, 3hrs, 24hrs and 5 days probed for pCSES346, total CSE, pAMPK, AMPK and GAPDH.
  • Panel b Graphic representation of the densitometry quantification depicted in Panel A for pCSE346 and p-AMPK protein expression.
  • Panel c Representative western blots were performed from non-ischemic (NI) and ischemic (I) skeletal muscle (SkM) tissues collected from day 4 post femoral artery ligation.
  • Panel d Graphic representation of the densitometry quantification of pCSE346 and p-AMPK protein expression. Graphs represent densitometry quantification of phospho-molecules compared to GAPDH loading control.
  • Panel f Graphic representation of the densitometry quantification 1 depicted in Panel E for ischemic limb blood flow.
  • Figure 30 shows CSE role in sulfide and per-polysulfide.
  • Panels e-i CSE, CBS, MPST, CARS1 and CARS2 mRNA expressions; Panels j-n Representative western blots and quantitation of protein levels of CSE, CBS, MPST, CARS1 and CARS2, following transfection of HEK293 cells with respective siRNA. All the data were averaged from triplicates from each experiment with at least n 5. ****P ⁇ 0.0001; *P ⁇ 0.01.
  • Figure 31 shows CSE mutant conservation.
  • Figure 32 shows sulfide levels in normoxia with phospho negative mutants.
  • H2S levels quantified with fluorescent probe SF7 showed no significant difference.
  • Panels a and b Ability of cysteine as a substrate, and panels c and d show cystine as a substrate.
  • Figure 33 shows sulfide levels in normoxia with phosphomimetic mutants.
  • H2S levels quantified with fluorescent probe SF7 showed no significant difference.
  • Panels a and b show the ability of cysteine as a substrate, and panels c and d show cystine as a substrate.
  • Figure 34 shows per and polysulfide levels in CSE phospho mutants.
  • Abundance to protein fold change signal in MAECs transfected with either wild type CSE (WT) under normoxia (N- WT) or hypoxia (H-WT), phospho negative mutants S346A, T355A; phospho-mimetics mutant S346E or T355E under hypoxia showing Panel a Glutathionine persulfide (GSSH) and Panel b Glutathionine polysulfide (GSSSSH). All the data are averaged from triplicates from each experiment with at least n 3. ****P ⁇ 0.0001; ***P ⁇ 0.0002; **P ⁇ 0.003.
  • Figure 35 shows AMPK regulation of CSE and per-polysulfide in endothelial cells.
  • Panel a CSE phospho sites S346 and T355 showing modular signaling domains of protein Serine/Threonine kinases motif groups using Scansite 4.
  • MAECs treated with si-Con, siAMPKal or AMPKa2 checked for Panel f AMPKal mRNA expression Panel g AMPKa2 mRNA expression.
  • Panel h Representative blots of pCSES346, total CSE, pAMPK, AMPK and GAPDH from MAECs treated under Normoxia or hypoxia, hypoxia+AMPK-I Panel i Quantitation of pCSES346 and p-AMPK protein levels, respectively from western blots in Panel G.
  • Figure 36 shows polysulfide elevates under ischemia.
  • Total sulfide levels including free/acid labile pools (F/Al) and bound Sulfane sulfur levels at 0, 24hr and 5days ischemia from Panel a.
  • Figure 37 to Figure 44 shows Western blot analysis validating antibody specificity, antibody binding data.
  • preventing can refer to inhibiting the full development of a disease, such as tissue injury.
  • Treating can refer to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as tissue injury.
  • Treating refers to the reduction in the number or severity of signs or symptoms of a disease, such as tissue injury.
  • a “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing a pathology, such as tissue injury.
  • terapéutica in conjunction with a phosphospecific antibody, a CSE variant polypeptide or fragment, a CSE variant polynucleotide, a vector, or a cell that is disclosed herein refers to a phosphospecific antibody, a CSE variant polypeptide or fragment, a CSE variant polynucleotide, a vector, or a cell that is suitable for use in human treatment of tissue injury.
  • diagnosis can refer to assessing whether a subject is suffering from a disease, such as tissue injury.
  • administration can refer to the act of the attending physician or caregiver, prescribing the agent for administration and thereby causing the application of an agent to a subject, through ingestion, infusion, injection, or any other means, whether self-administered or administered by a clinician or other qualified care giver.
  • a “subject” can include both human patient and veterinary subjects, including human and non-human mammals. In embodiments, the subject or patient has or has a risk of tissue injury.
  • the term “effective amount” can refer to a quantity sufficient to achieve a desired effect in a subject. For instance, this can be the amount necessary to prevent, treat, or ameliorate a disease, for example, inhibiting or suppressing tissue injury. Efficacy is first evident in the cellular response, for which a variety of in vitro and cell assays are well-known to measure. Kristina V. Kitaeva et al., Cell Culture Based In vitro Test Systems for Anticancer Drug Screening, 8 Front. Bioeng. Biotechnol. 322(2020)). For example, efficacy can be evidenced by increasing or decreasing levels of hydropersulfide, polysulfide, or both after administration of the therapeutic.
  • an effective amount is the amount necessary to significantly increase or decrease inhibit or reduce hydropersulfide levels, polysulfide levels, or both.
  • An effective amount typically provides improvement in important endpoints, including Overall Survival (OS), Disease-Free Survival (DFS), Objective Response Rate, Complete Response Rate or Progression Free Survival (PFS). See Dept, of Health and Human Services, Food and Drug Admin, Clinical Trial Endpoints for the Approval of Cancer Drugs and Biologies: Guidance for Industry (2016); E.A. Eisenhauer et al., New Response Evaluation Criteria in Solid Tumours: Revised RECIST Guideline (Version 1. I), 45 Eur. J. Cancer 228 (2009).
  • tissue injury can refer to a tissue that is experiencing or has experienced cell death, loss of tissue function, reduction of tissue function, fibrosis, DNA damage, and the like. Tissue injury can result from, for example, ischemia or ischemia/reperfusion injury.
  • recombinant polypeptide can refer to a peptide, polypeptide, or protein that results from the expression of a recombinant nucleic acid (e.g., recombinant DNA) within living cell.
  • a recombinant nucleic acid e.g., recombinant DNA
  • synthetic polypeptide can refer to a peptide, polypeptide, or protein that is formed, in vitro, by joining amino acids or amino acid analogs in a particular order, using well known techniques of synthetic organic peptide synthesis to form the peptide bonds, e.g., via solid phase peptide synthesis.
  • fused indicates that at least two polypeptide chains have been operably linked and recombinantly expressed.
  • two polypeptide chains can be “fused” as a result of chemical synthesis.
  • conjugate or “conjugation” can denote that two molecular entities (e.g., two polypeptides, or a polypeptide and a polymer such as PEG) have been chemically linked.
  • the disclosure provides an isolated antibody or fragment thereof, wherein the antibody binds to cystathionine gamma lyase (CSE).
  • CSE cystathionine gamma lyase
  • the disclosure provides an antibody or fragment thereof that binds CSE phosphorylated at amino acid position 346.
  • an "antibody” can refer to an immunoglobulin molecule comprising two heavy chains (HCs) and two light chains (LCs) interconnected by disulfide bonds.
  • the amino terminal portion of each LC and HC includes a variable region of about 100-120 amino acids primarily responsible for antigen recognition via the complementary determining region (CDRs) contained therein.
  • CDRs complementary determining region
  • the CDRs are interspersed with regions that are well-known and generally conserved among and between species (e.g., mouse and human), which are termed framework regions (FRs).
  • FRs framework regions
  • the CDRs are interspersed with FRs.
  • Antibodies disclosed herein have four FRs, termed FR1, FR2, FR3, and FR4.
  • the FRs are human FRs (e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)).
  • the three CDRs of the LC are referred to as “LCDR1, LCDR2, and LCDR3,” and the three CDRs of the HC are referred to as “HCDR1, HCDR2, and HCDR3.”
  • the functional ability of an antibody to bind a particular antigen is largely determined by the six CDRs. Assignment of amino acids to CDR domains within the LCVR and HCVR regions of the antibodies of the present disclosure is based on known numbering conventions (Andrew Martin, Protein Sequence and Structure Analysis of Antibody Variable Domains in Antibody Engineering (Roland Kontermann and Stefan Diibel eds., 2d ed. 2010)).
  • the constant region of the antibody defines the isotype of an antibody.
  • the antibodies of the present disclosure include IgG. IgG antibodies can be further divided into subclasses, e.g., IgGl, IgG2, IgG3, IgG4.
  • the carboxy -terminal portion of each HC defines a constant region primarily responsible for effector function.
  • the antibodies of the present disclosure can have one or more modifications in the constant region of each HC that reduces effector function.
  • a protein is a conservative variant where it contains conservative amino acid substitutions that do not substantially affect or decrease the affinity of a protein.
  • an antibody that binds CSE phosphorylated at serine 346 can include at least 1, 2, 5, 10, or 15 conservative substitutions, for example, in a constant domain, and bind CSE.
  • Conservative amino acid substitution tables providing functionally similar amino acids are well-known to one of ordinary skill in the art.
  • the following groups are examples of amino acids that are considered conservative substitutions for one another: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).
  • a “degenerate variant” can refer to a polynucleotide encoding a polypeptide (such as an antibody or fragment thereof) that includes a sequence that is degenerate based on the genetic code (i.e., the 20 natural amino acids can be specified by more than one codon). All degenerate nucleotide sequences encoding the disclosed antibody and fragment polypeptide sequences are included.
  • sequence identity can refer to as the similarity between amino acid or nucleic acid sequences, which is expressed as the similarity between the sequences. Sequence identity is frequently measured as percent identity, in which two sequences are considered more similar the higher the percentage. Homologs or variants of a polypeptide or nucleic acid molecule possess a relatively high degree of sequence identity when aligned using standard methods, which are well-known.
  • binding can refer to the well understood interaction between and antibody and a target protein, peptide, or polysaccharide. Binding can be measured in a variety of ways (see, e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)).
  • binding can be measured by methods known to the skilled artisan], A particular antibody or protein binds to a particular target protein, peptide, or polysaccharide and does not bind in a significant amount to other proteins or polysaccharides present in a sample or subject disclosed herein. Binding occurs between the disclosed antibodies and fragments thereof and an epitope of CSE.
  • epitope can refer to discrete sites of an antigen recognized by the disclosed antibodies and fragments thereof. Epitopes may be linear or three-dimensional.
  • An antibody binds to a target protein when the interaction has a KD of less than 10' 6 molar, such as less than 10’ 7 molar, less than 10‘ 8 molar, less than 10’ 9 molar, or less than 10’ 10 molar.
  • the antibodies and fragments thereof disclosed herein can bind CSE phosphorylated at amino acid position 346. [000116] The antibodies and fragments thereof disclosed herein can be administered to subjects or patients.
  • the antibody or fragment disclosed herein can be a therapeutic antibody.
  • the term “therapeutic antibody” can refer to an antibody disclosed herein that is suitable for use in human treatment of tissue injury.
  • Such an antibody has a KD of less than 10' 6 molar, such as less than 10' 7 molar, less than 10' 8 molar, less than 10' 9 molar, or less than IO' 10 molar and any toxic or detrimental effects of the antibody are outweighed by the therapeutic beneficial effects.
  • diagnostic in conjunction with an antibody disclosed herein can refer to an antibody suitable for use in detecting and visualizing of its target antigen, such as phosphorylated CSE.
  • Diagnostic antibodies can be used, for example, in assay systems (e.g., ELISA) or for in vitro imaging.
  • the diagnostic antibody can be, for example, a labeled therapeutic antibody, such as an antibody linked to a detectable moiety.
  • detecttable moiety can be used interchangeably with the term “label” and can relate to any moiety capable of being detected (e.g., primary labels and secondary labels).
  • Primary labels include radioisotope-containing moieties (e.g., moieties that contain 32P, 33P, 35S, or 14C), mass-tags, and fluorescent labels, and are signal-generating reporter groups which can be detected without further modifications.
  • primary labels include those useful for positron emission tomography including molecules containing radioisotopes (e.g., 18 F) or ligands with bound radioactive metals (e.g., 62 Cu).
  • primary labels are contrast agents for magnetic resonance imaging such as gadolinium, gadolinium chelates, or iron oxide (e.g., Fe3O4 and Fe2Ch) particles.
  • semiconducting nanoparticles e.g., cadmium selenide, cadmium sulfide, cadmium telluride
  • Other metal nanoparticles e.g., colloidal gold also serve as primary labels.
  • “Secondary” labels include moieties such as biotin, or protein antigens, that require the presence of a second compound to produce a detectable signal.
  • the second compound may include streptavidin-enzyme conjugates.
  • the second compound may include an antibody-enzyme conjugate.
  • certain fluorescent groups can act as secondary labels by transferring energy to another compound or group in a process of nonradiative fluorescent resonance energy transfer (FRET), causing the second compound or group to then generate the signal that is detected.
  • FRET nonradiative fluorescent resonance energy transfer
  • mass-tag can refer to any compound that is capable of being uniquely detected by virtue of its mass using mass spectrometry (MS) detection techniques.
  • mass-tags include electrophore release tags such as N-[3-[4'-[(p- methoxytetrafluorobenzyl)oxy]phenyl]-3-methylglyceronyl]-isonipecotic acid, 4'-[2, 3,5,6- tetrafluoro-4-(pentafluorophenoxyl)]methyl acetophenone, and their derivatives.
  • electrophore release tags such as N-[3-[4'-[(p- methoxytetrafluorobenzyl)oxy]phenyl]-3-methylglyceronyl]-isonipecotic acid, 4'-[2, 3,5,6- tetrafluoro-4-(pentafluorophenoxyl)]methyl acetophenone, and their derivatives.
  • electrophore release tags such as N-[3-
  • mass-tags include, but are not limited to, nucleotides, dideoxynucleotides, oligonucleotides of varying length and base composition, oligopeptides, oligosaccharides, and other synthetic polymers of varying length and monomer composition.
  • a large variety of organic molecules, both neutral and charged (biomolecules or synthetic compounds) of an appropriate mass range (100-2000 Daltons) may also be used as masstags.
  • fluorescent label can refer to compounds or moieties that absorb light energy at a defined excitation wavelength and emit light energy at a different wavelength.
  • fluorescent compounds include, but are not limited to: Alexa Fluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), AMCA, AMCA-S, anthracene, BODIPY dyes (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), carbazole, Carboxyrhodamine 6G, carboxy -X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Coumarin 343, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5), Dansy
  • antibody fragments such as well-characterized Fabs (e.g., Fab, Fab 1 , F(ab')2,), Fvs (the variable region of the light chain and the variable region of the heavy chain expressed as two chains), and single-chain fragments e.g., single-chain variable region fragments, scFv, and single chain Fabs, scFab), which also bind to CSE.
  • Fabs e.g., Fab, Fab 1 , F(ab')2,
  • Fvs the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • single-chain fragments e.g., single-chain variable region fragments, scFv, and single chain Fabs, scFab
  • Methods of making these fragments are routine (see, e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)).
  • mAbs monoclonal antibodies
  • mAbs can be produced, for example, by hybridoma technologies, recombinant technologies, phage display technologies, synthetic technologies (e.g., CDR or specificity-determining residue, SDR, grafting), or combinations of such or other technologies known in the art.
  • mAbs are antibodies derived from a single copy or clone including, for example, any eukaryotic, prokaryotic or phage clone.
  • a variety of well-known methods and tools can be used for producing and purifying the mAbs disclosed herein, including vectors, for example, plasmids, virus, or other vehicles for polynucleotide insertion or expression, and hosts, for example, microbial, yeast, insect, and mammalian organisms (see, e.g., Process Scale Purification of Antibodies (Uwe Gottschalk, ed., 2d ed. 2017)).
  • the antibodies herein are recombinant antibodies.
  • “Recombinant antibodies” can refer to antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or antibodies prepared, expressed, created, or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • an animal e.g., a mouse
  • recombinant antibodies include humanized, CDR grafted, chimeric, in vitro generated (e.g., by phage display) antibodies, and can optionally include constant regions derived from human germline immunoglobulin sequences.
  • “recombinant antibody” can direct to a portion of an intact antibody, including, without limitation, Fv, Fab, Fab', F(ab')2, diabodies, scFv, and single domain antibodies (e.g., variable heavy domain (VHH)).
  • VHH variable heavy domain
  • antibodies having a complementary binding means can be prepared and screened by well-known methods, such as hybridoma, transgenic animals, and phage or yeast display (see, e.g., Monoclonal Antibodies: Methods and Protocols (Vincent Ossipow and Nicolas Fischer, eds., 2d ed. 2014)).
  • Antibodies having equivalent complementary binding means differ in their amino acid sequence but perform the same function of binding the target through CDR-target interaction acting as (inhibitor/agonist/antagonist) to achieve the same result (inhibiting tumor growth).
  • the complementary binding means functions through the same epitope as the disclosed antibodies.
  • the nucleic acids described herein can be found in a vector.
  • vector can refer to a nucleic acid molecule capable of transporting another nucleic acid molecule in a host cell.
  • examples of vectors include plasmids, viral vectors, naked DNA or RNA expression vectors, cosmid or phage vectors.
  • Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Some vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., non-episomal mammalian vectors).
  • Certain vectors are capable of directing the expression of genes to which they are operatively linked, and therefore may be referred to as “expression vectors.”
  • tissue injury can refer to a tissue that is experiencing or has experienced cell death, loss of tissue function, reduction of tissue function, fibrosis, DNA damage, and the like. Tissue injury can result from, for example, ischemia or ischemia/reperfusion injury.
  • the antibodies and fragments thereof disclosed herein can be used for diagnosing a subject afflicted with or at risk of a tissue injury.
  • the antibodies and fragments thereof disclosed herein can be used in a drug screening method.
  • the antibodies and fragments thereof disclosed herein can detect phosphorylated CSE, such as CSE phosphorylated at position 346, and thereby can be used in a drug screening method.
  • the antibodies and fragments thereof disclosed herein can be used in therapy.
  • the antibodies and fragments thereof disclosed herein can be used to treat, prevent (such as through prophylactic treatment), or ameliorate a tissue injury.
  • CSE Homo sapiens cystathionine gamma lyase
  • CSE is a cytoplasmic enzyme in the transsulfuration pathway that converts cystathione derived from methionine into cysteine. Glutathione synthesis in the liver is dependent upon the availability of cysteine. Mutations in this gene cause cystathioninuria. Alternative splicing of this gene results in three transcript variants encoding different isoforms.
  • Figure 27 shows the three-dimensional structure of CSE as a tetramer. Tetramer formation is critical for CSE catalytic activity and the production of hydropersulfide and polysulfide. As shown herein, phosphorylation at S346 and at T355 is implicated in tetramer formation.
  • cystathionine gamma lyase (CSE) phosphorylation due to hypoxia increases cellular and tissue hydropersulfide and polysulfide, critical redox regulators that antagonize oxidative stress.
  • hypoxia increases AMP kinase dependent CSE phosphorylation of Serine 346 and Threonine 355 that selectively increases CSE dependent hydropersulfide and polysulfide formation without increased hydrogen sulfide generation.
  • aspects of the invention are drawn to CSE variants (e.g., phosphomimetic
  • CSE variants that can modulate cellular hydropersulfide and poly sulfide in subjects who are in need thereof.
  • CSE variant can refer to molecules that can modulate cellular hydropersulfide levels, polysulfide levels, or both, in a cell or in a subject. Accordingly, the term encompasses, e.g., CSE variant polypeptides or fragments, polynucleotides encoding such CSE variant polypeptides or fragments, vectors (e.g., vectors comprising polynucleotides encoding CSE variant polypeptides or fragments), micelles (e.g., micelles comprising CSE variant polypeptides or fragments, polynucleotides or vectors), and/or cells (e.g., cell comprising CSE variant polypeptides or fragments, polynucleotides or vectors).
  • vectors e.g., vectors comprising polynucleotides encoding CSE variant polypeptides or fragments
  • micelles e.g., micelles comprising CSE variant polypeptides or fragments, polynucleo
  • the CSE variant can increase cellular hydropersulfide levels, polysulfide levels, or both, in a cell or in a subject.
  • the CSE variant can decrease cellular hydropersulfide levels, polysulfide levels, or both, in a cell or in a subject.
  • fragment or polypeptide fragment can refer to a polypeptide having one or more (several) amino acids deleted from the amino and/or carboxyl terminus of the mature polypeptide or a homologous sequence thereof.
  • the polypeptide fragment has activity as the mature polypeptide thereof.
  • the polypeptide fragment has activity that is different from the mature polypeptide thereof.
  • the activity of the polypeptide fragment can be inhibitory of the the activity of the mature polypeptide.
  • the fragment polypeptide can refer to a fragment polypeptide, so long as the fragment polypeptide comprises Ser346, Thr355, or both.
  • a fragment polypeptide can refer to a CSE fragment comprising amino acid positions 334-364 (LFTLAESLGGFESLAELPAIMTHASVLKNDR - SEQ ID NO: 2).
  • the phosphomimetic fragment polypeptide can inhibit CSE catalytic activity by inhibiting CSE tetramer formation.
  • the phosphomimetic fragment polypeptide can inhibit the interaction between adjacent CSE monomers, thereby inhibiting tetramer formation and subsequent catalytic activity. Accordingly, certain CSE phosphomimetic fragments may lower polysulfide levels, hydrosulfilde levels, or both.
  • Embodiments can comprise a polynucleotide encoding a CSE variant polypeptide (e.g., a recombinant polypeptide or a synthetic polypeptide) or fragment thereof comprising at least one phosphorylatable amino acid selected from position 346 and/or 355, wherein the at least one phosphorylatable amino acid (e.g., Ser 346 and/or Thr355) or a combination thereof has been replaced (i.e., substituted, or mutated) with a phosphomimetic amino acid (e g., Asp or Glu) or analog (e.g., a non-hydrolyzable analog).
  • a CSE variant polypeptide e.g., a recombinant polypeptide or a synthetic polypeptide
  • fragment thereof comprising at least one phosphorylatable amino acid selected from position 346 and/or 355, wherein the at least one phosphorylatable amino acid (e.g., Ser 346 and/or Thr35
  • CSE variant polypeptides or fragments comprising phosphomimicking amino acids would mimic CSE in a phosphorylated state, thereby modulating cellular and tissue levels of hydropersulfide and polysulfide.
  • these CSE variant polypeptides or fragments can increase cellular and tissue levels of hydropersulfide and polysulfide.
  • full length phosphomimetic CSE protein may mimic CSE in a phosphorylated state, thereby increasing cellular and tissue levels of hydropersulfide and polysulfide.
  • these CSE variant polypeptides, such as CSE variant fragment polypeptides can decrease cellular and tissue levels of hydropersulfide and polysulfide.
  • embodiments provide a phosphomimetic CSE variant polypeptide or fragment comprising a sequence according to SEQ ID NO: 1 and/or SEQ ID NO: 2, wherein the amino acid according to CSE wild-type position 346 is mutated from a serine to a phosphomimetic amino acid, wherein the amino acid according to CSE wild-type position 355 is mutated from a threonine to a phosphomimetic amino acid, or both.
  • phosphomimetic amino acid can refer to an ammo acid that mimics a phosphorylated amino acid.
  • proteins are commonly modified at serine, threonine, and tyrosine amino acids by adding a phosphate group.
  • some non-phosphorylated amino acids appear chemically similar to phosphorylated amino acids.
  • aspartic acid is chemically similar to phospho-serine.
  • an aspartic acid replaces a serine, it is a phosphomimetic of phospho-serine and can make the protein function like it was in its phosphorylated form.
  • the phosphomimetic amino acid is aspartic acid (D) or glutamic acid (E).
  • the aspartic acid is L aspartic acid.
  • the aspartic acid is D aspartic acid.
  • the glutamic acid is L glutamic acid.
  • the glutamic acid is D glutamic acid.
  • the phosphomimetic amino acid is phosphoserine or phosphothreonine.
  • the phosphomimetic amino acid is L phosphoserine, D phosphoserine, L phosphothreonine, or D phosphothreonine.
  • the phosphomimetic amino acid analog is a non-cleavable analog, i.e., an amino acid analog having a group mimicking a phosphate group, wherein the mimicking group cannot be hydrolyzed by phosphatases and/or other enzymes.
  • the non-cleavable analog is, c. ., a phosphoserine non-hydrolyzable analog.
  • the non-hydrolyzable analog of phosphoserine is, e.g., 2-amino-4-phosphobutyric acid.
  • the phosphomimetic amino acid analog is, e.g., a thiophosphate analog.
  • the thiophosphate analog is, e.g., thiophosphoserine.
  • the phosphomimetic amino acid analog is, e.g., a sulfate analog.
  • the sulfate analog is, e.g., sulfoserine.
  • the CSE variant polypeptide or fragment comprises an N-terminal capping modification, a C-terminal capping modification, or a combination thereof [x-polypeptide- Y], wherein “x” is an N-terminal modification, “y” is a C-terminal modification (e.g., a capping modification), and the polypeptide is a CSE variant polypeptide or fragment thereof.
  • x and “y” can be peptide endings different from those found in a naturally occurring isolated polypeptide.
  • Capping modifications can be introduced at the termini of chemically synthesized peptides to increase their resistance to proteolytic degradation.
  • the N-terminal modification is acetylation. See, e.g., Thomas (2011) PLOS Biol. 9; Wallace (1992) Br. J. Nutr. 68:365-72. This modification removes the positive charge of the N-terminal of peptides, thus mimicking natural proteins and increasing peptide stability by preventing N-terminal degradation.
  • the C-terminal modification is amidation. This modification neutralizes the negative charge created by the C-terminal COOH.
  • This modification is added to prevent enzyme degradation, to mimic native proteins, and in some cases to remove hydrogen bonding at the C-terminal of the peptides. See, e.g., Kim et al. (2001) Biotechnol. Bioprocess Eng. 6:244-51.
  • Non-limiting examples of N-terminal capping modifications comprises an N-terminal acetylation, formylation, acylation, pyroglutamylation, or carbamate, sulfonamide, or alkylamine modification.
  • Non-limiting examples of C-terminal capping modifications comprises a C-terminal amidation, N-alkyl amidation, aldehyde modification, or esterification.
  • the CSE variant polypeptide or fragment can be flanked on its N- terminus by 1, 2, 3, 4, 5, or more additional amino acids.
  • the additional N-terminal amino acids are corresponding native amino acids from the wild-type amino acid sequence of CSE. See Table 10, for example.
  • the CSE variant polypeptide or fragment comprises SEQ ID NO: 2 flanked on its N-terminus by 1, 2, 3, 4, 5, or more additional amino acids.
  • the CSE variant polypeptide or fragment can be flanked on its C- terminus by 1, 2, 3, 4, 5, or more additional amino acids.
  • the additional C-terminal amino acids are corresponding native amino acids from the wild-type amino acid sequence of CSE. See Table 10, for example.
  • the CSE variant polypeptide or fragment comprises SEQ ID NO: 2 flanked on its C-terminus by 1, 2, 3, 4, 5, or more additional amino acids.
  • the CSE variant polypeptide or fragment can be flanked by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
  • the CSE variant polypeptide or fragment comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the sequence as set forth in SEQ ID NO: 1, or a fragment thereof.
  • the CSE variant polypeptide or fragment consists of or consists essentially of the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, the CSE variant polypeptide or fragment consists of or consists essentially of the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2, except for 1, 2, 3, 4 or 5 amino acid substitutions, e.g., conservative amino acid substitutions.
  • the CSE variant polypeptide or fragment comprises at least 14 amino acids, at least 15 amino acids, at least 16 amino acids, at least 17 amino acids, at least 18 amino acids, at least 19 amino acids, at least 20 amino acids, at least 21 amino acids, at least 22 amino acids, at least 23 amino acids, or at least 24 amino acids in length.
  • the polypeptide has at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124
  • the CSE variant polypeptide or fragment comprises at least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, at least about 50 amino acids, at least about 60 amino acids, at least about 70 amino acids, at least about 80 amino acids, at least about 90 amino acids, at least about 100 amino acids, at least about 120 amino acids, at least about 140 amino acids, at least about 160 amino acids, at least about 180 amino acids, at least about 200 amino acids, at least about 220 amino acids, at least about 240 amino acids, at least about 260 amino acids, at least about 280 amino acids, at least about 300 amino acids, at least about 320 amino acids, at least about 340 amino acids, at least about 360 amino acids in length, or about 366 amino acids in length.
  • the CSE variant polypeptide or fragment comprises a fusion protein or conjugate comprising at least one heterologous moiety.
  • heterologous moiety can refer to any molecule (chemical or biological), e.g., a half-life extending moiety or a detectable moiety, that is different from a CSE variant polypeptide or fragment disclosed herein, and which is genetically fused, conjugated, and/or otherwise associated to the CSE variant polypeptide or fragment.
  • the heterologous moiety comprises a serum half-life extending moiety.
  • half-life extending moiety can refer to a pharmaceutically acceptable moiety, domain, or molecule covalently linked (“conjugated” or “fused”) to a CSE variant polypeptide or fragment, optionally via a non-naturally encoded amino acid, directly or via a linker, that prevents or mitigates in vivo proteolytic degradation or other activity-diminishing chemical modification of the CSE variant polypeptide or fragment, increases half-life, and/or improves or alters other pharmacokinetic or biophysical properties including but not limited to increasing the rate of absorption, reducing toxicity, improving solubility, reducing protein aggregation, increasing biological activity and/or target selectivity of the CSE variant polypeptide or fragment, increasing manufacturability, and/or reducing immunogenicity of the CSE variant polypeptide or fragment, compared to a reference compound such as a non-conjugated or non
  • the serum half-life extending moiety comprises an Fc region or portion thereof, albumin, albumin binding polypeptide, a fatty acid, PAS, a glycine-rich homo-amino- acid polymer (HAP), the R subunit of the C-terminal peptide (CTP) of human chorionic gonadotropin, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN, albumin-binding small molecules, or a combination thereof.
  • HAP glycine-rich homo-amino- acid polymer
  • CTP C-terminal peptide
  • PEG polyethylene glycol
  • HES hydroxyethyl starch
  • XTEN albumin-binding small molecules
  • fusion proteins comprising an Fc region of an immunoglobulin bestow several desirable properties on a fusion protein including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337:525) as well as binding to Fc receptors such as the neonatal Fc receptor (FcRn) (U.S. Pat. Nos. 6,086,875, 6,485,726, 6,030,613; WO 03/077834; US2003- 0235536A1).
  • FcRn neonatal Fc receptor
  • the half-life extension moiety linked, e.g., fused or conjugated, to a CSE variant polypeptide or fragment is an albumin binding moiety, which comprises an albumin binding peptide, a bacterial albumin binding domain, an albumin-binding antibody fragment, or any combinations thereof.
  • the albumin binding protein can be a bacterial albumin binding protein, an antibody or an antibody fragment including domain antibodies (see U.S. Pat. No. 6,696,245).
  • An albumin binding protein for example, can be a bacterial albumin binding domain, such as the one of streptococcal protein G (Konig, T. and Skerra, A. (1998) J. Immunol. Methods 218, 73- 83).
  • albumin binding peptides that can be used as conjugation partner are, for instance, those having a Cys-Xaa 1-Xaa 2-Xaa 3-Xaa 4-Cys consensus sequence, wherein Xaa 1 is Asp, Asn, Ser, Thr, or Trp; Xaa 2 is Asn, Gin, H is, He, Leu, or Lys; Xaa 3 is Ala, Asp, Phe, Trp, or Tyr; and Xaa 4 is Asp, Gly, Leu, Phe, Ser, or Thr as described in US patent application 2003/0069395 or Dennis et al. (Dennis et al. (2002) J. Biol. Chem. 277, 35035-35043).
  • the half-life extension moiety linked, e.g., fused or conjugated, to a CSE variant polypeptide or fragment is a PAS sequence.
  • a “PAS sequence” can refer to an amino acid sequence comprising mainly alanine and serine residues or comprising mainly alanine, serine, and proline residues, the amino acid sequence forming random coil conformation under physiological conditions. Accordingly, the PAS sequence is a building block, an amino acid polymer, or a sequence cassette comprising, consisting essentially of, or consisting of alanine, serine, and proline which can be used as a part of the heterologous moiety in the fusion protein. Exemplary PAS sequences are provided, e.g., in US Pat. Publ. No. 2010/0292130 Al and PCT Appl. Publ. No. WO 2008/155134 Al, both of which are incorporated by reference in their entireties.
  • the half-life extension moiety linked, e.g., fused or conjugated, to a CSE variant polypeptide or fragment is a glycine-rich homo-amino-acid polymer (HAP).
  • HAP sequence can comprise a repetitive sequence of glycine, which has at least 50 amino acids, at least 100 amino acids, 120 amino acids, 140 amino acids, 160 amino acids, 180 amino acids, 200 amino acids, 250 amino acids, 300 amino acids, 350 amino acids, 400 amino acids, 450 amino acids, or 500 amino acids in length.
  • Non-limiting examples of the HAP sequence includes, but are not limited to (Gly)n, (Gly4Ser)n or S(Gly4Ser)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
  • n is 20, 21, 22, 23, 24, 25, 26, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.
  • n is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200.
  • the half-life extension moiety linked, e.g., fused or conjugated, to a CSE variant polypeptide or fragment is a soluble polymer known in the art, including, but not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, or polyvinyl alcohol.
  • PEG polyethylene glycol
  • the soluble polymer can be attached to any positions within the sequence SETD7 modulator of the present disclosure or at either or both termini.
  • the soluble polymer can be attached at random positions within the CSE variant polypeptide or fragment or at predetermined positions within the CSE variant polypeptide or fragment sequence and may include one, two, three or more attached soluble polymer moieties.
  • the polymer can be attached to a side chain of a naturally occurring amino acid.
  • the polymer can be attached to a side chain of a non-naturally encoded amino acid, e.g., a phenylalanine derivative such as para-acetyl-L-phenylalanine.
  • the soluble polymer can be of any molecular weight, and can be branched or unbranched.
  • the half-life extension moiety linked, e.g., fused or conjugated, to a CSE variant polypeptide or fragment is hydroxyethyl starch (HES) or a derivative thereof.
  • HES hydroxyethyl starch
  • HES is a derivative of naturally occurring amylopectin and is degraded by alpha-amylase in the body.
  • HES is a substituted derivative of the carbohydrate polymer amylopectin, which is present in corn starch at a concentration of up to 95% by weight.
  • HES exhibits advantageous biological properties and is used as a blood volume replacement agent and in hemodilution therapy in the clinics (Sommermeyer et al., Whypharmazie, 8(8), 271-278 (1987); and Weidler et al., Arzneim. Deutschen/Drug Res., 41, 494-498 (1991)).
  • the half-life extension moiety linked, e.g., fused or conjugated, to a CSE variant polypeptide or fragment is a polysialic acid (PSA) or a derivative thereof.
  • PSAs are naturally occurring unbranched polymers of sialic acid produced by certain bacterial strains and in mammals in certain cells Roth J., et al. (1993) in Polysialic Acid: From Microbes to Man, eds Roth J., Rutishauser U., Troy F. A. (Birkhauser Verlag, Basel, Switzerland), pp 335-348.
  • the half-life extension moiety linked, e.g., fused or conjugated, to a CSE variant polypeptide or fragment is an XTEN sequence.
  • XTEN sequence refers to extended length polypeptides with non-naturally occurring, substantially non-repetitive sequences that are composed mainly of small hydrophilic amino acids, with the sequence having a low degree or no secondary or tertiary structure under physiologic conditions.
  • XTENs can serve as a carrier, conferring certain desirable pharmacokinetic, physicochemical and pharmaceutical properties when linked to a CSE variant polypeptide or fragment to create a fusion protein.
  • Such desirable properties include but are not limited to enhanced pharmacokinetic parameters and solubility characteristics.
  • Examples of XTEN sequences that can be used according to the present disclosure are disclosed in US Patent Publication Nos. 2010/0239554 Al, 2010/0323956 Al, 2011/0046060 Al, 2011/0046061 Al, 2011/0077199 Al, or 2011/0172146 Al, or International Patent Publication Nos. WO 2010091122 Al, WO 2010144502 A2, WO 2010144508 Al, WO 2011028228 Al, WO 2011028229 Al, or WO 2011028344 A2, all of which are herein incorporated by reference in their entireties.
  • the serum half-life of a CSE variant polypeptide or fragment comprising a half-time extending moiety is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% higher than the plasma half-life of a corresponding CSE variant polypeptide or fragment without serum half-life extending moiety.
  • the at least one heterologous moiety comprises a detectable moiety, e g., a radionuclide, a fluorescent molecule, or a contrast agent.
  • aspects of the invention are also drawn towards a nucleic acid encoding a CSE variant polypeptide or fragment (e.g., a CSE variant polynucleotide) as described herein.
  • the a CSE variant polynucleotide comprises or consists of a fragment or variant of the sequence as set forth in NCBI Reference Sequence NG_008041.1.
  • the CSE variant polynucleotide comprises a nucleic acid sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity the polynucleotide encoding a CSE variant polypeptide or fragment as described herein.
  • the nucleic acid encoding the CSE variant polypeptide or fragment comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (base substitutions).
  • the sequence is an RNA sequence.
  • the sequence is an RNAZDNA sequence.
  • the polynucleotide encoding the CSE variant is mRNA.
  • the polynucleotide encoding the CSE variant is a DNA.
  • the polynucleotide encoding the CSE variant is inserted in a vector.
  • the CSE variant polynucleotide comprises a “synthetic nucleic acid”.
  • a “synthetic nucleic acid” can refer to a nucleic acid that does not have the chemical structure or sequence of a natural nucleic acid.
  • the CSE variant polynucleotide comprises at least one chemically modified nucleobase, sugar, backbone, or any combination thereof.
  • the CSE variant polynucleotide can comprise one or more modifications.
  • the modification comprises the use of a pseudo-uracil or Nl-methyl pseudo-uracil. Such modifications decrease immune reactions, increases mRNA stability, and increased ability for mRNA translation.
  • the CSE variant polynucleotide comprises at least one nucleotide analogue.
  • the nucleotide analogue can be introduced by using IVT (in vitro transcription) or chemical synthesis.
  • the nucleotide analogue can be selected from the group consisting of a 2'-O-methoxyethyl-RNA (2'-M0E-RNA) monomer, a 2'-fluoro-DNA monomer, a 2'-O-alkyl- RNA monomer, a 2'-amino-DNA monomer, a locked nucleic acid (LNA) monomer, a cEt monomer, a cMOE monomer, a 5'-Me-LNA monomer, a 2'-(3-hydroxy)propyl-RNA monomer, an arabino nucleic acid (ANA) monomer, a 2'-fluoro-ANA monomer, an anhydrohexitol nucleic acid (ANA) monomer, a
  • the CSE variant polynucleotide can be chemically modified at terminal locations, for example by introducing M (2'-O-methyl), MS (2'-O-methyl 3' phosphorothioate), or MSP (2'-O-methy 3 'thioPACE, phosphonoacetate) modifications, or combinations thereof at positions 1, 2, 3 respect to the 5' and/or 3' termini.
  • Modified CSE variant polynucleotides need not be uniformly modified along the entire length of the molecule.
  • Different nucleotide modifications and/or backbone structures can exist at various positions in the nucleic acid.
  • nucleotide analogs or other modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid is not substantially decreased.
  • a modification may also be a 5' or 3' terminal modification.
  • the nucleic acids may contain at a minimum one and at maximum 100% modified nucleotides, or any intervening percentage, such as at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% modified nucleotides.
  • the CSE variant polynucleotide can include modifications to prevent rapid degradation by endo- and exo-nucleases.
  • Modifications include, but are not limited to, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, as well as (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages.
  • end modifications e.g., 5' end modifications (phosphorylation dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucle
  • synthetic, modified CSE variant polynucleotide include, but are not limited to, polynucleotides containing modified or non-natural intemucleoside linkages.
  • Synthetic, modified CSE variant polynucleotides having modified internucleoside linkages include, among others, those that do not have a phosphorus atom in the internucleoside linkage.
  • the synthetic, modified CSE variant polynucleotide has a phosphorus atom in its intemucleoside linkage(s).
  • Non-limiting examples of modified intemucleoside linkages include phosphorothioates, chiral phosphorothi oates, phosphorodithioates, phosphotri esters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 '-5' linkages, T-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or T-5' to 5'-T.
  • Various salts, mixed salts and free acid forms are also included.
  • Modified intemucleoside linkages that do not include a phosphorus atom therein have intemucleoside linkages that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH2 component parts.
  • the CSE variant polynucleotide can be codon optimized by introducing one or more synonymous codon changes.
  • codon optimization or “codon optimized” can refer to the modification of the primary sequence of a nucleic acid by replacing synonymous codons in order to increase its translational efficiency. Accordingly, codon optimization comprises switching the codons used in a SETD7 modulator polynucleotide of the present disclosure without changing the amino acid sequence that it encodes for, which typically dramatically increases the abundance of the protein the codon optimized gene encodes because it generally removes “rare” codons and replaces them with abundant codons, or removes codon with a low tRNA recharge rate with codon with high tRNA recharge rates.
  • Such codon optimization can, for example, (i) improve protein yield in recombinant protein expression, or (ii) improve the stability, half life, or other desirable property of an mRNA or a DNA encoding a binding molecule disclosed herein, wherein such mRNA or DNA is administered to a subject in need thereof.
  • a CSE variant polynucleotide can be codon optimized using any methods known in the art at the time the present application was filed.
  • a CSE variant polynucleotide has been sequence optimized.
  • sequence optimized can refer to the modification of the sequence of a nucleic acid by to introduce features that increase its translational efficiency, remove features that reduce its translational efficiency, or in general improve properties related to expression efficacy after administration in vivo.
  • properties include, but are not limited to, improving nucleic acid stability (e.g., mRNA stability), increasing translation efficacy in the target tissue, reducing the number of truncated proteins expressed, improving the folding or prevent misfolding of the expressed proteins, reducing toxicity of the expressed products, reducing cell death caused by the expressed products, or increasing and/or decreasing protein aggregation.
  • Embodiments also provide a vector comprising an isolated polynucleotide, e.g., a CSE variant polynucleotide disclosed herein.
  • the vector is viral vector.
  • the viral vector is an adenoviral vector or an adenoassociated viral vector.
  • the viral vector is a retroviral vector, e.g., a lenti viral vector.
  • the CSE variant polynucleotide comprises a virus, a plasmid, or a phagemid encoding a CSE variant.
  • the virus is selected from the group consisting of an adeno-associated virus (AAV), a retrovirus, a lentivirus, an adenovirus, an SV40-type virus, a polyomavirus, an Epstein-Barr virus, a papilloma virus, a herpes virus, a vaccinia virus, a polio virus, and an RNA virus.
