US20170114100A1 - Targeting dimerization of bax to modulate bax activity - Google Patents

Targeting dimerization of bax to modulate bax activity Download PDF

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US20170114100A1
US20170114100A1 US15/311,861 US201515311861A US2017114100A1 US 20170114100 A1 US20170114100 A1 US 20170114100A1 US 201515311861 A US201515311861 A US 201515311861A US 2017114100 A1 US2017114100 A1 US 2017114100A1
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bax
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Evripidis Gavathiotis
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Albert Einstein College of Medicine
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • Programmed cell death, or apoptosis is a fundamental process that regulates the critical balance between cellular life and death (1).
  • Dysregulation of apoptosis results in an imbalance of normal homeostasis contributing to diseases such as cancer and neurodegeneration (2,3).
  • the dysregulation of apoptosis is pivotal to a number of high mortality human diseases including cancer, cardiovascular diseases, and neurodegenerative diseases.
  • the BCL-2 family of proteins comprises a complex interaction network that regulates the commitment of the cell to apoptosis at the mitochondrial pathway (4,5).
  • the BCL-2 family includes both pro- and anti-apoptotic proteins.
  • pro-apoptotic BCL-2 proteins BCL-2-associated X-protein (BAX) and BCL-2 homologous Antagonist Killer (BAK)—induce mitochondrial outer-membrane permeabilization and represent the key gatekeepers and effectors of mitochondrial apoptosis.
  • BAX BCL-2-associated X-protein
  • BAK BCL-2 homologous Antagonist Killer
  • Pro-apoptotic BAX is a critical effector member of the BCL-2 family (4,5) that is predominantly in the cytosol of nonapoptotic cells (6).
  • BAX Upon activation, BAX translocates from the cytosol to the mitochondria to execute permeabilization of the outer mitochondrial membrane and release of apoptogens into the cytosol (7,8), the “point of no return” for mitochondrial dysfunction and apoptosis (9,10).
  • Pro-apoptotic BAX is a highly regulated protein that interacts with pro- and anti-apoptotic BCL-2 proteins that trigger or inhibit its activation.
  • Anti-apoptotic BCL-2 proteins such as BCL-2 and BCL-X L directly inhibit activated BAX whereas a subgroup of pro-apoptotic BCL-2 proteins, such as BIM and BID, use their BH3 domain to directly trigger BAX activation.
  • the cytosolic conformation of BAX has a BH3 trigger site located at the N-terminal surface of its structure. The interaction of BAX with a stapled BIM BH3 helix, through the N-terminal trigger site (helices ⁇ 1/ ⁇ 6), results in BAX activation involving a series of conformational changes.
  • BAX activation Despite remarkable progress in understanding BAX activation, current knowledge about the regulation mechanisms of BAX is limited. It is only understood how activated BAX is inhibited through the interaction of the BAX BH3 domain with the BH3 groove of the anti-apoptotic BCL-2 proteins. However, numerous proteins inhibiting cytosolic BAX have already been reported and posttranslational modifications have been studied which stabilize the cytosolic form of BAX. Emerging data suggest that BAX has a highly dynamic localization between cytosolic and mitochondrial compartments without requiring BAX activation. However, there is no structural evidence or any mechanistic understanding of how cytosolic BAX is kept under control. Current knowledge is limited to the structure of intact BAX, the NMR structure of which suggested that its ⁇ 9 conformation keeps the protein in an inactive cytosolic form preventing it from translocation to the mitochondrial membrane.
  • ⁇ diseases associated with premature or unwanted cell death and characterized by abnormal activation, or expression or function of BAX include: cardiovascular diseases and disorders (e.g., arteriosclerosis, heart failure, heart transplantation, aneurism, chronic pulmonary disease, ischemic heart disease, hypertension, thrombosis, cardiomyopathies), neurodegenerative diseases and neurological disorders (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, retinitis pigmentosa, spinal muscular atrophy, various forms of cerebellar degeneration, amyotrophic lateral sclerosis), immunological disorders (e.g., organ transplant rejection, arthritis, lupus, inflammatory bowel disease, Crohn's disease, asthma, multiple sclerosis, diabetes), ischemia (e.g., stroke, myocardial infarction and reperfusion injury), infertility (e.g., premature menopause, ovarian failure or follicular atresi
  • cardiovascular diseases and disorders e.g., arteriosclerosis, heart
  • BAX inhibitors may be particularly applicable in the treatment of cancer and could selectively overcome anti-apoptotic resistance of cancer cells and spare normal cells.
  • direct activators of BAX can be applied to promote apoptosis in autoimmune disease that are associated with uncontrolled production of immune cells.
  • BAX inhibitors may be particularly applicable to heart disease, CNS disorder diseases and diseases of the liver and kidney where cell death is abnormally excessive.
  • the present invention addressed the need for identifying inhibitors and activators of BAX for therapeutic treatments.
  • the present invention discloses assays for identifying agents that interfere with or promote dimerization of BAX, and/or bind to previously unidentified dimer binding sites of BAX.
  • the invention also provides for agents that promote or interfere with dimerization.
