WO2017053864A1 - Inhibiteurs de protéases à cystéine - Google Patents

Inhibiteurs de protéases à cystéine Download PDF

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WO2017053864A1
WO2017053864A1 PCT/US2016/053539 US2016053539W WO2017053864A1 WO 2017053864 A1 WO2017053864 A1 WO 2017053864A1 US 2016053539 W US2016053539 W US 2016053539W WO 2017053864 A1 WO2017053864 A1 WO 2017053864A1
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ome
tfa
compound
fmk
vad
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David Phelps
Gary Johnson
Mary Margaret BARTIK
Eric S. BENSEN
David J. HALVERSON
Matthew S. Bogyo
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Intracellular Technologies, Llc
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06052—Val-amino acid
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    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06104—Dipeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06104—Dipeptides with the first amino acid being acidic
    • C07K5/06113—Asp- or Asn-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu

Definitions

  • Apoptosis is a complex mechanism of programmed cell death that is controlled by multiple biochemical events leading to morphological cell changes and eventual cell death.
  • the apoptosis process begins when apoptotic signals cause regulatory proteins to initiate an apoptosis pathway.
  • the primary pathways targeted include mitochondrial functionality, transduced signals via adaptor proteins to the apoptotic mechanism, and drug induced increases in calcium within the cell.
  • Apoptosis culminates in coordinated cell death that requires energy and, unlike cell death occurring by necrosis, does not induce an inflammatory response.
  • Apoptosis is a critical event in numerous processes within the body. For example, embryonic development relies on apoptosis, and tissues that turn over rapidly require tight regulation to avoid serious pathological consequences.
  • Certain medical conditions, such as cancer are characterized by insufficient apoptosis (insufficient cell death) and uncontrolled cell proliferation brought on in part by the failure to regulate apoptosis.
  • apoptosis can be induced by various means.
  • chemical induction of apoptosis can be achieved by administering drugs such as chemotherapeutic agents that initiate apoptosis.
  • excess apoptosis can damage organs.
  • Apoptosis can also be indicative of tissue damage, such as damaged heart tissue following ischemia or reperfusion insults.
  • Inflammation is an organism's reaction to harmful stimuli, such as pathogens, damaged cells, or irritants, and an attempt by the organism (e.g. a human body) to initiate a healing process and remove the cause of the inflammation.
  • An inflammatory reaction typically involves an organism's local vascular system, immune system, and cells within the injured tissue.
  • Chronic inflammation is characterized by a shift in cell types at the site of inflammation and simultaneous destruction and healing of tissue.
  • Cellular pathways leading to both apoptosis and inflammation involve the activation of members of a family of proteases known as caspases. At least 14 members of the caspase family have been identified in vertebrates, and at least 8 are known to be involved in apoptotic cell death (see Saunders, et ah, Anal. Biochem., 284, 114-24 (2000)).
  • Caspases are a group of highly specific cysteine proteases that cleave aspartic acid peptide bonds within proteins. Caspases collaborate in the proteolytic cascade by activating themselves and each other.
  • Apoptosis-related caspases can be divided into two categories: "initiator” caspases (e.g., caspase-2, caspase-8, caspase-9 and caspase- 10), and downstream “effector” caspases (e.g., caspase-3, caspase-6, caspase-7 and caspase-14).
  • Initiator caspases mediate their oligomerization and autoactivation in response to specific upstream signals, and can activate effector caspases by cleaving their inactive pro-forms.
  • Activated effector caspases continue the apoptotic process by cleaving protein substrates within a cell.
  • Inhibitors of caspases can thus regulate the initiation and/or effector enzymes within the apoptotic caspase chain reaction by inhibiting these processes.
  • caspases e.g., caspase-1 , caspase-4, caspase-5, caspase-1 1 and caspase-13
  • caspase-1 inflammation-related caspase
  • caspase-1 inflammation-related caspase
  • ICE-1 converting enzyme ICE-1
  • the detection of active caspases involved in inflammatory pathways indicates an acute or chronic inflammatory response - e.g., inflammation associated with inflammatory diseases such as rheumatoid arthritis or atherosclerosis.
  • peptidic pan- caspase inhibitors such as Z-VAD-FMK have routinely been used in vitro in scientific research and drug development screening to block caspase activity.
  • a more potent caspase inhibitor may prove valuable for in vitro testing, in that it may allow for the use of less product and potentially shorten test duration.
  • caspase inhibitors have been shown in various animal models to inhibit post myocardial infarction apoptosis, to reduce lesion volume and neurological deficit resulting from stroke or ischemia, to reduce post-traumatic apoptosis and neurological deficit in traumatic brain injury (TBI), and to be effective in treating fulminant liver destruction, liver disease and sepsis (see e.g. Yaoita et al. (1998) Circulation, 97: 276-281 ; Endres et al. (1998) J. Cerebral Blood Flow and Metabolism, 18: 238-247; Cheng et al. (1998) J. Clin. Invest. 101 : 1992-1999; Yakovlev et al. (1997) J.
  • pan caspase inhibitor emricasan (a potent inhibitor of both apoptotic and inflammatory caspases) has recently been investigated as a therapeutic in various models of liver disease. Recent research efforts also include the use of caspase inhibitors in combination with various therapeutics for the treatment of cancer (ref: Brumatti et al., The Caspase-8 Inhibitor Emricasan Combines With the SMAC Mimetic
  • caspase inhibitors may someday be used in vivo as potential therapeutics for certain conditions involving apoptosis and/or inflammation, including (but not limited to) neurodegenerative diseases such as Alzheimer's and Multiple Sclerosis, liver disease, spinal atrophy, stroke, traumatic brain injury, myocardial infarction, fibrotic diseases (kidney fibrosis, idiopathic pulmonary fibrosis, diabetic nephropathy, liver fibrosis, non-alcoholic steatohepatitis (NASH), primary biliary cirrhosis (PBC), systemic sclerosis, corneal fibrosis), and inflammatory conditions related to metabolic disease.
  • neurodegenerative diseases such as Alzheimer's and Multiple Sclerosis, liver disease, spinal atrophy, stroke, traumatic brain injury, myocardial infarction, fibrotic diseases (kidney fibrosis, idiopathic pulmonary fibrosis, diabetic nephropathy, liver fibrosis, non-alcoholic steatohepatit
  • caspase inhibitors in vivo has met with considerable challenges involving undesirable pharmacological effects and cytotoxicity, due in part to the dose of inhibitor required to achieve a sufficient anti-apoptotic and/or anti-inflammatory effect.
  • Available information suggests the need for safe, stable, caspase-selective, cell-permeant, irreversible caspase inhibitors with increased potency (i.e. enhanced caspase binding affinity and kinetics), suitable for both in vitro and in vivo
  • Inflammatory pathways also involve the expression of a family of proteases called cathepsins.