  • AAV adeno-associated virus
  • retrovirus a retrovirus
  • a lentivirus an adenovirus
  • an SV40-type virus a polyomavirus
  • Epstein-Barr virus a papilloma virus
  • a herpes virus a vaccinia virus
  • polio virus RNA virus
  • a nucleic acid sequence encoding a CSE variant polypeptide or fragment can be inserted into the genome of a target cell or a host cell (e.g., a stem cell for transplantation to the target tissue) by using CRISPR/Cas systems and genome edition alternatives such as zine-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases (MNs).
  • ZFNs zine-finger nucleases
  • TALENs transcription activator-like effector nucleases
  • MNs meganucleases
  • embodiments herein also provide cells comprising a CSE variant polynucleotide, or a vector comprising a SETD7 modulator polynucleotide.
  • Embodiments can comprise the use of a gene editing tool to modulate (e.g., increase or decrease) the expression of a gene encoding a CSE variant polypeptide or fragment.
  • the gene editing tool that can be used as described herein comprises a CRISPR/Cas system.
  • CRISPR/Cas systems can employ, for example, a Cas9 nuclease, which in some instances, is codon-optimized for the desired cell type in which it is to be expressed (e.g., T cells, e.g., CAR-expressing T cells).
  • CRISPR/Cas systems use Cas nucleases, e.g., Cas9 nucleases, that are targeted to a genomic site by complexing with a synthetic guide RNA (gRNA) that hybridizes to a target DNA sequence immediately preceding an NGG motif recognized by the Cas nuclease, e.g., Cas9.
  • gRNA synthetic guide RNA
  • a unique capability of the CRISPR/Cas9 system is the ability to simultaneously target multiple distinct genomic loci by co-expressing a single Cas9 protein with two or more gRNAs (e.g., at least one, two, three, four, five, six, seven, eight, nine or ten gRNAs).
  • gRNA guide RNA
  • the two-molecule gRNA comprises a crRNA-like (“CRISPR RNA” or “targeter-RNA” or “crRNA” or “crRNA repeat”) molecule and a corresponding tracrRNA-like (“trans-acting CRISPR RNA” or “activator-RNA” or “tracrRNA” or “scaffold”) molecule.
  • CRISPR RNA or “targeter-RNA” or “crRNA” or “crRNA repeat”
  • a corresponding tracrRNA-like trans-acting CRISPR RNA” or “activator-RNA” or “tracrRNA” or “scaffold”
  • a crRNA comprises both the DNA-targeting segment (single stranded) of the gRNA and a stretch of nucleotides that forms one half of a double stranded RNA (dsRNA) duplex of the protein-binding segment of the gRNA.
  • a corresponding tracrRNA comprises a stretch of nucleotides that forms the other half of the dsRNA duplex of the protein-binding segment of the gRNA.
  • a stretch of nucleotides of a crRNA is complementary to and hybridizes with a stretch of nucleotides of a tracrRNA to form the dsRNA duplex of the protein-binding domain of the gRNA.
  • each crRNA can be said to have a corresponding tracrRNA.
  • the crRNA additionally provides the single stranded DNA-targeting segment.
  • a gRNA comprises a sequence that hybridizes to a target sequence (e.g., SETD7 mRNA), and a tracrRNA.
  • a crRNA and a tracrRNA hybridize to form a gRNA. If used for modification within a cell, the exact sequence and/or length of a given crRNA or tracrRNA molecule can be designed to be specific to the species in which the RNA molecules will be used (e.g., humans).
  • CRISPR RNAs differ depending on the Cas9 system and organism but often contain a targeting segment of between 21 to 72 nucleotides length, flanked by two direct repeats (DR) of a length of between 21 to 46 nucleotides (see, e.g., WO2014/131833).
  • DR direct repeats
  • the DRs are 36 nucleotides long and the targeting segment is 30 nucleotides long.
  • the 3' located DR is complementary to and hybridizes with the corresponding tracrRNA, which in turn binds to the Cas9 protein.
  • a CRISPR system used herein can further employ a fused crRNA- tracrRNA construct (i.e., a single transcript) that functions with the codon-optimized Cas9.
  • This single RNA is often referred to as a guide RNA or gRNA.
  • the crRNA portion is identified as the “target sequence” for the given recognition site and the tracrRNA is often referred to as the “scaffold.”
  • a short DNA fragment containing the target sequence is inserted into a guide RNA expression plasmid.
  • the gRNA expression plasmid comprises the target sequence (in some aspects around 20 nucleotides), a form of the tracrRNA sequence (the scaffold) as well as a suitable promoter that is active in the cell and necessary elements for proper processing in eukaryotic cells.
  • the scaffold a form of the tracrRNA sequence
  • a suitable promoter that is active in the cell and necessary elements for proper processing in eukaryotic cells.
  • Many of the systems rely on custom, complementary oligos that are annealed to form a double stranded DNA and then cloned into the gRNA expression plasmid.
  • the gRNA expression cassette and the Cas9 expression cassette are then introduced into the cell. See, for example, Mali P et al., (2013) Science 2013 Feb. 15; 339(6121):823-6; Jinek M et al., Science 2012 Aug. 17; 337(6096):816-21; Hwang W Y et al., Nat Biotechnol 2013 March; 31(3):227-9; Jiang W et al., Nat Biotechnol 2013 March; 31 (3):233-9; and Cong L et al., Science 2013 Feb. 15; 339(6121): 819-23, each of which is herein incorporated by reference in its entirety.
  • the gene editing tool that can be used as described herein comprises a nuclease agent, such as a Transcription Activator-Like Effector Nuclease (TALEN).
  • TAL effector nucleases are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a prokaryotic or eukaryotic organism.
  • TAL effector nucleases are created by fusing a native or engineered transcription activator-like (TAL) effector, or functional part thereof, to the catalytic domain of an endonuclease, such as, for example, Fokl.
  • the unique, modular TAL effector DNA binding domain allows for the design of proteins with potentially any given DNA recognition specificity.
  • the DNA binding domains of the TAL effector nucleases can be engineered to recognize specific DNA target sites and thus, used to make double-strand breaks at desired target sequences. See, WO 2010/079430; Morbitzer et al., (2010) PNAS 10.1073/pnas.l013133107; Scholze & Boch (2010) Virulence 1 :428-432; Christian et al., Genetics (2010) 186:757-761; Li et al., (2010) Nuc. Acids Res. (2010) doi: 10.1093/nar/gkg704; and Miller et al., (2011) Nature Biotechnology 29: 143-148; all of which are herein incorporated by reference in their entirety.
  • Non-limiting examples of suitable TAL nucleases, and methods for preparing suitable TAL nucleases are disclosed, e.g., in US Patent Application No. 2011/0239315 Al, 2011/0269234 Al, 2011/0145940 Al, 2003/0232410 Al, 2005/0208489 Al, 2005/0026157 Al, 2005/0064474 Al, 2006/0188987 Al, and 2006/0063231 Al (each hereby incorporated by reference).
  • TAL effector nucleases are engineered that cut in or near a target nucleic acid sequence in, e.g., a genomic locus of interest, wherein the target nucleic acid sequence is at or near a sequence to be modified by a targeting vector.
  • the TAL nucleases suitable for use with the various methods and compositions provided herein include those that are specifically designed to bind at or near target nucleic acid sequences to be modified by targeting vectors as described herein.
  • the gene editing tool that can be used as described herein comprises a nuclease agent, such as a zinc-finger nuclease (ZFN) system.
  • ZFN zinc-finger nuclease
  • Zinc finger-based systems comprise a fusion protein comprising two protein domains: a zinc finger DNA binding domain and an enzymatic domain.
  • a “zinc finger DNA binding domain,” “zinc finger protein,” or “ZFP” is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion.
  • the zinc finger domain by binding to a target DNA sequence, directs the activity of the enzymatic domain to the vicinity of the sequence and, hence, induces modification of the endogenous target gene in the vicinity of the target sequence.
  • a zinc finger domain can be engineered to bind to virtually any desired sequence.
  • the zinc finger domain binds a DNA sequence that encodes the SETD7 protein. Accordingly, after identifying a target genetic locus containing a target DNA sequence at which cleavage or recombination is desired (e.g., a target locus in a target gene referenced in Table 1), one or more zinc finger binding domains can be engineered to bind to one or more target DNA sequences in the target genetic locus. Expression of a fusion protein comprising a zinc finger binding domain and an enzymatic domain in a cell, effects modification in the target genetic locus. [000205] In embodiments, a zinc finger binding domain comprises one or more zinc fingers. Miller et al., (1985) EMBO J.
  • a single zinc finger domain is about 30 amino acids in length.
  • An individual zinc finger binds to a three-nucleotide (i.e., triplet) sequence (or a four-nucleotide sequence which can overlap, by one nucleotide, with the four-nucleotide binding site of an adjacent zinc finger). Therefore, the length of a sequence to which a zinc finger binding domain is engineered to bind (e.g., a target sequence) will determine the number of zinc fingers in an engineered zinc finger binding domain.
  • binding sites for individual zinc fingers (i.e., subsites) in a target site need not be contiguous, but can be separated by one or several nucleotides, depending on the length and nature of the amino acids sequences between the zinc fingers (i.e., the inter-finger linkers) in a multi-finger binding domain.
  • the DNA-binding domains of individual ZFNs comprise between three and six individual zinc finger repeats and can each recognize between 9 and 18 basepairs.
  • Zinc finger binding domains can be engineered to bind to a sequence of choice. See, for example, Beerli et al., (2002) Nature Biotechnol. 20: 135-141; Pabo et al., (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al., (2001) Nature Biotechnol. 19:656-660; Segal et al., (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al., (2000) Curr. Opin. Struct. Biol. 10:411-416.
  • An engineered zinc finger binding domain can have a new binding specificity, compared to a naturally-occurring zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection.
  • the gene editing tool that can be used as described herein comprises a meganuclease system. Meganucleases have been classified into four families based on conserved sequence motifs, the families are the “LAGLIDADG,” “GIY-YIG,” “H-N-H,” and “His-Cys box” families. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds.
  • HEases are notable for their long recognition sites, and for tolerating some sequence polymorphisms in their DNA substrates. Meganuclease domains, structure and function are known, see, for example, Guhan and Muniyappa (2003) Crit Rev Biochem Mol Biol 38: 199-248; Lucas et al., (2001) Nucleic Acids Res 29:960-9; Jurica and Stoddard, (1999) Cell Mol Life Sci 55: 1304-26; Stoddard, (2006) Q Rev Biophys 38:49-95; and Moure et al., (2002) Nat Struct Biol 9:764.
  • a naturally occurring variant, and/or engineered derivative meganuclease is used.
  • Methods for modifying the kinetics, cofactor interactions, expression, optimal conditions, and/or recognition site specificity, and screening for activity are known, see for example, Epinat et al., (2003) Nucleic Acids Res 31 :2952-62; Chevalier et al., (2002) Mol Cell 10:895-905; Gimble et al., (2003) Mol Biol 334:993-1008; Seligman et al., (2002) Nucleic Acids Res 30:3870-9; Sussman et al., (2004) J Mol Biol 342:31-41; Rosen et al., (2006) Nucleic Acids Res 34:4791-800; Chames et al., (2005) Nucleic Acids Res 33 :el78; Smith et al., (2006) Nucleic Acids Res 34:el49; Gruen et al., (2002)
  • Any meganuclease can be used herein, including, but not limited to, I-Scel, I-Scell, I- Scelll, 1-SceIV, I-SceV, I-SecVI, LSceVII, I-Ceul, LCeuAIIP, I-Crel, LCrepsblP, 1-CrepsbIIP, I- CrepsblllP, LCrepsblVP, I-Tlil, I-Ppol, PI-PspI, F-Scel, F-Scell, F-Suvl, F-TevI, F-TevII, I-Amal, I- Anil, I-Chul, I-Cmoel, I-Cpal, LCpall, I-CsmI, I-Cvul, LCvuAIP, LDdil, LDdill, LDirl, I-Dmol, I- Hmul, I-Hmu
  • compositions comprising a phosphospecific antibody, a CSE variant polypeptide or fragment, a CSE variant polynucleotide, a vector, or a cell that are suitable for administration to a subject.
  • Such pharmaceutical compositions can comprise a phosphospecific antibody, a CSE variant polypeptide or fragment, a CSE variant polynucleotide, a vector, or a cell and a pharmaceutically-acceptable excipient or carrier in a form suitable for administration to a subject.
  • Pharmaceutically acceptable excipients or carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition.
  • compositions comprising a CSE variant as described herein (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 18th ed. (1990)).
  • the pharmaceutical compositions are generally formulated sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • the phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell can be co-administered with of one or more additional therapeutic agents, in a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprising the phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell is administered prior to administration of the additional therapeutic agent(s).
  • the pharmaceutical composition comprising the phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell is administered after the administration of the additional therapeutic agent(s).
  • the pharmaceutical composition comprising the phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell is administered concurrently with the additional therapeutic agent(s).
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients (e.g., animals or humans) at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine,
  • Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin.
  • the use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell described herein, use thereof in the compositions is contemplated.
  • compositions described herein can be administered by parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intratumoral, intramuscular route or as inhalants.
  • Solutions or suspensions can include the following components: a sterile diluent such as water, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition is generally sterile and fluid to the extent that easy syringeability exists.
  • the carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic compounds e.g., sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride can be added to the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound which delays absorption, e.g., aluminum monostearate and gelatin.
  • compositions of the present disclosure can be sterilized by conventional, well known sterilization techniques.
  • Aqueous solutions can be packaged for use or fdtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • Sterile injectable solutions can be prepared by incorporating a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell in an effective amount and in an appropriate solvent with one or a combination of ingredients enumerated herein, as desired.
  • dispersions are prepared by incorporating a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell into a sterile vehicle that contains a basic dispersion medium and any desired other ingredients.
  • a pharmaceutical composition can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner to permit a sustained or pulsatile release of the phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell.
  • Administration of the pharmaceutical composition can also be by transmucosal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, e.g., for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of, e.g., nasal sprays.
  • compositions can be administered intravenously into a subject that would benefit from the pharmaceutical composition.
  • the composition is administered to the lymphatic system, e.g., by intralymphatic injection or by intranodal injection (see e.g., Senti et al., PNAS 105(46): 17908 (2008)), or by intramuscular injection, by subcutaneous administration, by intratumoral injection, by direct injection into the thymus, or into the liver.
  • the pharmaceutical composition can be administered as a liquid suspension.
  • the pharmaceutical composition is administered as a formulation that is capable of forming a depot following administration.
  • the depot slowly releases the micelles described herein into circulation, or remains in depot form.
  • compositions are highly purified to be free of contaminants, are biocompatible and not toxic, and are suited to administration to a subject. If water is a constituent of the carrier, the water is highly purified and processed to be free of contaminants, e.g., endotoxins.
  • the pharmaceutically-acceptable carrier can be lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and/or mineral oil, but is not limited thereto.
  • the pharmaceutical composition can further include a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, and/or a preservative.
  • Dosage forms are provided that comprise a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell as described herein.
  • the dosage form is formulated as a liquid suspension for intravenous injection.
  • Actual dosage levels of a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
  • the selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular therapeutic agent, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the severity of the condition, other health considerations affecting the subject, and the status of liver and kidney function of the subject. It also depends on the response to administration of the agents, including factors such as blood sugar level or the level of glycated hemoglobin, body mass index as well as levels of enzymes involved in liver disease such as ALT and AST. It also depends on the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular therapeutic agent employed, as well as the age, weight, condition, general health and prior medical history of the subject being treated, and like factors.
  • a phosphospecific antibody, a CSE variant polypeptide or fragment, a CSE variant polynucleotide, or a vector comprising the same can be administered with a delivery agent, e.g., a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric compound, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, a micelle, or a conjugate.
  • a delivery agent e.g., a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric compound, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, a micelle, or a conjugate.
  • embodiments described herein also provides for a pharmaceutical composition
  • a pharmaceutical composition comprising a phosphospecific antibody, a CSE variant polypeptide or fragment, a CSE variant polynucleotide, or a vector comprising the same, and a delivery agent, e.g., a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric compound, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, a micelle, or a conjugate.
  • a delivery agent e.g., a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric compound, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, a micelle, or a conjugate.
  • Embodiments of the invention are also drawn towards a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell for use as a medicament.
  • Embodiments of the invention are also drawn towards a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell for use in the treatment of tissue injury in a subject in need thereof.
  • a CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell as described herein can be administered to a subject, e.g., a human subject, to treat diseases or conditions disclosed herein, such as a tissue injury.
  • the CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell can be used to prevent symptoms or sequelae of diseases and conditions disclosed herein.
  • the CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell can be administered via intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.
  • the CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell can be used concurrently with other medicaments or treatment suitable for the treatment of the diseases and conditions disclosed herein.
  • a CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell as described herein can be used to increase cellular hydropersulfide, polysulfide, or both.
  • hydropersulfide, polysulfide, or both can be measured using analytical clinical methods known in the art. See, for example, Malaeb, Hind, et al. "Stable isotope dilution mass spectrometry quantification of hydrogen sulfide and thiols in biological matrices.” Redox Biology 55 (2022): 102401; Shen, Xinggui, et al.
  • kits or products of manufacture comprising a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell, and optionally instructions for use, e.g., instructions for use according to the methods disclosed herein.
  • the kit comprises a pharmaceutical composition comprising a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell as described herein.
  • the kit or product of manufacture comprises the phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell disclosed herein in one or more containers.
  • the kit or product of manufacture optionally comprises a brochure and/or instructions for use.
  • a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell can be readily incorporated into one of the established kit formats which are well known in the art.
  • the kit or product of manufacture comprises a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell, in dry form in a container (e.g., a glass vial), and optionally a vial with a solvent.
  • Example 1 CSE phosphorylation regulates hydropersulfide and polysulfide formation
  • CSE cystathionine gamma lyase
  • Hypoxia reduced oxygen tension
  • hydropersulfide and polysulfide act as critical redox regulators that antagonize oxidative stress. It remains unknown whether or how hypoxia could alter bioavailability of hydropersulfide and polysulfide.
  • hypoxia increases AMP Kinase dependent CSE phosphorylation of Serine 346 and Threonine 355 that selectively increases CSE dependent hydropersulfide and polysulfide formation without increased hydrogen sulfide generation.
  • This is an entirely new molecular pathway for regulating biologically potent sulfide metabolites.
  • embodiments provide phospho-specific antibodies against CSE phosphorylated Serine 346, which can be used as a biomarker or molecular readout indicating increased hydropersulfide and polysulfide formation. Such antibodies can be useful for tissue and disease diagnosis, screening drugs that could potentially activate this new pathway, and as a research tool for further studies in the field.
  • the genetic sequence of CSE Glutamic acid amino acid mutants of either Serine 346 or Threonine 355, or both can be used in mRNA or gene-based therapy to discretely increase cellular hydropersulfide and polysulfide in subjects who are deficient in these mediators.
  • genetic sequence of CSE Alanine amino acid mutants of either Serine 346 or Threonine 355, or both can be used in mRNA or gene-based therapy to discretely decrease cellular hydropersulfide and polysulfide in subjects who have elevated levels of these mediators.
  • CRISRP/Cas targeting technology could be used to perform mutation of these residues in endogenous subject genomes.
  • Hydrogen sulfide is a gaseous signaling molecule (e.g., a ‘gasotransmitter’) that plays critical roles in cy toprot ection and in regulating many other biological functions 1 .
  • a gasotransmitter e.g., a ‘gasotransmitter’
  • its pathophysiological roles including cardiovascular, neurological, inflammatory and immune systems, has been well established through experimental animal models and clinical studies 1- 3 .
  • H2S is produced by enzymes of the transsulfuration pathway including cystathionine -synthase (CBS) and cystathionine y-lyase (CTH, also abbreviated as CSE), as well as by 3 -mercaptopyruvate sulfurtransferase, which is involved in cysteine catabolism.
  • CBS cystathionine -synthase
  • CTH cystathionine y-lyase
  • 3 -mercaptopyruvate sulfurtransferase which is involved in cysteine catabolism.
  • sulfide chemical biology is present in three forms, including the readily available free sulfide, acid-labile sulfide (e.g. protein iron-sulfur clusters that are localized in active centers of respiratory enzymes), and as bound sulfane sulfur (e.g.
  • hydropersulfide, polysulfide, and others 1, 2 - 4 Additionally, free H2S can be released by acid-labile and bound sulfane sulfur under various conditions making them reservoirs of sulfide bioequivalents 1 ' 2 .
  • hydropersulfides and polysulfides have been identified as molecules with a higher number of inner sulfur atoms that are able to act as both nucleophiles or electrophiles depending on protonation states and local redox environment. Importantly, hydropersulfides and polysulfides serve as signaling molecules that can regulate various metabolic pathways and have pathophysiological implications 1, 4 ‘ 5 . Hydropersulfides and polysulfides can be synthesized independently of H2S.
  • CBS and CSE can use substrates such as cystine or glutathione disulfide, resulting in the formation of cysteine hydropersulfide or glutathione hydropersulfide as well as polysulfides, which predominantly are the forms of bound sulfane sulfur 1, 4 ’ 5 .
  • Protein persulfides can originate from cysteine persulfide integration during translation from cysteinyl-tRNA synthetases (CARSs) 6, 7 .
  • CBS and CSE can be promiscuous in utilizing different substrates such as cysteine, homocysteine or cystine in generation of sulfides h 8 .
  • the utilization of these substrates can be varied under intra or intercellular conditions, changes in pH or oxygen conditions such as hypoxia or ischemia that may contribute to biological levels of hydropersulfide and polysulfide availability.
  • changes in pH or oxygen conditions such as hypoxia or ischemia that may contribute to biological levels of hydropersulfide and polysulfide availability.
  • hypoxia or ischemia may contribute to biological levels of hydropersulfide and polysulfide availability.
  • the formation, kinetics and biological relevance of these per- and polysulfide compounds under various pathophysiological conditions remain largely unclear.
  • H2S is constantly maintained in the mammalian cells can be increased either through transcriptional regulation of CSE or transiently via persulfidation popularly called S-sulfhydration of cysteine residues on target proteins 8, 9 .
  • CSE/H2S regulates ischemic vascular remodeling mediated during hind limb ischemia via upregulation of H2S and NO- dependent monocyte recruitment and cytokine induction revealing a previously unknown mechanism of arteriogenesis 10 .
  • CSE deletion accelerated the development of endothelial dysfunction and atherosclerosis 12 .
  • AMPK Adenosine monophosphate-activated protein kinase
  • CSE cystathionine y-lyase
  • this example will validate that endothelial cell and monocyte CSE dependent polysulfide formation regulates ischemic vascular remodeling and NO bioavailability.
  • Three specific aims will be pursued to validate this: 1) validating the mechanisms of endothelial CSE regulation of ischemic vascular remodeling and how it controls vascular cell NO bioavailability, 2) validating the mechanisms of monocyte CSE regulation of arteriogenesis, and 3) validating mechanisms of CSE activity and expression in experimental models and clinical specimens.
  • Peripheral ischemic vascular diseases e.g. Peripheral Arterial Disease (PAD) and critical limb ischemia (CLI)
  • PAD Peripheral Arterial Disease
  • CLI critical limb ischemia
  • PED Peripheral Arterial Disease
  • CLI critical limb ischemia
  • Peripheral ischemic vascular diseases e.g. peripheral arterial disease- PAD and critical limb ischemia-CLI
  • peripheral arterial disease- PAD and critical limb ischemia-CLI are chronic tissue disorders that affect millions of Americans and others worldwide and are poised to further increase due to several risk factors.
  • therapeutic approaches for ischemic vascular diseases are not widely effective and typically provide short-term benefit highlighting the need for new molecular targets or treatment approaches involved in vascular remodeling.
  • H2S hydrogen sulfide
  • CSE cystathionine gamma lyase
  • sulfide metabolites such as polysulfides/hydropersulfide (a.k.a. bound sulfane sulfur)
  • endothelial e.g. NO generation
  • immune cell e.g. monocyte recruitment and polarization
  • Peripheral ischemic vascular disease e.g. Peripheral Arterial Disease (PAD) and Critical Limb Ischemia (CLI)
  • PAD Peripheral Arterial Disease
  • CLI Critical Limb Ischemia
  • H2S hydrogen sulfide
  • NO nitric oxide
  • Sulfide metabolites exist in different biochemical forms influenced by various stimuli that elicit diverse vascular cell responses 4 ' 5 ’ 7
  • specific sulfide metabolites such as persulfides or polysulfides contribute to vascular remodeling or how they affect NO metabolite bioavailability and biological actions.
  • H2S in the cardiovascular system has been reported to be predominantly produced by cystathionine-y-lyase (CSE) in different vascular cell types including endothelial and vascular smooth muscle cells using cystathionine or L-cysteine as substrates ( Figure 8) 8,9, 10 .
  • CSE cystathionine-y-lyase
  • Sulfide bioavailability may exist in different biochemical forms or ‘pools’ including free sulfide, acid labile sulfide, and bound sulfane sulfur 11, 12 .
  • a wide range of sulfide metabolites may be produced via CSE besides H2S alone with an interest on formation of persulfide and polysulfides 13 .
  • H2S may also contribute to the formation of per- and polysulfides through formation of thiyl radicals (HS»), which can react to form RSS(n)H species ( Figure 8) 14 .
  • HS thiyl radicals
  • CSE can also use cystine (CysSSCys) as a substrate forming cysteine persulfide (CysSSH) that may be a biologically important bound sulfane sulfur 15 .
  • CysSSCys cystine
  • CysSSH cysteine persulfide
  • this example will validate whether CSE forms per/polysulfides during ischemia/hypoxia as potent chemical mediators of vascular remodeling.
  • CSE may also have diverse effects in different cellular compartments (e g. vascular vs immune cells) that remain poorly understood during ischemic vascular remodeling that will be further revealed in our studies 16 ' 19 .
  • vascular vs immune cells e g. vascular vs immune cells
  • the sulfide field remains highly unclear with regard to how CSE generates various sulfide metabolites in specific cell compartments under ischemic vascular conditions.
  • Results from this example will reveal for the first time CSE/polysulfide regulation of ischemic vascular remodeling generating new models, tools, and mechanistic insights for the scientific research community. Many important questions remain unanswered regarding sulfide metabolism in discrete cellular compartments and associated molecular mechanisms involved in ischemic vascular remodeling. This project will surpass these obstacles through innovative approaches including: 1) use of new tissue specific mutant animal models of CSE within the vasculature and monocyte compartment, generation of new molecular gene constructs of CSE to determine how its regulated, employ advanced analytical chemistry methods using HPLC/MS validating key sulfide metabolites, 2) identification of specific H2S metabolite (e.g.
  • Endothelial cell-specific CSE genetic deletion (ecCSEKO): Genetic deficiency of CSE in mouse models is important in modulating several cardiovascular responses 5 . Our group and others, have employed global CSE gene targeted deficient mice to gain initial insight into the pathophysiological role of this molecule and its impact on sulfide metabolism 24 . Global CSE KO mice manifest defective vascular growth responses, as well as defective monocyte mediated arteriogenesis 5 ; however, effects of CSE in specific cellular tissues remain poorly understood during ischemic vascular remodeling.
  • vascular cell-specific CSE mutant mice using C57BL/6NTac-Cth trn, a(EUCOMM)Hrngll /Ieg (Cth being the gene abbreviation for CSE) strain crossed with B6.129S4-Gt(ROSA)26Sor tm2(FLP * )So 7J Flp deleter line and then backcrossed to the VE-Cad CRE driver line to obtain VECad CSEKO (referred to as ecCSEKO).
  • Figure 10 panel A reports the effect of 30 minutes of 1% 02 on wild type mouse aortic endothelial cell (MAEC) CSE protein expression and enzyme activity. Hypoxia did not significantly affect CSE protein expression (left panel, Figure 10, panel A) but quickly and robustly increased CSE enzyme activity ⁇ 2.5 fold compared to control ‘normoxic’ tissue culture conditions (right panel, Figure 10, panel A).
  • Figure 10, panel B shows that 30 minutes of hypoxia alters sulfide metabolite levels with reduced acid labile sulfide levels and significantly elevated bound sulfane sulfur levels within endothelial cells. This illustrates a totally unique and previously unknown response regarding changes in sulfide metabolite bioavailability.
  • H2S versus per/polysulfides differentially affect endothelial cell activation: Persulfides and polysulfides have become increasingly more appreciated for their role in modulating various responses in cell model systems 28,29 . However, little is known regarding differential endothelial cell responses to hydrogen sulfide versus per/polysulfides.
  • Figure 11 illustrates the effect of different sulfide compounds including sodium sulfide, disulfide, trisulfide, and tetrasulfide on wild type MAEC at a dose of 50 pM.
  • H2S robustly and preferentially increases hypoxic endothelial NO production independent of NOS:
  • Our laboratory and others have investigated the impact of hydrogen sulfide on NO bioavailability resulting in various insights into how H2S can regulate NO levels 30, 31 .
  • While laboratories have examined the direct effect of exogenous sulfide donors on endothelial NO production under normoxic conditions, our laboratory has keenly focused on how sulfide bioavailability during chronic tissue ischemia impacts NO levels involving NOS and non-NOS pathways.
  • H2S and CSE expression impact ischemic tissue ability to generate NO with only a partial role of eNOS activity 4 ’ 5 .
  • Figure 12 further demonstrates that human umbilical vein endothelial cells exposed to varying concentrations of H2S preferentially causes NO production under hypoxic versus normoxic conditions ( Figure 12, panels A and B). NOS inhibition with L-NAME did not attenuate H2S dependent NO production in hypoxic HUVEC, but Febuxostat blockade of XO significantly blunted NO production, indicating that XO dependent NO2' reduction to NO is involved.
  • LysM Cre/CTH A/ A mice significantly reduces bone marrow monocyte CSE activity (Figure 13, panel D) compared to spleen monocytes ( Figure 13, panel C).
  • CSE/H2S regulates myeloid cell infiltration and subsequently tissue perfusion and ischemic vascular remodeling 5 .
  • Figure 14 shows that myeloid cell CSE knockout (cpCSEKO) mice significantly impairs recovery of ischemic limb blood flow and angiogenic index observed at day 7 after FAL. Without wishing to be bound by theory, these data indicate that monocyte/macrophage CSE expression can regulate vessel remodeling and blood flow during ischemia.
  • CSE genetic deficiency blunts monocyte recruitment: Having observed significant changes in monocyte CSE expression, activity, and H2S production, we examined the impact of CSE deficiency on bone marrow monocyte recruitment.
  • Figure 15 shows that genetic deficiency of CSE in either myeloid cells (cpCSEKO, upper panel) or endothelial cells (ecCSEKO, lower panel) decreases the monocyte recruitment (MAC-2) to the site of ischemia.
  • SA3 will examine molecular mechanisms regulating increased CSE activity and sulfide metabolite production through enzyme posttranslational modifications, cofactor bioavailability, substrate specificity, and also evaluate these parameters in clinical vascular disease specimens. These experiments address important unknown questions in the field of ischemic vascular remodeling and will provide new insight into molecular mechanisms of CSE functions.
  • Changes in limb perfusion and vascular contrast imaging will be performed using the PeriCam PSI laser speckle contrast imager and the SPY imaging system, respectively.
  • the PeriCam PSI system will enable accurate longitudinal hind limb blood flow measurement due to its depth of penetration reflecting changes in the microcirculation
  • SPY imaging will enable longitudinal arteriogram imaging using indocyanine green (ICG) contrast agent reflecting changes in arteriogenesis 5 ’ 37 ( Figure 18, panel A). Imaging will be performed pre and post-ligation and at days 1, 3, 5, 7, 14 and 21. In a separate cohort of mice, Microfil vascular casting will be performed in control and mutant mice at pre and post ligation, and days 3, 7, and 14 as we previously reported 5 ( Figure 18, panel, B).
  • gastrocnemius and adductor muscle will be collected at pre and post-ligation, and days 1, 5, 7, 14, and 21 for histopathology analysis including CD31 and a-SMA staining, as well as free hydrogen sulfide, acid-labile sulfide, and bound sulfane sulfur metabolite measurements via LC- MS/MS, as we’ve previously reported 1 L 12 ' 20 and shown in Figure 19.
  • Cells will be cultured under hypoxic conditions at various time points including 0, 0.25, 0.5, 1, 2, 4, 8, and 16 hours. Batches of cells will be harvested at each time point to measure bound polysulfide metabolites using the monobromobimane (MBB) assay as others and we’ve reported 15, 27 A separate cohort of cells will also be analyzed for NO metabolite levels using NOA chemiluminescent analysis for NOx, nitrosoheme, and nitrosothiols27. Other experiments will be performed using wild type MAEC treated with CSE siRNA or the pharmacological inhibitor PPG to measure hypoxia bound sulfane sulfur and NO levels. Together, these experiments will provide important insight regarding the importance of CSE for regulation of polysulfide levels that may influence NO bioavailability.
  • hypoxic wild type MAEC will be collected, placed in the NOA sparger, and treated with either sodium sulfide (H2S donor), hydrogen persulfide (H2S2), hydrogen trisulfide (H2S3), hydrogen tetrasulfide (H2S4), diallyl trisulfide (DATS), or diallyl disulfide (DADS).
  • H2S donor sodium sulfide
  • H2S2S2 hydrogen persulfide
  • H2S3 hydrogen trisulfide
  • H2S4 diallyl trisulfide
  • DATS diallyl trisulfide
  • DADS diallyl disulfide
  • Each of the compounds will be tested in a dose dependent manner including 0, 0.1, 0.5, 1, 10, 50, and 100 pM concentrations. This will enable wide range comparisons between the various compounds allowing us to validate different electrochemical properties based on the relative nucleophilic strength of bound sulfur compound species.
  • An analogous series of experiments will also be performed using CSE knockout MAEC that will allow deeper understanding of CSE sulfide dependent NO production.
  • Mechanisms of polysulfide dependent NO production will be validated using various inhibitors we’ve shown to be important for hydrogen sulfide mediated NO formation in hypoxic cells including, L-NAME (to block eNOS), cPTIO (NO scavenger), NEM (thiol blocker), paraformaldehyde (protein crosslinker), or febuxostat (XO inhibitor) to elucidate specific biochemical mechanisms4. Studies will also be performed examining eNOS phosphorylation at classical inhibitor/activator residues (Ser 1177, Thr495 etc.) along with XO/uric acid enzyme activity assays.
  • Hind limb perfusion and histological measurements will be performed using a PeriCam PSI laser speckle contrast imager.
  • histopathology measurements including capillary to myofiber ratio and vascular index will be made at pre, post, 1, 3, 5, 7, 14, and 21 days. Briefly, 5 pm frozen muscle tissue sections will be cut and fixed with ethanol/acetic acid and dual stained with anti-CD31 and Ki67 along with DAPI nuclear stain. Vascular density and cell proliferation index ratios will be validated by quantitative morphometric analysis to DAPI stain using Simple PCI software as we’ve reported 4 ’ 5 ’ 37 . Capillary to myofiber ratio will be performed using formalin fixed, paraffin embedded sections stained with CD31 and counterstained with hematoxylin, and number of capillaries per myocyte counted.
  • Collateral remodeling measurements will be performed as we’ve reported 37 .
  • SPY imaging will be performed in a longitudinal manner using ICG dye injection via retro-orbital injection. Still images are extracted to measure changes in vascular length, diameter, and branching. Microfil vascular casting and morphometric measurements will be performed as we’ve reported 37 .
  • LC-MS/MS measurement of polysulfide species and bioavailability Sulfide metabolite bioavailability will be measured as we’ve reported n ’ 12, 20 - 39 .
  • Blood and tissue specimens will be derivatized with MBB in 1% oxygen for 30 minutes at room temperature and stopped using 50 pL of 200 mM ice-cold sulfosalicylic acid solution.
  • Samples will be measured by LC-MS/MS using a Acquity UPLC system coupled to XEVO TQ (Thermo Scientific) with electrospray ionization (ESI(+)).
  • NO production from hypoxic or normoxic endothelial cells in response to exogenous sulfides will be done by chemiluminescent nitric oxide analysis as we’ve reported 4 ’ 27 .
  • Sulfide compounds including sodium sulfide (Na2S), sodium disulfide (Na2S2), sodium trisulfide (Na2S3), sodium tetrasulfide (Na2S4), diallyl disulfide (DADS), and diallyl trisulfide (DATS) will be used at various concentrations as we’ve reported 28 .
  • Inhibitor studies will also be performed using L-NAME, cPTIO, NEM, paraformaldehyde, or febuxostat as we’ve previously reported4. Endothelial cell proliferation studies described above will be performed as we’ve reported 5 .
  • Bone marrow monocytes will be isolated from WT, CSE KO, (pCSEKO male and female mice, cultured with M-CSF, and then treated with LPS and fFN-y for Ml or IL -4 for M2 phenotypes as previously reported46.
  • Ml vs M2 macrophage phenotypes will be validated via flow cytometry and gene expression changes in collaboration with Dr. Matthew Woolard.
  • a separate series of experiments will also be performed using H2S vs per/polysulfides donors to validate the impact of sulfide metabolite restoration.
  • CSE may be involved in macrophage movement in myocardium during ischemia-reperfusion injury consistent with our findings of deficient monocyte infiltration into chronically ischemic tissue 5 ’ 43 .
  • CSE expression is important for actual monocyte adhesion and recruitment responses that are critical for arteriogenesis.
  • experiments will be performed to validate whether monocyte deficiency of CSE alters adhesion and recruitment to mouse aortic and microvascular endothelial cells under hydrodynamic flow conditions.
  • Parallel plate flow chambers will be used to validate shear mediated detachment of WT or cpCSEKO monocyte rolling and firm adhesion to TNF- a activated MAEC or MVEC at a range of shear stresses (2- 15 dynes/cm2) as we’ve reported 47, 48, 48 . Results from these experiments will provide cellular mechanisms regarding the importance of monocyte CSE in regulating recruitment responses.
  • Bone marrow monocytes will be isolated using the EasySep mouse monocyte isolation kit as we’ve reported 5 .
  • Monocyte rolling and firm adhesion will be examined using established MAEC and MVEC lines in our laboratory with parallel plate flow chambers as we’ve reported 47, 48 .
  • Monocyte adhesion strength will be validated by adhesion detachment assays in response to increasing shear stress as we’ve reported 49 .
  • CSE deficient monocytes will be pretreated with different sulfide donors (10 and 50 iiM) for 4 hours then used in cell adhesion studies.
  • Macrophage polarization studies will be performed in conjunction with the Woolard Lab. Bone marrow monocytes will be expanded with recombinant murine M-CSF followed by 10 ng/ml LPS and 20 ng/ml IFN-y for Ml or 40 ng/ml IL-4 for M2 phenotypes.
  • Ml macrophage markers used will be TNF-a, iNOS, CCR7, COX-2, IL-12a, and CD38.
  • M2 macrophage markers used will be IL-10, Arg-1, CD206, YMl(Chil3), COX-1, and Egr2.
  • RNA will be isolated using the Qiagen RNeasy isolation midi-kit and subsequent RNA used with murine iNOS cyber green qRT-PCR with the following primers: iNOS- forward GCTGTTAGAGACACTTCTGAG, reverse CACTTTGGTAGGATTTGACTTTG.
  • Transduced cells will be exposed to normoxic or hypoxic cell culture conditions over 30 minutes, as we’ve previously reported 5 .
  • a similar set of experiments will be performed using CSE null bone marrow monocytes where we previously reported hypoxic CSE dependent activity and H2S production. Together, these studies will provide important new information regarding phosphoprotein regulation of CSE enzyme activity.
  • the resulting data will be compared to cells under normoxic conditions at similar time points to validate changes in substrate levels. These measurements will be also be extended to ischemic versus nonischemic limbs at 0, 1, 3, 6, 12, and 24 hours post femoral artery ligation allowing comparisons between in vitro vs. in vivo conditions. These data will validate the regulation of AMPK, PKC and ATR active sites on phosphorylation and CSE enzyme function.
  • studies will be performed using wild type or CSE null endothelial cells or monocytes quantifying the levels of substrates cystathionine, cysteine, cystine and PLP bioavailability under normoxic and hypoxic tissue culture conditions.
  • PLP levels will be measured via HPLC as reported and shown in figure 1651.
  • PLP binding to CSE will also be validated using the various phosphomutants described in SA3a.
  • PLP binding studies will be performed using FLAG immuno-enriched CSE followed by incubation with PLP (100 ng/ml). Results from these experiments will provide new information regarding the role of PLP cofactor bioavailability and function for CSE enzymatic activity during hypoxia and tissue ischemia.
  • Cells will be incubated at 1% 02 for 0, 0.5, 1, 2, 4, 8, 16, and 24 hours and subsequently collected for substrate measurement. The resulting data will be compared to cells under normoxic conditions at similar time points to validate changes in substrate levels. Substrate bioavailability will also be validated in ischemic versus nonischemic limbs at 0, 1, 3, 6, 12, and 24 hours post femoral artery ligation allowing substrate comparisons between in vitro vs. in vivo conditions. These data will validate substrate bioavailability impacting CSE enzyme function.
  • CSE characteristics in clinical tissue will be validated from femoral and carotid endarterectomy specimens of patients with vascular disease or non-diseased vascular tissue from bypass procedures.
  • LC-MS/MS procedures small tissue samples can be used to measure various per/polysulfide pools, CSE substrate and cofactor bioavailability, and CSE posttranslational modifications.
  • a total of 64 endarterectomy specimens from carotid and femoral vessels will be collected with 8 specimens from each of the following: carotid- african American male, african american female, Caucasian male, Caucasian female; femoral- african american male, African american female, Caucasian male, Caucasian female. Equal numbers of control vessel tissues will also be obtained. The numbers chosen enable detection of a minimal 40% difference with a power of 0.8. [000337] SA3 General Methods
  • CSE enzyme activity will be measured using different substrates of cystathionine, cysteine, or cystine as we’ve previously reported 5.
  • Cystathionine, cysteine, and cystine will be measured by LC-MS/MS. Samples will be injected into an Acquity UPLC system coupled to XEVO TQ (Thermo Scientific) with electrospray ionization (ESI(+)) in the multiple reaction monitoring (MRM) mode as described above.
  • PLP bioavailability will be measured by HPLC as previously reported51.
  • PLP bound to CSE protein will be identified via immuno-pulldown of FLAG tagged CSE, digestion with MS grade trypsin, and peptides analyzed using a nano flow ultrainterfaced to nano-electrospray quadrupole time-of-flight MS (nanoAcquity UPLC-Synapt HDMS, Waters Corporation, MA) as described previously 53 .
  • Data is acquired using MassLynxTM 4.1 software in an automatic data dependent acquisition mode.
  • MS-TOF scans are acquired from m/z 300 to m/z 1500, and up to three precursor ions are selected for subsequent MS/MS scans from m/z 50 to 1000 using charge state-dependent collision energy ramps to promote fragmentation.
  • Mass spectrometry data is processed using PEAKs Studio 7.5 software (Bioinformatics Solutions Inc., Canada).
