Classifications

• • 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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