  • the cysetine cathepsins in particular (cathepsins B, C, F, H, K, L, O, S, W, X/Z) are often highly upregulated or overexpressed during conditions where inflammation is present, such as cancer (especially with tumor invasion, angiogenesis, metastasis, or tumor associated macrophages (TAMS) in the tumor microenvironment), auto-immune diseases (e.g. lupus, psoriasis, Crohn's disease, Sjogren's syndrome, celiac disease), neurodegenerative diseases (e.g.
  • Alzheimers traumatic brain injury, arthritis, hepatitis (including alcohol-related and NASH), pancreatitis, liver fibrosis and steatosis (including HCV-associated), pulmonary fibrosis, renal fibrosis and cardiac fibrosis (ref: Golde et al., Science 255: 728-730, 1992; Munger et al., Biochem. J. 31 1 : 299-305, 1995; Iwata et al., Arthritis and Rheumatism 40: 499-509, 1997; Yan et al, Biol. Chem.
  • cysteine cathepsins (like caspases) play a critical role in a number of diseases, they are increasingly becoming attractive targets for numerous therapeutic agents.
  • cathepsin expresion involves the use of peptidic or synthetic cathepsin inhibitors which bind to (and as a result, inhibit the activity of) cysteine cathepsins.
  • cysteine cathepsin inhibitors are used in vitro in scientific research and drug development screening.
  • cathepsin inhibitors such as Z-FA-FMK (for cathepsin B) are widely available and have been used extensively for in vitro applications.
  • more potent cathepsin inhibitors may prove valuable for in vitro testing, in that they may allow for the use of less product and potentially shorten test duration.
  • cysteine cathepsins in vivo for therapeutic purposes is a continuing effort, with several commercial projects currently focusing on inhibition of cathepsin B, L, S or K for various clinical applications such as neuropathic pain and Alzheimers, liver fibrosis (associated with HCV, non-alcoholic steatohepatititis or "NASH", alcoholic steatohepatitis, non-alcoholic fatty liver disease), cirrhosis, and various conditions associated with metabolic disease.
  • HCV non-alcoholic steatohepatititis or "NASH”
  • alcoholic steatohepatitis non-alcoholic fatty liver disease
  • cirrhosis various conditions associated with metabolic disease.
  • cysteine cathepsins are also upregulated in cells within the tumor environment, such as tumor associated macrophages (TAMs).
  • TAMs tumor associated macrophages
  • Cathepsin inhibitors which can also target upregulation of cathepsins in the tumor microenvironment might someday represent a viable approach to cancer prevention or treatment, and cathepsin inhibitor compounds designed to target both the tumor and tumor microenvironment have already been proposed (ref: Mikhaylov, et al. Ferri- Liposomes as a Novel MRI- Visible Drug Delivery System for Targeting Tumours and their Microenvironment, Nat.
  • Cathepsin inhibitors have also been used as anti-viral agents. Examples of cathepsin inhibitors used to block virus replication were illustrated in Van der Linden, et al., Cysteine Cathepsins as Anti-Ebola Agents. ACS Infect. Dis., 2016, 2 (3), pp 173-179 and in US2009/0203629A1 (Hepatitis C related). Recent studies suggest that blocking cathepsin activity may not only address the inflammation and tissue injury associated with some viruses such as HCV, but also the overall viral burden itself.
  • cathepsin inhibitors As with caspase inhibitors, there is a need for safe, stable, selective, cell-permeant, cathepsin inhibitors with increased potency (i.e. enhanced cathepsin binding affinity and kinetics), suitable for both in vitro and in vivo applications.
  • the present invention provides compositions and methods for caspase and cysteine cathepsin inhibition.
  • a new class of highly potent, cell membrane permeant, anti- apoptotic and/or anti-inflammatory peptide based caspase and cathepsin inhibitors is provided.
  • the compounds of this invention are capable of forming irreversible covalent bonds to the active site of a caspase or cysteine cathepsin and inhibiting the activity of that enzyme.
  • HTS high throughput screening
  • HTS high throughput screening
  • Another potential application is the inhibition of caspase or cysteine cathepsin activity in a cell-free system by adding any one of the compounds described in the invention to the purified caspase(s) or cathepsin(s) that it targets.
  • Another potential application is in the treatment of a variety of mammalian disease states or conditions associated with an increase in cellular apoptosis and/or inflammation, including (but not limited to) myocardial infarction, stroke, traumatic brain injury, fulminant liver destruction, endotoxic shock, sepsis, septic shock, chronic hepatitis (including virus related hepatitis, alcoholic steatohepatitis and non-alcoholic steatohepatitis or "NASH”), pancreatitis, viral infection, fibrosis, implant or transplant rejection, auto-immune diseases, arthritis, neurological conditions (e.g. Alzheimer's Disease), cancer, and ototoxicity.
  • myocardial infarction stroke, traumatic brain injury, fulminant liver destruction, endotoxic shock, sepsis, septic shock, chronic hepatitis (including virus related hepatitis, alcoholic steatohepatitis and non-alcoholic steatohepatitis or "NASH
  • the invention provides a compound of formula (I):
  • Ui is absent or is a fluorinated group comprising 1-10 carbon atoms and one or more fluorine atoms
  • U 2 is absent or is a fluorinated group comprising 1 -10 carbon atoms and one or more fluorine atoms; wherein at least one of Ui and U 2 is present;
  • X is a molecule that will recognize a cysteine protease
  • J is a reactive group that binds to a cysteine protease.
  • One aspect of the invention concerns a method for preventing and/or treating a caspase- mediated or cathepsin-associated disease or condition in a subject in need thereof, comprising administering to said subject an effective amount of a compound represented by any of the above molecules.
  • Another related aspect of the invention concerns the use of a compound represented by any of the above molecules for the manufacture of a medication for the prevention and/or treatment of caspase-mediated or cathepsin-associated diseases or conditions in a subject in need thereof.
  • One aspect of the invention concerns a method of treating excessive apoptosis or inflammation affected by caspase activity in a cell or a tissue, the method comprising: contacting the cell or tissue with an effective amount of one or more compounds represented by any of the above formulas.
  • a final aspect of the invention concerns the use of a compound represented by any of the above molecules to inhibit caspase or cathepsin activity in a cell-free system.
  • FIG. 1 TFA-VAD(OMe)-FMK vs Z-VAD(OMe)-FMK vs Q-VD-OPH, Percent Inhibition of Caspase 3 at Varying Inhibitor Concentration.
  • Competition Assay using active Caspase-3 and Ac- V AD- AFC Enzo Lifesciences. Fluorescence kinetic reads performed with a Microplate Reader (M2e, Molecular Devices).
  • FIG. 3 Structure of TFA-VAD(OMe)-FMK (aka Trifluoroacetyl-L-valyl-L-alanyl-L- aspartic acid methyl ester fluoromethyl ketone).
  • TFA-VAD(OMe)-FMK is more potent at inhibiting staurosporine-induced caspase activity compared to Z-VAD(OMe)-FMK and Q-VD(OMe)-FMK.