  • Reactive sulfur species are important for numerous biological functions and pathophysiological roles in cardiovascular, neurological, inflammatory, and immune systems, and in other disease states 1 ’ 3 .
  • Hydrogen sulfide (H2S) has received predominant attention among RSS and is produced by enzymes in the trans-sulfuration pathway, cystathionine P-synthase (CBS) and cystathionine y-lyase (CSE/CTH), as well as by 3 -mercaptopyruvate sulfurtransferase, which is involved in cysteine catabolism.
  • endogenous sulfide may be present in three biochemically important forms, including, the readily available free sulfide, acid-labile (e.g. iron-sulfur clusters that are localized in active centers of respiratory enzymes), and as bound sulfane sulfur forms (e.g. hydropersulfides and hydropolysulfides) 1,2,4 .
  • Free H2S can be released by acid-labile and bound sulfane sulfur under acidic or reducing conditions, respectively 1,2 .
  • RSS are constantly maintained in mammalian cells and can be influenced either through transcriptional regulation of sulfide synthesis enzymes or via persulfidation popularly called S-sulfhydration of cysteine residues on target proteins 5,6 .
  • CSE/H2S regulates ischemic vascular remodeling mediated during hind limb ischemia via upregulation of sulfide metabolites and NO-dependent cytoprotective responses revealing important roles for cardiovascular function 7 .
  • Hydropersulfides and polysulfides can be synthesized independently of H2S.
  • Polysulfide release from cysteinyl-tRNA synthetases (CARSs) has been demonstrated 8,9 and both CBS and CSE utilize substrates such as cystine or glutathione disulfide, to catalyze formation of cysteine hydropersulfide or glutathione hydropersulfide, and other polysulfides 2,4,9,1 °.
  • CARSs cysteinyl-tRNA synthetases
  • siRNA against known sulfur metabolite producing enzymes, including CSE, CBS, MPST, CARS1 or CARS2, and then exposed murine endothelial cells to hypoxia and measured per-polysulfides using SSP4 (Fig. ID), and H2S using SF7 ( Figure 30, panel D).
  • SSP4 sulfur metabolite producing enzymes
  • SF7 H2S using SF7
  • Significant decreases in mRNA and protein levels of siRNA treatment for CSE, CBS, MPST CARS1 and CARS2 was confirmed by qRT-PCR and western blot ( Figure 30, panels E-I and J-N).
  • CSE is a promiscuous enzyme where based on its substrate utilization the kinetics can vary with change in the microenvironmentl5.
  • Wild type and CSE phospho-negative or mimetic constructs were FLAG tagged and used for transient transfection studies in HEK cells. Wild type, phospho-negative, or phospho-mimetic CSE was transfected into HEK and confirmed by FLAG immunoblotting for uniform expression ( Figure 31, panels B and C).
  • CSE can also use cystine or glutathione disulfide to form hydropersulfide and polysulfides 8 ’ 16 .
  • cystine substrate utilization under hypoxia could differentially influence per-polysulfide levels in endothelial cells.
  • Mouse aortic endothelial cells were transfected with wild type, S346A, T355A, S346E or T355E CSE constructs and exposed to hypoxia with cystine substrate followed by derivatization with HPE-IAM for per-polysulfide LC/MS analyses 17 .
  • Significant increases in GSSH persulfide and GSSSSH polysulfide with wild type CSE were observed ( Figure 34, panelsA and B).
  • T355A - H55D, R62D, R119A Figure 27, panel D, inset i
  • pT355B - R119B Figure 4D, inset ii
  • pT355D - Q323D, M354D, H356D, S358D, V359D Figure 27, panel D, inset iii
  • pT355C - R119C, H356C, R62B, N241B Figure 27, panel D, inset iv.
  • Adenosine 5'- monophosphate-activated protein kinase plays a key role in maintaining cellular energetic balance and metabolism 19. Increasing evidence indicates AMPK as a new modulator in multiple pathological conditions, including non-metabolic processes and diseases 19,20 .
  • AMPK AMPK regulates hypoxic CSE phosphorylation and subsequent increases in per-polysulfide using the ATP competitive AMPK inhibitor, dorsomorphin (AMPK-I).
  • AMPK-I dorsomorphin
  • Dorsomorphin significantly blunted hypoxia mediated pAMPK and pCSES346 ( Figure 28, panel C and D) that was associated with a significant reduction in hypoxia dependent SSP4 fluorescence ( Figure 28, panel E).
  • AMPK is a heterotrimeric protein kinase composed of a catalytic a subunit (al or a2) subunit 21,22 .
  • a catalytic a subunit al or a2 subunit 21,22 .
  • AMPK a subunit inhibition on pCSE and per-polysulfide levels using siRNA treatment of mouse aortic endothelial cells.
  • Individual knockdown of AMPKal Figure 35, panel F
  • a2 Figure 35, panel G
  • FAL femoral artery ligation
  • Ischemia increased CSE phosphorylation (pCSES346) rapidly (3 hours) in the gastrocnemius muscle that was sustained over 5 days ( Figure 29, panel A and B).
  • ischemic tissue AMP kinase phosphorylation beginning at 3 hours lasting to 5 days.
  • mice were treated with AMPK-I dorsomorphin (lOmg/kg) for 5 days during FAL.
  • Skeletal muscle tissues including from non-ischemic (NI), ischemic (Isch) or Isch+ AMPK-I groups were probed for pCSE and pAMPK protein levels (Figure 29, panel C).
  • NI non-ischemic
  • Isch ischemic
  • Isch+ AMPK-I groups were probed for pCSE and pAMPK protein levels (Figure 29, panel C).
  • Polysulfides can liberate free sulfide, contribute sulfur to metal complexes, serve as potent nucleophilic and electrophilic redox mediators, and are more biologically stable forms of RSS than H2S alone 4,12,24,25 .
  • CSE CBS, MPST, and cysteinyl-tRNA synthetases
  • CARSs cysteinyl-tRNA synthetases
  • CSE can in fact utilize cystine or cysteine as substrates apart from cystathionine to produce per-polysulfides under hypoxic conditions. Moreover, production of these sulfane sulfur metabolites appears more abundant with cystine than cysteine or cystathionine under hypoxia when CSE is phosphorylated. Possible presence of low cystathionine and cysteine levels under hypoxia may also favor utilization of cystine as a substrate thus serving as an alternative pathway to salvage intracellular cysteine and H2S. These results indicate that CSE utilization of cystine or cysteine to generate per-polysulfides under hypoxia (rather than H2S) may be a metabolic and redox pathway to maintain cellular function during stress.
  • CSE is a PLP-dependent enzyme consisting of 393 amino acid residues and one PLP moiety and is arranged as a tetramer 18 .
  • Pyridoxal phosphate is bound in the active site by Lys21228.
  • PLP is anchored by strong hydrogen bonds between the phosphate moieties, including Gly90, Leu91, Thr211 and Arg62, that can contribute towards stability of its binding with the CSE enzyme28.
  • Presented molecular modeling data demonstrates that phosphorylation of CSE significantly changes the molecular dynamics of CSE via electrostatic interactions, including inter- and intramolecular binding between monomers of CSE tetramer via S346 and T355.
  • H55 is also a part of a loop (residues D28 - S63) that is disordered in the absence of PLP.
  • residues T355 - V366 There is no electron density in the apo structure for residues T355 - V366, indicating that this region is disordered. Although this region does not directly interact with PLP, it is theorized that it could stabilize one side of the PLP binding pocket.
  • the T355 - V366 region of the CSE:PLP complex is occupied by the Ml 10 - N118 loop28.
  • N241 plays an important role in the inter-molecular stabilization of PLP in the active site of CSE29.
  • Supplement video 1 shows non-phosphorylated CSE tetramer molecular interactions over 300 nanoseconds
  • supplement videos 2 and 3 illustrate phospho-S346 CSE or phospho-T355 CSE molecular interactions, respectively.
  • Molecular model simulations clearly demonstrate that phospho- S346 engages in horizontal intermolecular monomer interactions, whereas phospho-T355 predominantly engages in vertical intra- and intermolecular monomer interactions.
  • phosphorylation of S346 and T355 transition the CSE tetramer from an open to closed conformation, particularly within the PLP binding domain.
  • 17p-estradiol can enhance endothelial CSE gene expression and H2S release by promoting phosphorylation of CSE via ERot-Spl interaction with binding sites in CSE gene promoter, subsequently improving endothelial function33,34.
  • this study is predominantly applicable to females and doesn’t identify how S56 effects CSE enzyme function.
  • Cisi sulfide levels are reduced under disturbed shear stress via phosphorylation of serine 377 thereby inhibiting CSE, due to increased IL-ipi3.
  • these mechanisms explain the inhibitory regulation of CSE alone.
  • Our data reveals increased CSE enzyme function under hypoxia via S346 and T355 phosphorylation.
  • AMP-activated protein kinase is a key regulatory molecule for endothelial function, redox homeostasis, and importantly, hypoxia and ischemia/reperfusion injury 35 .
  • AMPK has been reported to mediate myocardial protection from I/R injury 36 .
  • AMPK prevents apoptosis and reduces oxidative stress and inflammation upon ER injury, which is mediated via AMPK phosphorylation 37 .
  • AMPK protects from myocardial I/R injury via phosphorylation of AMPK at Thrl72 in ischemic heart tissues 38 .
  • AMPK regulation of CSE and subsequent sulfide metabolite levels have not been known.
  • AMPK/pAMPK can regulate CSE by increasing its activity via phosphorylation (S346) under hypoxia/ischemia conditions. This increase in CSE phosphorylation subsequently induced per-polysulfide generation, which is significantly blunted upon inhibition of AMPK using dorsomorphin or siRNA.
  • AMPK is a heterotrimeric complex of catalytic a-subunits, regulatory P and y-subunits.
  • AMPK catalytic al and 2 isoforms are activated in cardiac ischemia irrespective of regulatory subunits 40 .
  • AMPK can be activated by LKB1 and CaMKK pathways that elicits protective and adaptive mechanism to various cellular stress conditions in the endothelial cells, including hypoxia 41,42 .
  • hypoxia 41,42 the molecular regulation and the roles of specific AMPK subunit isoforms is not well defined.
  • Our data demonstrates that under hypoxia, molecular inhibition of either AMPKal or AMPKa2 in endothelial cells significantly blunts AMPK activity, hypoxia mediated CSE phosphorylation (pCSES346), and subsequent per-polysulfide production.
  • SSP4 Sulfane Sulfur Probe 4
  • Na2S3 sodium trisulfide
  • Anhydrous sodium sulfide was purchased from Alfa-Aesar Inc.
  • DATS Diallyl trisulfide
  • WSP-1 and SF-7 probes were purchased from Cayman Chemicals, MI, USA.
  • P-(4- Hydroxyphenyl)ethyl iodoacetamide (HPE-IAM) obtained from Chem-Impex.
  • the anti-CSE antibody was from Proteintech (IL, USA), anti-CBS, anti-3MST were from Abeam, MA, USA. All secondary fluorophore-labeled antibodies were obtained from Jackson Immunoresearch Inc (West Grove, PA, USA). Chemicals and tissue culture reagents were obtained from Sigma unless otherwise noted. [000378] Cell culture and treatments: Human embryonic kidney 293 cells (HEK) were purchased from ATCC, Virginia, USA and Human umbilical vein endothelial cells (HUVECs) were obtained from Lifeline Cell Technology, CA, USA (Cat# FC-0044). Mouse aortic endothelial cells were isolated in our COBRE molecular core and cultured in our laboratory.
  • HEK Human embryonic kidney 293 cells
  • UAVECs Human umbilical vein endothelial cells
  • HUVECs were cultured in VascuLife® Basal Medium (Cat# LM-0002) supplemented with the appropriate LifeFactors® Kit (Cat# LL-0003). All cells were grown in tissue culture flasks under normoxic conditions at 37°C, 5% CO2, and 21% 02. All media was changed every 2-3 days. Cells were transfected with transfection media (Lipofectamine 2000)
  • hypoxia treatment the cells were incubated in the hypoxic chamber (5% CO2, 37°C, 1% 02) for 30min. HUVECs from passages 2-4 was used in the experiments.
  • Site-directed mutagenesis of hCSE Phosphorylation mutants of hCSE of WT, constitutively active phospho negative alanine mutants S346A or T355A; phosphomimetic mutants with glutamate substitution, S346E or T355E.
  • the Site-Directed Mutagenesis kit from Agilent Technologies (Catalog #200522) was utilized. We performed PCR using two synthetic oligonucleotide primers, designed to incorporate the desired mutation. After thermal cycling, DPN I digestion was employed to selectively amplify PCR products containing the mutation.
  • nicked vector DNA now carrying the desired mutations, was transformed into competent cells. Plasmids were extracted from the resulting colonies and subjected to Sanger sequencing, thereby confirming the presence of the intended mutants. These mutated plasmids have been transfected into either HEKs, MAECS or HUVECs for various assays as mentioned in the manuscript.
  • H2S was measured using monobromobimane by RP-HPLC as we previously reported and described in detail elsewhere43. Briefly, plasma or tissue samples were collected and stabilized in vials containing 100 mM Tris-HCl buffer (pH 9.5, 0.1 mM DTP A) in a ratio of 1:5 (v/v) and snap-frozen until further analysis.
  • Fluorescent detection of poly sulfide metabolite Poly sulfide was detected using a fluorescent probe SSP4 as described earlier44. SSP4 was dissolved in DMSO to obtain a 1 mM stock solution. CTAB, a surfactant was dissolved in ethanol to yield a 5 mM stock solution. CTAB and SSP4 (5 pM) were dissolved in HBSS buffer and added to the samples for a total of lOOptl in a black polystyrene, flat-bottomed clear 96-well plate and incubated in the dark at room temperature for 20 min. Samples were measured on a TECAN fluorescence spectrophotometer (emission 525 nm: excitation 485 nm).
  • the digested peptides (5.0 pl) were loaded onto a trap column (PepMap C18, 2 cm x 100 pm, 100 A) at a flow rate of 20 pl/min using 0.1% formic acid and separated on an analytical column (EasySpray 50 cm x 75 pm, C18 1.9 pm, 100 A) with a flow rate of 300 nl/min with a linear gradient of 5 to 45% solvent B (100% ACN, 0.1% formic acid) over a 120 min gradient. Both precursor and fragment ions were acquired in the Orbitrap mass analyzer. Precursor ions were acquired in m/z range of 375-1500 with a resolution of 120,000 (at m/z 200).
  • Precursor fragmentation was carried out using the higher-energy collisional dissociation method using normalized collision energy (NCE) of 32.
  • NCE normalized collision energy
  • the fragment ions were acquired at a resolution of 150,000 (at m/z 200).
  • the scans were arranged in top-speed method with 3 sec cycle time between MS and MS/MS. Ion transfer capillary voltage were maintained at 2.1 kV.
  • the raw mass spectrometry data were analyzed using Proteome Discover (version 2.5, Thermo Fisher Scientific) software package with SequestHT using species-specific fasta database and the Percolator peptide validator. Cysteine alkylation was set as a fixed modification.
  • Each system was neutralized by adding the appropriate number of Na+ ions. Energy minimization of each structure was followed by a two-step equilibration (i.e., NVT equilibration followed by NPT equilibration).
  • the temperature of each system was controlled through velocity rescaling50 at 300 K with a time constant of 0.1 picosecond.
  • the pressure of each system was controlled using the Parrinello-Rahman barostat51 and set to 1 bar.
  • the particle mesh Ewald algorithm52 was used to calculate long-range electrostatics while a cutoff of 1 .0 nM was used for short-range electrostatics and van der Waals’ interactions.
  • S346 was produced by Genscript, USA Inc.
  • the antibody against the consensus CTH substrate was raised against the following synthetic peptide antigen CAESLGGFE[pSer]LAE. Briefly, rabbits were immunized with a synthetic phosphorylated peptide (KLH (keyhole limpet hemocyanin) coupled) corresponding to residues surrounding phosphorylation sites. Antibodies are then purified by to isolate the IgG antibody fraction. Affinity chromatography was performed using peptides coupled to SulfoLink resin. Both phospho-peptide-containing resin and the corresponding non-phospho-peptide resin were prepared.
  • KLH keyhole limpet hemocyanin
  • phospho-specific antibody (bound to the resin) was eluted with 0.1m glycine, pH 2.7, and pooled fractions were neutralized with 1 m Tris-HCl, pH 9.5 ( ⁇ l-2% of the fraction volume). The eluted phospho-specific antibody was then dialyzed overnight in PBS at 4 °C. The synthetic phospho-peptide was conjugated to KLH and injected into rabbits. Once rabbits showed high phospho-specific titers, serum was then purified by protein A chromatography. Phospho-CSE substrate antibody was found to be highly phospho-specific as crude serum, so the elution from the protein A column was used directly for affinity chromatography on a phospho-peptide-containing column.
  • Proteins were separated using 10% SDS-PAGE (mini or midi Bio-Rad, Hercules, CA) and transferred onto PVDF membranes, and incubated with antibodies against CSE (Cat# 12217-1-AP, Fisher Scientific), total AMPKa (Cat# 5832S), p-AMPKa (Cat#2535S) and a/ -Tubulin (Cat#2148) from Cell Signaling Technology. Chemiluminescent bands were detected and quantified using NIH Image J software.
  • RNA concentration was evaluated and purity with a typical OD260 to OD280 ratio of RNA sample was approximately 2.0.
  • cDNA was synthesized using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA), from 1 pg of total RNA. Quantitative PCR reactions were performed using the universal SYBR Green Supermix (Bio-Rad, CA, USA) on a CFX96 thermal cycler with Bio-Rad CFX Manager software (Bio-Rad, Hercules, CA, USA). The mean threshold cycle (Ct) values were plotted against the logarithm of the cDNA dilution factor. Quantitative PCR primers for genes, including GAPDH, CSE, CBS, AMPK 1, AMPK 2 was used in this study.
  • mice Twelve-week-old male C57BL/6J (Strain #:000664) mice purchased from Jackson Laboratory were used in this study as reported earlier7. Mice were housed at the Louisiana State University Health Sciences Center- Shreveport animal resources facility, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All animal studies were approved by the LSU Institutional Animal Care and Use Committee (LSU IACUC Protocol # P-21-010) and in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Chronic hindlimb ischemia was induced in 12-16-week-old male C57BL6/J mice, as we have reported previously?.
  • AMPK inhibitor, Dorsomorphin (10 mg/kg) was administered i.p. once a day during the length of the study.
  • Novadaq SPY imaging analysis The SPY imaging device (Novadaq Technologies) was used to quantify collateral vessel perfusion, as we have previously describedl4. Briefly, a bolus injection of 30 pl ICG (IC-Green, Akorn Pharmaceutical, Lake Forest, IL) was administered retro- orbitally, and angiograms were captured by an array of light-emitting diodes at a wavelength of 806 nm and recorded for 1 min. Angiograms were taken before and post-ligation on days 0, 1, 3, and 5 and percent change in blush rates were calculated as mentioned 14 .
  • ICG IC-Green, Akorn Pharmaceutical, Lake Forest, IL

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Abstract

This invention pertains to anti-CSE phosphospecific antibodies, phosphomimetic mutant polypeptides and fragments, and uses thereof to diagnose and treat tissue injury.

Description

PHOSPHOMIMETIC MUTANTS, PHOSPHOSPECIFIC ANTIBODIES, AND USES THEREOF
[0001] This application claims priority to U.S. Provisional Application No. 63/442,960, filed on February 02, 2023, and U.S. Provisional Application No. 63/534,065, filed on August 22, 2023, the entire contents of each of which are incorporated herein by reference.
[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
GOVERNMENT INTERESTS
[0004] This invention was made with government support under HL149264 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD OF THE INVENTION
[0005] This invention is directed to CSE phosphomimetic mutants, phosphospecific antibodies against CSE, and methods of use thereof.
SEQUENCE LISTING
[0006] The instant application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated by reference in its entirety. Said XML copy, created on [date], is named [file name] and is [size] bytes in size.
BACKGROUND
[0007] Hydrogen sulfide synthesis and metabolism is an important participant in cardiovascular health and function. Hydrogen sulfide can be generated by mammalian cells via a variety of enzymes, including cystathionine -synthase (CBS), cystathionine y-lyase (CSE), and 3 -mercaptopyruvate sulfur-transferase (MPST). Cystathionine y-lyase (CSE).
SUMMARY OF THE INVENTION
[0008] An aspect of the invention is directed to an isolated antibody or fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR).
[0009] In embodiments, the HCVR comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is SGGIS, the amino acid sequence of HCDR2 is SISTSGNTYYASWAKG, the amino acid sequence ofHCDR3 is ALAGI, the amino acid sequence of LCDR1 is QASQSVSKNNRLA, the amino acid sequence of LCDR2 is SASTLAS, and the amino acid sequence of LCDR3 is LGGYDCKSADCYI; the amino acid sequence of HCDR1 is SYGMI, the amino acid sequence of HCDR2 is AISSSGNTYYAKWAKG, the amino acid sequence of HCDR3 is NHYGSGDI, the amino acid sequence of LCDR1 is QSSQSVYDANRLA, the amino acid sequence of LCDR2 is GASTLDS, and the amino acid sequence of LCDR3 is QGYYSGYIYT; the amino acid sequence of HCDR1 is SYNMQ, the amino acid sequence of HCDR2 is IISSSDNTYYASWAKG, the amino acid sequence of HCDR3 is GRGYSSTTV, the amino acid sequence of LCDR1 is QASQSISSYLA, the amino acid sequence of LCDR2 is LASNMAS, and the amino acid sequence of LCDR3 is QCTYYHSSTSSTVGGA; the amino acid sequence of HCDR1 is SYGVT, the amino acid sequence of HCDR2 is WISTNSNAYYASWAKG, the amino acid sequence of HCDR3 is GIYSSGNI, the amino acid sequence of LCDR1 is QSSKSVYNNNKLS, the amino acid sequence of LCDR2 is STSSLAS, and the amino acid sequence of LCDR3 is AGGYSSNSDNT; the amino acid sequence of HCDR1 is SYTMS, the amino acid sequence of HCDR2 is YISRHGNTYYASWAKG, the amino acid sequence of HCDR3 is NIYSIDVI, the amino acid sequence of LCDR1 is QSSKSVGNNNRLS, the amino acid sequence of LCDR2 is GASTLAS, and the amino acid sequence of LCDR3 is LGAYSSSSDNS; the amino acid sequence of HCDR1 is SYAMG, the amino acid sequence of HCDR2 is YFSNNGNTYYANWAKG, the amino acid sequence of HCDR3 is SNL, the amino acid sequence of LCDR1 is QASQSVANNNRLS, the amino acid sequence of LCDR2 is YASTLAS, and the amino acid sequence of LCDR3 is LGSYDCRSADCYA; the amino acid sequence of HCDR1 is GSAVN, the amino acid sequence of HCDR2 is TISKNGNTYYATWAKG, the amino acid sequence of HCDR3 is RNPDTSGGLAL, the amino acid sequence of LCDR1 is QASQSVYSNNYLS, the amino acid sequence of LCDR2 is YASSLAS, and the amino acid sequence of LCDR3 is LGSYDCRAADCMA; or, the amino acid sequence of HCDR1 is SNGMS, the amino acid sequence of HCDR2 is YISSSGNVYYASWAKG, the amino acid sequence of HCDR3 is VLYSNGNI, the amino acid sequence of LCDR1 is QSSQSVYNNNRLS, the amino acid sequence of LCDR2 is EASKLAS, and the amino acid sequence of LCDR3 is LGGYSSNSDNA, or sequences that are at least 80% identical thereto.
[00010] In embodiments, the antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the amino acid sequence of the LCVR is AQVLTQTPSSVSAAVGSTVTINCQASQSVSKNNRLAWFQQKPGQPPKGLIYSASTLASGVSS RFKGSGSGTQFTLTISDVQCDDAATYYCLGGYDCKSADCYIFGGGTEVVVK, and the amino acid sequence of the HCVR is
QSVEESGGRLVTPGTPLTLTCTVSGIDLSSGGISWVRQAPGKGLEWIGSISTSGNTYYASWA KGRFTISKTSSTTVDLKMTSPTTEDTATYFCARALAGIWGPGTLVTVSS; the amino acid sequence of the LCVR is
AQVLTQTASSVSAAVGGTVTISCQSSQSVYDANRLAWYQQKPGQPPKRLIYGASTLDSGVS SRFKGSGSGTQFTLTISEVQCDDAATYYCQGYYSGYIYTFGGGTEVVVK, and the amino acid sequence of the HCVR is
QSVEESGGRLVTPGGSLTLTCTVSGIDLSSYGMIWVRQAPGEGLEWIGAISSSGNTYYAKWA KGRFPISRTSTTVDLKMTSLTASDTATYFCARNHYGSGDIWGPGTLVTVSS; the amino acid sequence of the LCVR is
DVVMTQTPASVSEPVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYLASNMASGVPSRF SGSGYGTEFTLTISGVQCEDAATYYCQCTYYHSSTSSTVGGAFGGGTEVVVK, and the amino acid sequence of the HCVR is
QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYNMQWVRQAPGKGLEYIGIISSSDNTYYASWA KGRFTISKTSSTTVDLRVTSLTTEDTATYFCARGRGYSSTTVWGPGTLVTVAS; the amino acid sequence of the LCVR is
AAVLTQTPSPVSAAVGGTVSISCQSSKSVYNNNKLSWFQQKPGQPPKQLIYSTSSLASGVPS RFSGSGSGTQFTLTISDVQCDDAATYYCAGGYSSNSDNTFGGGTEVVVK, and the amino acid sequence of the HCVR is
QSVKESGGRLVTPGTPLTLTCTVSGFSLSSYGVTWVRQAPGKGLEWIGWISTNSNAYYASW AKGRFTVSKTSSTTVTLTITSPTTEDTATYFCARGIYSSGNIWGPGTLVTVSS; the amino acid sequence of the LCVR is
AQVLTQTPSSVSAAVGGTVTINCQSSKSVGNNNRLSWYQQKPGQPPKQLIYGASTLASGVP SRFSGSGSGTQFTLTISDVQCDDAATYYCLGAYSSSSDNSFGGGTEVVVK, and the amino acid sequence of the HCVR is
QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYTMSWVRQAPGKGLEYIGYISRHGNTYYASW AKGRFTISKTSSTTVDLKTTSPTTEDTATYFCGRNIYSIDVIWGPGTLVTVSS; the amino acid sequence of the LCVR is
AQVLTQTASPVSAAVGGTVTINCQASQSVANNNRLSWFQQKPGQPPKLLIYYASTLASGVS SRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYDCRSADCYAFGGGTEVVVK, and the amino acid sequence of the HCVR is
QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYAMGWFRQAPGKGLEWIGYFSNNGNTYYAN WAKGRFTISKTSTTVDLKITRPTTEDTATYFCGRSNLWGQGTLVAVSS; the amino acid sequence of the LCVR is
AQVLTQTPSSVSAAVGGTVTINCQASQSVYSNNYLSWFQQKPGQPPKLLIYYASSLASGVPS RFSGSGSGTRFTLTISDVQCDDAAAYYCLGSYDCRAADCMAFGGGTEVVVR, and the amino acid sequence of the HCVR is
QSVEESGGRLVTPGTPLTLTCAVSGFSVSGSAVNWVRQAPGEGLEWIGTISKNGNTYYATW AKGRFTISKTSSTTVDLRMTTLTTEDTATYFCAGRNPDTSGGLALWGQGTLVTVSS; or the amino acid sequence of the LCVR is
AAVLTQTPSPVSAAVGGIVSISCQSSQSVYNNNRLSWFQKKPGQPPKLLIYEASKLASGVPP RFSGSGSGTQFTLTISGVQCDDAATYYCLGGYSSNSDNAFGGGTEVVVK, and the amino acid sequence of the HCVR is
QSVEESGGRLVTPGTPLTLTCTVSGIDLSSNGMSWVRQAPGKGLEWIGYISSSGNVYYASW AKGRFTISKTSTTVDLKITSPTTEDTATYFCARVLYSNGNIWGPGTLVTVSS, or sequences that are at least 80% identical thereto. [00011] In embodiments, the antibody binds to cystathionine gamma lyase (CSE). [00012] In embodiments, the antibody binds CSE phosphorylated at amino acid position serine 346 according to the sequence in Table 10. [00013] In embodiments, the antibody comprises a wildtype Fc or a modified Fc. [00014] In embodiments, the antibody fragment comprises an Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody. [00015] In embodiments, the antibody further comprises a detectable moiety.
[00016] Aspects of the invention are further directed to a nucleic acid encoding the antibody as described herein.
[00017] In embodiments, the nucleic acid sequence encoding LCVR is GCCCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAAGCACAGTCAC CATCAATTGCCAGGCCAGTCAGAGTGTTTCTAAGAACAACCGCTTAGCCTGGTTTCAGC AGAAACCAGGGCAGCCTCCCAAAGGCCTGATCTATTCTGCATCCACTCTGGCATCTGGG GTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGA CGTGCAGTGTGACGATGCTGCCACTTACTATTGTCTAGGCGGTTATGATTGTAAAAGTG CTGATTGTTATATTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCA CCTGCACAGTCTCTGGAATCGACCTCAGTAGTGGTGGAATAAGTTGGGTCCGCCAGGCT CCAGGGAAGGGGCTGGAATGGATCGGATCTATTAGTACTAGTGGTAACACATACTACG CGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTG AAAATGACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGCCCTGG CTGGCATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA, or a degenerate variant thereof; the nucleic acid sequence encoding LCVR is
GCGCAAGTGCTGACCCAGACTGCATCGTCCGTGTCTGCAGCTGTGGGAGGCACAGTCAC CATCAGTTGCCAGTCCAGTCAGAGTGTTTATGATGCCAACCGCTTAGCCTGGTATCAGC AGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTATGGTGCATCCACTCTGGATTCTGGG GTCTCATCACGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGA AGTACAGTGTGACGATGCTGCCACTTACTACTGTCAAGGCTATTATAGTGGTTATATTTA TACTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTAACGCCTGGAGGATCCCTGACACTCA CCTGCACAGTCTCTGGAATCGACCTCAGTAGCTATGGAATGATCTGGGTCCGCCAGGCT CCAGGGGAGGGGCTGGAATGGATCGGAGCCATTAGTAGTAGTGGTAACACATACTACG CGAAGTGGGCAAAAGGCCGATTCCCCATCTCCAGAACCTCGACCACGGTGGATCTGAA AATGACCAGTCTGACAGCCTCGGACACGGCCACCTATTTCTGTGCCAGAAATCATTATG GTAGCGGTGACATCTGGGGCCCGGGCACTTTGGTCACCGTCTCCTCA, or a degenerate variant thereof; the nucleic acid sequence encoding LCVR is GATGTTGTGATGACCCAGACTCCAGCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCAC CATCAAGTGCCAGGCCAGTCAGAGCATTAGTAGCTACTTAGCCTGGTATCAACAGAAAC CAGGGCAGCCTCCCAAGCTCCTGATCTATCTGGCATCCAATATGGCATCTGGGGTCCCA TCGCGGTTCAGCGGCAGTGGATATGGGACAGAGTTCACTCTCACCATCAGCGGCGTGCA GTGTGAAGATGCTGCCACTTATTATTGTCAATGTACTTATTATCATAGTAGTACTAGTAG TACTGTTGGGGGGGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCA CCTGCACAGTCTCTGGAATCGACCTCAGTAGCTACAACATGCAATGGGTCCGCCAGGCT CCAGGGAAGGGGCTGGAATACATCGGAATCATTAGTAGTAGTGATAACACATACTACG CGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTG AGAGTGACCAGTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGGGCGTG GTTATAGTAGTACTACTGTCTGGGGCCCAGGCACCCTGGTCACCGTCGCCTCA, or a degenerate variant thereof; the nucleic acid sequence encoding LCVR is GCCGCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGAGGCACAGTCAG CATCAGTTGCCAGTCCAGTAAGAGTGTTTATAATAACAACAAATTATCCTGGTTTCAGC AGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATTCTACATCCAGTCTGGCATCTGGG GTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGA CGTGCAGTGTGACGATGCTGCCACTTACTACTGTGCAGGCGGTTATAGTAGTAATAGTG ATAATACTTTCGGCGGAGGGACCGAGGTGGTCGTCAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is
CAGTCAGTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCA CCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATGGAGTGACCTGGGTCCGCCAGGCT CCAGGGAAGGGACTGGAATGGATCGGATGGATTAGTACTAATAGTAACGCATACTATG CGAGCTGGGCGAAAGGCCGATTCACCGTCTCCAAAACCTCGTCGACCACGGTGACTCTG ACAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGGGGTATTTA TAGTAGTGGTAACATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA, or a degenerate variant thereof; the nucleic acid sequence encoding LCVR is GCCCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCAC CATCAATTGCCAGTCCAGTAAGAGTGTTGGTAATAACAACCGCTTATCCTGGTATCAGC AGAAACCAGGGCAGCCTCCCAAGCAACTGATCTATGGTGCATCCACTCTGGCATCTGGG GTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGA CGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGCTTATAGTAGTAGTAGTG ATAATTCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCA CCTGCACAGTCTCTGGATTCTCCCTCAGTAGCTATACAATGAGTTGGGTCCGCCAGGCT CCAGGGAAGGGGCTGGAATACATCGGATACATTAGTAGGCATGGTAACACATACTACG CGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAGACCTCGTCGACCACGGTGGATCTG AAAACCACCAGTCCGACAACCGAGGACACGGCCACTTATTTCTGTGGCAGAAATATTTA TAGTATTGATGTCATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA, or a degenerate variant thereof; the nucleic acid sequence encoding LCVR is GCCCAAGTGCTGACCCAGACTGCATCCCCCGTGTCTGCGGCTGTTGGAGGCACAGTCAC CATCAATTGCCAGGCCAGTCAGAGTGTTGCTAATAACAACCGCTTATCCTGGTTTCAGC AGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTATTATGCATCCACTCTGGCATCTGGG GTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGA CGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCAGTTATGATTGTCGTAGTGC TGATTGTTATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCA CCTGCACAGTCTCTGGAATCGACCTCAGTAGCTATGCAATGGGCTGGTTCCGCCAGGCT CCAGGGAAGGGGCTGGAATGGATCGGATATTTTAGTAATAATGGTAACACATACTACG CGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAA AATCACCCGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGGCAGAAGCAACTTGT GGGGCCAAGGCACCCTGGTCGCCGTCTCCTCA, or a degenerate variant thereof; the nucleic acid sequence encoding LCVR is
GCTCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTCAC CATCAATTGCCAGGCCAGTCAGAGTGTTTATAGTAACAACTACTTATCCTGGTTTCAGC AGAAACCAGGGCAGCCTCCCAAACTCCTGATCTATTATGCATCCAGTCTGGCATCTGGG GTCCCATCGCGGTTCAGCGGCAGTGGATCTGGGACACGGTTCACTCTCACCATCAGCGA
CGTGCAATGTGACGATGCTGCCGCTTACTACTGTCTAGGCAGTTATGATTGTAGGGCTG CTGATTGTATGGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAGA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCA CCTGCGCAGTCTCTGGATTCTCCGTCAGTGGTAGTGCAGTGAACTGGGTCCGCCAGGCT CCAGGGGAGGGGCTGGAATGGATCGGGACAATTAGTAAGAATGGTAACACATACTACG CGACCTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGTGGATCTG AGAATGACCACTCTGACAACCGAGGACACGGCCACCTATTTCTGTGCCGGGCGCAATCC TGATACTAGTGGTGGTTTGGCCTTGTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCA, or a degenerate variant thereof; the nucleic acid sequence encoding LCVR is GCCGCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGGGAGGCATAGTCAG CATCAGTTGCCAGTCCAGTCAGAGTGTTTATAATAACAACCGCTTATCCTGGTTTCAGA AGAAACCAGGACAGCCTCCCAAGCTCCTGATCTACGAAGCATCCAAACTGGCATCTGG GGTCCCACCGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCG GCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCGGTTATAGTAGTAATAGT GATAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCA CCTGCACAGTCTCTGGAATCGACCTCAGTAGCAATGGAATGAGCTGGGTCCGCCAGGCT CCAGGGAAGGGGCTGGAATGGATCGGATATATTAGTAGTAGTGGTAACGTATACTACG CGAGCTGGGCAAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAA AATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGAGTTCTTTATA GTAATGGTAACATCTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA, or a degenerate variant thereof, or sequences that are at least 80% identical thereto.
[00018] Embodiments further comprise a vector encoding the nucleic acid as described herein, and a cell comprising the vector as described herein.
[00019] Aspects of the invention are drawn towards a pharmaceutical composition comprising the antibody as described herein, the nucleic acid as described herein, or the cell as described herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
[00020] Aspects of the invention are drawn towards a method of detecting phosphorylated CSE in a subject in need thereof. In embodiments, the method comprises contacting a biological sample with the antibody as described herein, wherein the antibody specifically binds to phosphorylated CSE. [00021] In embodiments, phosphorylated CSE is phosphorylated at amino acid position serine 346.
[00022] Aspects of the invention are drawn towards the use of an antibody as described herein, for detecting phosphorylated CSE in a subject.
[00023] In embodiments, the phosphorylated CSE is phophosphorylated at amino acid position 346.
[00024] Aspects of the invention are drawn towards a method of diagnosing a tissue injury using the antibody as described herein.
[00025] Aspects of the invention are drawn towards the use of an antibody as described herein for diagnosing a tissue injury.
[00026] Aspects of the invention are drawn towards a synthetic nucleic acid encoding a CSE mutant, wherein the CSE mutant mimicks phosphorylation at Serine 346, Threonine 355, or both. [00027] In embodiments, the synthetic nucleic acid is an mRNA molecule.
[00028] Aspects of the invention are drawn towards a pharmaceutical composition comprising a nucleic acid as described herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
[00029] Aspects of the invention are drawn towards a method of treating tissue injury in a subject. For example, the method comprises administering to a subject a nucleic acid encoding a CSE mutant, wherein the CSE mutant mimicks phosphosporylation at Serine 346, Threonine 355, or both. [00030] In embodiments, the nucleic acid increases cellular hydropersulfide, polysulfide, or both.
[00031] In embodiments, the nucleic acid comprises a synthetic nucleic acid expression construct, wherein the expression construct comprises a promoter operatively linked to the nucleic acid encoding a CSE mutant.
[00032] Aspects of the invention are drawn towards a method of increasing cellular hydropersulfide, polysulfide, or both. In embodiments, the method comprises administering to a subject a nucleic acid encoding a CSE mutant, wherein the CSE mutant mimicks phosphosporylation at Serine 346, Threonine 355, or both.
[00033] Other objects and advantages of this invention will become readily apparent from the ensuing description. BRIEF DESCRIPTION OF THE FIGURES
[00034] Figure 1 shows changes in sulfide metabolite levels under hypoxia and mass spec analyses depicting CSE (cystathionine gamma lyase) phosphorylation. Panel A shows mouse aortic endothelial cells (MAECs) exposed to either normoxia (21% oxygen) or hypoxic (1% oxygen) conditions for 30min and analyzed for various sulfide metabolites, including free, acid-labile sulfide and bound sulfane sulfur (that includes persulfide and polysulfides) using MBB/HPLC method developed in our lab. A significant increase in bound sulfane sulfur (persulfide and poly sulfide) was observed under hypoxia compared to normoxia. Panel B illustrates per- and polysulfides quantified using the fluorescent probe Sulfane Specific Probe 4 (SSP4) in MAECs treated with either normoxia (21% oxygen) or hypoxia (1% oxygen) for 30min. MAECs treated under hypoxia showed a significant 50% increase in per-polysulfide production compared to normoxia. Panel C illustrates that MAECs show a significant increase in cystathionine (a classical CSE substrate) consumption revealing an increase in CSE enzyme activity under hypoxia compared to normoxia. Panel D shows MAECs transfected with either mock, CSE, CBS (cystathionine beta synthase), MPST (3 -mercaptopyruvate sulfurtransferase), CARS-1 (cysteinyl-tRNA synthetase 1) or CARS-2 (cysteinyl-tRNA synthetase 2) siRNAs to reduce respective enzyme expression and then respectively were probed with SSP4 under hypoxic conditions. siCSE siRNA showed significant reduction in polysulfide generation compared to mock siRNA, demonstrating a dominant CSE role in hypoxia poly sulfide generation. siRNA to CARS1 and CARS2 also showed significant reduction in SSP4 compared to mock sima; however, they were significantly higher than siCSE revealing a lesser role of these enzymes in hypoxic per- polysulfide generation. Panel E shows LC/MS HCD fragmentation spectrum of trypsin digested human CSE that was purified from normoxic versus hypoxic treated cells. An over two-fold significant increase in CSE amino acid fragment 334-364 (LFTLAESLGGFESLAELPAIMTHASVLKNDR) phosphorylation was observed, which were identified by Protein Discoverer 2.5. (i) HCD spectra of native Human CSE [334-364] peptide, (ii) HCD spectra of singly phosphorylated Human CSE [334-364] at Ser346. (iii) HCD spectra of singly phosphorylated Human CSE [334-364] at T355. Panel F shows various human control, WT, or CSE phosphonegative alanine mutant constructs of S346A or T355A were transfected into HEK293 cells and cystathionine consumption measured. Panel G shows HEK293 cells transfected with either Control, WT, CSE glutamic acid (E) phospho-mimetics, S346E or T355E. Cells with either S346E or T355E showed a significant increase in cystathionine consumption under hypoxia. All the data are averaged from triplicates from each experiment with at least n=5. ****P<0.0001; ***p< 0.0002; **P<0.003; *P<0.01.
[00035] Figure 2 shows hydrogen sulfide (SF7 fluorophore) or per-polysulfide (SSP4 fluorophore) signal in HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-negative mutant S346A or T355A or S346A/T355A combined under normoxia. H2S levels quantified with fluorescent probe SF7 showed no significant difference. Panels A and B show the ability of cysteine as a substrate, and panels C and D show cystine as a substrate. Per-polysulfide levels measured with fluorescent probe SSP4 were significantly reduced in single phospho negative mutants S346A, T355A and S346A/T355A compared to WT CSE combined with cysteine (panel B) or cystine (panel D) substrates. All the data were averaged from triplicates from each experiment with at least n=5. ****P<0.0001; **P<0.003; *P<0.01.
[00036] Figure 3 shows hydrogen sulfide (SF7 fluorophore) or per-polysulfide (SSP4 fluorophore) signal in HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-mimetics mutant S346E or T355E or S346E/T355E combined under normoxia. H2S levels quantified with fluorescent probe SF7 showed no significant difference. Panels A and B show the ability of cysteine as a substrate, and panels C and D show cystine as a substrate. Per-polysulfide levels measured with fluorescent probe SSP4 were significantly greater in single phospho-mimetic mutants S346E, T355E and S346E/T355E combined compared to WT CSE with cysteine (panel B) or cystine (panel D) substrates. All the data were averaged from triplicates from each experiment with at least n=5. ****P<0.0001; **P<0.003; *P<0.01.