  • Methods are provided for identifying an agent as a candidate agent for promoting or inhibiting cell death comprising (a) contacting the agent with BCL-2-associated X-protein (BAX) monomers or portions thereof, and/or with BAX dimers, and (b) measuring if the agent inhibits or promotes dimerization of BAX; wherein a decrease in binding of BAX monomers to other BAX monomers or to portions of BAX monomers in the presence of the agent compared to in the absence of the agent indicates the agent inhibits dimerization of BAX, wherein an agent that agent inhibits dimerization of BAX is a candidate agent for promoting cell death; and wherein an increase in binding of BAX monomers or portions thereof to other BAX monomers or portions thereof in the presence of the agent compared to in the absence of the agent indicates that the agent promotes dimerization of BAX, wherein an agent that agent promotes dimerization of BAX is a candidate agent for inhibiting cell death.
  • BAX BCL-2-associated X-protein
  • Methods are also provided for identifying an agent that modulates the activity of BAX comprising contacting ⁇ 9 helix peptide of BAX with N-terminal binding site of BAX in the presence of the agent and in the absence of the agent; and measuring binding between the ⁇ 9 helix peptide of BAX and the N-terminal binding site of BAX, wherein decreased binding in the presence of the agent compared to binding in the absence of the agent indicates that the agent is a modulator of the activity of BAX.
  • Peptides are provided consisting of:
  • Methods are provided for modulating BAX and for treating diseases and disorders associated with blockade or unwanted cell death and characterized by abnormal activation, expression or function of BAX comprising contacting BAX with the any of the peptides disclosed herein.
  • FIG. 1A-1D BAX forms an inactive dimer conformation.
  • A Representative size exclusion chromatography (SEC) trace of recombinant BAX analyzed by Superdex 200 (HR 10/30) gel filtration column showing eluted monomer (M) and dimer (D) peaks at ⁇ 15.8 ml and ⁇ 14.2 ml, respectively.
  • B Recombinant BAX (rec. BAX), cytosolic extract of WT MEF and DKO MEF, and cytosolic extract from WT MEF treated with 1% Triton X-100 (cyt. BAX+1% Triton) were analyzed by Superdex 200 (HR 10/30) gel filtration.
  • FIG. 2A-2B Crystal structure of the inactive BAX dimer.
  • A Ribbon representation of the BAX dimer crystal structure. Dimerization interaction interfaces are shown for each BAX protomer. Helices ( ⁇ ) and loops (L) are depicted on the structures. Secondary structure representation cartoon showing the location of the BCL-2 homology domains (BH), the transmembrane region (TM), and the dimerization interaction interfaces.
  • B Ribbon representation of each BAX protomer in view related to the representation in (A) at 90 degrees rotation around a vertical axis, showing the N-terminal and C-terminal dimerization interfaces.
  • FIG. 3A-3E Structural details of the BAX dimerization mechanism.
  • A Calculated vacuum electrostatics of the C-terminal and N-terminal interaction surfaces of each BAX protomer in the dimer showing the position of complementary hydrophobic, polar and charged residues.
  • B Cartoon representation of the BAX dimer showing the protomers labelled by their dimer interaction interface.
  • the interacting residues are shown in sticks and are highlighted in three different regions of the dimer interaction interface: (C) hydrophobic core of the dimerization interface involving ⁇ 9 residues 1175 and F176 of one protomer and residues M20 and A24 from ⁇ 1, M137 and L141 from ⁇ 6 and L47 and V50 from ⁇ 1- ⁇ 2 loop, (D) hydrogen bonds between the following residue pairs of different BAX protomer: R145 of ⁇ 6 and Q171 of ⁇ 9, El7 of ⁇ 1 and M74 and E75 of ⁇ 3 and salt bridge between K21 of ⁇ 1 and E75 of ⁇ 3 residue pair and (E) hydrogen bonds between the following residue pairs of different BAX protomer: A46 of ⁇ 1- ⁇ 2 loop and Y164 of ⁇ 8, E44 of ⁇ 1- ⁇ 2 loop and W107 of ⁇ 5, D48 of ⁇ 1- ⁇ 2 loop and N106 of ⁇ 5 and salt bridge between D48 of ⁇ 1- ⁇ 2 loop and R109 of ⁇ 4- ⁇ 5 loop residue pair.
  • FIG. 4A-4D Autoinhibited dimer of BAX regulates BAX activation and apoptosis.
  • A Dimerization of purified monomeric recombinant BAX WT and mutants was analyzed by SEC and quantified by integration of areas under observed monomer and dimer peaks.
  • B Protein lysates of untreated DKO MEFs reconstituted with BAX WT and mutants were subjected to separation of cytosol from mitochondria, followed by SEC using Superdex 200 (HR 10/30).
  • C Viability assay of transient transduced DKO MEFs with human BAX WT and mutants as measured by annexin-V binding. P values ⁇ 0.05 for all mutants compared to BAX WT.