  • Inhibitors (10 uM) were added to Jurkat cells (human T lymphocyte cell line) for 15 minutes. 1 uM Staurosporine (protein kinase inhibitor) was then added for 3.5 hours to induce apoptosis. After 3.5 hours, CAS-MAP active caspase labeling reagent (FAM-VAD(OMe)-FMK) was added for 20 minutes. Cells were then analyzed by flow cytometry. An increase in FAM-VAD-FMK fluorescence intensity correlates with caspase activity.
  • TFA-VAD(OMe)-FMK is more potent than Z-VAD(OMe)-FMK at inhibiting apoptosis.
  • Jurkat cells were incubated with the indicated concentrations of TFA-VAD(OMe)- FMK or Z-VAD(OMe)-FMK for 15 minutes prior to stimulation with 1 ⁇ Staurosporine for 4 hours, 5 ⁇ Camptothecin (topoisomerase I inhibitor) for 4 hours or 100 ng/ml anti-TRAIL R2 agonist antibody for 24 hours.
  • Cells were labeled with Annexin V - Alexa Fluor 488 and a cell impermeable DNA dye (SYTOX AADvanced) and analyzed by flow cytometry.
  • TFA-VAD(OMe)-FMK inhibits staurosporine-induced apoptosis after 24 hours in cell culture.
  • Jurkat cells were incubated with 10 ⁇ TFA-VAD(OMe)-FMK or 10 ⁇ TFA-VAD(OMe)-FMK for 24 hours in RPMI 1640 media with 10% FBS. After 24 hours, cells were stimulated for 4 hours with 1 ⁇ staurosporine to induce apoptosis followed by labeling with Annexin V - Alexa Fluor 488 and a cell impermeable DNA dye (SYTOX AADvanced). Stained cells were analyzed by flow cytometry. % Apoptosis was calculated by adding the percentage of cells in the Annexin V positive/SYTOX negative and Annexin V positive/SYTOX positive populations.
  • TFA-VAD(OMe)-FMK, TFA-VAD(OMe)-TPH, TFA-VD(OMe)-TPH, TFA- EVD-TPH and TFA-6E8D-TPH inhibit caspase-3/7 activity in Hep G2 cells (human hepatocyte carcinoma cell line).
  • Hep G2 cells were incubated with the indicated concetrations of caspase inhibitor for 15 minutes prior to a 24 hour treatment with 2 ⁇ g/ml anti-TRAIL R2 agonist antibody to induce caspase activity.
  • a caspase-3/7 specific fluorescent substrate MP39 was added to each sample in a duel function cell lysis/caspase activity buffer.
  • TFA-VAD(OMe)-FMK, TFA-VAD(OMe)-TPH, TFA-VD(OMe)-TPH, TFA- EVD-TPH and TFA-6E8D-TPH inhibit staurosporine-induced apoptosis in Jurkat cells.
  • Jurkat cells were incubated with 5 ⁇ of the indicated caspase inhibitor for 15 minutes prior to stimulation with 1 ⁇ staurosporine for 4 hours.
  • EVD-TPH and TFA-6E8D-TPH inhibit anti-TRAIL R2 antibody-induced apoptosis in Jurkat cells.
  • Jurkat cells were incubated with the indicated concentrations of caspase inhibitor for 15 minutes prior to stimulation with 100 ng/ml anti-TRAIL R2 agonist antibody for 24 hours.
  • Cells were labeled with Annexin V - Alexa Fluor 488 and a cell impermeable DNA dye (SYTOX AADvanced) and analyzed by flow cytometry.
  • FIG. 10 RAW 264.7 cells were incubated with TFA-FK-TPH, TFA-FR-TPH or E64d at the indicated concentrations for 1 hour prior to a 2 hour incubation with 1 ⁇ BMV109. Cells were then collected, lysed in hypotonic lysis buffer and protein concentrations were determined. 50 ⁇ g of cell lysate was separated by SDS-PAGE on 15% Mini-PROTEAN TGX precast gels (Bio Rad). Gels were scanned using a Typhoon FLA 9500 (Cy5).
  • the present disclosure relates to cysteine protease inhibitors that covalently bind to cysteine proteases (e.g. active caspases or cathepsins), and methods of using such inhibitors to block caspase-mediated apoptosis, caspase-mediated inflammation, and cathepsin expression associated with any of the cathepsin related conditions mentioned herein.
  • the present disclosure further relates to kits comprising the present cysteine protease inhibitors and instructions for their use.
  • F is a small and dense atom, only about 10% bigger in diameter than hydrogen and fluorine-containing molecules are hydrophobic.
  • biological sample refers to any type of material of biological origin, including but not limited to a blood sample, tissue sample, cell suspension, cellular extract, or tissue homogenate.
  • cysteine protease includes enzymes that degrade proteins using a nucleophilic cysteine thiol.
  • the cysteine protease is a caspase or cysteine cathepsin.
  • the cysteine protease is a caspase.
  • the cysteine protease is a cysteine cathepsin.
  • in vitro refers to processes or procedures performed on a biological sample outside a living organism.
  • in vitro administration includes administering (i.e., delivering, applying, etc.) a cysteine protease inhibitor to a biological sample that is outside a living organism.
  • m vivo refers to processes or procedures performed inside a living, multicellular organism.
  • in vivo administration includes administering a cysteine protease inhibitor to a living subject (e.g., a mammal, such as a human).
  • incidence refers to the occurrence rate, frequency of an event, or the quantifiable degree to which an event occurs.
  • incubating when used with respect to incubating a sample with a cysteine protease inhibitor, refers to exposure conditions (e.g., time, temperature, pH, etc.) sufficient for the formation of caspase-inhibitor or cathepsin-inhibitor complexes.
  • N-terminal group refers to a moiety attached to the N- terminal position of the recognition sequence of the cysteine protease inhibitor.
  • the N-terminal group in the caspase inhibitors (and certain cathepsin inhibitors) of this invention is a
  • C-terminal group refers to a moiety attached to the C-terminal position of the recognition sequence of the cysteine protease inhibitor.
  • reactive group that binds to a cysteine protease includes groups that are capable of interacting with or reacting with the catalytic site of a cysteine protease.
  • a reactive group is capable of interacting with or reacting with the catalytic site of a caspase or cysteine cathepsin. In one embodiment, a reactive group is capable of forming a covalent bond with a cysteine protease. In one embodiment, a reactive group is capable of forming a covalent bond with a caspase or cysteine cathepsin. In one embodiment, a reactive group is a leaving group that can be displaced to form a covalent bond. In one embodiment, for example, the reactive group can be FMK, PMK, OPH, or TPH.
  • the reactive group can be a halogen atom, a substituted or unsubstituted phenol group, a substituted or unsubstituted benzoylate group, a substituted or unsubstituted BMK group, a substituted or unsubstituted FMK group, and a substituted or unsubstituted PMK group.
  • the reactive group can be a halogen atom (e.g. F or CI) or a (C 1 -C4) alkyl group that is substituted with one or more halogen atoms.
  • the reactive group can be a portion of a cysteine protease inhibitor that covalently binds to the active catalytic site of a cysteine protease.