[00037] Figure 4 shows hydrogen sulfide (SF7 fluorophore) or per-polysulfide (SSP4 fluorophore) signal in HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-negative mutant S346A or T355A or S346A/T355A combined under hypoxia (1% oxygen) for 30 minutes. H2S levels quantified with fluorescent probe SF7 showed no significant difference. Panels A and B show the ability of cysteine as a substrate, and panels C and D show cystine as a substrate. Per-polysulfide levels measured with fluorescent probe SSP4 were significantly reduced in single phospho negative mutants S346A, T355A and S346A/T355A compared to WT CSE combined with cysteine (panel B) or cystine (panel D) substrates. All the data were averaged from triplicates from each experiment with at least n=5. ****P<0.0001; **P<0.003; *P<0.01
[00038] Figure 5 shows hydrogen sulfide (SF7 fluorophore) or per-polysulfide (SSP4 fluorophore) signal in HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-mimetics mutant S346E or T355E or S346E/T355E combined under hypoxia (1% oxygen). H2S levels quantified with fluorescent probe SF7 showed no significant difference. Panels A and B show the ability of cysteine as a substrate, and panels C and D show cystine as a substrate. Per- polysulfide levels measured with fluorescent probe SSP4 were significantly greater in single phosphomimetic mutants S346E, T355E and S346E/T355E combined compared to WT CSE with cysteine (panel B) or cystine (panel D) substrates. All the data were averaged from triplicates from each experiment with at least n=5. ****P<0.0001; **P<0.003; *P<0.01.
[00039] Figure 6 shows the effect of cellular hypoxia or tissue ischemia on phosphorylation of CSE and its regulation in vitro and in vivo. Panel A shows SSP4 polysulfide levels were significantly reduced upon inhibition of AMPK (AMPK-I; Dorsomorphin) compared to control (mock). Panel B illustrates representative western blots of p-AMPK and GAPDH from MAECs treated under normoxia or hypoxia for 30 minutes with a graph representing the quantification shown below. Phospho- AMPK (pAMPK) was significantly increased under hypoxic conditions. Panel C shows western blots of pCSE346 and GAPDH from MAECs treated under normoxia or hypoxia. Graphic representation of the densitometry quantification depicted below shows a significant increase in pCSE346 protein expression under 30 minutes of hypoxia. Panel D illustrates representative blots of pCSE346 and GAPDH from MAECs treated under normoxia, hypoxia, or hypoxia+ AMPK-I. Graphic representation of the densitometry quantification shows a significant increase in pCSE346 protein expression under hypoxia, which is significantly reduced with AMPK-I. pAMPK and pCSE346 were next examined in muscle tissue from mice subject to the femoral artery ligation (FAL) model that establishes tissue ischemia. Western blots were performed from non-ischemic (NI) and ischemic (I) skeletal muscle (SkM) tissues collected from day 4 post femoral artery ligation. Graphs represent densitometry quantification of phospho-molecules compared to GAPDH loading control. Panel E shows representative western blots of pAMPK and GAPDH blots with pAMPK significantly increased in ischemic SkM tissues. Panel F demonstrates representative western blots of pCSE346 and GAPDH showing a significant increase in pCSE346 in ischemic SkM tissues. All the data were representative of at least n=3. **P<0.003; *P<0.01.
[00040] Figure 7 shows schematic depicting embodiments of the invention.
[00041] Figure 8 shows CSE Sulfide metabolite formation. CSE uses cysteine or cystathionine as substrates to make H2S but can also use cystine (CysSSCys) to generate cysteine persulfide (CysSSH). H2S can form HS* radical leading to hydropersulfide or polysulfide. [00042] Figure 9 shows endothelial specific CSE genetic deficiency. Panel A shows creation of CSE tmlc conditional mutant allele from the European Mutant Mouse Archive (EMMA). Tmla mice were bred with Flp recombinase mice creating the tmlc allele and subsequently bred with VE- Cad Cre- driver mice to create the VE-Cad cre/CTHA/A. Panel B shows the PCR genotyping of the CTHA/A mutant allele. Panel C shows representative images of gastrocnemius muscle tissue from control and VE-Cad/CTHA/A mice co-stained with CSE, CD31 and DAPI shows significant reduction in CSE expression. Panel D and E shows significant reduction in CSE mRNA in skeletal muscle and aorta respectively. Panel F and G represents reduction in Total sulfide levels of plasma and skeletal muscle tissues in ecCSEKO compared to the control. *p<0.01 ecCSEKO vs control.
[00043J Figure 10 shows endothelial CSE and ischemic vascular remodeling. Panel A reports a decrease in blood flow in CSEKO and ecCSEKO compared to WT. Panel B shows representative heatmap images of blood flow in WT and ecCSEKO at pre, post and at day7 after ligation respectively. Panel C and D shows representative images and quantification of skeletal muscle tissues showing reduced vascular angiogenic index and arteriole density in ecCSEKO ischemic tissue. *p<0.01 ecCSEKO vs WT.
[00044] Figure 11 shows hypoxia CSE and sulfide metabolism responses. Panel A shows 30 min of hypoxia does not change CSE protein levels, but CSE enzyme activity was increased >2 fold. Panel B reports a significant decrease in acid labile sulfide and increase in bound sulfane sulfur. *p<0.01 hypoxia vs control.
[00045] Figure 12 shows Per/polysulfide effects on MAEC proliferation and permeability. Panel A shows increases in BrdU incorporation due to per and polysulfide treatment. Panel B shows the effect of per/polysulfides on albumin permeability across MAEC monolayers. *p<0.05, **p<0.01, ***p< 001 vs vehicle.
[00046] Figure 13 shows H2S increases hypoxic HUVEC NO formation. Panels A & B show the amount of NO produced in response to increasing amounts of H2S in normoxic and hypoxic HUVEC, respectively. Panel C illustrates the amount of NO that was produced from normoxic and hypoxic HUVEC with various sulfide levels. Treatment with NOS inhibitor L-NAME did not, but the XO inhibitor febuxostat did blunt sulfide dependent NO formation. *p<0.01 hypoxic vs normoxic; #p<0.01 vs 100 uM sulfide alone.
[00047] Figure 14 shows CSE expression and activity in mouse spleen or bone marrow monocytes upon femoral artery ligation. Panels A & B report qRT-PCR CSE mRNA in monocytes from different tissue niches. Panels C & D demonstrate the monocyte CSE activity in WT control and (pCSEKO.
[00048] Figure 15 shows myeloid CSE and ischemic vascular remodeling. Panel A reports a significant decrease in blood flow in CSEKO and (pCSEKO compared to WT. Panel B shows a representative heatmap images of blood flow in WT control and (pCSEKO at pre, post and at day7 after ligation respectively. Panel C and D shows representative images and corresponding quantification of skeletal muscle tissues showing reduced vascular angiogenic index and arteriole density in (pCSEKO compared to WT. *p<0.01 (pCSEKO vs WT.
[00049] Figure 16 shows CSE deficiency inhibits myeloid recruitment in ischemic tissues. Representative images of ischemic skeletal muscle tissues of A. Control B. (pCSEKO C. VE-Cad Control and D. ecCSEKO, co-stained with DAPI (blue) and MAC-2 (green) reports a significant decrease in MAC-2 staining. Corresponding quantification is shown in panels E and F. *p<0.01 WT vs cell specific CSE KO.
[00050] Figure 17 shows identification of CSE phosphorylation by mass spectrometry. Panels A and B illustrates peptide motifs of human CSE under normoxic and hypoxic conditions identifying phospho-S346, T355, and S358. Panel C illustrates CSE activity via cystathionine consumption in response to normoxia vs hypoxia (*p<0.01). Panel D reports sulfide metabolite levels between cells under normoxic vs hypoxic treatments with a significant increase in bound sulfide (*p<0.01).
[00051] Figure 18 shows inhibition of regulators of CSE affects activity under hypoxia. A significant reduction in CSE activity was observed with inhibition of AMPK, PKC and ATR. *p<0.01 inhibitors vs control.
[00052] Figure 19 shows models of in situ vascular remodeling. Panel A shows SPY angiographic image showing tissue perfusion and collateral arterioles in mouse hindlimbs. Panel B shows microfil vascular casting image of ischemic gracillis arterioles at day 7 following FAL.
[00053] Figure 20 shows LC-MS/MS detection of different sulfide metabolites. Panels A, B and C illustrates MS of Sdibimane for H2S, SS-dibimane for H2S2 and SSSdibimane for H2S3, respectively. This LC-MS/MS method allows accurate detection of multiple sulfide species.
[00054] Figure 21 shows increased Ml and reduced M2 macrophage signature in CSE KO and (pCSEKO mice. Ischemic gastrocnemius muscle tissue was harvested 5 days post FAL in WT, CSEKO, (pCSEKO and ecCSEKO mice for Ml vs M2 marker measurement. *p<0.01 control vs (pCSEKO. [00055] Figure 22 shows specific site mutations of CSE phospho-amino acids for dominant negative or constitutively active analysis. Mutations of Ser346, Thr355, Ser358, or various combinations will be made to analyze effects on CSE enzyme activity and sulfide metabolite formations.
[00056] Figure 23 shows measurement of PLP by HPLC analysis. Analytical PLP measurement is performed by HPLC achieving nanomole detection sensitivity. Panel A illustrates PLP standard curve; Panel B reports PLP bioavailability in mouse plasma with or without spiked 20 nM PLP. **p<0.01 versus plasma alone.
[00057] Figure 24 shows hypoxic per-polysulfide formation and CSE phosphorylation. Panel a shows MAECs exposed to either normoxia (21% oxygen) or hypoxic (1% oxygen) conditions for 30min and analyzed for various sulfide metabolites, including free, acid-labile sulfide and bound sulfane sulfur (including persulfide and polysulfide) using MBB/HPLC method. Panel b Per- polysulfides levels in MAECs treated with either normoxia or hypoxia for 30min using the fluorescent probe SSP4. Panel c CSE enzyme activity under hypoxia compared to normoxia in MAECs. Panel d Per-polysulfide levels in MAECs transfected with either mock, CSE, CBS, MPST, CARS-1 or CARS- 2 siRNAs and then respectively were probed with SSP4 under hypoxic conditions. Panel e LC/MS HCD fragmentation spectrum of trypsin digested human CSE purified from normoxic versus hypoxic treated HEK cells. CSE amino acid fragment 334-364 (LFTLAESLGGFESLAELPAIMTHASVLKNDR) was identified by Protein Discoverer 2.5. (i) HCD spectra of native Human CSE [334-364] peptide, (ii) HCD spectra of singly phosphorylated Human CSE [334-364] at Ser346. (iii) HCD spectra of singly phosphorylated Human CSE [334-364] at T355. Panel f Phospho peptide ratios comparing CSE [334-364] and CSE[365-395] from LC/MS HCD fragmentation spectrum of trypsin digested human CSE purified from hypoxia. Panel g CSE activity of Control (Con), WT, or CSE phosphonegative alanine mutant constructs of S346A or T355A transfected into HEK293 cells. Panel h CSE activity of HEK293 cells transfected with either Control, WT, CSE glutamic acid (E) phospho-mimetics, S346E or T355E, under hypoxia. All the data are averaged from triplicates from each experiment with at least n=5. ****P<0.0001; ***P< 0.0002; **P<0.003; *P<0.01.
[00058] Figure 25 shows phospho-negative mutants reduces per-polysulfide levels under hypoxia. HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-negative mutant S346A or T355A under hypoxia (1% oxygen) for 30 minutes and probed for per-polysulfide (SSP4 fluorophore) or hydrogen sulfide (SF7 fluorophore) signal. Panels a and b show the ability of cysteine as a substrate, panels c and d show cystine as a substrate, whereas panels e and f show cystine as a substrate for hydrogen sulfide or per-polysulfide levels respectively. All the data were averaged from triplicates from each experiment with at least n=5. ****P<0.0001; **P<0.003; *P<0.01
[00059] Figure 26 shows phospho-mimetics induces per-polysulfide levels under hypoxia. HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-mimetic mutants S346E or T355E under hypoxia (1% oxygen) for 30 minutes and probed for per-polysulfide (SSP4 fluorophore) or hydrogen sulfide (SF7 fluorophore) signal. Panels a and b show the ability of cysteine as a substrate, panels c and d show cystine as a substrate, whereas panels e and f show cystine as a substrate for hydrogen sulfide or per-polysulfide levels respectively. All the data were averaged from triplicates from each experiment with at least n=5. ****P<0.0001; **P<0.003; *P<0.01
[00060] Figure 27 shows molecular dynamics simulations of cystathionine gamma lyase. Three-dimensional structure of CSE as a tetramer and the respective composite monomers generated by AlphaFold. Molecular dynamics simulations of 350 ns were performed with linear constraint solver (LINCS) constraints for all bonds for a. WT CSE, and b. CSE with phospho sites S346 (purple), and T355 (gold). Molecular dynamics simulations Phosphorylation of residues S346 and T355 modeled in Pymol using the PyTMs plugin. All were performed using GROMACS 2019 software with the GROMOS 54A7 force field and SPC216 water model. Frames were recorded every 2 ps. Panel c The 300 ns simulation showing extensive intra- and inter-molecular contacts induced by phosphorylation of 346. Each p346 monomer were color coded belongs to and kept the scheme as shown panel A to illustrate which contacts are intra vs inter-molecular contacts. The backbone RMSD was monitored over the production run of each protein to ensure the stability and convergence of the simulated trajectories. p346 leads to a new interaction between monomer A (p346) and B (K260, R257), monomer B (p346, H217, E345) and monomer A (K260, R257), monomer C (p346, T336) and monomer A (K48), and monomer D (p346, E345, E381) and monomer C (K260). Panel d Intra- and inter-molecular electrostatic interactions formed by phosphorylation of T355. Interactions to pT355A - H55D, R62D, R119A (Fig 4D, inset i); pT355B - R119B (Fig 4D, inset ii); pT355D - Q323D, M354D, H356D, S358D, V359D (Fig 4d, inset iii); and pT355C -R119C, H356C, R62B, N241B (Fig 4d, inset iv). Panel e HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-mimetic mutants S346E or T355E with or without PAG under hypoxia (1% oxygen) for 30 minutes and probed for per-polysulfide (SSP4 fluorophore) Panel F HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-negative mutants S346A or T355A with or without PAG under hypoxia (1% oxygen) for 30 minutes and probed for per-polysulfide (SSP4 fluorophore). All the data are averaged from triplicates from each experiment with at least n=5. ****P<0.0001; ***P< 0.0002; *P<0.01.
[00061] Figure 28 shows hypoxia induces per-polysulfide via AMPK-mediated phosphorylation of CSE. Panel a Representative blots of MAECs treated under hypoxia for 0, 5, 15, 30, 60 and 90 min probed for pCSES346, total CSE, pAMPK, AMPK and GAPDH. Panel b Quantitation of pCSES346 and p-AMPK protein levels, respectively from western blots in Panel A. Panel c Representative blots of MAECs treated with mock or AMPK inhibitor (AMPK-I), Dorsomorphin under hypoxia for 30min followed by per-polysulfide (SSP4 fluorophore) signal. Panel d Quantitation of pCSES346 and pAMPK protein levels, from western blots in Panel c. Panel e MAECs treated with mock or AMPK inhibitor (AMPK-I), Dorsomorphin under hypoxia for 30min followed by per-polysulfide (SSP4 fluorophore) signal. Panel f Representative blots from HUVECs treated under Normoxia or hypoxia or hypoxia+ AMPK-I analyzed for protein levels of pCSES346, total CSE, pAMPK, AMPK and GAPDH. Panel g HUVECs treated with mock or AMPK inhibitor (AMPK-I), Dorsomorphin under hypoxia for 30min followed by per-polysulfide (SSP4 fluorophore) signal. Panel h HUVECs treated under Normoxia or hypoxia or hypoxia+ AMPK-I for 30min followed by per-polysulfide (SSP4 fluorophore) signal. All the data are averaged from triplicates from each experiment with at least n=5. ****P<0.0001; ***P< 0.0002; *P<0.01.
[00062] Figure 29 shows AMPK inhibition reduces CSE phosphorylation, per-polysulfide and ischemic blood flow. Panel a Representative blots of ischemic gastrocnemius muscle tissues from mice subject to the femoral artery ligation (FAL) Ohrs, 3hrs, 24hrs and 5 days probed for pCSES346, total CSE, pAMPK, AMPK and GAPDH. Panel b Graphic representation of the densitometry quantification depicted in Panel A for pCSE346 and p-AMPK protein expression. Panel c Representative western blots were performed from non-ischemic (NI) and ischemic (I) skeletal muscle (SkM) tissues collected from day 4 post femoral artery ligation. Panel d Graphic representation of the densitometry quantification of pCSE346 and p-AMPK protein expression. Graphs represent densitometry quantification of phospho-molecules compared to GAPDH loading control. Panel e Representative heat map images of ischemic hindlimbs showing blood flow. All the data were representative of at least n=5. **P<0.003; *P<0.01. Panel f Graphic representation of the densitometry quantification 1 depicted in Panel E for ischemic limb blood flow. Panel g Plasma sulfide levels, including free, acid labile pools (F/ALP) and bound sulfide. All the data were representative of at least n=5. ****P<0.0001 [00063] Figure 30 shows CSE role in sulfide and per-polysulfide. Panel a Hydrogen sulfide (SF7 fluorophore) signal in HEK293 cells with either Control (Con), Na2S or Na2S3 treatments. Panel b Per-polysulfide levels (SSP4) in HEK293 cells with either Control (Con), DATS or Na2S3 treatments. Panel c Hydrogen sulfide (SF7) fold change in HEK293 cells treated under normoxia or hypoxia. Panel d Hydrogen sulfide (SF7) levels in MAECs transfected with either mock, CSE, CBS, MPST, CARS-1 or CARS-2 siRNAs and then respectively were probed with SF7 under hypoxic conditions. Panels e-i CSE, CBS, MPST, CARS1 and CARS2 mRNA expressions; Panels j-n Representative western blots and quantitation of protein levels of CSE, CBS, MPST, CARS1 and CARS2, following transfection of HEK293 cells with respective siRNA. All the data were averaged from triplicates from each experiment with at least n=5. ****P<0.0001; *P<0.01.
[00064] Figure 31 shows CSE mutant conservation. Panel a Conservation sequence of human CSE at S346 and T355 compared across various species, including human, monkey, Danio rerio (Zebra fish), Xenopus tropicalis (frog), rat, mouse, Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. Representative blots of Flag, CSE and GAPDH and quantification of protein levels from HEKs transfected with Panel b Veh, WT, phosphonegative mutants, S346A or T355A Panel c phospho mimetics S346E or T355E. All the data are averaged from triplicates from each experiment with at least n=3. ****P<0.0001
[00065] Figure 32 shows sulfide levels in normoxia with phospho negative mutants. Per- polysulfide (SSP4 fluorophore) or hydrogen sulfide (SF7 fluorophore) signal in HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-negative mutant S346A or T355A under normoxia. H2S levels quantified with fluorescent probe SF7 showed no significant difference. Panels a and b Ability of cysteine as a substrate, and panels c and d show cystine as a substrate. Per-polysulfide levels measured with fluorescent probe SSP4 in single phospho negative mutants S346A or T355A compared to WT CSE combined with cysteine (panel A) or cystine (panel C) substrates. All the data were averaged from triplicates from each experiment with at least n=5. ****p<0.0001; **P<0.003; *P<0.01.
[00066] Figure 33 shows sulfide levels in normoxia with phosphomimetic mutants. Per- polysulfide (SSP4 fluorophore) or hydrogen sulfide (SF7 fluorophore) signal in HEK293 cells transfected with either Control (Con), wild type CSE (WT), phospho-mimetics mutant S346E or T355E under normoxia. H2S levels quantified with fluorescent probe SF7 showed no significant difference. Panels a and b show the ability of cysteine as a substrate, and panels c and d show cystine as a substrate. Per-polysulfide levels measured with fluorescent probe SSP4 were significantly greater in single phospho-mimetic mutants S346E and T355E compared to WT CSE with cysteine (panel A) or cystine (panel C) substrates. All the data were averaged from triplicates from each experiment with at least n=5. ****P<0.0001; **P<0.003; *P<0.01.
[00067] Figure 34 shows per and polysulfide levels in CSE phospho mutants. Abundance to protein fold change signal in MAECs transfected with either wild type CSE (WT) under normoxia (N- WT) or hypoxia (H-WT), phospho negative mutants S346A, T355A; phospho-mimetics mutant S346E or T355E under hypoxia showing Panel a Glutathionine persulfide (GSSH) and Panel b Glutathionine polysulfide (GSSSSH). All the data are averaged from triplicates from each experiment with at least n=3. ****P<0.0001; ***P< 0.0002; **P<0.003.
[00068] Figure 35 shows AMPK regulation of CSE and per-polysulfide in endothelial cells. Panel a CSE phospho sites S346 and T355 showing modular signaling domains of protein Serine/Threonine kinases motif groups using Scansite 4. Panel b SF7 fold change in MAECs treated with mock or AMPK-I. Panel c SF7 levels from HUVECs under normoxia vs hypoxia. Panel d SSP4 levels from HUVECs under normoxia vs hypoxia. Panel e SSP4 levels from HUVECs under hypoxia vs hypoxia+ AMPK-I. MAECs treated with si-Con, siAMPKal or AMPKa2 checked for Panel f AMPKal mRNA expression Panel g AMPKa2 mRNA expression. Panel h. Representative blots of pCSES346, total CSE, pAMPK, AMPK and GAPDH from MAECs treated under Normoxia or hypoxia, hypoxia+AMPK-I Panel i Quantitation of pCSES346 and p-AMPK protein levels, respectively from western blots in Panel G. Panel j. SSP4 levels from MAECs transfected with siCon or AMPKal or AMPKa2 under hypoxia. All the data are averaged from triplicates from each experiment with at least n=5. ****P<0.0001; ***P< 0.0002; **P<0.003; *P<0.01.
[00069] Figure 36 shows polysulfide elevates under ischemia. Total sulfide levels, including free/acid labile pools (F/Al) and bound Sulfane sulfur levels at 0, 24hr and 5days ischemia from Panel a. Plasma Panel b ischemic skeletal muscle tissues. All the data are averaged from triplicates from each experiment with at least n=5. ***p< 0.0002; **P<0.003
[00070] Figure 37 to Figure 44 shows Western blot analysis validating antibody specificity, antibody binding data. DETAILED DESCRIPTION OF THE INVENTION
[00071] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.
[00072] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
[00073] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.
[00074] The term substantially allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.
[00075] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.
[00076] The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).
[00077] The term "preventing" a disease can refer to inhibiting the full development of a disease, such as tissue injury. "Treating" can refer to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as tissue injury. "Ameliorating" refers to the reduction in the number or severity of signs or symptoms of a disease, such as tissue injury. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing a pathology, such as tissue injury.
[00078] The term “therapeutic” in conjunction with a phosphospecific antibody, a CSE variant polypeptide or fragment, a CSE variant polynucleotide, a vector, or a cell that is disclosed herein refers to a phosphospecific antibody, a CSE variant polypeptide or fragment, a CSE variant polynucleotide, a vector, or a cell that is suitable for use in human treatment of tissue injury.
[00079J The term “diagnosing” a disease can refer to assessing whether a subject is suffering from a disease, such as tissue injury.
[00080] The term “administration” can refer to the act of the attending physician or caregiver, prescribing the agent for administration and thereby causing the application of an agent to a subject, through ingestion, infusion, injection, or any other means, whether self-administered or administered by a clinician or other qualified care giver. Herein, a “subject” can include both human patient and veterinary subjects, including human and non-human mammals. In embodiments, the subject or patient has or has a risk of tissue injury.
[00081] The term “effective amount” can refer to a quantity sufficient to achieve a desired effect in a subject. For instance, this can be the amount necessary to prevent, treat, or ameliorate a disease, for example, inhibiting or suppressing tissue injury. Efficacy is first evident in the cellular response, for which a variety of in vitro and cell assays are well-known to measure. Kristina V. Kitaeva et al., Cell Culture Based In vitro Test Systems for Anticancer Drug Screening, 8 Front. Bioeng. Biotechnol. 322(2020)). For example, efficacy can be evidenced by increasing or decreasing levels of hydropersulfide, polysulfide, or both after administration of the therapeutic. In embodiments, an effective amount is the amount necessary to significantly increase or decrease inhibit or reduce hydropersulfide levels, polysulfide levels, or both. An effective amount typically provides improvement in important endpoints, including Overall Survival (OS), Disease-Free Survival (DFS), Objective Response Rate, Complete Response Rate or Progression Free Survival (PFS). See Dept, of Health and Human Services, Food and Drug Admin, Clinical Trial Endpoints for the Approval of Cancer Drugs and Biologies: Guidance for Industry (2018); E.A. Eisenhauer et al., New Response Evaluation Criteria in Solid Tumours: Revised RECIST Guideline (Version 1. I), 45 Eur. J. Cancer 228 (2009).
[00082] The term “tissue injury” can refer to a tissue that is experiencing or has experienced cell death, loss of tissue function, reduction of tissue function, fibrosis, DNA damage, and the like. Tissue injury can result from, for example, ischemia or ischemia/reperfusion injury.
[00083] The term “recombinant polypeptide” can refer to a peptide, polypeptide, or protein that results from the expression of a recombinant nucleic acid (e.g., recombinant DNA) within living cell.
[00084] The term synthetic polypeptide can refer to a peptide, polypeptide, or protein that is formed, in vitro, by joining amino acids or amino acid analogs in a particular order, using well known techniques of synthetic organic peptide synthesis to form the peptide bonds, e.g., via solid phase peptide synthesis.
[00085] The terms “fused” or “fusion” indicate that at least two polypeptide chains have been operably linked and recombinantly expressed. In embodiments, two polypeptide chains can be “fused” as a result of chemical synthesis. The terms “conjugate” or “conjugation” can denote that two molecular entities (e.g., two polypeptides, or a polypeptide and a polymer such as PEG) have been chemically linked.
[00086]
[00087] Phosphospecific antibodies
[00088] The disclosure provides an isolated antibody or fragment thereof, wherein the antibody binds to cystathionine gamma lyase (CSE). In embodiments, the disclosure provides an antibody or fragment thereof that binds CSE phosphorylated at amino acid position 346.
[00089] Used herein, an "antibody" can refer to an immunoglobulin molecule comprising two heavy chains (HCs) and two light chains (LCs) interconnected by disulfide bonds. The amino terminal portion of each LC and HC includes a variable region of about 100-120 amino acids primarily responsible for antigen recognition via the complementary determining region (CDRs) contained therein. The CDRs are interspersed with regions that are well-known and generally conserved among and between species (e.g., mouse and human), which are termed framework regions (FRs). [00090] In embodiments, the CDRs are interspersed with FRs. Antibodies disclosed herein have four FRs, termed FR1, FR2, FR3, and FR4. In embodiments, the FRs are human FRs (e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)).
[00091] The three CDRs of the LC are referred to as "LCDR1, LCDR2, and LCDR3," and the three CDRs of the HC are referred to as "HCDR1, HCDR2, and HCDR3." The functional ability of an antibody to bind a particular antigen is largely determined by the six CDRs. Assignment of amino acids to CDR domains within the LCVR and HCVR regions of the antibodies of the present disclosure is based on known numbering conventions (Andrew Martin, Protein Sequence and Structure Analysis of Antibody Variable Domains in Antibody Engineering (Roland Kontermann and Stefan Diibel eds., 2d ed. 2010)).
[00092] FR1 -CDR1-FR2-CDR2-FR3 -CDR3-FR4
[00093] Table 1A: Nucleic acid sequence of antibody 1A5-1
[00094] Table IB: Amino Acid sequence of antibody 1A5-1
[00095] Table 2A: Nucleic acid sequence of antibody 4B7-1
[00096] Table 2B: Amino Acid sequence of antibody 4B7-1
[00097] Table 3A: Nucleic acid sequence of antibody 7A5-2
[00098] Table 3B: Amino Acid sequence of antibody 7A5-2
[00099] Table 4A: Nucleic acid sequence of antibody 7F7-2
[000100] Table 4B: Amino Acid sequence of antibody 7F7-2
[000101] Table 5 A: Nucleic acid sequence of antibody 9H9-2
[000102] Table 5B: Amino Acid sequence of antibody 9H9-2
[000103] Table 6 A: Nucleic acid sequence of antibody 10D1-1
[000104] Table 6B: Amino Acid sequence of antibody 10D1-1
[000105] Table 7 A: Nucleic acid sequence of antibody 14C7-1
[000106] Table 7B: Amino Acid sequence of antibody 14C7-1
[000108] Table 8B: Amino Acid sequence of antibody 28C6-1
[000109] Table 9: Amino Acid sequences of CDRs
[000110] The constant region of the antibody defines the isotype of an antibody. The antibodies of the present disclosure include IgG. IgG antibodies can be further divided into subclasses, e.g., IgGl, IgG2, IgG3, IgG4. The carboxy -terminal portion of each HC defines a constant region primarily responsible for effector function. In embodiments, the antibodies of the present disclosure can have one or more modifications in the constant region of each HC that reduces effector function.
[000111] Contemplated herein are conservative variants of the disclosed antibodies and fragments thereof. A protein is a conservative variant where it contains conservative amino acid substitutions that do not substantially affect or decrease the affinity of a protein. For example, an antibody that binds CSE phosphorylated at serine 346 can include at least 1, 2, 5, 10, or 15 conservative substitutions, for example, in a constant domain, and bind CSE. Conservative amino acid substitution tables providing functionally similar amino acids are well-known to one of ordinary skill in the art. The following groups are examples of amino acids that are considered conservative substitutions for one another: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).
[000112] Herein, a "degenerate variant" can refer to a polynucleotide encoding a polypeptide (such as an antibody or fragment thereof) that includes a sequence that is degenerate based on the genetic code (i.e., the 20 natural amino acids can be specified by more than one codon). All degenerate nucleotide sequences encoding the disclosed antibody and fragment polypeptide sequences are included.
[000113] Further contemplated are variants of the disclosed antibodies and fragments thereof with a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of the sequences according to Tables 1-9. Herein, “sequence identity can refer to as the similarity between amino acid or nucleic acid sequences, which is expressed as the similarity between the sequences. Sequence identity is frequently measured as percent identity, in which two sequences are considered more similar the higher the percentage. Homologs or variants of a polypeptide or nucleic acid molecule possess a relatively high degree of sequence identity when aligned using standard methods, which are well-known. Ceslovas Venclovas, Methods for Sequence-Structure Alignment in Homology Modeling: Methods and Protocols, 55-82 (Andrew Orry and Ruben Abagyan, eds., 2012)). [000114] Herein, “binding” (or “binds”) can refer to the well understood interaction between and antibody and a target protein, peptide, or polysaccharide. Binding can be measured in a variety of ways (see, e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)). In embodiments, binding can be measured by methods known to the skilled artisan], A particular antibody or protein binds to a particular target protein, peptide, or polysaccharide and does not bind in a significant amount to other proteins or polysaccharides present in a sample or subject disclosed herein. Binding occurs between the disclosed antibodies and fragments thereof and an epitope of CSE. Herein, "epitope" can refer to discrete sites of an antigen recognized by the disclosed antibodies and fragments thereof. Epitopes may be linear or three-dimensional. An antibody binds to a target protein when the interaction has a KD of less than 10'6 molar, such as less than 10’7 molar, less than 10‘8 molar, less than 10’9 molar, or less than 10’10 molar.
[000115] The antibodies and fragments thereof disclosed herein can bind CSE phosphorylated at amino acid position 346. [000116] The antibodies and fragments thereof disclosed herein can be administered to subjects or patients.
[000117] The antibody or fragment disclosed herein can be a therapeutic antibody. For example, the term “therapeutic antibody” can refer to an antibody disclosed herein that is suitable for use in human treatment of tissue injury. Such an antibody has a KD of less than 10'6 molar, such as less than 10'7 molar, less than 10'8 molar, less than 10'9 molar, or less than IO'10 molar and any toxic or detrimental effects of the antibody are outweighed by the therapeutic beneficial effects.
[000118] The term “diagnostic” in conjunction with an antibody disclosed herein can refer to an antibody suitable for use in detecting and visualizing of its target antigen, such as phosphorylated CSE. Diagnostic antibodies can be used, for example, in assay systems (e.g., ELISA) or for in vitro imaging. The diagnostic antibody can be, for example, a labeled therapeutic antibody, such as an antibody linked to a detectable moiety.
[000119] The term “detectable moiety” can be used interchangeably with the term “label” and can relate to any moiety capable of being detected (e.g., primary labels and secondary labels).
[000120] “Primary” labels include radioisotope-containing moieties (e.g., moieties that contain 32P, 33P, 35S, or 14C), mass-tags, and fluorescent labels, and are signal-generating reporter groups which can be detected without further modifications.
[000121] Other primary labels include those useful for positron emission tomography including molecules containing radioisotopes (e.g., 18F) or ligands with bound radioactive metals (e.g., 62Cu). In other embodiments, primary labels are contrast agents for magnetic resonance imaging such as gadolinium, gadolinium chelates, or iron oxide (e.g., Fe3O4 and Fe2Ch) particles. Similarly, semiconducting nanoparticles (e.g., cadmium selenide, cadmium sulfide, cadmium telluride) are useful as fluorescent labels. Other metal nanoparticles (e.g., colloidal gold) also serve as primary labels.
[000122] “Secondary” labels include moieties such as biotin, or protein antigens, that require the presence of a second compound to produce a detectable signal. For example, in the case of a biotin label, the second compound may include streptavidin-enzyme conjugates. In the case of an antigen label, the second compound may include an antibody-enzyme conjugate. Additionally, certain fluorescent groups can act as secondary labels by transferring energy to another compound or group in a process of nonradiative fluorescent resonance energy transfer (FRET), causing the second compound or group to then generate the signal that is detected. [000123] The term “mass-tag” as used herein can refer to any compound that is capable of being uniquely detected by virtue of its mass using mass spectrometry (MS) detection techniques. Examples of mass-tags include electrophore release tags such as N-[3-[4'-[(p- methoxytetrafluorobenzyl)oxy]phenyl]-3-methylglyceronyl]-isonipecotic acid, 4'-[2, 3,5,6- tetrafluoro-4-(pentafluorophenoxyl)]methyl acetophenone, and their derivatives. The synthesis and utility of these mass-tags is described in U.S. Pat. Nos. 4,650,750, 4,709,016, 5,360,8191, 5,516,931, 5,602,273, 5,604,104, 5,610,020, and 5,650,270. Other examples of mass-tags include, but are not limited to, nucleotides, dideoxynucleotides, oligonucleotides of varying length and base composition, oligopeptides, oligosaccharides, and other synthetic polymers of varying length and monomer composition. A large variety of organic molecules, both neutral and charged (biomolecules or synthetic compounds) of an appropriate mass range (100-2000 Daltons) may also be used as masstags.
[000124] The terms “fluorescent label”, “fluorescent group”, “fluorescent compound”, “fluorescent dye”, and “fluorophore”, as used herein, can refer to compounds or moieties that absorb light energy at a defined excitation wavelength and emit light energy at a different wavelength. Examples of fluorescent compounds include, but are not limited to: Alexa Fluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), AMCA, AMCA-S, anthracene, BODIPY dyes (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), carbazole, Carboxyrhodamine 6G, carboxy -X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Coumarin 343, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5), Dansyl, Dapoxyl, Dialkylaminocoumarin, 4',5'-Dichloro-2',7'-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin, Fluorescein, FAM, Hydroxy coumarin, IRDyes (IRD40, IRD 700, IRD 800), JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin, Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol Green, 2',4',5',7'-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR), Carboxytetramethylrhodamine (TAMRA), Texas Red, and Texas Red-X
[000125] Further disclosed herein are antibody fragments, such as well-characterized Fabs (e.g., Fab, Fab1, F(ab')2,), Fvs (the variable region of the light chain and the variable region of the heavy chain expressed as two chains), and single-chain fragments e.g., single-chain variable region fragments, scFv, and single chain Fabs, scFab), which also bind to CSE. Methods of making these fragments are routine (see, e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)).
[000126] The antibodies herein are monoclonal antibodies ("mAbs"). mAbs can be produced, for example, by hybridoma technologies, recombinant technologies, phage display technologies, synthetic technologies (e.g., CDR or specificity-determining residue, SDR, grafting), or combinations of such or other technologies known in the art. mAbs are antibodies derived from a single copy or clone including, for example, any eukaryotic, prokaryotic or phage clone. A variety of well-known methods and tools can be used for producing and purifying the mAbs disclosed herein, including vectors, for example, plasmids, virus, or other vehicles for polynucleotide insertion or expression, and hosts, for example, microbial, yeast, insect, and mammalian organisms (see, e.g., Process Scale Purification of Antibodies (Uwe Gottschalk, ed., 2d ed. 2017)).
[000127] The antibodies herein are recombinant antibodies. “Recombinant antibodies” can refer to antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or antibodies prepared, expressed, created, or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies include humanized, CDR grafted, chimeric, in vitro generated (e.g., by phage display) antibodies, and can optionally include constant regions derived from human germline immunoglobulin sequences. Also, “recombinant antibody” can direct to a portion of an intact antibody, including, without limitation, Fv, Fab, Fab', F(ab')2, diabodies, scFv, and single domain antibodies (e.g., variable heavy domain (VHH)).
[000128] Further antibodies having a complementary binding means can be prepared and screened by well-known methods, such as hybridoma, transgenic animals, and phage or yeast display (see, e.g., Monoclonal Antibodies: Methods and Protocols (Vincent Ossipow and Nicolas Fischer, eds., 2d ed. 2014)). Antibodies having equivalent complementary binding means differ in their amino acid sequence but perform the same function of binding the target through CDR-target interaction acting as (inhibitor/agonist/antagonist) to achieve the same result (inhibiting tumor growth). In embodiments, the complementary binding means functions through the same epitope as the disclosed antibodies. [000129] The nucleic acids described herein can be found in a vector. The term “vector” can refer to a nucleic acid molecule capable of transporting another nucleic acid molecule in a host cell. Examples of vectors include plasmids, viral vectors, naked DNA or RNA expression vectors, cosmid or phage vectors. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Some vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., non-episomal mammalian vectors). Certain vectors are capable of directing the expression of genes to which they are operatively linked, and therefore may be referred to as “expression vectors.”
[000130] The antibodies and fragments thereof disclosed herein can be used for detecting phosphorylated CSE in a sample. Phosphorylated CSE, such as CSE phosphorylated at Serine 346, is a marker of tissue injury. The term “tissue injury” can refer to a tissue that is experiencing or has experienced cell death, loss of tissue function, reduction of tissue function, fibrosis, DNA damage, and the like. Tissue injury can result from, for example, ischemia or ischemia/reperfusion injury.
[000131] The antibodies and fragments thereof disclosed herein can be used for diagnosing a subject afflicted with or at risk of a tissue injury.
[000132] The antibodies and fragments thereof disclosed herein can be used in a drug screening method. For example, the antibodies and fragments thereof disclosed herein can detect phosphorylated CSE, such as CSE phosphorylated at position 346, and thereby can be used in a drug screening method. [000133] The antibodies and fragments thereof disclosed herein can be used in therapy. In embodiments, the antibodies and fragments thereof disclosed herein can be used to treat, prevent (such as through prophylactic treatment), or ameliorate a tissue injury.
[000134]
[000135] CSE variant polypeptides
[000136] Homo sapiens cystathionine gamma lyase (CSE) is a cytoplasmic enzyme in the transsulfuration pathway that converts cystathione derived from methionine into cysteine. Glutathione synthesis in the liver is dependent upon the availability of cysteine. Mutations in this gene cause cystathioninuria. Alternative splicing of this gene results in three transcript variants encoding different isoforms.
[000137] Table 10 - Amino acid sequence of CSE, isoform 1 (SEQ ID NO: 1):
1 MQEKDASSQGFLPHFQHFATQAIHVGQDPEQWTSRAWPPI SLSTTFKQG 51 APGQHSGFE YSRSGNPTRNCLEKAVAALDGAKYCLAFASGLAATVT I THL 101 LKAGDQI ICMDDVYGGTNRYFRQVASEFGLKISFVDCSKIKLLEAAITPE 151 TKLVWIETPTNPTQKVIDIEGCAHIVHKHGDI ILWDNTFMSPYFQRPLA 201 LGADISMYSATKYMNGHSDWMGLVSVNCESLHNRLRFLQNSLGAVPSPI 251 DCYLCNRGLKTLHVRMEKHFKNGMAVAQFLESNPWVEKVI YPGLPSHPQH 301 ELVKRQCTGCTGMVTFYIKGTLQHAEI FLKNLKLFTLAESLGGFESLAEL 351 PAIMTHASVLKNDRDVLGISDTLIRLSVGLEDEEDLLEDLDQALKAAHPP 401 SGSHS
[000138] Figure 27 shows the three-dimensional structure of CSE as a tetramer. Tetramer formation is critical for CSE catalytic activity and the production of hydropersulfide and polysulfide. As shown herein, phosphorylation at S346 and at T355 is implicated in tetramer formation.
[000139] As described herein, cystathionine gamma lyase (CSE) phosphorylation due to hypoxia increases cellular and tissue hydropersulfide and polysulfide, critical redox regulators that antagonize oxidative stress. Specifically, hypoxia increases AMP kinase dependent CSE phosphorylation of Serine 346 and Threonine 355 that selectively increases CSE dependent hydropersulfide and polysulfide formation without increased hydrogen sulfide generation.
[000140] Accordingly, aspects of the invention are drawn to CSE variants (e.g., phosphomimetic
CSE variants) that can modulate cellular hydropersulfide and poly sulfide in subjects who are in need thereof.
[000141] The term “CSE variant” can refer to molecules that can modulate cellular hydropersulfide levels, polysulfide levels, or both, in a cell or in a subject. Accordingly, the term encompasses, e.g., CSE variant polypeptides or fragments, polynucleotides encoding such CSE variant polypeptides or fragments, vectors (e.g., vectors comprising polynucleotides encoding CSE variant polypeptides or fragments), micelles (e.g., micelles comprising CSE variant polypeptides or fragments, polynucleotides or vectors), and/or cells (e.g., cell comprising CSE variant polypeptides or fragments, polynucleotides or vectors). For example, the CSE variant can increase cellular hydropersulfide levels, polysulfide levels, or both, in a cell or in a subject. For example, the CSE variant can decrease cellular hydropersulfide levels, polysulfide levels, or both, in a cell or in a subject. [000142] The term “fragment” or “polypeptide fragment” can refer to a polypeptide having one or more (several) amino acids deleted from the amino and/or carboxyl terminus of the mature polypeptide or a homologous sequence thereof. In embodiments, the polypeptide fragment has activity as the mature polypeptide thereof. In embodiments, the polypeptide fragment has activity that is different from the mature polypeptide thereof. For example, the activity of the polypeptide fragment can be inhibitory of the the activity of the mature polypeptide. [000143] In embodiments, the fragment polypeptide can refer to a fragment polypeptide, so long as the fragment polypeptide comprises Ser346, Thr355, or both. For example, a fragment polypeptide can refer to a CSE fragment comprising amino acid positions 334-364 (LFTLAESLGGFESLAELPAIMTHASVLKNDR - SEQ ID NO: 2).