  • FIG. 5 Anti-apoptotic BCL-2, BCL-X L and MCL-1 proteins, established to interact with BAX, are in very low levels in the cytosol compared to BAX and predominantly reside at the mitochondria. Protein levels of BAX, BCL-2, BCL-X L and MCL-1 in cytosolic and mitochondrial fractions of MEFs as determined by immunodetection. Separation of the cytosolic from the mitochondrial fraction of MEFs is confirmed with b-tubulin and VDAC antibodies respectively.
  • FIG. 6A-6B Cytosolic BAX in dimer conformation is inactive as determined by immunoprecipitation assay with the 6A7 antibody that specifically recognizes the active conformation of BAX.
  • A Size-exclusion chromatography analysis of cytosolic extracts of MEFs using Superdex 200 10/300 GL gel filtration column, indicating the elution profile of BAX WT in dimer size
  • B Fractions 6 and 8 of BAX WT are 6A7 negative and failed to co-immunoprecipitate with the 6A7 antibody. As a positive control, fractions were incubated with 1% Octylglucoside detergent that activates BAX and exposes the 6A7 epitope on BAX.
  • FIG. 7 BAX dimerization is detected using a crosslinking approach. Coomassie stains of BAX in polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with 20 ⁇ BMH crosslinking reagent for 15 min at indicated BAX concentrations.
  • FIG. 8 Protein sequence alignment of BAX sequences from indicated species. Protein sequence alignment of BAX sequences showing identical residues shaded in light grey; conserved residues, similar residues and different residues shaded in white. Asterisks denote the positions of residues in each BAX protomer that is involved in interactions at the interface of the BAX dimer structure. Secondary structure symbols of helices and loops are based on the crystal structure of BAX dimer From top to bottom of sequences: SEQ ID NO:3 ( Homo sapiens ) to SEQ ID NO:10 (consensus).
  • FIG. 9A-9C Crystal structure of mutant BAX G67R dimer indicates the same dimerization mechanism of BAX as determined in the structure of the BAX P168G dimer.
  • A Structural alignment of the crystal structure of BAX G67R dimer (light grey) and BAX P168G dimer (darker grey).
  • B Structural alignment of BAX G67R protomer (light grey) and BAX P168G protomer (darker grey) crystal structures in view centered around helix ⁇ 5.
  • C Structural alignment of BAX G67R protomer (light grey) and BAX P168G protomer (darker grey) crystal structures in view centered around the N-terminal trigger site.
  • FIG. 10A-10C Structural insights of cytosolic BAX regulation mechanisms.
  • the hydrophobic surface of the BAX N-terminal trigger site comprising residues A24, L25, L27 of ⁇ 1 and L141, W139, G138, M137 of ⁇ 6, forms extended hydrophobic interactions with conserved hydrophobic residues F159, 1155, L152, and A149 of BIM BH3 (also FIG. 11B ). Additional stabilizing interactions occur between Q28 and Q32 of BAX and N160 of BIM BH3 and complementary charge interactions between residues K21, R134 and E131 of BAX and E158E, D157 and R153 of BIM BH3 ( FIG. 10B and FIG. 11B ).
  • FIG. 11A-11B Structural differences of the BAX's N-terminal binding site bound to ⁇ 9 (taken from BAX P168G dimer structure) and BIM BH3 (PDB ID: 2KW7) peptides provide structural insights of cytosolic BAX regulation. N-terminal surfaces of BAX shown in grey and hydrophobic (lighter grey), positively charged (K21, R134, and R145) and negatively charged (D48 and E131) residues of the BAX trigger site and ⁇ 1- ⁇ 2 loop are highlighted.
  • BAX ⁇ 9 binding with its hydrophobic residues make contacts with hydrophobic residues of the ⁇ 1, ⁇ 6 and ⁇ 1- ⁇ 2 loop, maintaining the structure of BAX in inactive conformation and ⁇ 1- ⁇ 2 loop in closed conformation.
  • B BIM BH3 binding and its hydrophobic residues make contacts with hydrophobic residues of ⁇ 1 and ⁇ 6, ensuing a conformational change in ⁇ 1- ⁇ 2 loop to an open conformation and change in the orientation of ⁇ 1 and ⁇ 6 residues. Furthermore, BIM BH3 has favorable electrostatic interactions with the charged residues of ⁇ 1 and ⁇ 6 at the periphery of the hydrophobic site.
  • FIG. 12 Structural analysis of the asymmetric BAX dimer crystal structure revealed two novel BAX interaction surfaces forming an autoinhibited dimer conformation.
  • the C-terminal helix ⁇ 9 of the BAX monomer binds with its solvent inaccessible surface the canonical BH3 pocket ( ⁇ 9 helix on right) and with its solvent accessible surface the BH3 trigger site of another BAX molecule ( ⁇ 9 helix* on left).
  • FIG. 13 A model of inactive BAX dimerization that regulates the BAX activation pathway triggered by select BH3-only proteins. Dimerization of inactive and cytosolic BAX provides an off pathway to BAX activation. Direct activation of BAX is initiated by direct interaction with BH3-only proteins and engagement of the trigger site (helices ⁇ 1 and ⁇ 6 indicated) of BAX monomers. Conformational change, mitochondrial translocation and autoactivation of BAX proceed and propagate BAX monomers to assemble an active dimer within the outer mitochondrial membrane and a homooligomeric pore to release key apoptogenic factors such as cytochrome c.