  • recognition sequence refers to a portion of the cysteine protease inhibitors comprising a sequence of 1 to 8 natural or unnatural amino acids or synthetic analogs thereof, which is selective for one or more cysteine proteases.
  • amino acid refers to a (chemically) substituted natural amino acid, an unsubstituted unnatural amino acid, or a substituted unnatural amino acid.
  • unnatural amino acid refers to an amino acid (or conformation thereof) not normally found in nature (i.e. not found in the genetic code of any organisms, not encoded into proteins). Synthetic analogs are a type of unnatural amino acid.
  • substituted means the group is substituted with one or more (e.g., 1 , 2, 3, 4 or 5) substituents independently selected from halo, cyano, nitro, carboxy, (C 1 -C 4 ) alkyl, (C 1 -C 4 ) haloalkyl, (C 1 -C 4 ) alkoxy, (C] -C 4 ) haloalkoxy, (Ci-C 4 ) alkanoyl, (CrC 4 ) alkoxycarbonyl, (CrC 4 ) alkanoyloxy, amino, (CrC 4 ) alkylamino, and ((C ! -C 4 )alkyl) 2 amino.
  • substituents independently selected from halo, cyano, nitro, carboxy, (C 1 -C 4 ) alkyl, (C 1 -C 4 ) haloalkyl, (C 1 -C 4 ) alkoxy, (C] -C 4 )
  • AOMK acyloxymethyl ketone
  • BMK benzoyloxymethyl ketone
  • DE L-aspartyl-L-glutamic acid
  • FK phenylalyl lysine
  • LAD L-leucyl-L-alanyl-L-aspartic acid
  • TD L-threonyl-L-aspartic acid
  • VAD L-valyl-L-alanyl-L-aspartic acid
  • VD L-valyl-L-aspartic acid
  • the N-terminal group may serve to protect the recognition sequence during synthesis of a cysteine protease inhibitor.
  • an N-terminal protecting group may be present during synthesis of an intermediate precursor of a cysteine protease inhibitor, but may be removed and replaced with a different N-terminal moiety - i.e., the N-terminal group of a cysteine protease inhibitor intermediate may be different from the N-terminal group of a final cysteine protease inhibitor.
  • the N-terminal group on final caspase inhibitors and some final cysteine cathepsin inhibitors described in this invention is a trifluoroacetyl (TFA) group.
  • the trifluoroacetyl (TFA) group is located on the C-terminal end of the Recognition Sequence. It is believed that a TFA group on the N-Terminal or C-Terminal position of a peptide based cysteine protease inhibitor enhances binding to the enzyme and/or enhances cell membrane permeability of the cysteine protease inhibitor.
  • Each recognition sequence comprises one or more natural or unnatural, synthetically modified amino acids and is able to bind to one or more caspases or cysteine cathepsins.
  • amino acids of the recognition sequence may be L, D, or D/L racemates.
  • a recognition sequence may allow the cysteine protease inhibitor to target structurally similar caspases or cysteine cathepsins with the same or different affinities and kinetics.
  • the recognition sequence VAD valine-alanine-aspartic acid
  • VAD valine-alanine-aspartic acid
  • caspase inhibitors containing the VAD recognition sequence will covalently bind to all or most active caspases.
  • the caspase inhibitor is designed to react with multiple caspases to allow inhibition of all or most caspases.
  • the FK recognition sequence is designed to react with multiple cysteine cathepsins (B/L/S/X/Z). Accordingly, the FK containing cysteine cathepsin inhibitors described herein are considered "poly" cysteine cathepsin inhibitors.
  • the caspase inhibitor is designed to react selectively with caspase- 1, thus allowing for detection of inflammation in particular.
  • caspase inhibitors containing the recognition sequence WEHD (SEQ ID NO:l) or YVAD (SEQ ID NO:2) will bind covalently to caspase-1 over other apoptosis-associated caspases.
  • the caspase inhibitor is designed to react with two caspases that have similar substrate selectivity.
  • caspase inhibitors containing the recognition sequence DEVD SEQ ID NO:3 will recognize and allow detection of both caspase-3 and caspase-7.
  • the caspase inhibitor recognition sequence is selected from inhibitor recognition sequences listed in Table 1.
  • Table 1 identifies well known recognition sequences of caspase inhibitors disclosed herein and their corresponding ability to selectively bind to all or most active caspases (i.e., poly-caspase), inflammation-related active caspases (e.g., caspase-1), or apoptosis-related active caspases (e.g., caspase-3/7 or caspase-8).
  • the molecule that will recognize a cysteine protease is selected from:
  • Examples of synthetic or "unnatural” amino acids include (but are not limited to):
  • the cysteine cathepsin inhibitor recognition sequence is FK, FR, GGR (including synthetic analogs thereof). Reactive Group - Binding Mechanism to Active Caspase (Illustrative Example)
  • a multi-step binding mechanism results in the formation of a covalent bond between the reactive group and the -SH moiety of the cysteine residue in the active catalytic site of the caspase.
  • the caspase inhibitor binds irreversibly and covalently to the caspase.
  • the caspase inhibitors comprise a reactive group that enables the inhibitor to covalently bind to an active caspase.
  • the reactive group includes a moiety that leaves as the reactive group reacts with the active caspase.
  • the reactive group can be tailored to recognize and bind to specific types of caspases, such as caspases associated with apoptosis, caspases associated with inflammation, or caspases associated with both.
  • a representative example of the multi-step binding mechanism is illustrated below for caspase inhibitors that comprise a ketone that reacts with the cysteine residue in the catalytic site of an active caspase.
  • This same general mechanism applies to cysteine cathepsin inhibitors described in this invention.
  • the epoxysuccinyl group irreversibly binds to an active thiol group on target cysteine cathepsins to form a thioether linkage.
  • the caspase inhibitors (and select cysteine cathepsin inhibitors) of the present disclosure have the following general structure: (U)-X-J-(U), where U must be present on at least one end of the molecule and may be attached directly or indirectly (i.e. via linker) to the C-terminal and/or N-terminal end of the molecule, and where U is a fluorinated group or perfluoroalkyl group comprising 1-10 carbon atoms and one or more fluorine atoms (e.g. a trifluoroacetyl (TFA) group); X is any molecule that will recognize a cysteine protease (e.g.
  • J is any reactive group that binds to a caspase or cysteine cathepsm (e.g. a halo-methyl ketone such as fluoro-methyl ketone (FMK), a phenoxy-methyl ketone (PMK), or a substituted phenoxy-methyl ketone (such as a 2,3,5,6 tetrafluoro phenoxy- methyl ketone a.k.a. "TPH” or a 2,6 difluoro phenoxy-methyl ketone a.k.a. "OPH”), an acyloxy- methyl ketone a.k.a. "AOMK” or an epoxide).