[000144] In embodiments, the phosphomimetic fragment polypeptide can inhibit CSE catalytic activity by inhibiting CSE tetramer formation. Without wishing to be bound by theory, for example, the phosphomimetic fragment polypeptide can inhibit the interaction between adjacent CSE monomers, thereby inhibiting tetramer formation and subsequent catalytic activity. Accordingly, certain CSE phosphomimetic fragments may lower polysulfide levels, hydrosulfilde levels, or both.
[000145] Embodiments can comprise a polynucleotide encoding a CSE variant polypeptide (e.g., a recombinant polypeptide or a synthetic polypeptide) or fragment thereof comprising at least one phosphorylatable amino acid selected from position 346 and/or 355, wherein the at least one phosphorylatable amino acid (e.g., Ser 346 and/or Thr355) or a combination thereof has been replaced (i.e., substituted, or mutated) with a phosphomimetic amino acid (e g., Asp or Glu) or analog (e.g., a non-hydrolyzable analog). These CSE variant polypeptides or fragments comprising phosphomimicking amino acids would mimic CSE in a phosphorylated state, thereby modulating cellular and tissue levels of hydropersulfide and polysulfide. In certain embodiments, these CSE variant polypeptides or fragments can increase cellular and tissue levels of hydropersulfide and polysulfide. For example, full length phosphomimetic CSE protein may mimic CSE in a phosphorylated state, thereby increasing cellular and tissue levels of hydropersulfide and polysulfide. In certain embodiments, these CSE variant polypeptides, such as CSE variant fragment polypeptides, can decrease cellular and tissue levels of hydropersulfide and polysulfide.
[000146] Thus, embodiments provide a phosphomimetic CSE variant polypeptide or fragment comprising a sequence according to SEQ ID NO: 1 and/or SEQ ID NO: 2, wherein the amino acid according to CSE wild-type position 346 is mutated from a serine to a phosphomimetic amino acid, wherein the amino acid according to CSE wild-type position 355 is mutated from a threonine to a phosphomimetic amino acid, or both.
[000147] The term phosphomimetic amino acid can refer to an ammo acid that mimics a phosphorylated amino acid. Within cells, proteins are commonly modified at serine, threonine, and tyrosine amino acids by adding a phosphate group. However, some non-phosphorylated amino acids appear chemically similar to phosphorylated amino acids. For example, aspartic acid is chemically similar to phospho-serine. Thus, when an aspartic acid replaces a serine, it is a phosphomimetic of phospho-serine and can make the protein function like it was in its phosphorylated form.
[000148] In embodiments, the phosphomimetic amino acid is aspartic acid (D) or glutamic acid (E). In embodiments, the aspartic acid is L aspartic acid. In other aspects, the aspartic acid is D aspartic acid. In embodiments, the glutamic acid is L glutamic acid. In other embodiments, the glutamic acid is D glutamic acid. In embodiments, the phosphomimetic amino acid is phosphoserine or phosphothreonine. In embodiments, the phosphomimetic amino acid is L phosphoserine, D phosphoserine, L phosphothreonine, or D phosphothreonine.
[000149] In embodiments, the phosphomimetic amino acid analog is a non-cleavable analog, i.e., an amino acid analog having a group mimicking a phosphate group, wherein the mimicking group cannot be hydrolyzed by phosphatases and/or other enzymes. In embodiments, the non-cleavable analog is, c. ., a phosphoserine non-hydrolyzable analog. In embodiments, the non-hydrolyzable analog of phosphoserine is, e.g., 2-amino-4-phosphobutyric acid.
[000150] In embodiments, the phosphomimetic amino acid analog is, e.g., a thiophosphate analog. In embodiments, the thiophosphate analog is, e.g., thiophosphoserine. In embodiments, the phosphomimetic amino acid analog is, e.g., a sulfate analog. In embodiments, the sulfate analog is, e.g., sulfoserine.
[000151] In embodiments, the CSE variant polypeptide or fragment comprises an N-terminal capping modification, a C-terminal capping modification, or a combination thereof [x-polypeptide- Y], wherein “x” is an N-terminal modification, “y” is a C-terminal modification (e.g., a capping modification), and the polypeptide is a CSE variant polypeptide or fragment thereof. For example, “x” and “y” can be peptide endings different from those found in a naturally occurring isolated polypeptide.
[000152] Capping modifications can be introduced at the termini of chemically synthesized peptides to increase their resistance to proteolytic degradation. In some aspects, the N-terminal modification is acetylation. See, e.g., Thomas (2011) PLOS Biol. 9; Wallace (1992) Br. J. Nutr. 68:365-72. This modification removes the positive charge of the N-terminal of peptides, thus mimicking natural proteins and increasing peptide stability by preventing N-terminal degradation. In embodioments, the C-terminal modification is amidation. This modification neutralizes the negative charge created by the C-terminal COOH. This modification is added to prevent enzyme degradation, to mimic native proteins, and in some cases to remove hydrogen bonding at the C-terminal of the peptides. See, e.g., Kim et al. (2001) Biotechnol. Bioprocess Eng. 6:244-51.
[000153] Non-limiting examples of N-terminal capping modifications comprises an N-terminal acetylation, formylation, acylation, pyroglutamylation, or carbamate, sulfonamide, or alkylamine modification. Non-limiting examples of C-terminal capping modifications comprises a C-terminal amidation, N-alkyl amidation, aldehyde modification, or esterification.
[000154] In embodiments, the CSE variant polypeptide or fragment can be flanked on its N- terminus by 1, 2, 3, 4, 5, or more additional amino acids. In embodiments, the additional N-terminal amino acids are corresponding native amino acids from the wild-type amino acid sequence of CSE. See Table 10, for example. For example, the CSE variant polypeptide or fragment comprises SEQ ID NO: 2 flanked on its N-terminus by 1, 2, 3, 4, 5, or more additional amino acids.
[000155] In embodiments, the CSE variant polypeptide or fragment can be flanked on its C- terminus by 1, 2, 3, 4, 5, or more additional amino acids. In embodiments, the additional C-terminal amino acids are corresponding native amino acids from the wild-type amino acid sequence of CSE. See Table 10, for example. For example, the CSE variant polypeptide or fragment comprises SEQ ID NO: 2 flanked on its C-terminus by 1, 2, 3, 4, 5, or more additional amino acids.
[000156] In embodiments, the CSE variant polypeptide or fragment can be flanked by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,
88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,
111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 additional amino acids on its N-terminus and/or C-terminus.
[000157] In embodiments, the CSE variant polypeptide or fragment comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the sequence as set forth in SEQ ID NO: 1, or a fragment thereof.
[000158] In embodiments, the CSE variant polypeptide or fragment consists of or consists essentially of the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In embodiments, the CSE variant polypeptide or fragment consists of or consists essentially of the amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2, except for 1, 2, 3, 4 or 5 amino acid substitutions, e.g., conservative amino acid substitutions.
[000159] In embodiments, the CSE variant polypeptide or fragment comprises at least 14 amino acids, at least 15 amino acids, at least 16 amino acids, at least 17 amino acids, at least 18 amino acids, at least 19 amino acids, at least 20 amino acids, at least 21 amino acids, at least 22 amino acids, at least 23 amino acids, or at least 24 amino acids in length. In some aspects, the polypeptide has at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, or 155 amino acids in length. In embodiments, the CSE variant polypeptide or fragment consists of or consists essentially of the amino acid sequence as set forth in SEQ ID NO: 1, or a fragment thereof.
[000160] In embodiments, the CSE variant polypeptide or fragment comprises at least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, at least about 50 amino acids, at least about 60 amino acids, at least about 70 amino acids, at least about 80 amino acids, at least about 90 amino acids, at least about 100 amino acids, at least about 120 amino acids, at least about 140 amino acids, at least about 160 amino acids, at least about 180 amino acids, at least about 200 amino acids, at least about 220 amino acids, at least about 240 amino acids, at least about 260 amino acids, at least about 280 amino acids, at least about 300 amino acids, at least about 320 amino acids, at least about 340 amino acids, at least about 360 amino acids in length, or about 366 amino acids in length.
[000161] In embodiments, the CSE variant polypeptide or fragment comprises a fusion protein or conjugate comprising at least one heterologous moiety. The term “heterologous moiety” can refer to any molecule (chemical or biological), e.g., a half-life extending moiety or a detectable moiety, that is different from a CSE variant polypeptide or fragment disclosed herein, and which is genetically fused, conjugated, and/or otherwise associated to the CSE variant polypeptide or fragment.
[000162] In embodiments, the heterologous moiety comprises a serum half-life extending moiety. The term “half-life extending moiety” can refer to a pharmaceutically acceptable moiety, domain, or molecule covalently linked (“conjugated” or “fused”) to a CSE variant polypeptide or fragment, optionally via a non-naturally encoded amino acid, directly or via a linker, that prevents or mitigates in vivo proteolytic degradation or other activity-diminishing chemical modification of the CSE variant polypeptide or fragment, increases half-life, and/or improves or alters other pharmacokinetic or biophysical properties including but not limited to increasing the rate of absorption, reducing toxicity, improving solubility, reducing protein aggregation, increasing biological activity and/or target selectivity of the CSE variant polypeptide or fragment, increasing manufacturability, and/or reducing immunogenicity of the CSE variant polypeptide or fragment, compared to a reference compound such as a non-conjugated or non-fused form of the CSE variant polypeptide or fragment.
[000163J In embodiments, the serum half-life extending moiety comprises an Fc region or portion thereof, albumin, albumin binding polypeptide, a fatty acid, PAS, a glycine-rich homo-amino- acid polymer (HAP), the R subunit of the C-terminal peptide (CTP) of human chorionic gonadotropin, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN, albumin-binding small molecules, or a combination thereof.
[0001641 For example, fusion proteins comprising an Fc region of an immunoglobulin bestow several desirable properties on a fusion protein including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337:525) as well as binding to Fc receptors such as the neonatal Fc receptor (FcRn) (U.S. Pat. Nos. 6,086,875, 6,485,726, 6,030,613; WO 03/077834; US2003- 0235536A1).
[000165] In certain aspects, the half-life extension moiety linked, e.g., fused or conjugated, to a CSE variant polypeptide or fragment is an albumin binding moiety, which comprises an albumin binding peptide, a bacterial albumin binding domain, an albumin-binding antibody fragment, or any combinations thereof. For example, the albumin binding protein can be a bacterial albumin binding protein, an antibody or an antibody fragment including domain antibodies (see U.S. Pat. No. 6,696,245). An albumin binding protein, for example, can be a bacterial albumin binding domain, such as the one of streptococcal protein G (Konig, T. and Skerra, A. (1998) J. Immunol. Methods 218, 73- 83). Other examples of albumin binding peptides that can be used as conjugation partner are, for instance, those having a Cys-Xaa 1-Xaa 2-Xaa 3-Xaa 4-Cys consensus sequence, wherein Xaa 1 is Asp, Asn, Ser, Thr, or Trp; Xaa 2 is Asn, Gin, H is, He, Leu, or Lys; Xaa 3 is Ala, Asp, Phe, Trp, or Tyr; and Xaa 4 is Asp, Gly, Leu, Phe, Ser, or Thr as described in US patent application 2003/0069395 or Dennis et al. (Dennis et al. (2002) J. Biol. Chem. 277, 35035-35043). [000166] In embodiments, the half-life extension moiety linked, e.g., fused or conjugated, to a CSE variant polypeptide or fragment is a PAS sequence. A “PAS sequence” can refer to an amino acid sequence comprising mainly alanine and serine residues or comprising mainly alanine, serine, and proline residues, the amino acid sequence forming random coil conformation under physiological conditions. Accordingly, the PAS sequence is a building block, an amino acid polymer, or a sequence cassette comprising, consisting essentially of, or consisting of alanine, serine, and proline which can be used as a part of the heterologous moiety in the fusion protein. Exemplary PAS sequences are provided, e.g., in US Pat. Publ. No. 2010/0292130 Al and PCT Appl. Publ. No. WO 2008/155134 Al, both of which are incorporated by reference in their entireties.
[000167] In embodiments, the half-life extension moiety linked, e.g., fused or conjugated, to a CSE variant polypeptide or fragment is a glycine-rich homo-amino-acid polymer (HAP). The HAP sequence can comprise a repetitive sequence of glycine, which has at least 50 amino acids, at least 100 amino acids, 120 amino acids, 140 amino acids, 160 amino acids, 180 amino acids, 200 amino acids, 250 amino acids, 300 amino acids, 350 amino acids, 400 amino acids, 450 amino acids, or 500 amino acids in length. Non-limiting examples of the HAP sequence includes, but are not limited to (Gly)n, (Gly4Ser)n or S(Gly4Ser)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In one aspect, n is 20, 21, 22, 23, 24, 25, 26, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In another aspect, n is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. [000168] In embodiments, the half-life extension moiety linked, e.g., fused or conjugated, to a CSE variant polypeptide or fragment is a soluble polymer known in the art, including, but not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, or polyvinyl alcohol. The soluble polymer can be attached to any positions within the sequence SETD7 modulator of the present disclosure or at either or both termini. The soluble polymer can be attached at random positions within the CSE variant polypeptide or fragment or at predetermined positions within the CSE variant polypeptide or fragment sequence and may include one, two, three or more attached soluble polymer moieties. In embodiments, the polymer can be attached to a side chain of a naturally occurring amino acid. In other embodiments, the polymer can be attached to a side chain of a non-naturally encoded amino acid, e.g., a phenylalanine derivative such as para-acetyl-L-phenylalanine. The soluble polymer can be of any molecular weight, and can be branched or unbranched. [000169] In embodiments, the half-life extension moiety linked, e.g., fused or conjugated, to a CSE variant polypeptide or fragment is hydroxyethyl starch (HES) or a derivative thereof. HES is a derivative of naturally occurring amylopectin and is degraded by alpha-amylase in the body. HES is a substituted derivative of the carbohydrate polymer amylopectin, which is present in corn starch at a concentration of up to 95% by weight. HES exhibits advantageous biological properties and is used as a blood volume replacement agent and in hemodilution therapy in the clinics (Sommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987); and Weidler et al., Arzneim. Forschung/Drug Res., 41, 494-498 (1991)).
[000170] In embodiments, the half-life extension moiety linked, e.g., fused or conjugated, to a CSE variant polypeptide or fragment is a polysialic acid (PSA) or a derivative thereof. PSAs are naturally occurring unbranched polymers of sialic acid produced by certain bacterial strains and in mammals in certain cells Roth J., et al. (1993) in Polysialic Acid: From Microbes to Man, eds Roth J., Rutishauser U., Troy F. A. (Birkhauser Verlag, Basel, Switzerland), pp 335-348.
[000171] In embodiments, the half-life extension moiety linked, e.g., fused or conjugated, to a CSE variant polypeptide or fragment is an XTEN sequence. As used here “XTEN sequence” refers to extended length polypeptides with non-naturally occurring, substantially non-repetitive sequences that are composed mainly of small hydrophilic amino acids, with the sequence having a low degree or no secondary or tertiary structure under physiologic conditions. As a fusion protein partner, XTENs can serve as a carrier, conferring certain desirable pharmacokinetic, physicochemical and pharmaceutical properties when linked to a CSE variant polypeptide or fragment to create a fusion protein. Such desirable properties include but are not limited to enhanced pharmacokinetic parameters and solubility characteristics. Examples of XTEN sequences that can be used according to the present disclosure are disclosed in US Patent Publication Nos. 2010/0239554 Al, 2010/0323956 Al, 2011/0046060 Al, 2011/0046061 Al, 2011/0077199 Al, or 2011/0172146 Al, or International Patent Publication Nos. WO 2010091122 Al, WO 2010144502 A2, WO 2010144508 Al, WO 2011028228 Al, WO 2011028229 Al, or WO 2011028344 A2, all of which are herein incorporated by reference in their entireties.
[000172] In embodiments, the serum half-life of a CSE variant polypeptide or fragment comprising a half-time extending moiety is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% higher than the plasma half-life of a corresponding CSE variant polypeptide or fragment without serum half-life extending moiety.
[000173] In embodiments, the at least one heterologous moiety comprises a detectable moiety, e g., a radionuclide, a fluorescent molecule, or a contrast agent.
[000174] Aspects of the invention are also drawn towards a nucleic acid encoding a CSE variant polypeptide or fragment (e.g., a CSE variant polynucleotide) as described herein.
[000175] In embodiments, the a CSE variant polynucleotide comprises or consists of a fragment or variant of the sequence as set forth in NCBI Reference Sequence NG_008041.1.
[000176] In embodiments, the CSE variant polynucleotide comprises a nucleic acid sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity the polynucleotide encoding a CSE variant polypeptide or fragment as described herein. For example, the nucleic acid encoding the CSE variant polypeptide or fragment comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations (base substitutions). [000177] In embodiments, the sequence is an RNA sequence. In some embodiments, the sequence is an RNAZDNA sequence. In embodiments, the polynucleotide encoding the CSE variant is mRNA. In some embodiments, the polynucleotide encoding the CSE variant is a DNA. In some embodiments, the polynucleotide encoding the CSE variant is inserted in a vector.
[000178] In embodiments, the CSE variant polynucleotide comprises a “synthetic nucleic acid”. A “synthetic nucleic acid” can refer to a nucleic acid that does not have the chemical structure or sequence of a natural nucleic acid.
[000179] In embodiments, the CSE variant polynucleotide comprises at least one chemically modified nucleobase, sugar, backbone, or any combination thereof. Thus, the CSE variant polynucleotide can comprise one or more modifications. For example, the modification comprises the use of a pseudo-uracil or Nl-methyl pseudo-uracil. Such modifications decrease immune reactions, increases mRNA stability, and increased ability for mRNA translation.
[000180] In embodiments, the CSE variant polynucleotide comprises at least one nucleotide analogue. For example, the nucleotide analogue can be introduced by using IVT (in vitro transcription) or chemical synthesis. For example, the nucleotide analogue can be selected from the group consisting of a 2'-O-methoxyethyl-RNA (2'-M0E-RNA) monomer, a 2'-fluoro-DNA monomer, a 2'-O-alkyl- RNA monomer, a 2'-amino-DNA monomer, a locked nucleic acid (LNA) monomer, a cEt monomer, a cMOE monomer, a 5'-Me-LNA monomer, a 2'-(3-hydroxy)propyl-RNA monomer, an arabino nucleic acid (ANA) monomer, a 2'-fluoro-ANA monomer, an anhydrohexitol nucleic acid (HNA) monomer, an intercalating nucleic acid (INA) monomer, or a combination of two or more of said nucleotide analogues. In embodiments, the optimized nucleic acid molecule comprises at least one backbone modification, for example, a phosphorothioate internucleotide linkage.
[000181] In embodiments, the CSE variant polynucleotide can be chemically modified at terminal locations, for example by introducing M (2'-O-methyl), MS (2'-O-methyl 3' phosphorothioate), or MSP (2'-O-methy 3 'thioPACE, phosphonoacetate) modifications, or combinations thereof at positions 1, 2, 3 respect to the 5' and/or 3' termini.
[000182] Modified CSE variant polynucleotides need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structures can exist at various positions in the nucleic acid. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid is not substantially decreased. A modification may also be a 5' or 3' terminal modification. The nucleic acids may contain at a minimum one and at maximum 100% modified nucleotides, or any intervening percentage, such as at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% modified nucleotides.
[000183] In embodiments, the CSE variant polynucleotide can include modifications to prevent rapid degradation by endo- and exo-nucleases. Modifications include, but are not limited to, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, as well as (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. [000184] Specific examples of synthetic, modified CSE variant polynucleotide include, but are not limited to, polynucleotides containing modified or non-natural intemucleoside linkages. Synthetic, modified CSE variant polynucleotides having modified internucleoside linkages include, among others, those that do not have a phosphorus atom in the internucleoside linkage. In other embodiments, the synthetic, modified CSE variant polynucleotide has a phosphorus atom in its intemucleoside linkage(s).
[000185] Non-limiting examples of modified intemucleoside linkages include phosphorothioates, chiral phosphorothi oates, phosphorodithioates, phosphotri esters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 '-5' linkages, T-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or T-5' to 5'-T. Various salts, mixed salts and free acid forms are also included.
[000186] Modified intemucleoside linkages that do not include a phosphorus atom therein have intemucleoside linkages that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
[000187] In embodiments, the CSE variant polynucleotide can be codon optimized by introducing one or more synonymous codon changes. As used herein, the terms “codon optimization” or “codon optimized” can refer to the modification of the primary sequence of a nucleic acid by replacing synonymous codons in order to increase its translational efficiency. Accordingly, codon optimization comprises switching the codons used in a SETD7 modulator polynucleotide of the present disclosure without changing the amino acid sequence that it encodes for, which typically dramatically increases the abundance of the protein the codon optimized gene encodes because it generally removes “rare” codons and replaces them with abundant codons, or removes codon with a low tRNA recharge rate with codon with high tRNA recharge rates. Such codon optimization can, for example, (i) improve protein yield in recombinant protein expression, or (ii) improve the stability, half life, or other desirable property of an mRNA or a DNA encoding a binding molecule disclosed herein, wherein such mRNA or DNA is administered to a subject in need thereof.
[000188] A CSE variant polynucleotide can be codon optimized using any methods known in the art at the time the present application was filed.
[000189] In embodiments, a CSE variant polynucleotide has been sequence optimized. As used herein, the term “sequence optimized” can refer to the modification of the sequence of a nucleic acid by to introduce features that increase its translational efficiency, remove features that reduce its translational efficiency, or in general improve properties related to expression efficacy after administration in vivo. Such properties include, but are not limited to, improving nucleic acid stability (e.g., mRNA stability), increasing translation efficacy in the target tissue, reducing the number of truncated proteins expressed, improving the folding or prevent misfolding of the expressed proteins, reducing toxicity of the expressed products, reducing cell death caused by the expressed products, or increasing and/or decreasing protein aggregation.
[000190] Embodiments also provide a vector comprising an isolated polynucleotide, e.g., a CSE variant polynucleotide disclosed herein.
[000191] In embodiments, the vector is viral vector. In embodiments, the viral vector is an adenoviral vector or an adenoassociated viral vector. In other embodiments, the viral vector is a retroviral vector, e.g., a lenti viral vector.
[000192] In embodiments, the CSE variant polynucleotide comprises a virus, a plasmid, or a phagemid encoding a CSE variant. In embodiments, the virus is selected from the group consisting of an adeno-associated virus (AAV), a retrovirus, a lentivirus, an adenovirus, an SV40-type virus, a polyomavirus, an Epstein-Barr virus, a papilloma virus, a herpes virus, a vaccinia virus, a polio virus, and an RNA virus.
[000193] In embodiments, a nucleic acid sequence encoding a CSE variant polypeptide or fragment can be inserted into the genome of a target cell or a host cell (e.g., a stem cell for transplantation to the target tissue) by using CRISPR/Cas systems and genome edition alternatives such as zine-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases (MNs). Thus, embodiments herein also provide cells comprising a CSE variant polynucleotide, or a vector comprising a SETD7 modulator polynucleotide. [000194] Embodiments can comprise the use of a gene editing tool to modulate (e.g., increase or decrease) the expression of a gene encoding a CSE variant polypeptide or fragment.
[000195] In embodiments, the gene editing tool that can be used as described herein comprises a CRISPR/Cas system. Such systems can employ, for example, a Cas9 nuclease, which in some instances, is codon-optimized for the desired cell type in which it is to be expressed (e.g., T cells, e.g., CAR-expressing T cells). CRISPR/Cas systems use Cas nucleases, e.g., Cas9 nucleases, that are targeted to a genomic site by complexing with a synthetic guide RNA (gRNA) that hybridizes to a target DNA sequence immediately preceding an NGG motif recognized by the Cas nuclease, e.g., Cas9. This results in a double-strand break three nucleotides upstream of the NGG motif. A unique capability of the CRISPR/Cas9 system is the ability to simultaneously target multiple distinct genomic loci by co-expressing a single Cas9 protein with two or more gRNAs (e.g., at least one, two, three, four, five, six, seven, eight, nine or ten gRNAs). Such systems can also employ a guide RNA (gRNA) that comprises two separate molecules. In certain aspects, the two-molecule gRNA comprises a crRNA-like (“CRISPR RNA” or “targeter-RNA” or “crRNA” or “crRNA repeat”) molecule and a corresponding tracrRNA-like (“trans-acting CRISPR RNA” or “activator-RNA” or “tracrRNA” or “scaffold”) molecule.
[000196] A crRNA comprises both the DNA-targeting segment (single stranded) of the gRNA and a stretch of nucleotides that forms one half of a double stranded RNA (dsRNA) duplex of the protein-binding segment of the gRNA. A corresponding tracrRNA (activator-RNA) comprises a stretch of nucleotides that forms the other half of the dsRNA duplex of the protein-binding segment of the gRNA. Thus, a stretch of nucleotides of a crRNA is complementary to and hybridizes with a stretch of nucleotides of a tracrRNA to form the dsRNA duplex of the protein-binding domain of the gRNA. As such, each crRNA can be said to have a corresponding tracrRNA. The crRNA additionally provides the single stranded DNA-targeting segment. Accordingly, a gRNA comprises a sequence that hybridizes to a target sequence (e.g., SETD7 mRNA), and a tracrRNA. Thus, a crRNA and a tracrRNA (as a corresponding pair) hybridize to form a gRNA. If used for modification within a cell, the exact sequence and/or length of a given crRNA or tracrRNA molecule can be designed to be specific to the species in which the RNA molecules will be used (e.g., humans).
[000197] Genes encoding the three elements (Cas9, tracrRNA and crRNA) are typically organized in operon(s). CRISPR RNAs differ depending on the Cas9 system and organism but often contain a targeting segment of between 21 to 72 nucleotides length, flanked by two direct repeats (DR) of a length of between 21 to 46 nucleotides (see, e.g., WO2014/131833). In the case of S. pyogenes, the DRs are 36 nucleotides long and the targeting segment is 30 nucleotides long. The 3' located DR is complementary to and hybridizes with the corresponding tracrRNA, which in turn binds to the Cas9 protein.
[000198] Alternatively, a CRISPR system used herein can further employ a fused crRNA- tracrRNA construct (i.e., a single transcript) that functions with the codon-optimized Cas9. This single RNA is often referred to as a guide RNA or gRNA. Within a gRNA, the crRNA portion is identified as the “target sequence” for the given recognition site and the tracrRNA is often referred to as the “scaffold.” Briefly, a short DNA fragment containing the target sequence is inserted into a guide RNA expression plasmid. The gRNA expression plasmid comprises the target sequence (in some aspects around 20 nucleotides), a form of the tracrRNA sequence (the scaffold) as well as a suitable promoter that is active in the cell and necessary elements for proper processing in eukaryotic cells. Many of the systems rely on custom, complementary oligos that are annealed to form a double stranded DNA and then cloned into the gRNA expression plasmid.
[000199] The gRNA expression cassette and the Cas9 expression cassette are then introduced into the cell. See, for example, Mali P et al., (2013) Science 2013 Feb. 15; 339(6121):823-6; Jinek M et al., Science 2012 Aug. 17; 337(6096):816-21; Hwang W Y et al., Nat Biotechnol 2013 March; 31(3):227-9; Jiang W et al., Nat Biotechnol 2013 March; 31 (3):233-9; and Cong L et al., Science 2013 Feb. 15; 339(6121): 819-23, each of which is herein incorporated by reference in its entirety. See also, for example, WO/2013/176772 Al, WO/2014/065596 Al, WO/2014/089290 Al, WO/2014/093622 A2, WO/2014/099750 A2, and WO/2013142578 Al, each of which is herein incorporated by reference in its entirety.
[000200] In some aspects, the gene editing tool that can be used as described herein comprises a nuclease agent, such as a Transcription Activator-Like Effector Nuclease (TALEN). TAL effector nucleases are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a prokaryotic or eukaryotic organism. TAL effector nucleases are created by fusing a native or engineered transcription activator-like (TAL) effector, or functional part thereof, to the catalytic domain of an endonuclease, such as, for example, Fokl.
[000201] The unique, modular TAL effector DNA binding domain allows for the design of proteins with potentially any given DNA recognition specificity. Thus, the DNA binding domains of the TAL effector nucleases can be engineered to recognize specific DNA target sites and thus, used to make double-strand breaks at desired target sequences. See, WO 2010/079430; Morbitzer et al., (2010) PNAS 10.1073/pnas.l013133107; Scholze & Boch (2010) Virulence 1 :428-432; Christian et al., Genetics (2010) 186:757-761; Li et al., (2010) Nuc. Acids Res. (2010) doi: 10.1093/nar/gkg704; and Miller et al., (2011) Nature Biotechnology 29: 143-148; all of which are herein incorporated by reference in their entirety.
[000202] Non-limiting examples of suitable TAL nucleases, and methods for preparing suitable TAL nucleases, are disclosed, e.g., in US Patent Application No. 2011/0239315 Al, 2011/0269234 Al, 2011/0145940 Al, 2003/0232410 Al, 2005/0208489 Al, 2005/0026157 Al, 2005/0064474 Al, 2006/0188987 Al, and 2006/0063231 Al (each hereby incorporated by reference).
[000203] In embodiments, TAL effector nucleases are engineered that cut in or near a target nucleic acid sequence in, e.g., a genomic locus of interest, wherein the target nucleic acid sequence is at or near a sequence to be modified by a targeting vector. The TAL nucleases suitable for use with the various methods and compositions provided herein include those that are specifically designed to bind at or near target nucleic acid sequences to be modified by targeting vectors as described herein. [000204] In embodiments, the gene editing tool that can be used as described herein comprises a nuclease agent, such as a zinc-finger nuclease (ZFN) system. Zinc finger-based systems comprise a fusion protein comprising two protein domains: a zinc finger DNA binding domain and an enzymatic domain. A “zinc finger DNA binding domain,” “zinc finger protein,” or “ZFP” is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The zinc finger domain, by binding to a target DNA sequence, directs the activity of the enzymatic domain to the vicinity of the sequence and, hence, induces modification of the endogenous target gene in the vicinity of the target sequence. A zinc finger domain can be engineered to bind to virtually any desired sequence. As disclosed herein, in some aspects, the zinc finger domain binds a DNA sequence that encodes the SETD7 protein. Accordingly, after identifying a target genetic locus containing a target DNA sequence at which cleavage or recombination is desired (e.g., a target locus in a target gene referenced in Table 1), one or more zinc finger binding domains can be engineered to bind to one or more target DNA sequences in the target genetic locus. Expression of a fusion protein comprising a zinc finger binding domain and an enzymatic domain in a cell, effects modification in the target genetic locus. [000205] In embodiments, a zinc finger binding domain comprises one or more zinc fingers. Miller et al., (1985) EMBO J. 4: 1609-1614; Rhodes (1993) Scientific American February:56-65; U.S. Pat. No. 6,453,242. Typically, a single zinc finger domain is about 30 amino acids in length. An individual zinc finger binds to a three-nucleotide (i.e., triplet) sequence (or a four-nucleotide sequence which can overlap, by one nucleotide, with the four-nucleotide binding site of an adjacent zinc finger). Therefore, the length of a sequence to which a zinc finger binding domain is engineered to bind (e.g., a target sequence) will determine the number of zinc fingers in an engineered zinc finger binding domain. For example, for ZFPs in which the finger motifs do not bind to overlapping subsites, a six- nucleotide target sequence is bound by a two-finger binding domain; a nine-nucleotide target sequence is bound by a three-finger binding domain, etc. Binding sites for individual zinc fingers (i.e., subsites) in a target site need not be contiguous, but can be separated by one or several nucleotides, depending on the length and nature of the amino acids sequences between the zinc fingers (i.e., the inter-finger linkers) in a multi-finger binding domain. In some aspects, the DNA-binding domains of individual ZFNs comprise between three and six individual zinc finger repeats and can each recognize between 9 and 18 basepairs.
[000206] Zinc finger binding domains can be engineered to bind to a sequence of choice. See, for example, Beerli et al., (2002) Nature Biotechnol. 20: 135-141; Pabo et al., (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al., (2001) Nature Biotechnol. 19:656-660; Segal et al., (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al., (2000) Curr. Opin. Struct. Biol. 10:411-416. An engineered zinc finger binding domain can have a new binding specificity, compared to a naturally-occurring zinc finger protein. Engineering methods include, but are not limited to, rational design and various types of selection.
[000207] In embodiments, the gene editing tool that can be used as described herein comprises a meganuclease system. Meganucleases have been classified into four families based on conserved sequence motifs, the families are the “LAGLIDADG,” “GIY-YIG,” “H-N-H,” and “His-Cys box” families. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds.
[000208] HEases are notable for their long recognition sites, and for tolerating some sequence polymorphisms in their DNA substrates. Meganuclease domains, structure and function are known, see, for example, Guhan and Muniyappa (2003) Crit Rev Biochem Mol Biol 38: 199-248; Lucas et al., (2001) Nucleic Acids Res 29:960-9; Jurica and Stoddard, (1999) Cell Mol Life Sci 55: 1304-26; Stoddard, (2006) Q Rev Biophys 38:49-95; and Moure et al., (2002) Nat Struct Biol 9:764.
[000209] In some examples a naturally occurring variant, and/or engineered derivative meganuclease is used. Methods for modifying the kinetics, cofactor interactions, expression, optimal conditions, and/or recognition site specificity, and screening for activity are known, see for example, Epinat et al., (2003) Nucleic Acids Res 31 :2952-62; Chevalier et al., (2002) Mol Cell 10:895-905; Gimble et al., (2003) Mol Biol 334:993-1008; Seligman et al., (2002) Nucleic Acids Res 30:3870-9; Sussman et al., (2004) J Mol Biol 342:31-41; Rosen et al., (2006) Nucleic Acids Res 34:4791-800; Chames et al., (2005) Nucleic Acids Res 33 :el78; Smith et al., (2006) Nucleic Acids Res 34:el49; Gruen et al., (2002) Nucleic Acids Res 30:e29; Chen and Zhao, (2005) Nucleic Acids Res 33:el54; W02005105989; W02003078619; W02006097854; W02006097853; W02006097784; and W02004031346; each of which is herein incorporated by reference in its entirety.
[000210] Any meganuclease can be used herein, including, but not limited to, I-Scel, I-Scell, I- Scelll, 1-SceIV, I-SceV, I-SecVI, LSceVII, I-Ceul, LCeuAIIP, I-Crel, LCrepsblP, 1-CrepsbIIP, I- CrepsblllP, LCrepsblVP, I-Tlil, I-Ppol, PI-PspI, F-Scel, F-Scell, F-Suvl, F-TevI, F-TevII, I-Amal, I- Anil, I-Chul, I-Cmoel, I-Cpal, LCpall, I-CsmI, I-Cvul, LCvuAIP, LDdil, LDdill, LDirl, I-Dmol, I- Hmul, I-HmuII, LHsNIP, I-Llal, I-Msol, I-Naal, I-NanI, I-NcIIP, I-NgrIP, I-Nitl, I-Njal, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, LPgrlP, LPoblP, 1-PorIIP, LPbpIP, I-SpBetaTP, I-Scal, I-SexIP, 1-SneIP, LSpoml, LSpomCP, 1-SpomIP, LSpomllP, I-SquIP, I-Ssp6803I, LSthPhiJP, I- SthPhiST3P, I-SthPhiSTe3bP, 1-TdeIP, LTevI, I-TevII, I-TevIII, LUarAP, LUarHGPAIP, I- UarHGPA13P, I-VinIP, LZbilP, PLMtuI, PLMtuHIP, PLMtuHIIP, PLPfuI, Pl-PfuII, PLPkoI, PL PkoII, PI-Rma43812IP, PLSpBetalP, Pl-Scel, PI-Tful, PI-TfuII, PLThyl, PLTlil, PI-Tlill, or any active variants or fragments thereof.
[000211]
[000212] Pharmaceutical Compositions
[000213] Aspects of the invention provide pharmaceutical compositions comprising a phosphospecific antibody, a CSE variant polypeptide or fragment, a CSE variant polynucleotide, a vector, or a cell that are suitable for administration to a subject. Such pharmaceutical compositions can comprise a phosphospecific antibody, a CSE variant polypeptide or fragment, a CSE variant polynucleotide, a vector, or a cell and a pharmaceutically-acceptable excipient or carrier in a form suitable for administration to a subject. Pharmaceutically acceptable excipients or carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition.
[000214] There is a wide variety of suitable formulations of pharmaceutical compositions comprising a CSE variant as described herein (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 18th ed. (1990)). The pharmaceutical compositions are generally formulated sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
[000215] In embodiments, the phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell can be co-administered with of one or more additional therapeutic agents, in a pharmaceutically acceptable carrier. In embodiments, the pharmaceutical composition comprising the phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell is administered prior to administration of the additional therapeutic agent(s). In other embodiments, the pharmaceutical composition comprising the phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell is administered after the administration of the additional therapeutic agent(s). In further embodiments, the pharmaceutical composition comprising the phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell is administered concurrently with the additional therapeutic agent(s).
[000216] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients (e.g., animals or humans) at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn- protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). [000217] Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell described herein, use thereof in the compositions is contemplated.
[000218] Typically, a pharmaceutical composition is formulated to be compatible with its intended route of administration. Pharmaceutical compositions described herein can be administered by parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intratumoral, intramuscular route or as inhalants.
[000219] Solutions or suspensions can include the following components: a sterile diluent such as water, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
[000220] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The composition is generally sterile and fluid to the extent that easy syringeability exists. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. If desired, isotonic compounds, e.g., sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride can be added to the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound which delays absorption, e.g., aluminum monostearate and gelatin.
[000221] Pharmaceutical compositions of the present disclosure can be sterilized by conventional, well known sterilization techniques. Aqueous solutions can be packaged for use or fdtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
[000222] Sterile injectable solutions can be prepared by incorporating a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell in an effective amount and in an appropriate solvent with one or a combination of ingredients enumerated herein, as desired. Generally, dispersions are prepared by incorporating a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell into a sterile vehicle that contains a basic dispersion medium and any desired other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A pharmaceutical composition can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner to permit a sustained or pulsatile release of the phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell.
[000223] Administration of the pharmaceutical composition can also be by transmucosal means. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, e.g., for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of, e.g., nasal sprays.
[000224] In embodiments, pharmaceutical compositions can be administered intravenously into a subject that would benefit from the pharmaceutical composition. In certain other aspects, the composition is administered to the lymphatic system, e.g., by intralymphatic injection or by intranodal injection (see e.g., Senti et al., PNAS 105(46): 17908 (2008)), or by intramuscular injection, by subcutaneous administration, by intratumoral injection, by direct injection into the thymus, or into the liver.
[000225] In embodiments, the pharmaceutical composition can be administered as a liquid suspension. In embodiments, the pharmaceutical composition is administered as a formulation that is capable of forming a depot following administration. In certain preferred aspects, the depot slowly releases the micelles described herein into circulation, or remains in depot form.
[000226] Typically, pharmaceutically-acceptable compositions are highly purified to be free of contaminants, are biocompatible and not toxic, and are suited to administration to a subject. If water is a constituent of the carrier, the water is highly purified and processed to be free of contaminants, e.g., endotoxins.
[000227] The pharmaceutically-acceptable carrier can be lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and/or mineral oil, but is not limited thereto. The pharmaceutical composition can further include a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, and/or a preservative.
[000228] Dosage forms are provided that comprise a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell as described herein. In embodiments, the dosage form is formulated as a liquid suspension for intravenous injection. Actual dosage levels of a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular therapeutic agent, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the severity of the condition, other health considerations affecting the subject, and the status of liver and kidney function of the subject. It also depends on the response to administration of the agents, including factors such as blood sugar level or the level of glycated hemoglobin, body mass index as well as levels of enzymes involved in liver disease such as ALT and AST. It also depends on the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular therapeutic agent employed, as well as the age, weight, condition, general health and prior medical history of the subject being treated, and like factors. Methods for determining optimal dosages are described in the art, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000. [000229] In embodiments, a phosphospecific antibody, a CSE variant polypeptide or fragment, a CSE variant polynucleotide, or a vector comprising the same can be administered with a delivery agent, e.g., a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric compound, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, a micelle, or a conjugate. Thus, embodiments described herein also provides for a pharmaceutical composition comprising a phosphospecific antibody, a CSE variant polypeptide or fragment, a CSE variant polynucleotide, or a vector comprising the same, and a delivery agent, e.g., a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric compound, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, a micelle, or a conjugate.
[000230] Embodiments of the invention are also drawn towards a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell for use as a medicament. Embodiments of the invention are also drawn towards a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell for use in the treatment of tissue injury in a subject in need thereof.
[000231]
[000232] Methods of Treatment
[000233] A CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell as described herein can be administered to a subject, e.g., a human subject, to treat diseases or conditions disclosed herein, such as a tissue injury.
[000234] In embodiments, the CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell can be used to prevent symptoms or sequelae of diseases and conditions disclosed herein.
[000235] In embodiments, the CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell can be administered via intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.
[000236] In embodiments, the CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell can be used concurrently with other medicaments or treatment suitable for the treatment of the diseases and conditions disclosed herein.
[000237] A CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell as described herein can be used to increase cellular hydropersulfide, polysulfide, or both. In embodiments, hydropersulfide, polysulfide, or both can be measured using analytical clinical methods known in the art. See, for example, Malaeb, Hind, et al. "Stable isotope dilution mass spectrometry quantification of hydrogen sulfide and thiols in biological matrices." Redox Biology 55 (2022): 102401; Shen, Xinggui, et al. "Measurement of H2S in vivo and in vitro by the monobromobimane method." Methods in enzymology. Vol. 554. Academic Press, 2015. 31-45; Disbrow, Elizabeth, et al. "Plasma hydrogen sulfide: a biomarker of Alzheimer's disease and related dementias." Alzheimer's & Dementia 17.8 (2021): 1391-1402; Dominic, Paari, et al. "Decreased availability of nitric oxide and hydrogen sulfide is a hallmark of COVID-19." Redox biology 43 (2021): 101982; and Reekes, Tyler H., et al. "Elevated plasma sulfides are associated with cognitive dysfunction and brain atrophy in human Alzheimer's disease and related dementias." Redox Biology 62 (2023): 102633.
[000238]
[000239] Kits
[000240] Aspects of the invention are also drawn towards kits or products of manufacture, comprising a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell, and optionally instructions for use, e.g., instructions for use according to the methods disclosed herein.