  • FIG. 14A-14B The autoinhibited dimeric form of BAX dissociates to BAX monomer before is activated by high doses of the activator BIM SHAB.
  • BIM SHAB Inactive BAX 4MA dimer is competed and dissociated to monomer by the trigger site binder, BIM SHAB, at high doses.
  • BIM SHAB can activate the 4MA monomer to induce BAX oligomerization.
  • Samples were analyzed by Superdex 75 (HR 10/30) gel filtration chromatography and quantified by integration of areas under observed monomer peaks.
  • B Liposome permeabilization assay of purified BAX WT dimer and BAX P168G dimer upon treatment with increasing doses of BIM SAHB A .
  • BAX P168G dimer is more resistant to activation by BIM SHAB compared to BAX WT.
  • the doses that are required for BIM SHAB A to activate the BAX WT dimer are significantly higher than the dose used to fully activate the BAX WT monomer in FIG. 1 .
  • Bar graphs indicate maximum release at 90 min. Data are mean+SD from experiments performed in triplicate and repeated three times.
  • FIG. 15 Structure of the ⁇ 9 helix peptide with labeled residues that interact with N-terminal binding site of BAX.
  • a method for identifying an agent as a candidate agent for promoting or inhibiting cell death comprising
  • BAX BCL-2-associated X-protein
  • an increase in binding of BAX monomers or portions thereof to other BAX monomers or portions thereof in the presence of the agent compared to in the absence of the agent indicates that the agent promotes dimerization of BAX, wherein an agent that agent promotes dimerization of BAX is a candidate agent for inhibiting cell death.
  • the agent can bind to amino acid residues of the N-terminal binding site and/or C-terminal binding site of BAX.
  • the agent can bind to the N-terminal of BAX at one or more of binding site residues S16, E17, Q18, 119, M20, K21, T22, G23, A24, L25, L26, 127, Q28, G29, F30, I31, Q32, D33, R34, A35, G36, R37, M38, G39, G40, E41, A42, P43, E44, L45, A46, L47, D48, P49, V50, P51, Q52, D53, A54, V129, P130, E131, L132, I133, R134, T135, I136, M137, G138, W139, T140, L141, D142, F143, L144, R145, E146, and R147.
  • the agent can bind to the C-terminal of BAX at one or more of binding site residues N73, M74, E75, L76, D98, M99, F100, S101, D102, G103, N104, F105, N106, W107, G108, R109, I152, Q153, D154, Q155, G156, G157, W158, D159, G160, L161, L162, 5163, Y164, F165, G166, T167, P168, T169, W170, Q171, T172, V173, T174, I175, F176, V177, A178, G179, V180, L181, T182, A183, S184, L185, T186, I187, W188, K189 K190, M191, and G192.
  • measurement of BAX as a dimer or monomer is carried out using one or more of a fluorescent polarization assay, size exclusion chromatography (SEC), polyacrylamide gel electrophoresis, dynamic light scattering, and an antibody that specifically binds to either BAX dimer or BAX monomer.
  • SEC size exclusion chromatography
  • polyacrylamide gel electrophoresis polyacrylamide gel electrophoresis
  • dynamic light scattering and an antibody that specifically binds to either BAX dimer or BAX monomer.
  • a method for identifying an agent that modulates the activity of BAX comprising
  • the agent is an inhibitor of BAX.
  • the agent may be an activator of BAX.
  • the ⁇ 9 peptide of BAX has the amino acid sequence TWQTVTIFVAGVLTASLTIWKKMG (SEQ ID NO:11).
  • the ⁇ 9 helix peptide of BAX is illustrated in FIG. 15 .
  • the ⁇ 9 helix peptide of BAX or the N-terminal binding site of BAX can be immobilized on a solid substrate. Binding between the ⁇ 9 helix peptide of BAX and the N-terminal binding site of BAX can be measured, for example, using a fluorescence assay or a surface plasmon resonance assay or a co-immunoprecipitation assay or an NMR assay.
  • Methods are also disclosed for identifying an agent as a modulator of a BCL-2-associated X-protein (BAX) comprising contacting the agent with the BAX and measuring if the agent inhibits or promotes the dimerization of BAX with another BAX or portion thereof, wherein a decrease in binding in the presence of the agent compared to in the absence of the agent indicates that the agent inhibits dimerization of BAX and an increase in binding of BAX to another BAX molecule in the presence of the agent compared to the absence of the agent indicates that the agent promotes dimerization of BAX.
  • BAX BCL-2-associated X-protein
  • the BAX can be a human BAX.
  • the agent can be, for example, a small molecule, an isolated peptide, a synthetic peptide, a peptide-based agent with natural or non-natural amino acids or combinations, a hydrocarbon stapled peptide, a constrained peptide, a macrocycle, a peptoid, a peptidomimetic, a foldamer, an aptamer, an antibody, a monobody, a nanobody, or combinations thereof.
  • an agent is a peptidomimetic of the ⁇ 9 helix of BAX.