  • a halo-methyl ketone such as fluoro-methyl ketone (FMK), a phenoxy-methyl ketone (PMK), or a substituted phenoxy-methyl ketone (such as a 2,3,5,6 tetrafluoro phenoxy- methyl ketone
  • the caspase inhibitors of the present disclosure are further described by the following formula:U-X-NH-CH(R)-CO-CH 2 -J,where U is any fluorinated group or perfluoroalkly group (e.g. a trifluoroacetyl (TFA) group), X is any molecule that will recognize an active caspase (e.g.
  • R is CH2- C02-CH3 for live cells and in vivo applications or CH2-C02-H for cell-free systems
  • J is any molecule that will react with active caspases; for example, a halo-methyl ketone such as fluoro-methyl ketone (FMK), a phenoxy-methyl ketone (PMK), or a substituted phenoxy-methyl ketone (such as a 2,3,5,6 tetrafluoro phenoxy-methyl ketone a.k.a. "TPH” or a 2,6 difluoro phenoxy-methyl ketone a.k.a. "OPH”), an acyloxy-methyl ketone a.k.a. "AOMK” or an epoxide).
  • FMK fluoro-methyl ketone
  • PMK phenoxy-methyl ketone
  • PH 2,6 difluoro phenoxy-methyl ketone
  • U comprises the N-terminal group
  • X-NH-CH(R)-CO-CH 2 comprises the recognition sequence
  • J comprises the reactive group
  • X is selected from one or more naturally occurring amino acids, one or more synthetic amino acids and combinations thereof.
  • X may be one or more amino acids selected from alanine, aspartic acid, glutamic acid, histidine, glycine, arginine, lysine, methionine, proline, isoleucine, phenylalanine, leucine, threonine, tryptophan, valine, tyrosine, glutamine, serine, pyrrolysine, selenocysteine, norvaline, norleucine and synthetic analogs thereof (where applicable).
  • J is capable of binding at least one caspase selected from caspase- 1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase- 10, caspase- 1 1, caspase- 12, caspase- 13 and caspase- 14.
  • J may be selected from BMK, FMK, PMK, OPH, TPH and AOMK.
  • J is capable of binding at least one caspase selected from caspase- 1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase- 10, caspase- 1 1, caspase- 12, caspase- 13 and caspase- 14.
  • J may be selected from FMK, PMK, OPH, TPH and AOMK.
  • J is capable of binding to at least one cysteine cathepsin selected from Cathepsins B, C, F, H, K, L, O, S, W, X/Z. J may be selected from FMK, PMK, OPH, TPH and AOMK.
  • J is selected from a halogen, a phenol group, a benzoylate group, a BMK group, an FMK group, and a PMK group, in which the groups mentioned herein may be substituted or unsubstituted.
  • J is selected from BMK, FMK, and PMK.
  • the J halogen is F, CI, or Br.
  • the J phenol is a compound of Formula A:
  • X 1? X 2 , X 3 , X 4 , and X 5 is each individually selected from H, F, CI, alkyl, aryl, aralkyl, amino, nitro, and carboxy.
  • at least one of Xj, X 2 , X 3 , X , and X5 is H.
  • alkyl is C 1-10 alkyl, for example, Ci -6 alkyl.
  • the J benzoylate is a compound of Formula B:
  • X 1? X 2 , X 3 , X 4 , and X 5 is each individually selected from H, F, CI, alkyl, aryl, aralkyl, amino, nitro, or carboxy.
  • at least one of X], X 2 , X 3 , X 4 , and X 5 is H.
  • alkyl is C 1-10 alkyl, for example, Ci -6 alkyl.
  • the cysteine protease inhibitor has a molecular weight of about 300- 1500 Daltons.
  • the cysteine protease inhibitors are typically cell permeant (i.e., exhibit good cell membrane permeability) and can typically selectively target cysteine proteases (i.e. caspases and cysteine cathepsins) of interest inside the cells of a living organism or a biological sample.
  • cysteine proteases i.e. caspases and cysteine cathepsins
  • the cysteine protease inhibitors do not undergo facile metabolism, and possess a long half-life (e.g., more than 6 hours) throughout the life of the permeated cell.
  • each cysteine protease inhibitor forms a metabolite having the formula U-X-NH-CH(R)-CO-CH 2 -S-Cys-Enzyme, wherein U, X, and R are as defined above.
  • caspase inhibitors of the present disclosure includes, but is not limited to TFA-VAD(OMe)-FMK:
  • cysteine cathepsin inhibitors of the present disclosure include, but are not limited to:
  • cysteine protease inhibitors examples include, but are not limited to: eye, breast, heart, brain and central nervous system (CNS), kidneys, lungs, liver, skin, pancreas, skeletal system, connective tissue (e.g., joints), stomach, upper gastrointestinal tract, lower gastrointestinal tract, circulatory system (e.g., blood), lymphatic system, sexual organs (male and female), prostate, embryologic tissue, muscular system, and gallbladder.
  • the cysteine protease inhibitor is delivered in vitro to a biological sample (e.g., a blood sample, tissue sample, cell suspension, cellular extract, or tissue homogenate) by direct application of the inhibitor to the sample.
  • a biological sample e.g., a blood sample, tissue sample, cell suspension, cellular extract, or tissue homogenate
  • the cysteine protease inhibitors exhibit good cell permeability, there is no need to use additional reagents to facilitate the entry of the inhibitors into cells.
  • the cysteine protease inhibitor is reconstituted in DMSO, further diluted in phosphate buffered saline (PBS) or cell culture media and applied directly to cells in a cell culture dish.
  • PBS phosphate buffered saline
  • the cysteine protease inhibitor is allowed to incubate with the cells under the conditions sufficient for the formation of caspase-inhibitor complexes.
  • the cysteine protease inhibitor is administered in vivo to a subject (e.g., animal or human) intravenously, intraperitoneally, intramuscularly, subcutaneously, topically, and/or by direct application to a target organ.
  • the cysteine protease inhibitor is diluted with a suitable excipient (in some cases a pharmaceutically acceptable excipient) and administered in an effective amount, which is an amount that is sufficient to provide meaningful results with respect to the intended purpose - e.g., therapeutic, drug development, etc.
  • Suitable excipients include, but are not limited to, adhesives, binders, bulking agents, carriers, colors, diluents, disintegrating agents, fillers, glidants, granulating agents, lubricating agents, polymers, preservatives, wetting agents, and combinations thereof.
  • One or more excipients may be selected from sucrose, lactose, cellulose, methyl cellulose, gelatin, polyvinylpyrrolidone, polyethylene glycol and water. Kits
  • kits of the present disclosure are used for blocking apoptotic activity and/or conditions associated with inflammatory activity in a biological sample.
  • the kit may comprise one or more cysteine protease inhibitors.
  • the kit may further include packaging materials with instructions for using the components of the kit, such as how to use the cysteine protease inhibitors provided in the kit, storage conditions, etc.
  • Components of the kit may be provided in separate containers (e.g., vials) or combined.