[000241] In embodiments, the kit comprises a pharmaceutical composition comprising a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell as described herein. In embodiments, the kit or product of manufacture comprises the phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell disclosed herein in one or more containers.
[000242] In embodiments, the kit or product of manufacture optionally comprises a brochure and/or instructions for use. One skilled in the art will readily recognize that a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell can be readily incorporated into one of the established kit formats which are well known in the art.
[000243] In embodiments, the kit or product of manufacture comprises a phosphospecific antibody, CSE variant polypeptide or fragment, CSE variant polynucleotide, vector, or cell, in dry form in a container (e.g., a glass vial), and optionally a vial with a solvent.
Examples [000244] Example 1 — CSE phosphorylation regulates hydropersulfide and polysulfide formation
[000245] Aspects of the invention provides compositions and methods to increase cellular and tissue hydropersulfide and polysulfide by cystathionine gamma lyase (CSE) phosphorylation due to increased AMP kinase activity due to hypoxia. Hypoxia (reduced oxygen tension) causes increased oxidative stress and cellular injury. Also, hydropersulfide and polysulfide act as critical redox regulators that antagonize oxidative stress. It remains unknown whether or how hypoxia could alter bioavailability of hydropersulfide and polysulfide. Described herein, hypoxia increases AMP Kinase dependent CSE phosphorylation of Serine 346 and Threonine 355 that selectively increases CSE dependent hydropersulfide and polysulfide formation without increased hydrogen sulfide generation. This is an entirely new molecular pathway for regulating biologically potent sulfide metabolites. Moreover, embodiments provide phospho-specific antibodies against CSE phosphorylated Serine 346, which can be used as a biomarker or molecular readout indicating increased hydropersulfide and polysulfide formation. Such antibodies can be useful for tissue and disease diagnosis, screening drugs that could potentially activate this new pathway, and as a research tool for further studies in the field. [000246] In embodiments, the genetic sequence of CSE Glutamic acid amino acid mutants of either Serine 346 or Threonine 355, or both can be used in mRNA or gene-based therapy to discretely increase cellular hydropersulfide and polysulfide in subjects who are deficient in these mediators. Conversely, genetic sequence of CSE Alanine amino acid mutants of either Serine 346 or Threonine 355, or both can be used in mRNA or gene-based therapy to discretely decrease cellular hydropersulfide and polysulfide in subjects who have elevated levels of these mediators. Likewise, CRISRP/Cas targeting technology could be used to perform mutation of these residues in endogenous subject genomes.
[000247]
[000248] Example 2
[000249] Hydrogen sulfide is a gaseous signaling molecule (e.g., a ‘gasotransmitter’) that plays critical roles in cy toprot ection and in regulating many other biological functions1. In the past few decades, its pathophysiological roles, including cardiovascular, neurological, inflammatory and immune systems, has been well established through experimental animal models and clinical studies1- 3. H2S is produced by enzymes of the transsulfuration pathway including cystathionine -synthase (CBS) and cystathionine y-lyase (CTH, also abbreviated as CSE), as well as by 3 -mercaptopyruvate sulfurtransferase, which is involved in cysteine catabolism. Endogenously, sulfide chemical biology is present in three forms, including the readily available free sulfide, acid-labile sulfide (e.g. protein iron-sulfur clusters that are localized in active centers of respiratory enzymes), and as bound sulfane sulfur (e.g. hydropersulfide, polysulfide, and others)1, 2- 4 Additionally, free H2S can be released by acid-labile and bound sulfane sulfur under various conditions making them reservoirs of sulfide bioequivalents1' 2.
[000250] Over the last decade, hydropersulfides and polysulfides have been identified as molecules with a higher number of inner sulfur atoms that are able to act as both nucleophiles or electrophiles depending on protonation states and local redox environment. Importantly, hydropersulfides and polysulfides serve as signaling molecules that can regulate various metabolic pathways and have pathophysiological implications1, 4‘ 5. Hydropersulfides and polysulfides can be synthesized independently of H2S. CBS and CSE can use substrates such as cystine or glutathione disulfide, resulting in the formation of cysteine hydropersulfide or glutathione hydropersulfide as well as polysulfides, which predominantly are the forms of bound sulfane sulfur1, 4’ 5. Protein persulfides can originate from cysteine persulfide integration during translation from cysteinyl-tRNA synthetases (CARSs)6, 7. Additionally, CBS and CSE can be promiscuous in utilizing different substrates such as cysteine, homocysteine or cystine in generation of sulfides h 8. Without wishing to be bound by theory, the utilization of these substrates can be varied under intra or intercellular conditions, changes in pH or oxygen conditions such as hypoxia or ischemia that may contribute to biological levels of hydropersulfide and polysulfide availability. However, the formation, kinetics and biological relevance of these per- and polysulfide compounds under various pathophysiological conditions remain largely unclear.
[000251] H2S is constantly maintained in the mammalian cells can be increased either through transcriptional regulation of CSE or transiently via persulfidation popularly called S-sulfhydration of cysteine residues on target proteins8, 9. Our lab has previously demonstrated that CSE/H2S regulates ischemic vascular remodeling mediated during hind limb ischemia via upregulation of H2S and NO- dependent monocyte recruitment and cytokine induction revealing a previously unknown mechanism of arteriogenesis10. We also demonstrated how changes in per/polysulfides regulate cellular permeability and barrier function11. Additionally, we also identified CSE deletion accelerated the development of endothelial dysfunction and atherosclerosis12. Similarly, H2S levels were reduced under disturbed shear stress via phosphorylation of serine 377 thereby inhibiting CSE, due to increased IL-ip13. Although there are some insights into CSE regulation by transcriptional regulation via Activating Transcription Factor 4 (ATF4)14 and specificity protein-1 (Spl)13; how CSE is regulated and how this regulates per- and polysulfide generation remains completely unknown.
[000252] Described herein, we demonstrate the mechanisms underlying CSE regulation, specifically via phosphorylation of CSE at Serine 346 (S346) and Threonine 355 (T355) that enhances its activity and polysulfide generation under hypoxia/ischemia. We have used a phospho-negative and phosphomimetic mutants under normoxia and hypoxic conditions to demonstrate their effects on CSE regulation and subsequent polysulfide generation. Adenosine monophosphate-activated protein kinase (AMPK) is a key regulatory molecule for endothelial function, redox homeostasis, and importantly, hypoxia and ischemia/reperfusion injury16. Our studies further revealed a key regulatory role of p- AMPK in regulating CSE activity and phosphorylation under hypoxia/ischemia. Furthermore, we have generated a polyclonal antibody that selectively detects phospho-CSE S346 and used this to identify in vitro (normoxia and hypoxia) and in vivo (chronic hindlimb ischemia) hypoxia responses.
[000253] References Cited in this example:
[000254] 1. Kolluru GK, Shackelford RE, Shen X, Dominic P and Kevil CG. Sulfide regulation of cardiovascular function in health and disease. Nat Rev Cardiol. 2023;20:109-125.
[000255] 2. Kimura H. Hydrogen sulfide and polysulfides as signaling molecules. Proc Jpn
Acad Ser B Phys Biol Sci. 2015;91 : 131-59.
[000256] 3 Polhemus DJ and Lefer DJ. Emergence of hydrogen sulfide as an endogenous gaseous signaling molecule in cardiovascular disease. Circ Res. 2014; 114:730-7.
[000257] 4. Fukuto JM, Ignarro LJ, Nagy P, Wink DA, Kevil CG, Feelisch M, Cortese-
Krott MM, Bianco CL, Kumagai Y, Hobbs AJ, Lin J, Ida T and Akaike T. Biological hydropersulfides and related polysulfides - a new concept and perspective in redox biology. FEBS Lett. 2018;592:2140- 2152.
[000258] 5. Akaike T, Ida T, Wei FY, Nishida M, Kumagai Y, Alam MM, Ihara H, Sawa
T, Matsunaga T, Kasamatsu S, Nishimura A, Morita M, Tomizawa K, Nishimura A, Watanabe S, Inaba K, Shima H, Tanuma N, Jung M, Fujii S, Watanabe Y, Ohmuraya M, Nagy P, Feelisch M, Fukuto JM and Motohashi H. Cysteinyl-tRNA synthetase governs cysteine polysulfidation and mitochondrial bioenergetics. Nat Commun. 2017;8: 1177.
[000259] 6. Akaike T, Ida T, Wei F-Y, Nishida M, Kumagai Y, Alam MM, Ihara H, Sawa
T, Matsunaga T, Kasamatsu S, Nishimura A, Morita M, Tomizawa K, Nishimura A, Watanabe S, Inaba K, Shima H, Tanuma N, Jung M, Fujii S, Watanabe Y, Ohmuraya M, Nagy P, Feelisch M, Fukuto JM and Motohashi H. Cysteinyl-tRNA synthetase governs cysteine polysulfidation and mitochondrial bioenergetics. Nature Communications. 2017;8:1177.
[000260] 7. Ida T, Sawa T, Ihara H, Tsuchiya Y, Watanabe Y, Kumagai Y, Suematsu M,
Motohashi H, Fujii S, Matsunaga T, Yamamoto M, Ono K, Devarie-Baez NO, Xian M, Fukuto JM and Akaike T. Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling. Proc Natl Acad Sci U S A. 2014;111 :7606-11.
[000261] 8. Baneijee R. Catalytic promiscuity and heme-dependent redox regulation of
H2S synthesis. Curr Opin Chem Biol. 2017;37: 115-121.
[000262] 9. Paul BD and Snyder SH. Protein sulfhydration. Methods in enzymology.
2015;555:79-90.
[000263] 10. Kolluru GK, Bir SC, Yuan S, Shen X, Pardue S, Wang R and Kevil CG.
Cystathionine y-lyase regulates arteriogenesis through NO-dependent monocyte recruitment. Cardiovasc Res. 2015;107:590-600.
[000264] 11. Yuan S, Pardue S, Shen X, Alexander JS, Orr AW and Kevil CG. Hydrogen sulfide metabolism regulates endothelial solute barrier function. Redox Biol. 2016;9: 157-166.
[000265] 12. Yuan S, Yurdagul A, Jr., Peretik JM, Alfaidi M, Al Yafeai Z, Pardue S, Kevil
CG and Orr AW. Cystathionine '/-Lyase Modulates Flow-Dependent Vascular Remodeling. Arterioscler Thromb Vase Biol. 2018;38:2126-2136.
[000266] 13. Bibli SI, Hu J, Sigala F, Wittig I, Heidler J, Zukunft S, Tsilimigras DI,
Randriamboavonjy V, Wittig J, Kojonazarov B, Schtirmann C, Siragusa M, Siuda D, Luck B, Abdel Malik R, Filis KA, Zografos G, Chen C, Wang DW, Pfeilschifter J, Brandes RP, Szabo C, Papapetropoulos A and Fleming I. Cystathionine y Lyase Sulfhydrates the RNA Binding Protein Human Antigen R to Preserve Endothelial Cell Function and Delay Atherogenesis. Circulation. 2019;139: 101-114.
[000267] 14. Dickhout JG, Carlisle RE, Jerome DE, Mohammed-Ali Z, Jiang H, Yang G,
Mani S, Garg SK, Banerjee R, Kaufman RJ, Maclean KN, Wang R and Austin RC. Integrated stress response modulates cellular redox state via induction of cystathionine y-lyase: cross-talk between integrated stress response and thiol metabolism. J Biol Chem. 2012;287:7603-14. [000268] 15. Yang G, Pei Y, Teng H, Cao Q and Wang R. Specificity protein-1 as a critical regulator of human cystathionine gamma-lyase in smooth muscle cells. J Biol Chem. 2011;286:26450- 60.
[000269] 16. Rodriguez C, Munoz M, Contreras C and Prieto D. AMPK, metabolism, and vascular function. Febsj. 2021;288:3746-3771.
[000270]
[000271] Example 3 - CSE regulation of vascular remodeling
[000272] Hydrogen sulfide synthesis and metabolism is an important participant in cardiovascular health and function. Specifically, our laboratory has shown that cystathionine y-lyase (CSE) expression and function play a critical role in ischemic vascular remodeling responses of arteriogenesis and angiogenesis. Moreover, our group has revealed important chemical biology and pathophysiological relationships between sulfide and nitric oxide metabolites, which may be important for cooperative regulation of ischemic vascular remodeling. However, numerous molecular and cellular mechanisms remain unknown in these responses including: how chronic ischemia quickly increases sulfide metabolite bioavailability, the role of specific cell populations in producing discrete sulfide species during ischemic vascular remodeling, and how different sulfide metabolites modulate nitric oxide (NO) bioavailability through various enzymatic and non-enzymatic pathways. This example will use new tissue specific CSE mutant mouse models, cutting edge analytical chemistry measurement methods of sulfide and NO species, and cellular and molecular methods to validate posttranslational regulation of CSE protein activity in response to hypoxia. Using the models and tools above, this example will validate that endothelial cell and monocyte CSE dependent polysulfide formation regulates ischemic vascular remodeling and NO bioavailability. Three specific aims will be pursued to validate this: 1) validating the mechanisms of endothelial CSE regulation of ischemic vascular remodeling and how it controls vascular cell NO bioavailability, 2) validating the mechanisms of monocyte CSE regulation of arteriogenesis, and 3) validating mechanisms of CSE activity and expression in experimental models and clinical specimens.
[000273] Peripheral ischemic vascular diseases (e.g. Peripheral Arterial Disease (PAD) and critical limb ischemia (CLI)) are chronic disorders associated with reduced blood flow to the extremities that results in serious consequences including limb amputation and increased risk of cardiovascular death. Work from our laboratory indicates that hydrogen sulfide metabolites such as hydropersulfide and polysulfide may be important for vascular growth and remodeling during limb ischemia. However, mechanisms involved in this response are unknown and are important for understanding pathophysiological processes involved in ischemic vascular disease. This example will validate key contributions of the sulfide-generating enzyme (CSE) in both endothelial cells and monocytes during ischemic vascular remodeling. Cell and molecular biology studies will also determine how CSE activity is regulated during ischemia. Together, these results will validate new molecular targets for therapeutic interventions directed at peripheral ischemic vascular disease.
[000274] Specifics Aims
[000275] Peripheral ischemic vascular diseases (e.g. peripheral arterial disease- PAD and critical limb ischemia-CLI) are chronic tissue disorders that affect millions of Americans and others worldwide and are poised to further increase due to several risk factors. Importantly, therapeutic approaches for ischemic vascular diseases are not widely effective and typically provide short-term benefit highlighting the need for new molecular targets or treatment approaches involved in vascular remodeling.
[000276] Experimental evidence has revealed that hydrogen sulfide (H2S) chemical biology and cystathionine gamma lyase (CSE) serves important roles in vascular growth and remodeling during tissue ischemia. Also, sulfide metabolites, such as polysulfides/hydropersulfide (a.k.a. bound sulfane sulfur), exist as potent signal mediators beyond that of H2S itself. However, the role and pathophysiological importance of these sulfide metabolites for ischemic vascular remodeling in different cellular compartments remains unknown. Lastly, little information exists regarding key specific cell biology responses and mechanisms for sulfide dependent ischemic vascular remodeling involving endothelial (e.g. NO generation) and immune cell (e.g. monocyte recruitment and polarization), respectively.
[000277] My research team has revealed that CSE and associated changes in H2S bioavailability are protective against chronic tissue ischemia involving increased arteriogenesis and angiogenesis. However, it is completely unclear how CSE function is regulated at the cellular and molecular level and how it influences diverse sulfide metabolites (e.g. per & polysulfides). Data herein reveals new findings in CSE activity under hypoxia with preferential formation of endothelial bound sulfane sulfur metabolites. We also show differential sulfide metabolite effects on hypoxic endothelium that alters proliferation, permeability, and NO formation. However, the role and mechanisms of hydropersulfide and polysulfide metabolites in regulating ischemic endothelial responses also remains completely unknown. New findings herein also show that CSE activity and function also significantly influences macrophage activation and cytokine production that is necessary for vascular remodeling. Thus, this proposal will validate new information on critical unanswered questions regarding CSE and hydrogen sulfide metabolite functions by investigating whether endothelial cell and monocyte cystathionine y- lyase (CSE) dependent polysulfide formation regulates ischemic vascular remodeling and NO bioavailability. Without wishing to be bound by theory, this will be validated through the pursuit of three specific aims that are illustrated in Figure 7.
[000278] Specific Aim 1- Validate the mechanisms of endothelial CSE regulation of ischemic vascular remodeling and how it controls vascular cell NO bioavailability. This aim will use endothelial specific CSE gene deficient mice created in our laboratory to reveal endothelial CSE functions during ischemic vascular remodeling and the effect of polysulfide generation and function on NO formation through various pathways.
[000279] Specific Aim 2- Validate the mechanisms of monocyte CSE regulation of arteriogenesis. This aim will use monocyte specific CSE gene deficient mice created in our lab to validate how monocyte CSE regulates arteriogenesis and angiogenesis, along with in vitro studies of monocyte recruitment and macrophage polarization.
[000280] Specific Aim 3- Validate mechanisms of CSE activity and expression in experimental models and clinical specimens. This aim will reveal molecular mechanisms controlling CSE activity and expression during hypoxia/ischemia including post-translational modifications, substrate and PLP cofactor bioavailability studies.
[000281] Research Plan
[000282] A) Scientific Premise and Significance
[000283] Importance of the Problem & Scientific Premise
[000284] Ischemic vascular disease - a growing problem with few solutions: Peripheral ischemic vascular disease (e.g. Peripheral Arterial Disease (PAD) and Critical Limb Ischemia (CLI)) are vascular disorders associated with reduced tissue blood flow to organs and extremities, which results in serious complications such as defective wound healing, limb amputation and significantly increased mortality over a five year period1. In 2010, approximately 10-12 million U.S. citizens suffered from some form of peripheral vascular disease (PAD) and this number is projected to double by 20302. Worldwide, the incidence of peripheral vascular diseases is growing rapidly, presenting significant health burdens in both developed and growing countries3. No FDA approved therapeutic is available to effectively promote ischemic revascularization of chronically ischemic tissue. Work from our laboratory has revealed that hydrogen sulfide (H2S) and nitric oxide (NO) metabolic pathways play important pathophysiological roles in vascular growth and remodeling that are compromised during ischemic vascular disease4'6. These findings indicate that H2S and NO metabolic pathways are important for vascular remodeling; however, specific cellular mechanisms and metabolites involved are unclear. Sulfide metabolites exist in different biochemical forms influenced by various stimuli that elicit diverse vascular cell responses 4' 5’ 7 Unfortunately, no information exists with regard to how specific sulfide metabolites such as persulfides or polysulfides contribute to vascular remodeling or how they affect NO metabolite bioavailability and biological actions.
[000285] Hydrogen sulfide chemical biology and metabolism: H2S in the cardiovascular system has been reported to be predominantly produced by cystathionine-y-lyase (CSE) in different vascular cell types including endothelial and vascular smooth muscle cells using cystathionine or L-cysteine as substrates (Figure 8)8,9, 10. Sulfide bioavailability may exist in different biochemical forms or ‘pools’ including free sulfide, acid labile sulfide, and bound sulfane sulfur11, 12. A wide range of sulfide metabolites may be produced via CSE besides H2S alone with an interest on formation of persulfide and polysulfides13. Moreover, H2S may also contribute to the formation of per- and polysulfides through formation of thiyl radicals (HS»), which can react to form RSS(n)H species (Figure 8)14. However, the formation, chemical biology, and signaling roles of these molecules remain completely unknown during ischemic vascular remodeling responses. Moreover, it has been shown that CSE can also use cystine (CysSSCys) as a substrate forming cysteine persulfide (CysSSH) that may be a biologically important bound sulfane sulfur15. As illustrated in Figure 8, this example will validate whether CSE forms per/polysulfides during ischemia/hypoxia as potent chemical mediators of vascular remodeling. Moreover, CSE may also have diverse effects in different cellular compartments (e g. vascular vs immune cells) that remain poorly understood during ischemic vascular remodeling that will be further revealed in our studies16'19. In summary, the sulfide field remains highly unclear with regard to how CSE generates various sulfide metabolites in specific cell compartments under ischemic vascular conditions.
[000286] Barriers to Progress & Potential to Advance Knowledge in the Field
[000287] Sulfide metabolite measurement challenges and discrete cell biology function: While H2S has emerged as an important participant in various cardiovascular functions; much less certainty exists regarding precise levels of different sulfide metabolite biochemical forms (e.g. hydropersulfides and polysulfides); their biological effects within discrete cellular compartments; and regulation of their formation. Our laboratory has pioneered analytical chemistry techniques to measure all sulfide metabolites via HPLC and LC-MS/MS approaches enabling highly accurate detection11, 20. We’ve also applied these techniques to both basic experimental and human research studies validating the ability to discern key metabolic responses21-23. Lastly, until very recently it was challenging to examine the cell biology function of different sulfide metabolites due to lack of stable products. This has been resolved through commercially available per- and polysulfide species along with our analytical methods to measure these products. Importantly, the biological effects of per/polysulfide compared to H2S alone remain completely unknown. Also, insight into these mediators and CSE functions in vascular disease specimens remains completely unexplored representing a major gap in our knowledge. This example will reveal fundamental new information regarding cell specific CSE expression and function that mediates ischemic vascular remodeling, how CSE contributes to hydropersulfide and polysulfide formation and their influence on NO production and cell signaling, and how CSE activity is regulated under hypoxia and in cardiovascular disease tissue.
[000288] B) Innovation:
[000289] Results from this example will reveal for the first time CSE/polysulfide regulation of ischemic vascular remodeling generating new models, tools, and mechanistic insights for the scientific research community. Many important questions remain unanswered regarding sulfide metabolism in discrete cellular compartments and associated molecular mechanisms involved in ischemic vascular remodeling. This project will surpass these obstacles through innovative approaches including: 1) use of new tissue specific mutant animal models of CSE within the vasculature and monocyte compartment, generation of new molecular gene constructs of CSE to determine how its regulated, employ advanced analytical chemistry methods using HPLC/MS validating key sulfide metabolites, 2) identification of specific H2S metabolite (e.g. persulfide and polysulfide) bioavailability and their effect on endothelial cell functions including nitrite reduction to nitric oxide, 3) identification of critical cellular and molecular pathophysiological mechanisms governing ischemic vascular remodeling via vascular endothelium and monocyte immune cell responses, and 4) determine the effect of specific phospho-amino acid modification on CSE activity along with substrate and cofactor bioavailability, and bioavailability and identify these properties of CSE from diseased vascular tissues. [000290] Together, these results will significantly advance the field in understanding endogenous mechanisms of sulfide dependent vascular remodeling
[000291] C) Approach and Scientific Rigor [000292] Cl.
[000293] Endothelial cell-specific CSE genetic deletion (ecCSEKO): Genetic deficiency of CSE in mouse models is important in modulating several cardiovascular responses5. Our group and others, have employed global CSE gene targeted deficient mice to gain initial insight into the pathophysiological role of this molecule and its impact on sulfide metabolism24. Global CSE KO mice manifest defective vascular growth responses, as well as defective monocyte mediated arteriogenesis5; however, effects of CSE in specific cellular tissues remain poorly understood during ischemic vascular remodeling. To address this, we developed vascular cell-specific CSE mutant mice using C57BL/6NTac-Cthtrn, a(EUCOMM)Hrngll/Ieg (Cth being the gene abbreviation for CSE) strain crossed with B6.129S4-Gt(ROSA)26Sortm2(FLP*)So7J Flp deleter line and then backcrossed to the VE-Cad CRE driver line to obtain VECad CSEKO (referred to as ecCSEKO). This is a useful cell-specific model that clearly diminishes endothelial CSE protein, mRNA, and sulfide metabolites seen in Figure 9.
[000294] Blood flow and ischemic vascular remodeling responses decreases in ecCSEKO mice: Restoration of blood flow and mature blood vessel formation in ischemic tissues is crucial for limb regeneration. To understand the cell-specific roles of CSE in ischemic revascularization, we subjected WT and ecCSEKO to femoral artery ligation (FAL). Blood flow and perfusion rates of WT and ecCSEKO mice were measured using SPY angiogram at day 7 following FAL. Blood flow in ischemic tissue was significantly reduced in ecCSEKO mice compared with WT mice and global CSE KO (Figure 9, panels A and B). Mature vessel density and angiogenic index (dual staining with CD31 and a-SMA antibodies) in ecCSEKO mice were significantly decreased compared to WT control (Figure 9, panels C and D), indicating the crucial need of endothelial CSE expression for vessel remodeling under hind limb ischemia.
[000295] Hypoxia increases endothelial cell CSE activity and bound sulfane sulfur bioavailability: We previously reported that tissue ischemia due to femoral artery ligation quickly augmented CSE enzyme activity even before abundant increases in mRNA 5. Additionally, we established new analytical chemistry work flows to measure changes in free hydrogen sulfide, acid labile sulfide (e.g. Fe-S clusters), and bound sulfane sulfur (e.g. persulfide, polysulfides, polythionates, etc.) representing the total sulfide bioavailability flux in tissues ,2’ 23. However, it is not clear how hypoxia effects CSE enzyme expression and/or activity in endothelial cells. Figure 10, panel A reports the effect of 30 minutes of 1% 02 on wild type mouse aortic endothelial cell (MAEC) CSE protein expression and enzyme activity. Hypoxia did not significantly affect CSE protein expression (left panel, Figure 10, panel A) but quickly and robustly increased CSE enzyme activity ~2.5 fold compared to control ‘normoxic’ tissue culture conditions (right panel, Figure 10, panel A). Figure 10, panel B shows that 30 minutes of hypoxia alters sulfide metabolite levels with reduced acid labile sulfide levels and significantly elevated bound sulfane sulfur levels within endothelial cells. This illustrates a totally unique and previously unknown response regarding changes in sulfide metabolite bioavailability. The importance of this is further understood in context with the fact that we first reported CSE KO mouse aortic endothelial cells have a blunted capacity to generate bound sulfane sulfur molecules 25 that was also confirmed by Bibli et al 26 Without wishing to be bound by theory, this may be due to the fact that a deficiency in CSE is unable to utilize substrates like cystine to generate cysteine persulfide or polysulfides, which have recently been shown to be important intracellular signaling and redox regulatory molecules 15- 27 These data provide compelling evidence for further validation into the mechanisms whereby CSE regulates the formation of bound sulfides during hypoxia that may be important for multiple cellular responses.
[000296] H2S versus per/polysulfides differentially affect endothelial cell activation: Persulfides and polysulfides have become increasingly more appreciated for their role in modulating various responses in cell model systems 28,29. However, little is known regarding differential endothelial cell responses to hydrogen sulfide versus per/polysulfides. Figure 11 illustrates the effect of different sulfide compounds including sodium sulfide, disulfide, trisulfide, and tetrasulfide on wild type MAEC at a dose of 50 pM. Specifically, these compounds immediately generate the following upon hydration in buffer: Na2S-H2S, Na2S2-H2S2 (hydrogen persulfide), Na2S3-H2S3 (hydrogen trisulfide), and Na2S4-H2S4 (hydrogen tetrasulfide) with the last two being hydro-polysulfides. Figure 11, panel A shows that sodium disulfide most potently stimulated proliferation of serum starved MAEC. Interestingly, Figure 11, panel B shows the effect of the same donors on endothelial solute permeability to FITC albumin over 60 minutes. In this model, hydrogen trisulfide and hydrogen tetrasulfide were most potent for stimulating increased albumin permeability. Together, these data clearly demonstrate that per/polysulfides are more potent than hydrogen sulfide in activating various endothelial cell responses using concentrations of the per/polysulfides that are physiologically or pharmacologically realistic.
[000297] H2S robustly and preferentially increases hypoxic endothelial NO production independent of NOS: Our laboratory and others have investigated the impact of hydrogen sulfide on NO bioavailability resulting in various insights into how H2S can regulate NO levels 30, 31. While laboratories have examined the direct effect of exogenous sulfide donors on endothelial NO production under normoxic conditions, our laboratory has keenly focused on how sulfide bioavailability during chronic tissue ischemia impacts NO levels involving NOS and non-NOS pathways. In brief, we reported that H2S and CSE expression impact ischemic tissue ability to generate NO with only a partial role of eNOS activity 4’ 5. Importantly, we revealed that H2S could augment hypoxic MAEC NO formation involving XO activity and protein thiols4. Figure 12 further demonstrates that human umbilical vein endothelial cells exposed to varying concentrations of H2S preferentially causes NO production under hypoxic versus normoxic conditions (Figure 12, panels A and B). NOS inhibition with L-NAME did not attenuate H2S dependent NO production in hypoxic HUVEC, but Febuxostat blockade of XO significantly blunted NO production, indicating that XO dependent NO2' reduction to NO is involved.
[000298] Role of monocyte CSE expression, activity and H2S in ischemic vascular remodeling: Recent findings from our and other laboratories have revealed a role of H2S metabolism in various immune cell functions that participate in numerous pathophysiological responses 5- 32, 33. Figure 13, panels Aand B show that CSE mRNA expression is unaltered in spleen monocytes but is increased in bone marrow monocytes from mice subjected to FAL-mediated limb ischemia, respectively. Likewise, we previously reported that bone marrow monocytes from mice with hind limb ischemia display increased CSE activity and sulfide production compared to sham treated mice, which was defective in global CSE KO mice 5. Importantly, LysM Cre/CTH A/ A mice (referred to as cpCSEKO) significantly reduces bone marrow monocyte CSE activity (Figure 13, panel D) compared to spleen monocytes (Figure 13, panel C). As we have previously reported that CSE/H2S regulates myeloid cell infiltration and subsequently tissue perfusion and ischemic vascular remodeling 5. Figure 14 shows that myeloid cell CSE knockout (cpCSEKO) mice significantly impairs recovery of ischemic limb blood flow and angiogenic index observed at day 7 after FAL. Without wishing to be bound by theory, these data indicate that monocyte/macrophage CSE expression can regulate vessel remodeling and blood flow during ischemia.
[000299] CSE genetic deficiency blunts monocyte recruitment: Having observed significant changes in monocyte CSE expression, activity, and H2S production, we examined the impact of CSE deficiency on bone marrow monocyte recruitment. Figure 15 shows that genetic deficiency of CSE in either myeloid cells (cpCSEKO, upper panel) or endothelial cells (ecCSEKO, lower panel) decreases the monocyte recruitment (MAC-2) to the site of ischemia. These results (Figures 14 and 15) clearly demonstrate that monocyte recruitment into ischemic tissue is impaired in cpCSEKO mice, indicating that CSE expression is required for monocyte recruitment for arteriole remodeling. These data reveal that CSE activity modulates monocyte recruitment that will be studied in this proposal.
[000300] Identification of hypoxia dependent CSE phosphorylation: Data above and our previous report shows a unique and important finding that CSE enzyme activity quickly increases in ischemic tissue before significant increases in protein levels or gene expression5. However, mechanisms responsible for increased CSE activity remain poorly understood. While previous studies have revealed possible regulatory mechanisms associated with changes in CSE expression via miRNA, transcription factors, or changes in calcium levels, few studies have examined regulation of CSE enzyme activity under cell stress. Work from Yuan et al indicates that CSE may be subject to posttranslational regulation as PKG could phosphorylate S376 that constitutively inhibits H2S production in carotid body glomus cells thus serving an oxygen-sensing role34. Recently, Bibli et al. reported the role of inflammatory cytokine IL-1 P eliciting phosphorylation of serine 377 (S377) that attenuated CSE activity 26. However, no studies have described the mechanisms of CSE enzyme regulation or differential posttranslational modifications in response to hypoxia. Thus, we overexpressed human FLG tagged CSE in HEK294 cells, exposed them to 30 minutes of normoxia or hypoxia (1% 02), immunopurified CSE and performed LCMS/ MS analysis. Figure 16, panels A and B report MS profiles of CSE under normoxic and hypoxic conditions. Three CSE phospho amino acids were discovered under hypoxia including S346, T355, and S358. Figure 16, panel C shows cystathionine consumption indicating increased CSE activity along with Figure 16, panel D reporting increased bound sulfide (i.e. per/polysulfide) production. Together, these data provide fundamental new insight into posttranslational modifications that may regulate CSE activity.
[000301] Upstream mediators of CSE phosphorylation domain regulate CSE activity Recent studies show the role of phosphorylation in regulation of CSE activity26, 34 Our preliminary observations indicated an increase in CSE activity in endothelial cells under hypoxia (Figure 10, panel A). We next sought to understand the signaling mediator significance that could influence phosphorylation sites that we identified under hypoxia including S346, T355, and S358 (Figure 16). We used ScanSite 4.0 to identify the unique kinase phosphorylation motifs associated with identified sites. The domain organization and location of these phosphorylation sites is within the active site regions of AMPK and protein kinase C. It is known from the literature that AMPK can influence the biological functions mediated by H2S35. Additionally, our very recent work demonstrates that the ATR kinase initiates signaling cascades that regulate cellular H2S bioavailability and metabolism 36. Importantly, CSE activity was significantly reduced in human umbilical endothelial cells (HUVECs) under hypoxia upon individual inhibition of AMPK (Dorsomorphin), PKC a and P (Go 6976) or ATR (NU6027) inhibitors, respectively (Figure 17). These results indicate that these signaling kinases may play role in regulating the activation of CSE enzyme through phosphorylation that will be studied in this proposal.
[000302] C2. Research Design: Herein we describe examples to validate that tissue ischemia differentially increases CSE activity and polysulfide production in vascular and monocyte cells to effectively coordinate ischemic vascular remodeling of ischemic tissue. Experiments in SAI will validate how endothelial cell specific knockout of CSE regulates ischemic vascular remodeling responses using cell-specific mutagenesis models along with studies investigating polysulfide dependent NO production in hypoxic endothelial cells. Studies in SA2 will identify the importance of monocyte CSE expression for ischemic vascular remodeling using cell-specific mutagenesis approaches along with in vitro studies examining monocyte recruitment and differentiation responses. SA3 will examine molecular mechanisms regulating increased CSE activity and sulfide metabolite production through enzyme posttranslational modifications, cofactor bioavailability, substrate specificity, and also evaluate these parameters in clinical vascular disease specimens. These experiments address important unknown questions in the field of ischemic vascular remodeling and will provide new insight into molecular mechanisms of CSE functions.
[000303] Specific Aim 1- Validate the mechanisms of endothelial CSE regulation of ischemic vascular remodeling and how it controls vascular cell NO bioavailability.
[000304] Rationale: We’ve reported that global CSE genetic deficiency blunted ischemic vascular remodeling responses in the femoral artery ligation model involving defective arteriogenesis, angiogenesis, and NO production. However, these findings do not reveal which cellular compartments are involved in CSE dependent vascular remodeling or which CSE dependent sulfide metabolites augment NO production or the mechanisms involved. We’ve established new endothelial specific CSE mutant mice in our laboratory to examine the specific mechanisms of CSE expression and activity for vascular remodeling. We’ve also established mouse aortic endothelial cell (MAEC) lines from global CSE knockout and wild type mice to investigate molecular mechanisms of hydrogen sulfide dependent nitric oxide production in vitro. Experiments described hereinprovide new insight into endothelial CSE regulation of vascular remodeling in vivo and sulfide metabolite regulation of NO production. [000305] a) Validate the effect of vascular endothelial CSE genetic deficiency on ischemic vascular remodeling. Data in Figure 9 shows that tissue specific CSE mutant mice are readily available in the lab. These strains and appropriate littermate controls including VE-Cad and ecCSEKO will be used in the femoral artery ligation model. Briefly, ligation and vessel excision will be performed on the left femoral artery proximal to the profunda femoris, as we’ve reported 37. Changes in limb perfusion and vascular contrast imaging will be performed using the PeriCam PSI laser speckle contrast imager and the SPY imaging system, respectively. The PeriCam PSI system will enable accurate longitudinal hind limb blood flow measurement due to its depth of penetration reflecting changes in the microcirculation, and SPY imaging will enable longitudinal arteriogram imaging using indocyanine green (ICG) contrast agent reflecting changes in arteriogenesis 5’ 37 (Figure 18, panel A). Imaging will be performed pre and post-ligation and at days 1, 3, 5, 7, 14 and 21. In a separate cohort of mice, Microfil vascular casting will be performed in control and mutant mice at pre and post ligation, and days 3, 7, and 14 as we previously reported 5 (Figure 18, panel, B). In a separate cohort of animals, gastrocnemius and adductor muscle will be collected at pre and post-ligation, and days 1, 5, 7, 14, and 21 for histopathology analysis including CD31 and a-SMA staining, as well as free hydrogen sulfide, acid-labile sulfide, and bound sulfane sulfur metabolite measurements via LC- MS/MS, as we’ve previously reported 1 L 12' 20 and shown in Figure 19.
[000306] b) Validate the importance of endothelial CSE activity and polysulfide formation for endothelial NO production and bioavailability under hypoxic conditions. Data in Figure 10 demonstrates that hypoxia selectively increased bound sulfane sulfur while decreasing acid labile sulfide levels. However, the importance of CSE dependent bound sulfane sulfur generation for hypoxic endothelial NO production remains unknown. Experiments will be performed using wild type and CSE KO MAECs under normoxic and hypoxic conditions to quantify bound sulfane sulfur per /polysulfide species using LC-MS/MS methods we established in the lab (Figure 19). Cells will be cultured under hypoxic conditions at various time points including 0, 0.25, 0.5, 1, 2, 4, 8, and 16 hours. Batches of cells will be harvested at each time point to measure bound polysulfide metabolites using the monobromobimane (MBB) assay as others and we’ve reported 15, 27 A separate cohort of cells will also be analyzed for NO metabolite levels using NOA chemiluminescent analysis for NOx, nitrosoheme, and nitrosothiols27. Other experiments will be performed using wild type MAEC treated with CSE siRNA or the pharmacological inhibitor PPG to measure hypoxia bound sulfane sulfur and NO levels. Together, these experiments will provide important insight regarding the importance of CSE for regulation of polysulfide levels that may influence NO bioavailability.
[000307] c) Validate the effect of per/polysulfides versus free hydrogen sulfide compounds on NO production and endothelial cell proliferation. Association of per/polysulfides in H2S biosynthesis and signaling has been appreciated 14, 28; however, their influence on NO biosynthesis and cellular signaling remain elusive. Figure 12 reveals that exogenous hydrogen sulfide via sodium sulfide treatment increases NO production and bioavailability of hypoxic endothelial cells. Moreover, data in Figure 11 also reveal differential effects of per- vs. polysulfides on endothelial cell activation responses. Yet, it is not known whether hydrogen sulfide vs. polysulfides differentially influence NO synthesis. Experiments will be performed using wild type MAEC incubated under hypoxic conditions for 4 hours, as we’ve previously reported that these are ideal cell culture conditions that enable measurement of hydrogen sulfide dependent NO formation using NOA chemiluminescent detection. Briefly, hypoxic wild type MAEC will be collected, placed in the NOA sparger, and treated with either sodium sulfide (H2S donor), hydrogen persulfide (H2S2), hydrogen trisulfide (H2S3), hydrogen tetrasulfide (H2S4), diallyl trisulfide (DATS), or diallyl disulfide (DADS). Each of the compounds will be tested in a dose dependent manner including 0, 0.1, 0.5, 1, 10, 50, and 100 pM concentrations. This will enable wide range comparisons between the various compounds allowing us to validate different electrochemical properties based on the relative nucleophilic strength of bound sulfur compound species. An analogous series of experiments will also be performed using CSE knockout MAEC that will allow deeper understanding of CSE sulfide dependent NO production. Mechanisms of polysulfide dependent NO production will be validated using various inhibitors we’ve shown to be important for hydrogen sulfide mediated NO formation in hypoxic cells including, L-NAME (to block eNOS), cPTIO (NO scavenger), NEM (thiol blocker), paraformaldehyde (protein crosslinker), or febuxostat (XO inhibitor) to elucidate specific biochemical mechanisms4. Studies will also be performed examining eNOS phosphorylation at classical inhibitor/activator residues (Ser 1177, Thr495 etc.) along with XO/uric acid enzyme activity assays. Lastly, per- & polysulfide versus free sulfide treatments will be examined for MAPK dependent endothelial cell proliferation (BrdU assay) and phospho-Erkl/2 western blot in conjunction with MAPK inhibition using U0216 or PD98059. Together, these studies will elucidate specific molecular target mechanisms of per- & polysulfide endothelial activation responses.
[000308] SAI Methods [000309] a) Femoral artery ligation induction of hind limb ischemia. Femoral artery ligation and excision will be performed in male and female mice as we’ve previously reported 4’ 37 Recent work revealed sex specific response in female CSE null animals with substantial differences in the methylfolate trap 38. Thus, it is important to examine both male and female sex in this new animal model. While animal models of hind limb ischemia are not completely reflective of clinical conditions, nonetheless the FAL model is widely regarded as informative for ischemic vascular remodeling responses and underlying cellular and molecular mechanisms.
[000310] b) Hind limb perfusion and histological measurements. Hind limb perfusion measurements will be performed using a PeriCam PSI laser speckle contrast imager. Likewise, histopathology measurements including capillary to myofiber ratio and vascular index will be made at pre, post, 1, 3, 5, 7, 14, and 21 days. Briefly, 5 pm frozen muscle tissue sections will be cut and fixed with ethanol/acetic acid and dual stained with anti-CD31 and Ki67 along with DAPI nuclear stain. Vascular density and cell proliferation index ratios will be validated by quantitative morphometric analysis to DAPI stain using Simple PCI software as we’ve reported4’ 5’ 37. Capillary to myofiber ratio will be performed using formalin fixed, paraffin embedded sections stained with CD31 and counterstained with hematoxylin, and number of capillaries per myocyte counted.
[000311] c) Collateral remodeling measurements. Collateral artery remodeling measurements will be performed as we’ve reported37. SPY imaging will be performed in a longitudinal manner using ICG dye injection via retro-orbital injection. Still images are extracted to measure changes in vascular length, diameter, and branching. Microfil vascular casting and morphometric measurements will be performed as we’ve reported37.