  • the agent is a small molecule of 2000 daltons or less. In an embodiment of the methods described herein, the agent is a small molecule of 1500 daltons or less. In an embodiment of the methods described herein, the agent is a small molecule of 1000 daltons or less. In an embodiment of the methods described herein, the agent is a small molecule of 800 daltons or less. In an embodiment of the methods described herein, the agent is a small molecule of either 2000, 1500, 1000, 800, 700, 600, 500 or 400 daltons or less. In an embodiment of the methods described herein, the agent is a small organic molecule.
  • the methods disclosed herein can further comprise administering an agent identified as promoting dimerization of BAX or as inhibiting BAX to a subject with a disease or disorder associated with premature or unwanted cell death and characterized by abnormal activation, expression or function of BAX, and testing the efficacy of the agent in treating the disease.
  • the methods can further comprise administering an agent identified as inhibiting dimerization of BAX or as activating BAX to a subject with a cancer and testing the efficacy of the agent in treating cancer.
  • the peptide of 11-30 amino acid residues comprising the sequence TXQTXXIFXAG does not include any of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
  • the invention also provides a peptide of 15-30 amino acid residues comprising the sequence: TXQTXXIFXAGVXTA (SEQ ID NO:16) where “X” at any position can independently be any amino acid or unnatural amino acid.
  • the peptide of 15-30 amino acid residues comprising the sequence TXQTXXIFXAGVXTA does not include any of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
  • the invention also provides a peptide of 11-30 amino acid residues comprising the sequence Y1XY2Y3XXY4Y5XY6Y7 (SEQ ID NO:17), where Y1 is threonine or a conserved amino acid, or unnatural amino acid, Y2 is glutamine or a conserved residue or unnatural amino acid, where Y3 is threonine or a conserved amino acid, or unnatural amino acid, where Y4 is isoleucine or a conserved amino acid, or unnatural amino acid, where Y5 is phenylalanine or a conserved amino acid, or unnatural amino acid, where Y6 is alanine or a conserved amino acid, or unnatural amino acid, where Y7 is glycine or a conserved amino acid, or unnatural amino acid, where X at any position can independently be any amino acid, or natural or unnatural chemically modified amino acid.
  • At least one X or one Y in each sequence is an unnatural amino acid or a non-naturally occurring chemically modified amino acid.
  • 11-30 amino acids it is meant any number of amino acids between 11-30, i.e., 11, 12, 13, 14, . . . 29, or 30 amino acid residues.
  • the peptide consists of the sequence TXQTXXIFXAG (SEQ ID NO:15) or the sequence TXQTXXIFXAGVXTA (SEQ ID NO:16).
  • the interacting residues could be also mutated to conserved or similar residues that will maintain the interaction with BAX.
  • the peptides can be labeled with a label such as a fluorescent label or a radioactive label.
  • the peptides claimed herein are chemically synthesized peptides or peptides produced by recombinant DNA or cDNA.
  • the peptides can be made by liquid phase synthesis or by solid phase synthesis.
  • the peptides can be synthesized, e.g., using recombinant DNA or cDNA.
  • the peptides are not directly obtained from a larger naturally-occurring BAX protein, for example, by digestion of the protein.
  • the peptides are isolated and purified peptides.
  • the BAX can be in a living cell where preferably inhibition of BAX inhibits cell death.
  • the BAX can in a subject, such as a mammal such as a human, and the agent is administered to the subject.
  • the subject can have a disease or disorder associated with premature or unwanted cell death and characterized by abnormal activation, expression or function of BAX.
  • Cardiodegenerative diseases and disorders e.g. arteriosclerosis, heart failure, heart transplantation, aneurism, chronic pulmonary disease, ischemic heart disease, hypertension, thrombosis, cardiomyopathies
  • neurodegenerative and neurological diseases and disorders e.g. Alzheimer's disease, Parkinson's disease, Huntington's disease, retinitis pigmentosa, spinal muscular atrophy, various forms of cerebellar degeneration, amyotrophic lateral sclerosis
  • liver diseases and disorders e.g.
  • organ transplant rejection arthritis, lupus, IBD, Crohn's disease, asthma, multiple sclerosis, diabetes
  • ischemia e.g. stroke, myocardial infarction and reperfusion injury
  • infertility e.g. premature menopause, ovarian failure or follicular atresia
  • blood disorders e.g. fanconi anemia, aplastic anemia, thalassemia, congenital neutropenia, myelodysplasia
  • renal hypoxia hepatitis
  • asthma and AIDS Diseases associated with inhibition of cell death and characterized by abnormal inhibition, expression or function of BAX include, for example, cancer and autoimmune diseases.
  • Methods are also provided for identifying an agent as a candidate agent for inhibiting cell death comprising:
  • BCL-2-associated X-protein (BAX) with one or more of any of the peptides disclosed herein in the presence of the agent and in the absence of the agent, and
  • the peptides are fluorescently labeled, for example with fluorescein isothiocyanate (FITC).
  • FITC fluorescein isothiocyanate
  • BAX is BCL-2-associated X-protein.
  • the BAX is mammalian.