  • the invention relates to a kit containing instructions for use and at least one cysteine protease inhibitor compound of the following formula: (U)-X-J-(U), where at least one U must be present and where U may be attached either directly or indirectly (i.e. via linker) to the C-terminal or N-terminal end of the molecule, and where U is a fluorinated group or perfluoroalkyl group comprising 1-10 carbon atoms and one or more fluorine atoms (e.g. a trifluoroacetyl (TFA) group);
  • X is any molecule that will recognize a cysteine protease (e.g.
  • J is any reactive group that binds to a caspase or cysteine cathepsin (e.g. a halo-methyl ketone such as fluoro-methyl ketone (FMK), a phenoxy-methyl ketone (PMK), or a substituted phenoxy-methyl ketone (such as a 2,3,5,6 tetrafluoro phenoxy- methyl ketone a.k.a. "TPH” or a 2,6 difluoro phenoxy-methyl ketone a.k.a. "OPH”), an acyloxy- methyl ketone a.k.a. "AOMK” or an epoxide).
  • a halo-methyl ketone such as fluoro-methyl ketone (FMK), a phenoxy-methyl ketone (PMK), or a substituted phenoxy-methyl ketone (such as a 2,3,5,6 tetrafluoro phenoxy- methyl ketone
  • the invention relates to a kit containing instructions for use and at least one caspase inhibitor compound of the following formula: U-X-NH-CH(R)-CO-CH 2 -J, where U is any fluorinated group or perfluoroalkly group (e.g. a trifluoroacetyl (TFA) group), X is any molecule that will recognize an active caspase (e.g. a peptide having 1-8 amino acids or synthetic analogs thereof), R is CH2-C02-CH3 for live cells and in vivo applications or CH2- C02-H for cell-free systems, and J is any molecule that will react with active caspases, e.g.
  • U any fluorinated group or perfluoroalkly group (e.g. a trifluoroacetyl (TFA) group)
  • X is any molecule that will recognize an active caspase (e.g. a peptide having 1-8 amino acids or synthetic analogs thereof)
  • R is CH2-
  • a halo-methyl ketone such as fluoro-methyl ketone (FMK), a phenoxy-methyl ketone (PMK), or a substituted phenoxy-methyl ketone (such as a 2,3,5,6 tetrafluoro phenoxy-methyl ketone ("TPH”) or a 2,6 difluoro phenoxy-methyl ketone a.k.a. "OPH”), an acyloxy-methyl ketone a.k.a. "AOMK” or an epoxide).
  • FMK fluoro-methyl ketone
  • PMK phenoxy-methyl ketone
  • TPH 2,3,5,6 tetrafluoro phenoxy-methyl ketone
  • OHPH 2,6 difluoro phenoxy-methyl ketone
  • AOMK acyloxy-methyl ketone
  • epoxide an epoxide
  • the "X" for the caspase or cysteine cathepsin inhibitor comprises at least one amino acid selected from alanine, aspartic acid, glutamic acid, histidine, glycine, arginine, lysine, methionine, proline, isoleucine, phenylalanine, leucine, threonine, tryptophan, valine, tyrosine, glutamine, serine, pyrrolysine, selenocysteine, norvaline, norleucine and synthetic analogs thereof (where applicable).
  • the compound is selected from the group consisting of:
  • the compound is selected from the group consisting of:
  • the compound is selected from the group consisting of:
  • the compound is selected from the group consisting of:
  • the compound contains an epoxide based "J" reactive group and is selected from the group consisting of:
  • the compound contains at least one cathepsin inhibitor and is selected from the group consisting of:
  • the present invention relates to a composition
  • a composition comprising a caspase inhibitor or cysteine cathepsin inhibitor and an excipient (which may be selected from sucrose, lactose, cellulose, gelatin, polyvinylpyrrolidone, trehalose, cyclodextrin, hyaluronic acid, polyethylene glycol, DMSO and water.
  • an excipient which may be selected from sucrose, lactose, cellulose, gelatin, polyvinylpyrrolidone, trehalose, cyclodextrin, hyaluronic acid, polyethylene glycol, DMSO and water.
  • the present invention relates to a method of inhibiting apoptosis and/or inflammation in a living organism by administering in vivo a caspase inhibitor or cathepsin inhibitor to the living organism.
  • the present invention relates to a method of inhibiting apoptosis and/or inflammation in a cell population by administering in vitro a caspase inhibitor or cathepsin inhibitor to a biological sample and incubating the sample with the inhibitor under conditions sufficient to form caspase-inhibitor complexes.
  • the biological sample may be selected from a blood sample, tissue sample, cell suspension, cellular extract, or tissue homogenate.
  • the compound is a compound of formula (la): UrX-J (la).
  • the compound is a compound of formula (lb):
  • X is a molecule that will recognize a caspase or cysteine cathepsin.
  • X is a peptide that contains 1-8 amino acids.
  • X is a peptide that contains 2-5 amino acids.
  • X is a peptide selected from the group consisting of: VD, VAD, EVD, D-3-V-D, L-E-H-D (SEQ ID NO:9), L-E-T-D (SEQ ID NO: 10), D-E-V-D (SEQ ID NO:3), D-E-P-D (SEQ ID NO:7), D-29-V-D, D-34-V-D, 26-34-V-D, 26-3-V-D, 26-E-V-D, 31- E-T-D, 31-E-23-D, 29-E-T-D, 6-E-8-D, D-E-1 1-D, D-30-11-D, D-30-V-D, P-L-A-D (SEQ ID NO:8), I-L-A-D (SEQ ID NO:l 1), I-L-38-D, I-F-P-D (SEQ ID NO:12), D-3-V-D, D-34-V-D,
  • J is selected from the group consisting of AMOK, BMK, FMK, OPH, PMK, and TPH.
  • J is selected from the group consisting of AMOK, OPH, PMK, and TPH.
  • J is TPH.
  • the compound is selected from the group consisting of: TFA- EVD-TPH; TFA-6E8D-TPH; TFA-VAD(OMe)-TPH; TF A- VD(OMe)-TPH ; TFA-FK-TPH; and TFA-F -TPH.
  • the compound is a compound of formula (Id): (U)-X-J-(U) (Id)
  • U is present on at least one end of the molecule and consists of a fluorinated group comprising 1-10 carbon atoms and one or more fluorine atoms;
  • X is any molecule that will recognize a cysteine protease
  • J is any reactive group that binds to a caspase or cysteine cathepsin.
  • U is a trifluoroacetyl (TFA) group that may be connected directly or indirectly (i.e. via linker) to either the C-terminal end or to the N-terminal end J group.
  • TFA trifluoroacetyl
  • the compound is a compound of formula (Ie): (U)-X-NH-CH(R)-CO-CH 2 -J(U) (Ie)
  • U is a fluorinated group comprising 1-10 carbon atoms and one or more fluorine atoms
  • X is a peptide-based caspase recognition sequence comprising 1-8 amino acids;
  • R is -CH 2 C0 2 CH 3 , -CH 2 C0 2 H;
  • J is any reactive group that binds to a caspase.
  • U is a trifluoroacetyl (TFA) group that may be connected directly or indirectly (i.e. via linker) to either the C-terminal end or to the N-terminal end J group.