[000312] d) LC-MS/MS measurement of polysulfide species and bioavailability. Sulfide metabolite bioavailability will be measured as we’ve reported n’ 12, 20- 39. Blood and tissue specimens will be derivatized with MBB in 1% oxygen for 30 minutes at room temperature and stopped using 50 pL of 200 mM ice-cold sulfosalicylic acid solution. Samples will be measured by LC-MS/MS using a Acquity UPLC system coupled to XEVO TQ (Thermo Scientific) with electrospray ionization (ESI(+)). Data will be collected in MRM mode by screening parent and daughter ions simultaneously, cone voltage will be set depending upon each specific MRM for each metabolite, and the dwell time automatically set by MassLynxTM 4.1 software. Product ions for MBB derivatives of hydrogen sulfide (H2S), persulfide (HSSH) and polysulfide (HS-(S)n-SH) levels will be calculated using the peak area ratios of signature products (192.1 m/z) to corresponding stable 34S (internal standards). [000313] e) Measurement of polysulfide dependent NO production and cell proliferation. NO production from hypoxic or normoxic endothelial cells in response to exogenous sulfides will be done by chemiluminescent nitric oxide analysis as we’ve reported 4’ 27. Sulfide compounds including sodium sulfide (Na2S), sodium disulfide (Na2S2), sodium trisulfide (Na2S3), sodium tetrasulfide (Na2S4), diallyl disulfide (DADS), and diallyl trisulfide (DATS) will be used at various concentrations as we’ve reported 28. Inhibitor studies will also be performed using L-NAME, cPTIO, NEM, paraformaldehyde, or febuxostat as we’ve previously reported4. Endothelial cell proliferation studies described above will be performed as we’ve reported5.
[000314] f) Statistical analysis. All time course experiments will be statistically evaluated using two-way ANOVA between different mouse genotypes over time with Bonferroni post-testing. Quantitative histomorphological changes will be statistically evaluated using two-way ANOVA with Bonferroni post testing among different genotypes over time. Western blot and qRT-PCR data from different genotypes will be compared using two way ANOVA with Bonferroni post-test over time. Justification of animal numbers and associated power analysis are clarified in the vertebrate animals section.
[000315] SAI Milestones and Timeline
[000316] Validate the role of endothelial CSE for ischemic vascular remodeling responses using the FAL model.
[000317] ii) Validate the differences and mechanisms associated with hydrogen sulfide versus polysulfide dependent hypoxic endothelial cell NO production.
[000318] Specific Aim 2- Validate the mechanisms of monocyte CSE regulation of arteriogenesis. Rationale: It is well understood that monocytes play an important role in controlling arteriogenesis and vascular remodeling responses 40, 41. Surprisingly, we recently revealed an important role of CSE in regulating monocyte dependent arteriogenesis responses involving endothelial cell proliferations. However, a limitation of the previous study was the use of a global CSE knockout mouse that was unable to define discrete effects of CSE in different cellular compartments. Moreover, recent work also reveals that hydrogen sulfide synthesis and metabolism is involved in macrophage responses associated with myocardial ischemia-reperfusion 42’ 43. Thus, the studies outlined below will utilize new experimental models to examine the precise role of monocyte CSE for ischemic vascular remodeling in the femoral artery ligation model. [000319] a) Validate effect of monocyte CSE genetic deficiency on arteriogenesis. Findings from the previous funding period data revealed an important role of CSE in regulating monocyte dependent arteriogenesis. Yet, the pathophysiological role of monocyte CSE expression and sulfide metabolite formation for arteriogenesis and associated vascular remodeling is completely unknown. Should this occur, we have established CTH Tmlc allele mice bred with LysM-Cre driver mice to generate the LysM-Cre/CTHA/A (ipCSEKO) mice with CSE depletion in monocytes, which has been characterized (Figure 9). Using this line, ischemic vascular remodeling in the femoral artery ligation model will be performed analogous to that of SAI a. Changes in limb perfusion will be analyzed by laser speckle and SPY vascular angiogram imaging at pre and post-ligation and at days 1, 3, 5, 7, 14 and 21. In a separate cohort of mice, Microfil vascular casting will be performed in control and mutant mice at pre and post ligation, and days 3, 7, and 14 as we previously reported 5’39. Adductor and gastrocnemius muscle tissues will be collected at time points indicated in SAla and processed for H&E histopathology, CD31, and alpha SMA immunostaining as we’ve previously reported5. Together, these studies will reveal the importance of monocyte CSE expression for ischemic vascular remodeling responses.
[000320] b) Validate the effect of CSE genetic deficiency on macrophage polarization. It is known that Ml vs M2 macrophage types influence vascular remodeling44, 45. However, it is not clear if CSE influences this response. Figure 21, panel A show global CSE KO does effect on CD38 (Ml) and EGFR2 (M2) markers but not in ecCSE KO (Figure 21, panel B and C). Conversely, Figure 21, panel D and E show myeloid cell CSE regulates tissue M1/M2 phenotype differentiation. Studies will be performed to validate how CSE expression is involved in differentiation to specific macrophage phenotypes. Bone marrow monocytes will be isolated from WT, CSE KO, (pCSEKO male and female mice, cultured with M-CSF, and then treated with LPS and fFN-y for Ml or IL -4 for M2 phenotypes as previously reported46. Ml vs M2 macrophage phenotypes will be validated via flow cytometry and gene expression changes in collaboration with Dr. Matthew Woolard. A separate series of experiments will also be performed using H2S vs per/polysulfides donors to validate the impact of sulfide metabolite restoration.
[000321] c) Validate whether monocyte CSE function influences adhesion to endothelium and recruitment into tissues. Without wishing to be bound by theory, CSE may be involved in macrophage movement in myocardium during ischemia-reperfusion injury consistent with our findings of deficient monocyte infiltration into chronically ischemic tissue 5’ 43. However, no studies have been reported investigating whether CSE expression is important for actual monocyte adhesion and recruitment responses that are critical for arteriogenesis. Thus, experiments will be performed to validate whether monocyte deficiency of CSE alters adhesion and recruitment to mouse aortic and microvascular endothelial cells under hydrodynamic flow conditions. Parallel plate flow chambers will be used to validate shear mediated detachment of WT or cpCSEKO monocyte rolling and firm adhesion to TNF- a activated MAEC or MVEC at a range of shear stresses (2- 15 dynes/cm2) as we’ve reported 47, 48, 48. Results from these experiments will provide cellular mechanisms regarding the importance of monocyte CSE in regulating recruitment responses.
[000322] SA2 General Methods
[000323] a) Monocyte CSE mutant ischemic vascular remodeling studies. The effect of monocyte CSE deficiency on ischemic vascular remodeling will be validated as described in SAI methods.
[000324] b) Monocyte adhesion and recruitment studies. Bone marrow monocytes will be isolated using the EasySep mouse monocyte isolation kit as we’ve reported5. Monocyte rolling and firm adhesion will be examined using established MAEC and MVEC lines in our laboratory with parallel plate flow chambers as we’ve reported47, 48. Monocyte adhesion strength will be validated by adhesion detachment assays in response to increasing shear stress as we’ve reported49. In some experiments, CSE deficient monocytes will be pretreated with different sulfide donors (10 and 50 iiM) for 4 hours then used in cell adhesion studies.
[000325] c) Macrophage polarization studies. Established monocyte polarization assays will be performed in conjunction with the Woolard Lab. Bone marrow monocytes will be expanded with recombinant murine M-CSF followed by 10 ng/ml LPS and 20 ng/ml IFN-y for Ml or 40 ng/ml IL-4 for M2 phenotypes. Ml macrophage markers used will be TNF-a, iNOS, CCR7, COX-2, IL-12a, and CD38. M2 macrophage markers used will be IL-10, Arg-1, CD206, YMl(Chil3), COX-1, and Egr2. [000326] d) iNOS western blotting and qRT-PCR. Protein cell and tissue lysates will be prepared using RIPA lysis buffer containing protease inhibitors as we’ve reported 4 Western blotting for iNOS and beta actin will be done as we’ve reported 4 Western blot band densitometry will be validated using ImageJ software. Total RNA will be isolated using the Qiagen RNeasy isolation midi-kit and subsequent RNA used with murine iNOS cyber green qRT-PCR with the following primers: iNOS- forward GCTGTTAGAGACACTTCTGAG, reverse CACTTTGGTAGGATTTGACTTTG.
Quantitative differences in mRNA will be validated using the ACT method compared against GAPDH or P-actin as we’ve reported 4 [000327] e) Sulfide and NO metabolite measurements. Monocyte sulfide metabolites including specific per/polysulfides will be measured using MBB derivatization and LC-MS/MS detection as we’ve reported 11 and described in SAI methods. Monocyte NO metabolites including nitrite, nitrate, nitrosothiol, and nitrosoheme will be measured with GE Sievers 280i NO analyzer as we’ve reported 27
[000328] f) Statistical analysis. All time course experiments involving vascular remodeling will be statistically evaluated using two-way ANOVA between different mouse genotypes over time with Bonferroni post-testing as described in SAI. Sulfide metabolites, NO metabolites, western blot and qRT-PCR data from different genotypes will be compared using two-way ANOVA with Bonferroni post-test over time. Changes in monocyte detachment, recruitment under hydrodynamic flow, and Ml vs M2 phenotypic differentiation from monocyte genotypes will be compared using one-way ANOVA with Bonferroni post-test. Justification of animal numbers and associated power analysis are found in the vertebrate animals section.
[000329] SA2 Milestones and Timeline
[000330] i) Validate the role of monocyte CSE in regulating ischemic vascular remodeling in the FAL model.
[000331] (ii) Validate role of CSE deficiency on monocyte recruitment, NO production, and macrophage polarization.
[000332] Specific Aim 3- Validate mechanisms of CSE activity and expression in experimental models and clinical specimens. Rationale: We’ve previously reported that femoral artery ligation and subsequent hind limb ischemia results in robust tissue CSE enzyme activity. Moreover, an increase in CSE mRNA was found to lag the increase in enzyme activity indicating direct regulation of CSE activity plays a key role in modulating tissue sulfide bioavailability. However, regulation of CSE activity under conditions associated with ischemic vascular remodeling is not known. Experiments below will significantly advance the field’s understanding of CSE enzyme regulation and function, which is essential for understanding fundamental ischemic vascular remodeling responses.
[000333] a) Validate the impact of phosphorylation on CSE enzyme activity under normoxic and hypoxic conditions. In this sub aim, the three phospho-amino acid identified sites Ser346, Thr355, and Ser358 are mutated individually or in combination to phosphomimetic or dominant negative sites. Specifically, human CSE cDNA 3X FLAG tagged lentiviral construct will be used to mutate residues to either dominant negative Ala or constitutively active Glu sites as shown in Figure 21. CSE knockout endothelial cells and HEK293 cells will be transfected with various constructs and enzyme activity measured as we’ve reported5. Transduced cells will be exposed to normoxic or hypoxic cell culture conditions over 30 minutes, as we’ve previously reported5. A similar set of experiments will be performed using CSE null bone marrow monocytes where we previously reported hypoxic CSE dependent activity and H2S production. Together, these studies will provide important new information regarding phosphoprotein regulation of CSE enzyme activity.
[000334] b) Validate the impact of AMPK, PKC and ATR on phosphorylation and CSE enzyme activity in endothelial cells and whole tissues under ischemia. Our observations identify that inhibition of AMPK, PKC or ATR effects CSE enzyme activity (Figure 11). Likewise, these kinases may influence the phosphorylation and subsequent activity of CSE. Studies will be performed using wild type or CSE null endothelial cells with treatments of AMPK (Dorsomorphin), PKC a and 0 (Go 6976) or ATR (NU6027) inhibitors, respectively under normoxic and hypoxic tissue culture conditions at 1% 02 for 0, 0.5, 1, 2, 4, 8, 16, and 24 hours for CSE enzyme activity and phosphorylation. The resulting data will be compared to cells under normoxic conditions at similar time points to validate changes in substrate levels. These measurements will be also be extended to ischemic versus nonischemic limbs at 0, 1, 3, 6, 12, and 24 hours post femoral artery ligation allowing comparisons between in vitro vs. in vivo conditions. These data will validate the regulation of AMPK, PKC and ATR active sites on phosphorylation and CSE enzyme function.
[000335] c) Validate substrate bioavailability in endothelial cells, monocytes, and whole tissues under conditions associated with ischemic vascular remodeling. While enzyme use of cystathionine and cysteine result in H2S production, use of cystine results in the formation of per/polysulfidesl5. However, the predominant levels of these substrates remain completely unknown in specific cell compartments and whole tissues during chronic hypoxia and vascular remodeling. Pyridoxal-5 phosphate (PLP) is a critical cofactor that facilitates CSE conformation in its active tetrameric formation. Few studies have been reported examining PLP levels in ischemic tissue (with none in the ischemic hind limb model). Therefore, studies will be performed using wild type or CSE null endothelial cells or monocytes quantifying the levels of substrates cystathionine, cysteine, cystine and PLP bioavailability under normoxic and hypoxic tissue culture conditions. PLP levels will be measured via HPLC as reported and shown in figure 1651. PLP binding to CSE will also be validated using the various phosphomutants described in SA3a. PLP binding studies will be performed using FLAG immuno-enriched CSE followed by incubation with PLP (100 ng/ml). Results from these experiments will provide new information regarding the role of PLP cofactor bioavailability and function for CSE enzymatic activity during hypoxia and tissue ischemia. Cells will be incubated at 1% 02 for 0, 0.5, 1, 2, 4, 8, 16, and 24 hours and subsequently collected for substrate measurement. The resulting data will be compared to cells under normoxic conditions at similar time points to validate changes in substrate levels. Substrate bioavailability will also be validated in ischemic versus nonischemic limbs at 0, 1, 3, 6, 12, and 24 hours post femoral artery ligation allowing substrate comparisons between in vitro vs. in vivo conditions. These data will validate substrate bioavailability impacting CSE enzyme function.
[000336] d) Validate CSE functional characteristics from clinical samples of vascular disease. CSE characteristics in clinical tissue will be validated from femoral and carotid endarterectomy specimens of patients with vascular disease or non-diseased vascular tissue from bypass procedures. We will obtain fresh endarterectomy samples from femoral and carotid arteries, or nondiseased specimens from bypass procedures. With our LC-MS/MS procedures small tissue samples can be used to measure various per/polysulfide pools, CSE substrate and cofactor bioavailability, and CSE posttranslational modifications. A total of 64 endarterectomy specimens from carotid and femoral vessels will be collected with 8 specimens from each of the following: carotid- african American male, african american female, Caucasian male, Caucasian female; femoral- african american male, African american female, Caucasian male, Caucasian female. Equal numbers of control vessel tissues will also be obtained. The numbers chosen enable detection of a minimal 40% difference with a power of 0.8. [000337] SA3 General Methods
[000338] a) Generation of CSE phospho amino acid mutants. Site directed mutagenesis of specific amino acids will be performed in collaboration with COBRE Molecular core facility and confirmed by sequencing.
[000339] b) Measurement of CSE enzyme activity. CSE enzyme activity will be measured using different substrates of cystathionine, cysteine, or cystine as we’ve previously reported 5.
[000340] c) Measurement of CSE substrate bioavailability. Cystathionine, cysteine, and cystine will be measured by LC-MS/MS. Samples will be injected into an Acquity UPLC system coupled to XEVO TQ (Thermo Scientific) with electrospray ionization (ESI(+)) in the multiple reaction monitoring (MRM) mode as described above.
[000341] d) Measurement of Pyridoxal-5-phosphate bioavailability and binding to CSE. PLP bioavailability will be measured by HPLC as previously reported51. PLP bound to CSE protein will be identified via immuno-pulldown of FLAG tagged CSE, digestion with MS grade trypsin, and peptides analyzed using a nano flow ultrainterfaced to nano-electrospray quadrupole time-of-flight MS (nanoAcquity UPLC-Synapt HDMS, Waters Corporation, MA) as described previously53. Data is acquired using MassLynxTM 4.1 software in an automatic data dependent acquisition mode. MS-TOF scans are acquired from m/z 300 to m/z 1500, and up to three precursor ions are selected for subsequent MS/MS scans from m/z 50 to 1000 using charge state-dependent collision energy ramps to promote fragmentation. Mass spectrometry data is processed using PEAKs Studio 7.5 software (Bioinformatics Solutions Inc., Canada).
[000342] f) Statistical analysis. Statistical analysis will be performed as discussed in SAI and SA2.
[000343] SA3 Milestones and Timeline
[000344] i) Validate the effect of phospho amino acid mutations and signal kinases on CSE enzyme activity.
[000345] ii) Validate CSE substrate/cofactor bioavailability and differences between experimental and clinical samples.
[000346]
[000347] Example 4 - Hypoxia Increases Persulfide and Polysulfide Formation by AMP Kinase Dependent Cystathionine Gamma Lyase Phosphorylation
[000348] Abstract:
[000349] Hydropersulfide and hydropolysulfide metabolites are increasingly important reactive sulfur species (RSS) regulating numerous cellular redox dependent functions. Intracellular production of these species is known to occur through RSS interactions or through translational mechanisms involving cysteinyl t-RNA synthetases. However, regulation of these species under cell stress conditions, such as hypoxia, that are known to modulate RSS remain poorly understood. Here we define a crucial mechanism of increased persulfide and polysulfide production in substrate specific manner involving cystathionine gamma lyase (CSE) phosphorylation at serine 346 and threonine 355 under acute hypoxic conditions. Hypoxic phosphorylation of CSE occurs in an AMP kinase dependent manner increasing enzyme activity involving unique inter- and intramolecular interactions within the tetramer. Importantly, both cellular hypoxia and tissue ischemia result in AMP Kinase dependent CSE phosphorylation that regulates blood flow in ischemic tissues. Our findings reveal hypoxia molecular signaling pathways regulating CSE dependent persulfide and polysulfide production impacting tissue and cellular response to stress.
[000350] Introduction:
[000351] Reactive sulfur species (RSS) are important for numerous biological functions and pathophysiological roles in cardiovascular, neurological, inflammatory, and immune systems, and in other disease states1’3. Hydrogen sulfide (H2S) has received predominant attention among RSS and is produced by enzymes in the trans-sulfuration pathway, cystathionine P-synthase (CBS) and cystathionine y-lyase (CSE/CTH), as well as by 3 -mercaptopyruvate sulfurtransferase, which is involved in cysteine catabolism. However, endogenous sulfide may be present in three biochemically important forms, including, the readily available free sulfide, acid-labile (e.g. iron-sulfur clusters that are localized in active centers of respiratory enzymes), and as bound sulfane sulfur forms (e.g. hydropersulfides and hydropolysulfides)1,2,4. Free H2S can be released by acid-labile and bound sulfane sulfur under acidic or reducing conditions, respectively1,2. RSS are constantly maintained in mammalian cells and can be influenced either through transcriptional regulation of sulfide synthesis enzymes or via persulfidation popularly called S-sulfhydration of cysteine residues on target proteins5,6. We have previously demonstrated that CSE/H2S regulates ischemic vascular remodeling mediated during hind limb ischemia via upregulation of sulfide metabolites and NO-dependent cytoprotective responses revealing important roles for cardiovascular function7.
[000352] Hydropersulfides and polysulfides can be synthesized independently of H2S. Polysulfide release from cysteinyl-tRNA synthetases (CARSs) has been demonstrated8,9 and both CBS and CSE utilize substrates such as cystine or glutathione disulfide, to catalyze formation of cysteine hydropersulfide or glutathione hydropersulfide, and other polysulfides 2,4,9,1°. These various intracellular polysulfide/persulfide species together constitute the total concentration of sulfane sulfur inside the cell. Importantly, recent insights show a differential metabolism of these species are during changes in pH or oxygen (hypoxia or ischemia) that is linked to lipid peroxidation and ferroptosis 11,12. As such, polysulfide/persulfides are now considered critical mediators within cellular redox signaling networks that regulate diverse cellular functions. Recent study has reported an inhibitory phosphorylation site, serine 377 that inhibits CSE activity and decreases sulfide levels under disturbed shear stress 13. However, a limitation remains the lack of insight into specific mechanisms regulating formation per- and polysulfide compounds under various pathophysiological conditions. In the current study we reveal a unique phosphorylation-dependent mechanism of hypoxia/ischemia mediated increased CSE activity leading to specific generation of per/polysulfide with potent pathophysiological effects.
[000353] Results:
[000354] Hypoxia induces polysulfide levels via new CSE phosphorylation:
[000355] We previously reported an increase in CSE activity and sulfide metabolites under sustained tissue ischemia 7 14 but the mechanism(s) responsible for these effects remain unknown. Murine aortic endothelial cells were exposed to hypoxia and sulfide metabolites measured. We observed a selective three-fold increase in bound sulfane sulfur consisting of per- and polysulfide pools using the monobromobimane (MBB) RP-HPLC quantitative analytical measurement after 30 minutes of hypoxia; no changes in free H2S nor acid labile H2S was observed (Figure 24, panel A). We next utilized and standardized intracellular fluorescent probes to detect free H2S using SF7 (Figure 30, panel A), and per-polysulfide using SSP4 probe (Figure 30, panel B) and confirmed that hypoxia increased per-polysulfides (Figure 24, panel B) without changing free H2S levels (Figure 30, panel C). We observed that hypoxia significantly increased CSE activity supporting a primary role for this enzyme in observed per-polysulfide generation (Fig. 1C). Further, to determine the source of increased per-polysulfides and H2S, we used siRNA against known sulfur metabolite producing enzymes, including CSE, CBS, MPST, CARS1 or CARS2, and then exposed murine endothelial cells to hypoxia and measured per-polysulfides using SSP4 (Fig. ID), and H2S using SF7 (Figure 30, panel D). Significant decreases in mRNA and protein levels of siRNA treatment for CSE, CBS, MPST CARS1 and CARS2 was confirmed by qRT-PCR and western blot (Figure 30, panels E-I and J-N). In CSE siRNA cells, hypoxia dependent increased in SSP4 fluorescence was inhibited >70% (Figure 24, panel C); while a 30% reduction was observed in SF7 intensity. A moderate yet significant reduction in SSP4 intensity was also observed with CARS1 siRNA (Figure 24, panel C). Interestingly, this inhibition is significantly less compared to inhibition of both per-polysulfides (SSP4), and H2S (SF7), respectively via CSE siRNA. These findings are consistent with previous findings of these enzymes in per-polysulfide production.
[000356] We next examined CSE protein for post-translational modifications under normoxia versus hypoxia, wild type human CSE FLAG-tagged construct was expressed in murine endothelial cells, treated under normoxia or hypoxia, purified and trypsin digested for LC/MS HCD (higher- energy C-trap dissociation) fragmentation spectrum (Figure 24, panel E). A three-fold increase in phospho-peptide abundance ratio in HCD spectra for CSE amino acid fragment [334-364] under hypoxia compared to normoxia was observed. The LC/MS HCD fragmentation spectrum identified hypoxia dependent phosphorylation site at serine 346 (S346) and threonine 355 (T355) (Figure 24, panel E). Moreover, we observed that the abundance ratio of CSE[334-364] peptide fragment was 3 fold higher than CSE[365-395] peptide fragment containing the phospho-inhibitory serine 377 residue under hypoxia compared to normoxia conditions (Fig. IF). To check if these phosphorylation sites impact CSE activity, we generated phospho-mimetic glutamate or phospho-negative alanine mutants of either S346 or T355 and examined their enzyme activities in transfected HEK cells. A two-fold decrease in CSE enzyme activity with the phospho-negative S346A or T355A (Fig. 1G) was observed under hypoxia; whereas the phospho-mimetic S346E or T355E significantly increased CSE enzyme activity under hypoxia (Fig. 1H). Next, whether conservation of CSE S346 and T355 sequences are similar across different species was determined using the homology search tool OrthologR. Both the CSE phosphorylation sites S346 and T355 are conserved across species, including human, monkey, zebra fish, frog, rat, mouse, C. cerevisiae, D. melanogaster, and C. elegans (Figure 31, panel A) indicating a key role for phosphorylation in CSE activity regulation.
[0003571 Substrate influences CSE-mediated per-polysulfide generation under hypoxia:
[000358] CSE is a promiscuous enzyme where based on its substrate utilization the kinetics can vary with change in the microenvironmentl5. Next, we investigated the role of phosphorylation sites and their role in the subsequent production of per-polysulfide or free H2S levels using different substrates under hypoxia. Wild type and CSE phospho-negative or mimetic constructs were FLAG tagged and used for transient transfection studies in HEK cells. Wild type, phospho-negative, or phospho-mimetic CSE was transfected into HEK and confirmed by FLAG immunoblotting for uniform expression (Figure 31, panels B and C). Hypoxia dependent increases in per-polysulfide were completely blunted by either S346A or T355A using cysteine, cystine, or cystathionine substrates (Figure 25, panels A, C and E) as well as under normoxic conditions (Figure 32, panels A, C, and E). Free H2S levels were not significantly different with various substrates under hypoxia (Figure 25, panels B, D, and F) or normoxia (Figure 32, panels B, D, and F).
[000359] We next examined the effect of CSE phosphomimetic constructs S346E and T355E under hypoxia with various substrates. A significant 2.5- and 2.75-fold increase, respectively in per- polysulfides was observed with cysteine and cystine (Figure 26, panels A and C); but only a 1.5-fold increase with cystathionine (Figure 26, panel E) as a substrate. Additionally, S346E or T355E significantly increased SSP4 signal compared to wild type CSE but did not significantly change free H2S levels as measured by SF7 under hypoxia (Figure 26, panels B, D, and F) These data reveal that CSE enzyme favors hypoxic per-polysulfide production with cystine or cysteine as substrates versus cystathionine. Similar observations of substrate-specific per-polysulfide generation was observed with phosphomimetics S346E and T355E under normoxic conditions (Figure 33, panelsA, C, and E) although with remarkably reduced SSP4 signal compared to hypoxic conditions. Importantly, no significant changes were noticed with free H2S generation (Figure 33, panelsB, D, and F).
[000360] CSE can also use cystine or glutathione disulfide to form hydropersulfide and polysulfides 8’16. We further examined whether cystine substrate utilization under hypoxia could differentially influence per-polysulfide levels in endothelial cells. Mouse aortic endothelial cells were transfected with wild type, S346A, T355A, S346E or T355E CSE constructs and exposed to hypoxia with cystine substrate followed by derivatization with HPE-IAM for per-polysulfide LC/MS analyses17. Significant increases in GSSH persulfide and GSSSSH polysulfide with wild type CSE were observed (Figure 34, panelsA and B). BothS346A or T355 A significantly blunted hypoxic GSSH persulfide (Figure 34, panel A) and GSSSSH polysulfide (Figure 34, panel B) production. Conversely, S346E and T355E phospho-mimetics significantly increased GSSH persulfide and GSSSSH polysulfide under hypoxia and above that of wild type CSE (Figure 34, panels A and B).
[000361] Molecular modeling determines molecular stabilization of CSE via phosphorylation: [000362] CSE is arranged as a tetramer with D2 symmetry18; so, we questioned whether phosphorylation could modulate CSE activity via allosteric mechanisms. We confirmed these effects of phosphorylation sites S346 or T355 on human CSE tetrameric structure using molecular dynamics simulations (Figure 27, panels A and B). For reference, each monomer is labeled as hCSEA, hCSEB, hCSEC, and hCSED (Figure 27, panel A), and the location of S346 shown in purple and T355 in gold (Figure 27, panel B). As can be seen in Figure 27, panels C and D, snapshots of the trajectories reveal that phosphorylation of these two residues establishes several new electrostatic interactions within and between each monomer of the CSE tetramer. Phosphorylation of S346 induces new intra- and inter- molecular electrostatic interactions that include but are not limited to pS346A - E381A, K260B (Figure 27, panel C, inset i); pS346B - E381B, R257A, K260A (Figure 27, panel C, inset ii); p346D - T336D, K48A (Fig 4C, inset iii); and pS346C - S218C, T336C, E345C, E381C, K260D (Figure 27, panel C, inset iv). We also observed several new intra- and inter-molecular electrostatic interactions formed by phosphorylation of T355. These include but are not limited to pT355A - H55D, R62D, R119A (Figure 27, panel D, inset i); pT355B - R119B (Fig 4D, inset ii); pT355D - Q323D, M354D, H356D, S358D, V359D (Figure 27, panel D, inset iii); and pT355C - R119C, H356C, R62B, N241B (Figure 27, panel D, inset iv). We next tested whether phosphorylation modulated interactions with CSE and PLP using the inhibitor propargyl glycine (PAG). PAG treatment of HEK transfected with wild type, S346E, or T355E CSE constructs significantly blunted hypoxia and cystine dependent increases in SSP4 fluorescence (Figure 27, panel E). Conversely, PAG had no effect on hypoxia and cysteine dependent effects with S346A or T355A (Figure 27, panel F) indicating that S346 and T355 phosphorylation preferentially conveys tetramer structural organization enabling PLP binding.
[000363] Hypoxia dependent AMP Kinase regulation of CSE phosphorylation and polysulfide formation in endothelial cells:
[000364] Assessment of potential protein Serine/Threonine kinases (using Scansite 4) that may phosphorylate CSE at S346 and T355 identified AMP Kinase basophilic Serine/Threonine kinase group motifs (Figure 35, panel A). Mouse endothelial cells were exposed to hypoxia (0-90 min) and phosphorylation of CSE assessed by immunoblotting with pCSES346 polyclonal antibodies generated by phospho-peptide specific immunization. In parallel, pAMPK was also measured (Figure 28, panel A). We observed a significant two-fold increase in pCSE at 30, 60 and 90 minutes, along with a significant increase in pAMPK levels at 30 and 60min (Figure 28, panel B). Adenosine 5'- monophosphate-activated protein kinase (AMPK) plays a key role in maintaining cellular energetic balance and metabolism 19. Increasing evidence indicates AMPK as a new modulator in multiple pathological conditions, including non-metabolic processes and diseases19,20. We next examined whether AMPK regulates hypoxic CSE phosphorylation and subsequent increases in per-polysulfide using the ATP competitive AMPK inhibitor, dorsomorphin (AMPK-I). Dorsomorphin significantly blunted hypoxia mediated pAMPK and pCSES346 (Figure 28, panel C and D) that was associated with a significant reduction in hypoxia dependent SSP4 fluorescence (Figure 28, panel E). Similar effects were observed with HUVECs indicating this pathway is operational in human cells also (Figure 28, panels F, G, and H). We corroborated that hypoxia also increased per-polysulfide SSP4 signal in HUVECs (Figure 35, panel H); however, hypoxia did not significantly increase free H2S SF7 signal (Figure 35, panel D), although AMPK inhibition moderately reduced free H2S SF7 signal (Figure 35, panel E) under hypoxia.
[000365] AMPK is a heterotrimeric protein kinase composed of a catalytic a subunit (al or a2) subunit21,22. We further investigated the effects of specific AMPK a subunit inhibition on pCSE and per-polysulfide levels using siRNA treatment of mouse aortic endothelial cells. Individual knockdown of AMPKal (Figure 35, panel F) or a2 (Figure 35, panel G) selectively reduced al or a2 mRNA and significantly reduced hypoxic phosphorylation of AMP kinase and CSE (Figure 35, panels H and I). Lastly, siRNA inhibition of AMPKal or AMPKa2 significantly reduced hypoxia mediated increased per-polysulfide SSP4 fluorescence (Figure 35, panel J). Together, these data show that AMP Kinase activity critically regulates CSE phosphorylation and subsequent sulfane sulfur production.
[000366] Inhibition of AMP Kinase impairs ischemia dependent phosphorylation of CSE, per- polysulfide formation, and blood flow:
[000367] CSE and H2S regulate vascular dilation and regulate ischemic vascular remodeling involving arteriogenesis and angiogenic properties 7’23. We examined the effect of tissue ischemia on CSE and AMP kinase phosphorylation using the femoral artery ligation (FAL) model at various time points (0, 3hrs, 24hrs and 5 days). We also quantified total sulfide levels in plasma and skeletal muscle tissues. Ischemia increased CSE phosphorylation (pCSES346) rapidly (3 hours) in the gastrocnemius muscle that was sustained over 5 days (Figure 29, panel A and B). Likewise, ischemic tissue AMP kinase phosphorylation beginning at 3 hours lasting to 5 days. Ischemia increased bound sulfane sulfur levels (containing per-polysulfides) in both plasma and skeletal muscle tissue (Figure 36, panels A and B). Finally, to assess whether AMPK modulate CSE phosphorylation and activity in vivo, mice were treated with AMPK-I dorsomorphin (lOmg/kg) for 5 days during FAL. Skeletal muscle tissues, including from non-ischemic (NI), ischemic (Isch) or Isch+ AMPK-I groups were probed for pCSE and pAMPK protein levels (Figure 29, panel C). We found a three-fold increase in pCSES346 and pAMPK protein levels, which was completely blunted with AMPK-I treatment (Figure 29, panel D). We also evaluated FAL physiological ischemic hindlimb blood flow changes between saline vehicle versus AMPK-I at day 5 (Figure 29, panel E). Laser speckle blood flow quantification shows that saline vehicle treatment results in the characteristic -60% reduction in hind limb blood flow after FAL but that AMPK-I treatment resulted in greater than 80% reduction in hind limb blood flow after FAL (Figure 29, panel F). Lastly, we examined AMPK-I treatment on FAL plasma per/polysulfide generation and found a profound and significant decrease in plasma bound sulfane sulfur with AMPK- I compared to saline vehicle (Figure 29, panel G).
[000368] Discussion:
[000369] The current investigation reveals a new molecular signaling pathway regulating sulfide metabolism involving hypoxia dependent phosphorylation of CSE and formation of sulfane sulfurs. We identified serine 346 (S346) and threonine 355 (T355) as two phosphorylation sites, which enhanced CSE activity under hypoxia/ischemia conditions. Molecular modeling revealed these posttranslational modifications likely stabilize tetramer organization and function due to numerous inter- and intramolecular interactions. This was confirmed by specific phospho-negative mutants that remove these modifications which inhibited hypoxia-mediated increase in CSE activity as well as decreased per-polysulfide generation. Whereas phosphomimetic mutant CSE constructs expressed under normoxia, or hypoxia significantly elevate per-polysulfide generation. Importantly, AMPK inhibition abolished hypoxia/ischemia induced CSE phosphorylation and per-polysulfide generation along with impairment of ischemic vascular responses highlighting a crucial role for cardiovascular and likely other pathophysiological responses.
[000370J Previous work has reported an increase in CSE expression and subsequent increase in total sulfide levels under tissue ischemia over prolonged time periods7; however, underlying molecular mechanisms remained unknown. We observed a preferential increase in sulfide as bound sulfane sulfur that includes per-polysulfides. Formation of per-polysulfide biochemical species under hypoxia may serve to is the most logical. Preferential formation of polysulfides under hypoxia as opposed to free H2S likely reflects cellular responses to ensure reactive sulfide availability. Polysulfides can liberate free sulfide, contribute sulfur to metal complexes, serve as potent nucleophilic and electrophilic redox mediators, and are more biologically stable forms of RSS than H2S alone4,12,24,25. Apart from CSE, CBS, MPST, and cysteinyl-tRNA synthetases (CARSs) have been reported to produce per- polysulfides8,16. Interestingly, apart from CARS1, no significant role for formation of per-polysulfides was observed under hypoxia with knockdown of CBS, MPST, or CARS2. This substantiates earlier observations of CSE-mediated sulfide release under hypoxia7, and identifies CSE as the predominant enzyme producing per-polysulfides in endothelium under hypoxia.
[000371] Dietary modifications, such as methionine restriction (MR) has received considerable attention as they can improve metabolism, including insulin sensitivity and longevity 26,27. Effects of MR are attributed, to increased CSE/H2S signaling that regulates substrates such as cysteine or cystine 27. CSE plays a critical role under MR with significant beneficial towards improved metabolism with substrate utilization 26. Additionally, CSE is known to be promiscuous for its substrate utilization that can change with its microenvironment6. With a decrease in cellular cysteine levels, CSE can catalyze P-elimination of cysteine persulfides from cystine, to produce H2S and cysteine 26. Interestingly, we observed that CSE, can in fact utilize cystine or cysteine as substrates apart from cystathionine to produce per-polysulfides under hypoxic conditions. Moreover, production of these sulfane sulfur metabolites appears more abundant with cystine than cysteine or cystathionine under hypoxia when CSE is phosphorylated. Possible presence of low cystathionine and cysteine levels under hypoxia may also favor utilization of cystine as a substrate thus serving as an alternative pathway to salvage intracellular cysteine and H2S. These results indicate that CSE utilization of cystine or cysteine to generate per-polysulfides under hypoxia (rather than H2S) may be a metabolic and redox pathway to maintain cellular function during stress.
[000372] CSE is a PLP-dependent enzyme consisting of 393 amino acid residues and one PLP moiety and is arranged as a tetramer18. Pyridoxal phosphate is bound in the active site by Lys21228. Apart from lysine, PLP is anchored by strong hydrogen bonds between the phosphate moieties, including Gly90, Leu91, Thr211 and Arg62, that can contribute towards stability of its binding with the CSE enzyme28. Presented molecular modeling data demonstrates that phosphorylation of CSE significantly changes the molecular dynamics of CSE via electrostatic interactions, including inter- and intramolecular binding between monomers of CSE tetramer via S346 and T355. Interestingly, a significant number of these new electrostatic interactions involve amino acids known to be functionally relevant for CSE activity. For instance, Arg62 and Lys212 to form a hydrogen bond with the phosphate moiety of PLP known to be functionally obligatory for CSE activity28,29. The unstructured loop containing R119 (residues G115 - Y120) forms a two-turn helix upon CSE binding PLP28. Furthermore, a recent study posits that the guanidinium moiety of R119 forms a hydrogen bond with the distal a-carboxylate of cystathionine while maintaining contact with the adjacent monomer via a second hydrogen bond with S24229. H55 is also a part of a loop (residues D28 - S63) that is disordered in the absence of PLP. There is no electron density in the apo structure for residues T355 - V366, indicating that this region is disordered. Although this region does not directly interact with PLP, it is theorized that it could stabilize one side of the PLP binding pocket. For the apo structure, the T355 - V366 region of the CSE:PLP complex is occupied by the Ml 10 - N118 loop28. Finally, N241 plays an important role in the inter-molecular stabilization of PLP in the active site of CSE29. Supplement video 1 shows non-phosphorylated CSE tetramer molecular interactions over 300 nanoseconds, whereas supplement videos 2 and 3 illustrate phospho-S346 CSE or phospho-T355 CSE molecular interactions, respectively. Molecular model simulations clearly demonstrate that phospho- S346 engages in horizontal intermolecular monomer interactions, whereas phospho-T355 predominantly engages in vertical intra- and intermolecular monomer interactions. Importantly, phosphorylation of S346 and T355 transition the CSE tetramer from an open to closed conformation, particularly within the PLP binding domain. We posit that phosphorylation of S346 and T355 induce changes in side-chain orientations of numerous functionally relevant residues, thereby altering the function of CSE as observed in our biological experiments. These results highlight the magnitude of conformational changes involved via phosphorylation sites (S346 and T355) towards structural stability of CSE tetramer as indicated by molecular modeling simulations and represent alternative molecular targets for enzyme inhibition beyond interfering with PLP binding.
[000373] Prevailing understanding of CSE regulation has predominantly focused on transcriptional regulation via Activating Transcription Factor 4 (ATF4)30 and specificity protein-1 (Spl)31 with Spl consensus binding sites present in the core promoter region of the human CSE gene31. Additionally, the transcription factor, activating transcription factor 4 (ATF4) regulates CSE under stress conditions30. Moreover, regulatory mechanisms governing CSE function and its production of per- and polysulfide generation has remained unknown. We have previously reported CSE regulation of flow-dependent vascular remodeling and endothelial activation preferentially involving altered sulfane sulfur levels32. 17p-estradiol (E2) can enhance endothelial CSE gene expression and H2S release by promoting phosphorylation of CSE via ERot-Spl interaction with binding sites in CSE gene promoter, subsequently improving endothelial function33,34. However, this study is predominantly applicable to females and doesn’t identify how S56 effects CSE enzyme function. Bibli et al. has also reported that sulfide levels are reduced under disturbed shear stress via phosphorylation of serine 377 thereby inhibiting CSE, due to increased IL-ipi3. However, these mechanisms explain the inhibitory regulation of CSE alone. Our data reveals increased CSE enzyme function under hypoxia via S346 and T355 phosphorylation. Interestingly, our results from LC/MS analysis of CSE posttranslational modification found a significant reduction in phospho peptides CSE[365-395] (containing S377) in comparison to CSE[334-364] (containing S346 and T355). This indicates an inhibition or reduction of phosphorylation of S377, a negative regulator of CSE13, under hypoxia that requires further study.
[000374] AMP-activated protein kinase (AMPK) is a key regulatory molecule for endothelial function, redox homeostasis, and importantly, hypoxia and ischemia/reperfusion injury35. AMPK has been reported to mediate myocardial protection from I/R injury36. AMPK prevents apoptosis and reduces oxidative stress and inflammation upon ER injury, which is mediated via AMPK phosphorylation37. Specifically, AMPK protects from myocardial I/R injury via phosphorylation of AMPK at Thrl72 in ischemic heart tissues38. While H2S -mediated induction of AMPK and subsequent pharmacological actions has been previously reported39, AMPK regulation of CSE and subsequent sulfide metabolite levels have not been known. We demonstrated that AMPK/pAMPK can regulate CSE by increasing its activity via phosphorylation (S346) under hypoxia/ischemia conditions. This increase in CSE phosphorylation subsequently induced per-polysulfide generation, which is significantly blunted upon inhibition of AMPK using dorsomorphin or siRNA. AMPK is a heterotrimeric complex of catalytic a-subunits, regulatory P and y-subunits. AMPK catalytic al and 2 isoforms are activated in cardiac ischemia irrespective of regulatory subunits40. AMPK can be activated by LKB1 and CaMKK pathways that elicits protective and adaptive mechanism to various cellular stress conditions in the endothelial cells, including hypoxia41,42. However, the molecular regulation and the roles of specific AMPK subunit isoforms is not well defined. Our data demonstrates that under hypoxia, molecular inhibition of either AMPKal or AMPKa2 in endothelial cells significantly blunts AMPK activity, hypoxia mediated CSE phosphorylation (pCSES346), and subsequent per-polysulfide production. Importantly, inhibition of AMPK significantly impairs ischemic vascular blood flow in mouse hindlimbs in a model of chronic ischemia that directly involves altered CSE phosphorylation and bound sulfane sulfur production. These results highlight a new and critical role of AMPK in regulation of CSE phosphorylation and pAMPK in regulating ischemic vascular blood flow.