  • the BAX is a human BAX.
  • the BAX comprises consecutive amino acid residues having the following sequence:
  • the methods are useful for identifying therapeutic cell death inhibitors. In an embodiment of the methods described herein, the methods are useful for identifying therapeutic cell death activators.
  • BIM SAHB A2 N-acetylated 145 EIWIAQELRS5IGDS5FNAYYA 164 -CONH 2 (SEQ ID NO:2), where S5 represents the non-natural amino acid inserted for olefin metathesis, was synthesized, purified and characterized as previously described by CPC Scientific (11).
  • Dynamic Light-Scattering The dynamic light scattering of various samples was measured using a DynaPro801 instrument from Wyatt Technology and DYNAMICS v.5.25.44 software for data collection and analysis. The protein samples after elution from the size-exclusion chromatography were centrifuged for 10 min at 13,000 rpm and loaded to plastic cuvettes. Typically, one hundred scans of five seconds was acquired for each 100 ⁇ l sample. Normalized intensity versus hydrodynamic radius (nm) measured in 20 mM HEPES pH 7.2, 150 mM KCl, 1 mM DTT buffer.
  • Diffraction quality rod-shaped crystals were generated in 0.1 M Bis-Tris pH 6.5, 1.5 M ammonium sulfate via the hanging-drop vapour-diffusion method using 24-well VDX plates (Hampton Research). 1 ⁇ l of protein solution and reservoir solution were mixed and equilibrated against 1 ml of reservoir solution. Crystals were cryoprotected by soaking for 5 s in 20 ⁇ l of cryoprotectant solution containing 0.1 M Bis-Tris pH 6.5, 1.5 M ammonium sulphate and 25% (v/v) glycerol, and flash-frozen in liquid nitrogen.
  • NMR samples and spectroscopy were prepared in 25 mM sodium phosphate, 50 mM NaCl solution at pH 6.0 in 5% D 2 O. Correlation 1 H- 15 N HSQC, 1 H- 15 N TROSY spectra and triple resonance spectra for backbone 1 H, 13 C, 15 N assignments of BAX P168G monomer: HNCO, HNCA, HNCOCA, HNCACB, HNCOCACB and N 15 NNH-NOESY were acquired at 25° C. on a Inova 600 MHz NMR spectrometer equipped with a cryogenic probe, processed using Topsin, and analyzed with CCPNMR (26). BAX wild type cross-peak assignments were applied as previously reported (11,16).
  • the weighted average chemical shift difference A at the indicated molar ratio was calculated as ⁇ ( ⁇ H 1 ) 2 +( ⁇ N 15 /5) 2 in p.p.m.
  • the absence of a bar indicates no chemical shift difference, or the presence of a proline or a residue that is overlapped and not used in the analysis.
  • the significance threshold for backbone amide chemical shift changes was calculated based on the average chemical shift across all residues plus the standard deviation, in accordance with standard methods.
  • BAX dimerization assays BAX wild type and mutants were concentrated to ⁇ 0.5 mM in BAX buffer (10 mM HEPES pH 7, 150 mM KCl, 1 mM DTT) and incubated at 20° C. for 1-3 days before diluting to a total volume of 250 ⁇ l and loaded onto a Superdex 75 HR 10/30 size exclusion column (GE Healthcare). These conditions allow a controlled dimerization process than aggregation due to BAX activation. Separation of monomeric and dimeric BAX was achieved using a flow rate of 0.5 ml/min at 4° C. The chromatogram traces show the monomeric and dimeric peaks at ⁇ 11.8 and ⁇ 10.4 ml, respectively. Protein standards (GE Healthcare) were used to calibrate the molecular mass of gel filtration peaks. Chromatogram traces are representative of several independent preparations of freshly SEC-purified monomeric BAX.
  • BAX crosslinking Dimerization was detected using a crosslinking approach by incubating BAX at indicated doses with 20 ⁇ BMH on ice for 15 min followed by quenching with 1 mM DTT. Samples were denatured at 90 degrees and analyzed with SDS-PAGE.
  • Liposomal permeabilization assay Liposomes were composed of the following molar percentages of lipids (Avanti Polar Lipids): phosphatidylcholine, 48%; phosphatidylethanolamine, 28%; phosphatidylinositol, 10%; dioleoyl phosphatidylserine, 10%; and tetraoleoyl cardiolipin, 4% and were loaded with ANTS/DPX (Molecular Probe) upon extrusion.
  • ANTS/DPX Molecular Probe
  • BAX 400 nM was combined with BIM SAHB A2 at the indicated concentrations in 96-well format (Corning) and then liposomes were added (10 ⁇ l from 50 mM total lipid stock) in assay buffer (10 mM HEPES, pH 7, 200 mM KCl, 5 mM MgCl 2 , and 0.2 mM EDTA) to a final volume of 100 ⁇ l.
  • assay buffer (10 mM HEPES, pH 7, 200 mM KCl, 5 mM MgCl 2 , and 0.2 mM EDTA
  • 1% Triton treatment is used to determine the maximum amount of liposomal release per assay, and this value sets the 100% value.