  • TFA trifluoroacetyl
  • X comprises at least one amino acid selected from alanine, aspartic acid, glutamic acid, histidine, glycine, arginine, lysine, methionine, proline, isoleucine, phenylalanine, leucine, threonine, tryptophan, valine, tyrosine, glutamine, serine, pyrrolysine, selenocysteine, norvaline, norleucine and synthetic analogs thereof (where applicable).
  • X comprises VAD(OMe), VD(OMe), VAD, VD,
  • X is VAD(OMe), VD(OMe), VAD, VD, E(OMe)VD(OMe), EVD, 6E8D, 6E(OMe)8D(OMe), FK, FR, GGR, WE(OMe)HD(OMe), IE(OMe)TD(OMe), D(OMe)E(OMe)VD(OMe), LE(OMe)HD(OMe), LE(OMe)TD(OMe), YVAD(OMe),
  • VD(OMe)VAD(OMe) or any of the following: Asp-3,4 Difluorophenylalanine- Val-Asp, Asp- Pentafluorophenylalanine- Val-Asp, (2-Oxoacetamido)(Propanamido)-4-Oxo-Pentanoic Acid, (Oxiran-2-yl) Carbonyl-L-Leucyl-3-(p-Hydroxyphenyl) Ethylamide.
  • J is any molecule that will react with active cysteine proteases, e.g.a halomethyl ketone (such as CMK, FMK), a substituted or unsubstituted phenoxymethyl ketone (PMK), an acyloxymethyl ketone (AOMK), or an epoxide.
  • active cysteine proteases e.g. a halomethyl ketone (such as CMK, FMK), a substituted or unsubstituted phenoxymethyl ketone (PMK), an acyloxymethyl ketone (AOMK), or an epoxide.
  • the compound is selected from:
  • TFA-GGR-TPH TFA-FK-TPH TFA-FR-TPH TFA-GGR-AOMK TFA-FK-AOMK TFA-FR-AOMK and the following compounds:
  • the compound is a compound of formula (If):
  • X is a peptide that will recognize a caspase or cysteine cathepsin.
  • the compound is a compound of formula (If):
  • X is a peptide that contains 1-8 amino acids.
  • the compound is a compound of formula (If):
  • X is a peptide that contains 2-5 amino acids.
  • the compound is a compound of formula (If):
  • X is a peptide selected from the group consisting of: E(OMe)-V-D, E(OMe)-V- D(OMe), E-V-D(OMe), 6-E(OMe)-8-D, 6-E(OMe)-8-D(OMe), 6-E-8D(OMe), V-A-D(OMe).
  • X is a peptide selected from the group consisting of:
  • E(OMe)-V-D E(OMe)-V-D(OMe), E-V-D(OMe), 6-E(OMe)-8-D, 6-E(OMe)-8-D(OMe), 6-E- 8D(OMe), V-A-D(OMe).
  • X is a peptide selected from the group consisting of:
  • X is a peptide selected from the group consisting of:
  • J is acyloxymethyl, benzoyloxymethyl, fluoromethyl, 2,6- difluorophenoxymethyl, phenoxymethyl, or 2,3,5,6-tetrafluorophenoxymethyl, which form AMOK, BMK, FMK, OPH, PMK, or THP, respectively, when attached to the carboxy carbonyl group of X in a compound of the invention.
  • a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable excipient, and optionally a therapeutic agent.
  • the excipient comprises sucrose, lactose, cellulose, gelatin, polyvinylpyrrolidone, trehalose, cyclodextrin, hyaluronic acid, polyethylene glycol or water.
  • the therapeutic agent is a cancer-targeted chemotherapeutic agent.
  • a method for inhibiting a cysteine protease in vitro or in vivo comprises contacting the cysteine protease with a compound of the invention.
  • a method for inhibiting a cysteine protease in vitro or in vivo comprises contacting the caspase with a compound of the invention.
  • the compound inhibits the cysteine protease by forming an irreversible covalent bond to the active site of a cysteine protease.
  • a method for treating a disease or condition associated with an increase in cellular apoptosis and/or inflammation in a subject comprises administering to the subject a compound of the invention.
  • the disease or condition is myocardial infarction, stroke, traumatic brain injury, spinal atrophy, auto-immune diseases (e.g.
  • liver disease such as chronic hepatitis (including virus-related hepatitis, alcoholic steatohepatitis and non-alcoholic steatohepatitis or "NASH"), pancreatitis, arthritis, viral infection, metabolic disease, cancer, implant or transplant rejection,
  • chronic hepatitis including virus-related hepatitis, alcoholic steatohepatitis and non-alcoholic steatohepatitis or "NASH”
  • pancreatitis arthritis
  • viral infection metabolic disease
  • cancer implant or transplant rejection
  • neurodegenerative diseases e.g. multiple sclerosis
  • pulmonary fibrosis pulmonary fibrosis
  • cardiac fibrosis pulmonary fibrosis
  • renal fibrosis pulmonary fibrosis
  • liver fibrosis cirrhosis or ototoxicity.
  • a method for inhibiting apoptosis and/or inflammation in another embodiment, a method for inhibiting apoptosis and/or inflammation
  • a subject e.g. animal or human
  • administering comprises administering to the subject a compound of the invention.
  • a method for inhibiting apoptosis and/or inflammation in a cell population comprises administering in vitro a compound of the invention to a biological sample to provide a resulting sample, and incubating the resulting sample under conditions sufficient to form caspase-inhibitor or cysteine cathepsin-inhibitor complexes.
  • the biological sample comprises blood sample, tissue, a cell suspension, a cellular extract, or a tissue homogenate.
  • a method for preventing and/or treating a caspase-mediated or cysteine cathepsin-associated disease or condition in an subject comprises administering to the subject a compound of the invention.
  • a kit comprising:
  • a kit comprising:
  • a method of inhibiting caspase or cysteine cathepsin activity in a cell-free system comprises adding a compound of the invention to the purified caspases.