[000375] Together, these results elucidate molecular mechanisms of increased CSE activity and per-polysulfide formation for tissue and cellular ischemic responses. Implications for this signaling pathway and mechanism are many with immediate relevancy to regulation of tissue growth, metabolism, and adaptation responses, carcinogenesis pathways, and reno-cardiovascular diseases. [000376] Methods:
[000377] Chemicals and reagents: Sulfane Sulfur Probe 4 (SSP4) and sodium trisulfide (Na2S3) were from Dojindo Molecular Technologies, Inc., MD, USA. Anhydrous sodium sulfide was purchased from Alfa-Aesar Inc., Diallyl trisulfide (DATS) was procured from Sigma-Aldrich, CA, USA. WSP-1 and SF-7 probes were purchased from Cayman Chemicals, MI, USA. P-(4- Hydroxyphenyl)ethyl iodoacetamide (HPE-IAM) obtained from Chem-Impex. The anti-CSE antibody was from Proteintech (IL, USA), anti-CBS, anti-3MST were from Abeam, MA, USA. All secondary fluorophore-labeled antibodies were obtained from Jackson Immunoresearch Inc (West Grove, PA, USA). Chemicals and tissue culture reagents were obtained from Sigma unless otherwise noted. [000378] Cell culture and treatments: Human embryonic kidney 293 cells (HEK) were purchased from ATCC, Virginia, USA and Human umbilical vein endothelial cells (HUVECs) were obtained from Lifeline Cell Technology, CA, USA (Cat# FC-0044). Mouse aortic endothelial cells were isolated in our COBRE molecular core and cultured in our laboratory. HUVECs were cultured in VascuLife® Basal Medium (Cat# LM-0002) supplemented with the appropriate LifeFactors® Kit (Cat# LL-0003). All cells were grown in tissue culture flasks under normoxic conditions at 37°C, 5% CO2, and 21% 02. All media was changed every 2-3 days. Cells were transfected with transfection media (Lipofectamine 2000)
[000379] For hypoxia treatment, the cells were incubated in the hypoxic chamber (5% CO2, 37°C, 1% 02) for 30min. HUVECs from passages 2-4 was used in the experiments.
[000380] Site-directed mutagenesis of hCSE: Phosphorylation mutants of hCSE of WT, constitutively active phospho negative alanine mutants S346A or T355A; phosphomimetic mutants with glutamate substitution, S346E or T355E. For the generation of hCSE alanine or glutamate mutants, the Site-Directed Mutagenesis kit from Agilent Technologies (Catalog #200522) was utilized. We performed PCR using two synthetic oligonucleotide primers, designed to incorporate the desired mutation. After thermal cycling, DPN I digestion was employed to selectively amplify PCR products containing the mutation. Subsequently, the nicked vector DNA, now carrying the desired mutations, was transformed into competent cells. Plasmids were extracted from the resulting colonies and subjected to Sanger sequencing, thereby confirming the presence of the intended mutants. These mutated plasmids have been transfected into either HEKs, MAECS or HUVECs for various assays as mentioned in the manuscript.
[000381] Measurement of biological pools of H2S: H2S was measured using monobromobimane by RP-HPLC as we previously reported and described in detail elsewhere43. Briefly, plasma or tissue samples were collected and stabilized in vials containing 100 mM Tris-HCl buffer (pH 9.5, 0.1 mM DTP A) in a ratio of 1:5 (v/v) and snap-frozen until further analysis. Samples were derivatized with monobromobimane (MBB) and analyzed using a reversed-phase high-performance liquid chromatography (RP-HPLC) system with an Agilent Eclipse XDB-C18 column (5 pm, 80 A, 4.6 mmx250 mm) for sulfide-dibimane fluorescence detection (excitation: 390 nm; emission: 475 nm). [000382] Measurement of CSE activity: CSE activity was measured as we previously described?. Briefly, samples were incubated with 2 mM cystathionine, 0.25 mM pyridoxal 5'-phosphate in 100 mM Tris-HCl buffer (pH 8.3) for 30 min at 37 °C. 20% Trichloroacetic acid was added into reaction mixture. After centrifugation, the supernatant was mixed with 2% ninhydrin reagent and incubated for 5 min at 105 °C then quickly cooled to 4 °C. Samples were then mixed with 97% ethanol and read at 455 nm using a spectrophotometer (Biotek Inc.). CSE activity was assessed by cystathionine consumption and enzyme activity expressed as fold change calculated from nanomoles of cystathionine consumed per mg of total protein.
[000383] Fluorescent detection of poly sulfide metabolite: Poly sulfide was detected using a fluorescent probe SSP4 as described earlier44. SSP4 was dissolved in DMSO to obtain a 1 mM stock solution. CTAB, a surfactant was dissolved in ethanol to yield a 5 mM stock solution. CTAB and SSP4 (5 pM) were dissolved in HBSS buffer and added to the samples for a total of lOOptl in a black polystyrene, flat-bottomed clear 96-well plate and incubated in the dark at room temperature for 20 min. Samples were measured on a TECAN fluorescence spectrophotometer (emission 525 nm: excitation 485 nm).
[000384] Identification of phosphorylation by LC-MS/MS: Human CSE from HEK sample cells was performed SDS-PAGE, and then stained with Imperial Protein Stain. The Gel lanes were cut into 2mm X 2mm cubes. The cubes underwent trypsin digestion, followed by disulfide bond reduction and cysteine alkylation with dithiothreitol and iodoacetamide, respectively. LC-MS/MS analysis was conducted by a 3000 RSLCnano system coupled via an EasySpray source to an Orbitrap Exploris 480 mass spectrometer as mentioned previously45. The digested peptides (5.0 pl) were loaded onto a trap column (PepMap C18, 2 cm x 100 pm, 100 A) at a flow rate of 20 pl/min using 0.1% formic acid and separated on an analytical column (EasySpray 50 cm x 75 pm, C18 1.9 pm, 100 A) with a flow rate of 300 nl/min with a linear gradient of 5 to 45% solvent B (100% ACN, 0.1% formic acid) over a 120 min gradient. Both precursor and fragment ions were acquired in the Orbitrap mass analyzer. Precursor ions were acquired in m/z range of 375-1500 with a resolution of 120,000 (at m/z 200). Precursor fragmentation was carried out using the higher-energy collisional dissociation method using normalized collision energy (NCE) of 32. The fragment ions were acquired at a resolution of 150,000 (at m/z 200). The scans were arranged in top-speed method with 3 sec cycle time between MS and MS/MS. Ion transfer capillary voltage were maintained at 2.1 kV. The raw mass spectrometry data were analyzed using Proteome Discover (version 2.5, Thermo Fisher Scientific) software package with SequestHT using species-specific fasta database and the Percolator peptide validator. Cysteine alkylation was set as a fixed modification. Phosphorylation on serine\threonine\tyrosine residues, and deamidation of asparagine residues were selected as variable modifications. [000385] Molecular dynamics simulations: The three-dimensional structure of cystathionine gamma lyase used for the molecular dynamics simulations were generated by using AlphaFold to predict the structural elements missing from the previously solved crystal structure (PBD: 3ELP)46. Phosphorylation of residues S346 and T355 was modeled in Pymol47 using the PyTMs plugin48. All molecular dynamics simulations were performed using GROMACS 2019 software with the GROMOS 54A7 force field49 and SPC216 water model. Specifically, each protein was solvated with 72,319 SPC216 explicit water molecules and places in the center of a cubic box of 136 136 136 A48.
Each system was neutralized by adding the appropriate number of Na+ ions. Energy minimization of each structure was followed by a two-step equilibration (i.e., NVT equilibration followed by NPT equilibration). The temperature of each system was controlled through velocity rescaling50 at 300 K with a time constant of 0.1 picosecond. The pressure of each system was controlled using the Parrinello-Rahman barostat51 and set to 1 bar. The particle mesh Ewald algorithm52 was used to calculate long-range electrostatics while a cutoff of 1 .0 nM was used for short-range electrostatics and van der Waals’ interactions. Molecular dynamics simulations of 350 ns were performed for WT, S346- PO4, and T355-PO4 with linear constraint solver (LINCS) constraints for all bonds53. Frames were recorded every 2 ps. The backbone RMSD was monitored over the production run of each protein to ensure the stability and convergence of the simulated trajectories.
[000386] Creation of pCSES346 antibody: Polyclonal antibodies to specific phosphorylation site
S346 was produced by Genscript, USA Inc. The antibody against the consensus CTH substrate was raised against the following synthetic peptide antigen CAESLGGFE[pSer]LAE. Briefly, rabbits were immunized with a synthetic phosphorylated peptide (KLH (keyhole limpet hemocyanin) coupled) corresponding to residues surrounding phosphorylation sites. Antibodies are then purified by to isolate the IgG antibody fraction. Affinity chromatography was performed using peptides coupled to SulfoLink resin. Both phospho-peptide-containing resin and the corresponding non-phospho-peptide resin were prepared. Following two rounds of subtractive purification using the non-phospho-peptide resin; protein A eluate was incubated with non-phospho-peptide resin by rotation in a sealed column at room temperature for 1 h to remove antibodies reactive with the non-phospho version of the protein antigen. The column was drained, and the flow-through (containing the desired antibody) was incubated with fresh non-phospho-peptide resin. The flow-through from this second subtractive step was next purified by incubation with phospho-peptide resin. After the phospho-peptide column was washed twice with PBS, phospho-specific antibody (bound to the resin) was eluted with 0.1m glycine, pH 2.7, and pooled fractions were neutralized with 1 m Tris-HCl, pH 9.5 (~l-2% of the fraction volume). The eluted phospho-specific antibody was then dialyzed overnight in PBS at 4 °C. The synthetic phospho-peptide was conjugated to KLH and injected into rabbits. Once rabbits showed high phospho-specific titers, serum was then purified by protein A chromatography. Phospho-CSE substrate antibody was found to be highly phospho-specific as crude serum, so the elution from the protein A column was used directly for affinity chromatography on a phospho-peptide-containing column.
[000387] Western blot analysis: Cells or mouse tissues were homogenized in a lysis solution (Thermofisher Scientific Inc.), containing a protease inhibitor cocktail (Roche, Indianapolis, IN), and phosphatase inhibitor cocktail type I and II (Sigma, Saint Louis, MO). Homogenates were centrifuged at 500*g for 15 min and supernatants were collected. Protein concentrations were analyzed using the Bradford protein assay (BioRad, Hercules, CA). Proteins were separated using 10% SDS-PAGE (mini or midi Bio-Rad, Hercules, CA) and transferred onto PVDF membranes, and incubated with antibodies against CSE (Cat# 12217-1-AP, Fisher Scientific), total AMPKa (Cat# 5832S), p-AMPKa (Cat#2535S) and a/ -Tubulin (Cat#2148) from Cell Signaling Technology. Chemiluminescent bands were detected and quantified using NIH Image J software.
[000388] Quantitative PCR: Tissue samples were stored in TRIzol reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) and RNA was isolated as previously reported54. RNA concentration was evaluated and purity with a typical OD260 to OD280 ratio of RNA sample was approximately 2.0. cDNA was synthesized using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA), from 1 pg of total RNA. Quantitative PCR reactions were performed using the universal SYBR Green Supermix (Bio-Rad, CA, USA) on a CFX96 thermal cycler with Bio-Rad CFX Manager software (Bio-Rad, Hercules, CA, USA). The mean threshold cycle (Ct) values were plotted against the logarithm of the cDNA dilution factor. Quantitative PCR primers for genes, including GAPDH, CSE, CBS, AMPK 1, AMPK 2 was used in this study.
[000389] Mouse hindlimb ischemia model and treatment routes: Twelve-week-old male C57BL/6J (Strain #:000664) mice purchased from Jackson Laboratory were used in this study as reported earlier7. Mice were housed at the Louisiana State University Health Sciences Center- Shreveport animal resources facility, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All animal studies were approved by the LSU Institutional Animal Care and Use Committee (LSU IACUC Protocol # P-21-010) and in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Chronic hindlimb ischemia was induced in 12-16-week-old male C57BL6/J mice, as we have reported previously?. After anesthesia with isoflurane (2-3%), the left femoral artery was dissected, separated, ligated and excised distal to profunda femoris artery to create the hindlimb ischemia model. Mice were randomly assigned to different experimental groups by one investigator and were treated and evaluated by a second blinded investigator. AMPK inhibitor, Dorsomorphin (10 mg/kg) was administered i.p. once a day during the length of the study.
[000390] Novadaq SPY imaging analysis: The SPY imaging device (Novadaq Technologies) was used to quantify collateral vessel perfusion, as we have previously describedl4. Briefly, a bolus injection of 30 pl ICG (IC-Green, Akorn Pharmaceutical, Lake Forest, IL) was administered retro- orbitally, and angiograms were captured by an array of light-emitting diodes at a wavelength of 806 nm and recorded for 1 min. Angiograms were taken before and post-ligation on days 0, 1, 3, and 5 and percent change in blush rates were calculated as mentioned14.
[000391] Statistics: All statistical analyses were performed using GraphPad Prism 9 software. Numerical values were shown as mean ±SEM. All data were tested for normality using the Kolmogorov-Smirnov test, and the data have passed the normality assumption Comparisons of two groups were analyzed using Student’s t test (two tailed) and post-hoc Bonferroni adjustment has been performed. For more than two groups we have performed a two-way ANOVA followed by post-hoc Bonferroni adjustment. We have also performed a post-hoc Tukey’s test. P < 0.05 was considered statistically significant. The nonsignificant difference between samples is indicated as “ns”; P values of significant differences are shown in the graphs.
[000392] References cited in this example:
[000393] 1. Kimura, H. Hydrogen sulfide and polysulfides as signaling molecules. Proc Jpn
Acad Ser B Phys Biol Sci 91, 131-59 (2015).
[000394] 2. Kolluru, G.K., Shackelford, R.E., Shen, X., Dominic, P. & Kevil, C.G. Sulfide regulation of cardiovascular function in health and disease. Nat Rev Cardiol 20, 109-125 (2023).
[000395] 3. Polhemus, D.J. & Lefer, D.J. Emergence of hydrogen sulfide as an endogenous gaseous signaling molecule in cardiovascular disease. Circ Res 114, 730-7 (2014).
[000396] 4. Fukuto, J.M. et al. Biological hydropersulfides and related polysulfides - a new concept and perspective in redox biology. FEBS Lett 592, 2140-2152 (2018). [000397] 5. Paul, B.D. & Snyder, S.H. Protein sulfhydration. Methods Enzymol 555, 79-90
(2015).
[000398] 6. Banerjee, R. Catalytic promiscuity and heme-dependent redox regulation of
H2S synthesis. Curr Opin Chem Biol 37, 115-121 (2017).
[000399] 7 Kolluru, G.K. et al. Cystathionine y-lyase regulates arteriogenesis through NO- dependent monocyte recruitment. Cardiovasc Res 107, 590-600 (2015).
[000400] 8. Ida, T. et al. Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling. Proc Natl Acad Sci U S A 111, 7606-11 (2014).
[000401] 9. Akaike, T. et al. Cysteinyl-tRNA synthetase governs cysteine poly sulfidation and mitochondrial bioenergetics. Nat Commun 8, 1177 (2017).
[000402] 10. Moutiez, M. et al. Reduction of a trisulfide derivative of glutathione by glutathione reductase. Biochem Biophys Res Commun 202, 1380-6 (1994).
[000403] 11 . Barayeu, U. et al. Hydropersulfides inhibit lipid peroxidation and ferroptosis by scavenging radicals. Nat Chem Biol 19, 28-37 (2023).
[000404] 12. Wu, Z. et al. Hydropersulfides Inhibit Lipid Peroxidation and Protect Cells from
Ferroptosis. Journal of the American Chemical Society 144, 15825-15837 (2022).
[000405] 13. Bibli, S.I. et al. Cystathionine y Lyase Sulfhydrates the RNA Binding Protein
Human Antigen R to Preserve Endothelial Cell Function and Delay Atherogenesis. Circulation 139, 101-114 (2019).
[000406] 14. Bir, S.C. et al. Hydrogen sulfide stimulates ischemic vascular remodeling through nitric oxide synthase and nitrite reduction activity regulating hypoxia-inducible factor- 1 alpha and vascular endothelial growth factor-dependent angiogenesis. J Am Heart Assoc 1, e004093 (2012). [000407] 15. Banerjee, R. Catalytic promiscuity and heme-dependent redox regulation of
H(2)S synthesis. Curr Opin Chem Biol 37, 115-121 (2017).
[000408] 16. Akaike, T. et al. Cysteinyl-tRNA synthetase governs cysteine polysulfidation and mitochondrial bioenergetics. Nature Communications 8, 1177 (2017).
[000409] 17. Kasamatsu, S. et al. High-Precision Sulfur Metabolomics Innovated by a New
Specific Probe for Trapping Reactive Sulfur Species. Antioxid Redox Signal 34, 1407-1419 (2021).
[000410] 18. Messerschmidt, A. et al. Determinants of enzymatic specificity in the Cys-Met- metabolism PLP-dependent enzymes family: crystal structure of cystathionine gamma-lyase from yeast and intrafamiliar structure comparison. Biol Chem 384, 373-86 (2003). [000411] 19. Tarasiuk, O., Miceli, M., Di Domizio, A. & Nicolini, G. AMPK and Diseases:
State of the Art Regulation by AMPK-Targeting Molecules. Biology (Basel) 11(2022).
[000412] 20. Jeon, S.-M. Regulation and function of AMPK in physiology and diseases.
Experimental & Molecular Medicine 48, e245-e245 (2016).
[000413] 21. Banko, M.R. et al. Chemical genetic screen for AMPKa2 substrates uncovers a network of proteins involved in mitosis. Mol Cell 44, 878-92 (2011).
[000414] 22. Schaffer, B.E. et al. Identification of AMPK Phosphorylation Sites Reveals a
Network of Proteins Involved in Cell Invasion and Facilitates Large-Scale Substrate Prediction. Cell Metab 22, 907-21 (2015).
[000415] 23. Pardue, S. et al. Hydrogen sulfide stimulates xanthine oxidoreductase conversion to nitrite reductase and formation of NO. Redox Biol 34, 101447 (2020).
[000416] 24. Yu, B., Yang, X., Yuan, Z. & Wang, B. Prodrugs of sulfide and persulfide species: Implications in their different pharmacological activities. Current Opinion in Chemical Biology 75, 102329 (2023).
[000417] 25. Noguchi, N., Saito, Y. & Niki, E. Actions of Thiols, Persulfides, and
Polysulfides as Free Radical Scavenging Antioxidants. Antioxid Redox Signal (2023).
[000418] 26. Jeitner, T.M. et al. Cystine rather than cysteine is the preferred substrate for p- elimination by cystathionine '/-lyase: implications for dietary methionine restriction. GeroScience (2023).
[000419] 27. Hine, C. et al. Endogenous hydrogen sulfide production is essential for dietary restriction benefits. Cell 160, 132-44 (2015).
[000420] 28. Sun, Q. et al. Structural basis for the inhibition mechanism of human cystathionine gamma-lyase, an enzyme responsible for the production of H(2)S. J Biol Chem 284, 3076-3085 (2009).
[000421] 29. Le Corre, L. & Padovani, D. Mechanism-based and computational modeling of hydrogen sulfide biogenesis inhibition: interfacial inhibition. Sci Rep 13, 7287 (2023).
[000422] 30. Dickhout, J.G. et al. Integrated stress response modulates cellular redox state via induction of cystathionine /-lyase: cross-talk between integrated stress response and thiol metabolism. J Biol Chem 287, 7603-14 (2012). [000423] 31. Yang, G., Pei, Y., Teng, H., Cao, Q. & Wang, R. Specificity protein-1 as a critical regulator of human cystathionine gamma-lyase in smooth muscle cells. J Biol Chem 286, 26450-60 (2011).
[000424] 32. Yuan, S. et al. Cystathionine y-Lyase Modulates Flow-Dependent Vascular
Remodeling. Arterioscler Thromb Vase Biol 38, 2126-2136 (2018).
[000425] 33. Xu, X. et al. 17p-Estradiol nongenomically induces vascular endothelial H(2)S release by promoting phosphorylation of cystathionine y-lyase. J Biol Chem 294, 15577-15592 (2019). [000426] 34. Fu, X. et al. 17P-estradiol attenuates atherosclerosis development: the possible role of hydrogen sulfide. Int J Cardiol 167, 1061-3 (2013).
[000427] 35. Rodriguez, C., Munoz, M., Contreras, C. & Prieto, D. AMPK, metabolism, and vascular function. Febsj 288, 3746-3771 (2021).
[000428] 36. Russell, R.R., 3rd et al. AMP-activated protein kinase mediates ischemic glucose uptake and prevents postischemic cardiac dysfunction, apoptosis, and injury. J Clin Invest 114, 495-503 (2004).
[000429] 37. Paskeh, M.D.A. et al. Targeting AMPK signaling in ischemic/reperfusion injury: From molecular mechanism to pharmacological interventions. Cellular Signalling 94, 110323 (2022).
[000430] 38. Shibata, R. et al. Adiponectin protects against myocardial ischemia-reperfusion injury through AMPK- and COX-2-dependent mechanisms. Nat Med 11, 1096-103 (2005).
[000431] 39. Wang, M., Tang, W. & Zhu, Y.Z. An Update on AMPK in Hydrogen Sulfide
Pharmacology. Front Pharmacol 8, 810 (2017).
[000432] 40. Li, J. et al. Activation of AMPK alpha- and gamma-isoform complexes in the intact ischemic rat heart. Am J Physiol Heart Circ Physiol 291, Hl 927-34 (2006).
[000433] 41. Nagata, D., Mogi, M. & Walsh, K. AMP-activated protein kinase (AMPK) signaling in endothelial cells is essential for angiogenesis in response to hypoxic stress. J Biol Chem 278, 31000-6 (2003).
[000434] 42. Zou, M.H. & Wu, Y. AMP-activated protein kinase activation as a strategy for protecting vascular endothelial function. Clin Exp Pharmacol Physiol 35, 535-45 (2008).
[000435] 43. Shen, X., Kolluru, G.K., Yuan, S. & Kevil, C.G. Measurement of H2S in vivo and in vitro by the monobromobimane method. Methods Enzymol 554, 31-45 (2015). [000436] 44. Shieh, M. et al. Shining a light on SSP4: A comprehensive analysis and biological applications for the detection of sulfane sulfurs. Redox Biol 56, 102433 (2022).
[000437] 45. Islam, M.Z., Shen, X., Pardue, S., Kevil, C.G. & Shackelford, R.E. The ataxiatelangiectasia mutated gene product regulates the cellular acid-labile sulfide fraction. DNA Repair (Amst) 116, 103344 (2022).
[000438] 46. Sun, Q. et al. Structural Basis for the Inhibition Mechanism of Human
Cystathionine y-Lyase, an Enzyme Responsible for the Production of H2S*. Journal of Biological Chemistry 284, 3076-3085 (2009).
[000439] 47. WL., D. The PyMOL Molecular Graphics System. . DeLano Scientific, San
Carlos, CA. (2002).
[000440] 48. Warnecke, A., Sandalova, T., Achour, A. & Harris, R.A. PyTMs: a useful
PyMOL plugin for modeling common post-translational modifications. BMC Bioinformatics 15, 370 (2014).
[000441] 49. Huang, W., Lin, Z. & van Gunsteren, W.F. Validation of the GROMOS 54A7
Force Field with Respect to (3-Peptide Folding. Journal of Chemical Theory and Computation 7, 1237- 1243 (2011).
[000442] 50. Bussi, G., Donadio, D. & Parrinello, M. Canonical sampling through velocity rescaling. J Chem Phys 126, 014101 (2007).
[000443] 51. Parrinello, M. & Rahman, A. Polymorphic transitions in single crystals: A new molecular dynamics method. Journal of Applied Physics 52, 7182-7190 (1981).
[000444] 52. Essmann, U. et al. A smooth particle mesh Ewald method. The Journal of
Chemical Physics 103, 8577-8593 (1995).
[000445] 53. Hess, B., Bekker, H., Berendsen, H.J.C. & Fraaije, J.G.E.M. LINCS: A linear constraint solver for molecular simulations. Journal of Computational Chemistry 18, 1463-1472 (1997).
[000446] 54. Kolluru, G.K. et al. Methamphetamine causes cardiovascular dysfunction via cystathionine gamma lyase and hydrogen sulfide depletion. Redox Biol 57, 102480 (2022).
[000447]
Equivalents
[000448] Various aspects of the present disclosure may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is, therefore, not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

Claims

We claim:
1. An isolated antibody or fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein a. the amino acid sequence of HCDR1 is SGGIS, the amino acid sequence of HCDR2 is SISTSGNTYYASWAKG, the amino acid sequence ofHCDR3 is ALAGI, the amino acid sequence of LCDR1 is QASQSVSKNNRLA, the amino acid sequence of LCDR2 is SASTLAS, and the amino acid sequence of LCDR3 is LGGYDCKSADCYI; b. the amino acid sequence of HCDR1 is SYGMI, the amino acid sequence of HCDR2 is AISSSGNTYYAKWAKG, the amino acid sequence of HCDR3 is NHYGSGDI, the amino acid sequence of LCDR1 is QSSQSVYDANRLA, the amino acid sequence of LCDR2 is GASTLDS, and the amino acid sequence of LCDR3 is QGYYSGYIYT; c. the amino acid sequence of HCDR1 is SYNMQ, the amino acid sequence of HCDR2 is IISSSDNTYYASWAKG, the amino acid sequence of HCDR3 is GRGYSSTTV, the amino acid sequence of LCDR1 is QASQSISSYLA, the amino acid sequence of LCDR2 is LASNMAS, and the amino acid sequence of LCDR3 is QCTYYHSSTSSTVGGA; d. the amino acid sequence of HCDR1 is SYGVT, the amino acid sequence of HCDR2 is WISTNSNAYYASWAKG, the amino acid sequence of HCDR3 is GIYSSGNI, the amino acid sequence of LCDR1 is QSSKSVYNNNKLS, the amino acid sequence of LCDR2 is STSSLAS, and the amino acid sequence of LCDR3 is AGGYSSNSDNT; e. the amino acid sequence of HCDR1 is SYTMS, the amino acid sequence of HCDR2 is YISRHGNTYYASWAKG, the amino acid sequence of HCDR3 is NIYS1DVI, the amino acid sequence of LCDR1 is QSSKSVGNNNRLS, the amino acid sequence of LCDR2 is GASTLAS, and the amino acid sequence of LCDR3 is LGAYSSSSDNS; f. the amino acid sequence of HCDR1 is SYAMG, the amino acid sequence of HCDR2 is YFSNNGNTYYANWAKG, the amino acid sequence of HCDR3 is SNL, the amino acid sequence of LCDR1 is QASQSVANNNRLS, the amino acid sequence of LCDR2 is YASTLAS, and the amino acid sequence of LCDR3 is LGSYDCRSADCYA; g. the amino acid sequence of HCDR1 is GSAVN, the amino acid sequence of HCDR2 is TISKNGNTYYATWAKG, the amino acid sequence of HCDR3 is RNPDTSGGLAL, the amino acid sequence of LCDR1 is QASQSVYSNNYLS, the amino acid sequence of LCDR2 is YASSLAS, and the amino acid sequence of LCDR3 is LGSYDCRAADCMA; or, h. the amino acid sequence of HCDR1 is SNGMS, the amino acid sequence of HCDR2 is YISSSGNVYYASWAKG, the amino acid sequence of HCDR3 is VLYSNGNI, the amino acid sequence of LCDR1 is QSSQSVYNNNRLS, the amino acid sequence of LCDR2 is EASKLAS, and the amino acid sequence of LCDR3 is LGGYSSNSDNA, or sequences that are at least 80% identical thereto.
2. The antibody of claim 1, comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein: a. the amino acid sequence of the LCVR is
AQVLTQTPSSVSAAVGSTVTINCQASQSVSKNNRLAWFQQKPGQPPK GLIYSASTLASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGGYD CKSADCYIFGGGTEVVVK, and the amino acid sequence of the HCVR is
QSVEESGGRLVTPGTPLTLTCTVSGIDLSSGGISWVRQAPGKGLEWIGS ISTSGNTYYASWAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCARAL AGIWGPGTLVTVSS; b. the amino acid sequence of the LCVR is
AQVLTQTASSVSAAVGGTVTISCQSSQSVYDANRLAWYQQKPGQPPK RLIYGASTLDSGVSSRFKGSGSGTQFTLTISEVQCDDAATYYCQGYYS GYIYTFGGGTEVVVK, and the amino acid sequence of the HCVR is
QSVEESGGRLVTPGGSLTLTCTVSG1DLSSYGM1WVRQAPGEGLEWIG AIS S SGNT YYAKWAKGRFPISRT STTVDLKMT SLTASDTAT YFC ARNH YGSGDIWGPGTLVTVSS; c. the amino acid sequence of the LCVR is DVVMTQTPASVSEPVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLI
YLASNMASGVPSRFSGSGYGTEFTLTISGVQCEDAATYYCQCTYYHSS TSSTVGGAFGGGTEVVVK, and the amino acid sequence of the HCVR is
QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYNMQWVRQAPGKGLEYIG IIS S SDNTYYASWAKGRFTISKTS STTVDLRVTSLTTEDTATYFC ARGR GYSSTTVWGPGTLVTVAS; d. the amino acid sequence of the LCVR is
AAVLTQTPSPVSAAVGGTVSISCQSSKSVYNNNKLSWFQQKPGQPPK
QLIYSTSSLASGVPSRFSGSGSGTQFTLTISDVQCDDAATYYCAGGYSS NSDNTFGGGTEVVVK, and the amino acid sequence of the HCVR is
QSVKESGGRLVTPGTPLTLTCTVSGFSLSSYGVTWVRQAPGKGLEWIG
WISTNSNAYYASWAKGRFTVSKTSSTTVTLTITSPTTEDTATYFCARGI YS SGNIWGPGTL VTVS S ; e. the amino acid sequence of the LCVR is
AQ VLTQTP S S VS A A VGGTVTINCQ S SK S VGNNNRL S W YQQKPGQPPK
QLIYGASTLASGVPSRFSGSGSGTQFTLTISDVQCDDAATYYCLGAYSS SSDNSFGGGTEVVVK, and the amino acid sequence of the HCVR is
QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYTMSWVRQAPGKGLEYIG
YISRHGNTYYASWAKGRFTISKTSSTTVDLKTTSPTTEDTATYFCGRNI YSIDVIWGPGTLVTVSS; f. the amino acid sequence of the LCVR is
AQVLTQTASPVSAAVGGTVTINCQASQSVANNNRLSWFQQKPGQPPK
LLIYYASTLASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYD CRSADCYAFGGGTEVVVK, and the amino acid sequence of the HCVR is
QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYAMGWFRQAPGKGLEWIG
YFSNNGNTYYANWAKGRFT1SKTSTTVDLKITRPTTEDTATYFCGRSN LWGQGTLVAVSS; g. the amino acid sequence of the LCVR is
AQVLTQTPSSVSAAVGGTVTINCQASQSVYSNNYLSWFQQKPGQPPK
LLIYYASSLASGVPSRFSGSGSGTRFTLTISDVQCDDAAAYYCLGSYDC RAADCMAFGGGTEVVVR, and the amino acid sequence of the HCVR is
QSVEESGGRLVTPGTPLTLTCAVSGFSVSGSAVNWVRQAPGEGLEWI GTISKNGNTYYATWAKGRFTISKTSSTTVDLRMTTLTTEDTATYFCAG RNPDTSGGLALWGQGTLVTVSS; or h. the amino acid sequence of the LCVR is AAVLTQTPSPVSAAVGGIVSISCQSSQSVYNNNRLSWFQKKPGQPPKL LIYEASKLASGVPPRFSGSGSGTQFTLTISGVQCDDAATYYCLGGYSSN SDNAFGGGTEVVVK, and the amino acid sequence of the HCVR is
QSVEESGGRLVTPGTPLTLTCTVSGIDLSSNGMSWVRQAPGKGLEWIG YISSSGNVYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARVLY SNGNIWGPGTLVTVSS, or sequences that are at least 80% identical thereto.
3. The antibody of claim 1 or claim 2, wherein the antibody binds to cystathionine gamma lyase (CSE).
4. The antibody of claim 3, wherein the antibody binds CSE phosphorylated at amino acid position serine 346 according to the sequence in Table 10.
5. The antibody of claim 1 or claim 2, wherein the antibody comprises a wildtype Fc or a modified Fc.
6. The antibody of claim 1 or claim 2, wherein the fragment comprises an Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody.
7. The antibody of claim 1 or claim 2, further comprising a detectable moiety.
8. A nucleic acid encoding the antibody of claim 1 or claim 2.
9. The nucleic acid of claim 5, wherein a. the nucleic acid sequence encoding LCVR is
GCCCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGG GAAGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTCTAA GAACAACCGCTTAGCCTGGTTTCAGCAGAAACCAGGGCAGCCTCC CAAAGGCCTGATCTATTCTGCATCCACTCTGGCATCTGGGGTCTCA TCGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCA TCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTATTGTCTAGG CGGTTATGATTGTAAAAGTGCTGATTGTTATATTTTCGGCGGAGGG ACCGAGGTGGTGGTCAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACA CCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTAGTG GTGGAATAAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAAT GGATCGGATCTATTAGTACTAGTGGTAACACATACTACGCGAGCTG GGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGT GGATCTGAAAATGACCAGTCCGACAACCGAGGACACGGCCACCTA TTTCTGTGCCAGAGCCCTGGCTGGCATCTGGGGCCCAGGCACCCTG GTCACCGTCTCCTCA, or a degenerate variant thereof; b. the nucleic acid sequence encoding LCVR is GCGCAAGTGCTGACCCAGACTGCATCGTCCGTGTCTGCAGCTGTGG GAGGCACAGTCACCATCAGTTGCCAGTCCAGTCAGAGTGTTTATGA TGCCAACCGCTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCC CAAGCGCCTGATCTATGGTGCATCCACTCTGGATTCTGGGGTCTCA TCACGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCA TCAGCGAAGTACAGTGTGACGATGCTGCCACTTACTACTGTCAAGG CTATTATAGTGGTTATATTTATACTTTCGGCGGAGGGACCGAGGTG GTGGTCAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTAACGCCTGGAGGA TCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTAGCT ATGGAATGATCTGGGTCCGCCAGGCTCCAGGGGAGGGGCTGGAAT GGATCGGAGCCATTAGTAGTAGTGGTAACACATACTACGCGAAGT GGGCAAAAGGCCGATTCCCCATCTCCAGAACCTCGACCACGGTGG ATCTGAAAATGACCAGTCTGACAGCCTCGGACACGGCCACCTATTT CTGTGCCAGAAATCATTATGGTAGCGGTGACATCTGGGGCCCGGGC
ACTTTGGTCACCGTCTCCTCA, or a degenerate variant thereof; c. the nucleic acid sequence encoding LCVR is
GATGTTGTGATGACCCAGACTCCAGCCTCCGTGTCTGAACCTGTGG GAGGCACAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAGTA GCTACTTAGCCTGGTATCAACAGAAACCAGGGCAGCCTCCCAAGC TCCTGATCTATCTGGCATCCAATATGGCATCTGGGGTCCCATCGCG GTTCAGCGGCAGTGGATATGGGACAGAGTTCACTCTCACCATCAGC GGCGTGCAGTGTGAAGATGCTGCCACTTATTATTGTCAATGTACTT ATTATCATAGTAGTACTAGTAGTACTGTTGGGGGGGCTTTCGGCGG AGGGACCGAGGTGGTGGTCAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACA CCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTAGCT ACAACATGCAATGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAAT ACATCGGAATCATTAGTAGTAGTGATAACACATACTACGCGAGCTG GGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGGT GGATCTGAGAGTGACCAGTCTGACAACCGAGGACACGGCCACCTA TTTCTGTGCCAGAGGGCGTGGTTATAGTAGTACTACTGTCTGGGGC
CCAGGCACCCTGGTCACCGTCGCCTCA, or a degenerate variant thereof; d. the nucleic acid sequence encoding LCVR is
GCCGCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGG GAGGCACAGTCAGCATCAGTTGCCAGTCCAGTAAGAGTGTTTATAA TAACAACAAATTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCC AAGCAACTGATCTATTCTACATCCAGTCTGGCATCTGGGGTCCCAT CGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCAT CAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTGCAGGC GGTTATAGTAGTAATAGTGATAATACTTTCGGCGGAGGGACCGAG
GTGGTCGTCAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is CAGTCAGTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACA CCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCT ATGGAGTGACCTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAAT GGATCGGATGGATTAGTACTAATAGTAACGCATACTATGCGAGCTG GGCGAAAGGCCGATTCACCGTCTCCAAAACCTCGTCGACCACGGT GACTCTGACAATCACCAGTCCGACAACCGAGGACACGGCCACCTA TTTCTGTGCCAGGGGTATTTATAGTAGTGGTAACATCTGGGGCCCA GGCACCCTGGTCACCGTCTCCTCA, or a degenerate variant thereof; e. the nucleic acid sequence encoding LCVR is
GCCCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGG GAGGCACAGTCACCATCAATTGCCAGTCCAGTAAGAGTGTTGGTA ATAACAACCGCTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTC CCAAGCAACTGATCTATGGTGCATCCACTCTGGCATCTGGGGTCCC ATCGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACC ATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAG GCGCTTATAGTAGTAGTAGTGATAATTCTTTCGGCGGAGGGACCGA GGTGGTGGTCAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACA CCCCTGACACTCACCTGCACAGTCTCTGGATTCTCCCTCAGTAGCT ATACAATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAAT ACATCGGATACATTAGTAGGCATGGTAACACATACTACGCGAGCT GGGCGAAAGGCCGATTCACCATCTCCAAGACCTCGTCGACCACGG TGGATCTGAAAACCACCAGTCCGACAACCGAGGACACGGCCACTT ATTTCTGTGGCAGAAATATTTATAGTATTGATGTCATCTGGGGCCC AGGCACCCTGGTCACCGTCTCCTCA, or a degenerate variant thereof; f. the nucleic acid sequence encoding LCVR is
GCCCAAGTGCTGACCCAGACTGCATCCCCCGTGTCTGCGGCTGTTG GAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTGCTA ATAACAACCGCTTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCC CAAGCTCCTGATCTATTATGCATCCACTCTGGCATCTGGGGTCTCAT CGCGGTTCAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCAT CAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGC AGTTATGATTGTCGTAGTGCTGATTGTTATGCTTTCGGCGGAGGGA CCGAGGTGGTGGTCAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACA CCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTAGCT ATGCAATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGGGCTGGAAT GGATCGGATATTTTAGTAATAATGGTAACACATACTACGCGAACTG GGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGA TCTGAAAATCACCCGTCCGACAACCGAGGACACGGCCACCTATTTC TGTGGCAGAAGCAACTTGTGGGGCCAAGGCACCCTGGTCGCCGTC TCCTCA, or a degenerate variant thereof; g. the nucleic acid sequence encoding LCVR is GCTCAAGTGCTGACCCAGACTCCATCCTCCGTGTCTGCAGCTGTGG GAGGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAG TAACAACTACTTATCCTGGTTTCAGCAGAAACCAGGGCAGCCTCCC AAACTCCTGATCTATTATGCATCCAGTCTGGCATCTGGGGTCCCAT
CGCGGTTCAGCGGCAGTGGATCTGGGACACGGTTCACTCTCACCAT CAGCGACGTGCAATGTGACGATGCTGCCGCTTACTACTGTCTAGGC AGTTATGATTGTAGGGCTGCTGATTGTATGGCTTTCGGCGGAGGGA CCGAGGTGGTGGTCAGA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACA CCCCTGACACTCACCTGCGCAGTCTCTGGATTCTCCGTCAGTGGTA GTGCAGTGAACTGGGTCCGCCAGGCTCCAGGGGAGGGGCTGGAAT GGATCGGGACAATTAGTAAGAATGGTAACACATACTACGCGACCT
GGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGTCGACCACGG TGGATCTGAGAATGACCACTCTGACAACCGAGGACACGGCCACCT ATTTCTGTGCCGGGCGCAATCCTGATACTAGTGGTGGTTTGGCCTT GTGGGGCCAAGGCACCCTGGTCACCGTCTCCTCA, or a degenerate variant thereof; h. the nucleic acid sequence encoding LCVR is
GCCGCCGTGCTGACCCAGACTCCATCTCCCGTGTCTGCAGCTGTGG GAGGCATAGTCAGCATCAGTTGCCAGTCCAGTCAGAGTGTTTATAA TAACAACCGCTTATCCTGGTTTCAGAAGAAACCAGGACAGCCTCCC AAGCTCCTGATCTACGAAGCATCCAAACTGGCATCTGGGGTCCCAC
CGCGGTTCAGCGGCAGTGGATCTGGGACACAGTTCACTCTCACCAT CAGCGGCGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGC GGTTATAGTAGTAATAGTGATAATGCTTTCGGCGGAGGGACCGAG GTGGTGGTCAAA, or a degenerate variant thereof; and the nucleic acid sequence encoding HCVR is
CAGTCGGTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACA CCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTAGCA ATGGAATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAAT GGATCGGATATATTAGTAGTAGTGGTAACGTATACTACGCGAGCTG
GGCAAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGA TCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACCTATTTC TGTGCCAGAGTTCTTTATAGTAATGGTAACATCTGGGGCCCAGGCA CCCTGGTCACCGTCTCCTCA, or a degenerate variant thereof, or sequences that are at least 80% identical thereto.
10. A vector encoding the nucleic acid of claim 5 or claim 6.
11. A cell comprising the vector of claim 7.
12. A pharmaceutical composition comprising the antibody of claim 1 or claim 2, the nucleic acid of claim 5 or claim 6, or the cell of claim 8, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
13. A method of detecting phosphorylated CSE in a subject in need thereof, the method comprising contacting a biological sample with the antibody of claim 1, wherein the antibody of claim 1 specifically binds to phosphorylated CSE.
14. The method of claim 13, wherein phosphorylated CSE is phosphorylated at amino acid position serine 346.
15. Use of the antibody of claim 1 or claim 2, for detecting phosphorylated CSE in a subject.
16. The method of claim 17, wherein phosphorylated CSE is phophosphorylated at amino acid position 346.
17. A synthetic nucleic acid encoding a CSE mutant, wherein the CSE mutant mimicks phosphorylation at Serine 346, Threonine 355, or both.
18. The nucleic acid of claim 20, wherein the nucleic acid is an mRNA molecule.
19. A synthetic polypeptide comprising SEQ ID NO: 1 or a fragment thereof, or a sequence at least 80% identical thereto.
20. The synthetic polypeptide of claim 1, wherein the synthetic polypeptide comprises a phosphomimetic amino acid at Ser346, The355, or both.
21. The synthetic polypeptide of claim 1, wherein the synthetic polypeptide comprises SEQ ID NO: 2.
22. A nucleic acid encoding the synthetic polypeptide of claim 19.
23. A pharmaceutical composition comprising the nucleic acid of claim 17 or the synthetic polypeptide of claim 19, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
24. A method of treating tissue injury in a subject, the method comprising: administering to a subject the nucleic acid of claim 17 or the synthetic polypeptide of claim 19.
25. The method of claim 20, wherein the nucleic acid increases cellular hydropersulfide, polysulfide, or both.
26. The method of claim 20, wherein the nucleic acid comprises a synthetic nucleic acid expression construct, wherein the expression construct comprises a promoter operatively linked to the nucleic acid encoding a CSE mutant.
27. A method of increasing cellular hydropersulfide, poly sulfide, or both, the method comprising: administering to a subject the nucleic acid of claim 17 or the synthetic polypeptide of claim 19.
EP24751139.7A 2023-02-02 2024-02-02 Phosphomimetic mutants, phosphospecific antibodies, and uses thereof Pending EP4658689A2 (en)

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