  • Subcellular fractionation MEFs were maintained in DMEM (Invitrogen) supplemented with 10% FBS, 100 U ml ⁇ 1 penicillin/streptomycin, 2 mM 1-glutamine, 0.1 mM MEM nonessential amino acids, and 50 ⁇ M ⁇ -mercaptoethanol. MEFs (20 ⁇ 10 6 per well) were seeded in a 150 mm dish for 12 hours. To isolate cytosol and mitochondrial fractions, cells were lysed by Dounce homogenizer in lysis buffer LB containing 10 mM Tris, pH 7.5, 1 mM EGTA, 200 mM Sucrose and Complete Protease Inhibitors.
  • the cell lysates were centrifuged at 700 ⁇ g for 10 min to remove unlysed cells and nuclei.
  • the supernatants were centrifuged at 12000 ⁇ g for 10 min at 4° C. and the resulting pellet was collected as the mitochondrial fraction.
  • the membrane pellet was resuspended in LB+1% CHAPS.
  • BAX conformational change assay Cytosolic fractions were subjected to immunoprecipitation followed by immunoblotting against total BAX. Briefly, 100-300 ⁇ g total protein was collected and incubated with pre-equilibrated protein G Sepharose beads (Santa Cruz Biotechnology Inc.) for 1 hour. The precleared samples were then incubated with the 6A7 antibody (6 ⁇ g/ml) (1:1,000, sc-23959, Santa Cruz Biotechnology) for four hours at 4° C. followed by the addition of pre-equilibrated protein G Sepharose beads for 1 hour. The beads were pelleted, washed with lysis buffer 3 times at 4° C., and protein eluted by heating the beads at 90° C. for 10 minutes in LDS/DTT loading buffer. The immunoprecipitates were subjected to electrophoresis and Western blot analysis using anti-BAX antibody (1:1,000) (Cell Signaling 2772S).
  • the current invention makes use of the discovery that cytosolic BAX forms a dimeric autoinhibited form that is mediated by the N-terminal trigger site of one protomer and a novel interacting C-terminal surface that includes ⁇ 9 of the second protomer.
  • These interacting protein surfaces of BAX provide important insights for understanding the structural basis of BAX inhibition and designing drugs for pharmacological modulation of BAX. It was found that monomeric wild type BAX (BAX WT), in the absence of a BH3 activating domain, reversibly forms a dimer over time as measured by size exclusion chromatography (SEC) and polyacrylamide gel electrophoresis (PAGE).
  • BAX P168G forms a more stable dimer, which persists several days at room temperature.
  • recombinant BAX and cytosolic BAX from mouse embryonic fibroblasts were investigated.
  • Recombinant full-length BAX was purified from E. coli extracts using chitin affinity chromatography followed by size-exclusion chromatography (SEC) (17).
  • SEC size-exclusion chromatography
  • Recombinant BAX elutes from SEC predominantly in a peak that corresponds to its monomeric form (11,17) and additionally in a second distinct peak that corresponds to a dimeric form ( FIG. 1A, 1B ).
  • Cultured MEFs were lysed, without detergents in the buffer, and cytosolic and mitochondrial fractions were separated followed by fractionation with SEC.
  • endogenous BAX eluted in fractions that corresponds to a BAX dimer using the same SEC conditions for recombinant BAX ( FIG. 1B ).
  • SEC was repeated with MEFs that are doubly deficient in BAX ⁇ / ⁇ and BAK ⁇ / ⁇ (DKO MEFs) and stably express human BAX at endogenous levels, and again cytosolic BAX appeared to be a dimer ( FIG. 1B ).
  • cytosolic BAX is a heterodimer with anti-apoptotic BCL-2 proteins in the cytosol
  • cytosolic and mitochondrial fractions were subjected to western blot analysis, confirming that anti-apoptotic proteins are in very low levels in the cytosol compared to BAX and predominantly reside at the mitochondria ( FIG. 5 ).
  • the cytosolic BAX dimer is in inactive conformation as proved by immunoprecipitation analysis using the 6A7 antibody that only recognizes the active BAX conformation ( FIG. 6 ) ( 18 ).
  • BAX appeared predominantly at high molecular weight, presumably forming the BAX oligomer.
  • the asymmetric unit contained two BAX molecules with excellent electron density map, in which all BAX residues could be traced except from residues of the N-terminal unstructured region (residues 1-13) and four residues of the unstructured loop between helices ⁇ 1 and ⁇ 2 (residues 37-40) ( FIG. 2 ).
  • the dimerization interface is formed by conserved residues and is extensive, burying almost 1900 ⁇ 2 surface area ( FIGS. 2A and 8 ).
  • the BAX dimer structure reveals a dimerization interface that includes the interaction of two structural surfaces critical for the activation of BAX; the N-terminal trigger site from one BAX protomer and a novel C-terminal surface from the second BAX protomer that includes the C-terminal ⁇ 9 helix ( FIGS. 2B, 14 ).
  • this dimeric structure and dimerization interface is irrelevant of the BAX P168G mutant as a BH3 residue mutant, G67R, that introduces a new hydrogen bond with helix 1, yields the same asymmetric dimer conformation as determined by X-ray crystallography at resolution of 3.3 ⁇ ( FIG. 9 , Table 2).
  • the BAX protomers within the asymmetric BAX dimer structure resemble the inactive monomeric BAX structure determined by solution NMR, however, they have noticeable differences in orientation of helices and conformation of loops (backbone r.m.s.d. of 2.1 ⁇ ) (17). Most pronounced differences correspond to residues located in the N-terminal trigger site surface, including helices ⁇ 1, ⁇ 6, ⁇ 2 and ⁇ 1- ⁇ 2 loop. Furthermore, the comparison with the structure of full length BAX bound to the BIM BH3 helix (backbone r.m.s.d.
  • the asymmetric BAX dimer conformation highlights two novel interaction surfaces of BAX ( FIG. 3 ).
  • the interaction surface of one BAX protomer includes mostly the BAX trigger site residues previously identified for binding to BIM SAHB A2 with a number of additional interactions at its periphery (11).
  • the interaction site at the C-terminal surface is a novel and unforeseen binding surface of BAX that includes the C-terminal helix ⁇ 9.
  • the C-terminal binding surface has a hydrophobic center from solvent exposed hydrophobic residues of ⁇ 9 that are surrounded by polar and charged residues of helices ⁇ 8, ⁇ 7, ⁇ 3, the ⁇ 4- ⁇ 5 loop and the ⁇ 8- ⁇ 9 loop ( FIG. 3A ).
  • the N-terminal binding surface has a hydrophobic center from solvent exposed hydrophobic residues of ⁇ 1, ⁇ 6 and the ⁇ 1- ⁇ 2 loop that are surrounded by charged residues, which complement charged residue of the C-terminal binding surface ( FIG. 3A ).
  • the core of the dimerization interface is hydrophobic ( FIG. 3B ,C).
  • Several residues form a network of hydrophobic interactions that complement the binding of helix ⁇ 9 to the N-terminal BH3-binding site ( FIG. 3C ).
  • a number of hydrogen bonds and salt bridges contribute to the dimer formation and stability ( FIG. 3B , D, E).
  • hydrophobic, polar interactions and salt bridges are involved in the extensive and highly complementary dimerization interface.
  • the ⁇ 1- ⁇ 2 loop Upon BH3 binding to the N-terminal trigger site, the ⁇ 1- ⁇ 2 loop is displaced into an open conformation, a conformational change essential for exposure of the 6A7 epitope (residues 12-24) and BAX activation (11,12,17,18) ( FIG. 10A ).
  • ⁇ 9 binds the N-terminal BH3-binding site in an orientation that preserves the conformation of the ⁇ 1- ⁇ 2 loop in a closed and inactive conformation ( FIG. 10A ).
  • BIM BH3 binding is mediated by exposed hydrophobic residues of ⁇ 1 and ⁇ 6, with conserved hydrophobic residues of BIM BH3 ( FIG.
  • the BIM SHAB A can also activate BAX 4MA monomer to induce BAX oligomerization.
  • BAX mutants with monomeric BAX showed significantly increased activity in cell death induction compared to BAX WT ( FIG. 4C ).
  • BAX P168G mutant cells with a more stable cytosolic dimer have impaired activity compared to BAX WT ( FIG. 4C ).
  • BAX P168G dimer was more resistant to undergo translocation and oligomerization in the outer mitochondrial membrane compared to BAX WT ( FIG. 4D ).
  • monomeric BAX E75K and S184L undergo faster activation and transformation to an oligomer at the outer mitochondrial membrane than the corresponding BAX WT dimer ( FIG. 4D ).
  • cytosolic BAX may switch its conformation from the autoinhibited dimer to a monomer and that monomeric BAX favors faster BAX activation and more potent cell death activity while the dimeric BAX hinders BAX activation and ultimately cell death activity.
  • cytosolic BAX is activated through an interaction of activator BH3-only proteins with the N-terminal trigger site, followed by the N-terminal conformational change and the displacement of ⁇ 9 from its C-terminal hydrophobic groove, in order to translocate to the mitochondria Moreover, mitochondrial attached BAX with the ⁇ 9 displaced from its hydrophobic groove undergoes N-terminal conformational change upon further activation by activator BH3-only proteins (13). Regardless of the step of BAX activation, conformational changes at the N-terminal and C-terminal surfaces are required for complete BAX activation leading to membrane permeabilization.
  • cytosolic BAX forms an autoinhibited dimer conformation to prevent either the N-terminal or C-terminal conformational change in each protomer and maintain BAX activation under control ( FIG. 13 ).
  • Induction of apoptosis in a cell should require commitment of pro-apoptotic signals (e.g. BH3-only proteins) beyond stochastic fluctuations of their expression levels or active state; therefore, BAX autoinhibition provides a mechanism for blocking BAX activation and preventing unwanted apoptosis.
  • pro-apoptotic signals e.g. BH3-only proteins

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US11174308B2 (en) 2015-09-10 2021-11-16 Albert Einstein College Of Medicine, Inc. Synthetic antibodies to BAX and uses thereof
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