  • the compounds of the invention e.g. the compounds of formula
  • N-AM-G-F 6 V-AD-BMK N-AM-G-F 6 V-AD-FMK
  • BODIPY-FL-D(OMe)E(OMe)VD(OMe)-FMK BODIPY-FL-IE(OMe)TD(OMe)-FMK
  • BODIPY-FL-LE(OMe)HD(O e)-FMK BODIPY-FL-LE(OMe)TD(OMe)-FMK
  • BODIPY-FL-VAD(OMe)-FMK BODIPY-FL-D(OMe)E(OMe)VD(OMe)-FMK
  • BODIPY-FL-D(OMe)E(OMe)VD(OMe)-PMK BODIPY-FL-IE(OMe)TD(OMe)-PMK, BODIPY-FL-LE(OMe)HD(OMe)-PMK, BODIPY-FL-LE(OMe)TD(OMe)-PMK, BODIPY-FL-VAD(OMe)-PMK,
  • BODIPY-FL-VD(OMe)VAD(OMe)-PMK BODIPY-FL-F 6 V-AD(OMe)-PMK
  • BODIPY-FL-D(OMe)E(OMe)VD(OMe)-BMK BODIPY-FL-IE(OMe)TD(OMe)-BMK
  • TFMCBZ-GWE(OMe)HD(OMe)-FMK TFMCBZ-GWE(OMe)HD(OMe)-BMK
  • TFMCBZ-GWE(OMe)HD(OMe)-PMK TFMCBZ-GWE(OMe)HD(OMe)-OPH
  • TFMCBZ-GYVAD(OMe)-FMK TFMCBZ-GYVAD(OMe)-BMK
  • TFMCBZ-GYVAD(OMe)-PM TFMCBZ-GYVAD(OMe)-OPH
  • the caspase inhibitors can be synthesized using liquid phase peptide synthesis (Bodanszky, PRINCIPLES OF PEPTIDE SYNTHESIS (1993) or solid phase peptide synthesis (Merrifield, J Amer Chem Soc 85(14):2149-54 (1963); Amblard et al., Mol. Biotechnol., 33(3):239-54 (2006)).
  • a suitable liquid phase peptide synthetic pathway is shown below, and generally involves building oligo peptides from the N-terminus of an amino acid by providing a first amino acid (1); protecting or blocking the N terminus of the amino acid using a protecting or blocking group (e.g., BOC or FMOC); coupling the first amino acid with a C-terminal ester of a second amino acid (2) by reacting the amino acids in the presence of a coupling agent (e.g., dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), usually in the presence of N-hydroxysuccinimide or 1 -hydroxybenzotriazole) to form a dipeptide (3); and deprotecting the dipeptide without removing any other protecting groups to yield the free dipeptide acid (4).
  • a coupling agent e.g., dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), usually in the presence of
  • Dipeptide (4) can be coupled to the suitably derivatized L- aspartic acid ⁇ -methyl ester to yield a desired caspase inhibitor or it can be coupled with a suitably protected amino acid to yield a fully protected tripeptide (5). If desired, by sequential deprotection of the C-terminus of 5 and analogous coupling to another suitably protected amino acid, a fully protected tetrapeptide may be constructed.
  • Synthesized caspase inhibitors disclosed herein may be in the form of a waxy material or lyophilized powder.
  • the caspase inhibitor may be prepared by finishing the peptide chain with an aspartic acid portion and adding a fluoromethyl ketone (FMK), phenoxymethyl ketone (PMK), an OPH (2,6-difluorophenoxy ketone or 2,6-difluorophenoxymethyl ketone) group, or an acyloxymethyl ketone (AOMK) to the end of the peptide chain as a leaving group that is part of the reactive group and is positioned at the C-terminus of the recognition sequence of the caspase inhibitor.
  • FMOC-L-aspartic acid ⁇ -methyl ester may be made. From these molecules, FMK/PMK/OPH/AOMK can be attached to the a-carboxylic acid via a methylene group, introduced using diazomethane chemistry.
  • Solubility 1 mg/mL in 10% Acetonitrile in H 2 0
  • TFA-VAD(OMe)-FMK is more potent than Z-VAD(OMe)-FMK and Q- VD(OMe)-OPH at inhibiting purified caspase-3.
  • Inhibitors and substrate were mixed in assay buffer (50 niM HEPES pH 7.2, 50 mM NaCl, 0.1% CHAPS, 10 mM EDTA, 5% glycerol, 10 mM DTT) in 96-well plates.
  • TFA-VAD(OMe)-FMK is more potent at inhibiting staurosporine-induced caspase activity compared to Z-VAD(OMe)-FMK and Q-VD(OMe)-FMK.
  • Inhibitors (10 ⁇ ) were added to 1 x 10 6 Jurkat cells (human T lymphocyte cell line) for 15 minutes in a total volume of 1 ml cell culture medium (RPMI + 10% FBS) at 37°C. 1 ⁇ Staurosporine (protein kinase inhibitor) was added and cells were incubated for 3.5 hours at 37°C to induce apoptosis. After 3.5 hours, CAS-MAP active caspase labeling reagent (FAM- VAD(OMe)-FMK, 0.75 ⁇ ) was added and cells were incubated for 20 minutes at 37°C to label active caspases.
  • FAM- VAD(OMe)-FMK 0.75 ⁇
  • TFA-VAD(OMe)-FM is a more potent than Z-VAD(OMe)-FMK at inhibiting apoptosis.
  • Jurkat cells (1 x 10 6 cells, 1 ml total volume in RPMI + 10% FBS) were incubated at 37°C with the indicated concentrations of TFA-VAD(OMe)-FMK or Z-VAD(OMe)-FMK for 15 minutes prior to apoptosis induction with 1 ⁇ Staurosponne (protein kinase inhibitor) for 4 hours at 37°C, 5 ⁇ Camptothecin (topoisomerase I inhibitor) for 4 hours at 37°C or 100 ng/ml anti-TRAIL R2 agonist antibody for 24 hours at 37°C.
  • Staurosponne protein kinase inhibitor
  • Camptothecin topoisomerase I inhibitor
  • annexin V binding buffer 140 mM NaCl, 2.5 mM CaC12, 10 mM HEPES pH 7.4
  • annexin V - Alexa Fluor 488 was added to each sample and cells were incubated in the dark, at room temperature for 15 minutes.
  • 1 ml of annexin V binding buffer was added to each sample followed by 1 ⁇ SYTOX AADvanced (Thermo Scientific).
  • TFA-VAD(OMe)-FMK inhibits staurosporine-induced apoptosis after 24 hours in cell culture.
  • Jurkat cells (1 x 10 6 cells, 1 ml total volume in RPMI + 10% FBS) were incubated with 10 ⁇ TFA-VAD(OMe)-FMK or 10 ⁇ Z-VAD(OMe)-FMK for 24 hours at 37°C. After 24 hours, apoptosis was induced with 1 ⁇ staurosporine for 4 hours at 37°C.
  • Cells were washed with annexin V binding buffer (140 mM NaCl, 2.5 mM CaC12, 10 mM HEPES pH 7.4) and resuspended in 100 ⁇ annexin V binding buffer.
  • annexin V binding buffer 140 mM NaCl, 2.5 mM CaC12, 10 mM HEPES pH 7.4
  • Annexin V - Alexa Fluor 488 was added to each sample and cells were incubated in the dark, at room temperature for 15 minutes. 1 ml of annexin V binding buffer was added to each sample followed by 1 ⁇ SYTOX AADvanced (Thermo Scientific). Cells were incubated in the dark, at room temperature
  • NxT acoustic focusing cytometer (488 nm excitation laser, 530 nm 30 nm band pass emission filter for annexin V- Alexa Fluor 488 and 488 nm excitation laser, 695 nm/40 nm band pass emission filter for SYTOX AADvanced).
  • % Apoptosis was calculated by adding the percentage of cells in the Annexin V positive/SYTOX negative and Annexin V positive/SYTOX positive populations. ( Figure 6)
  • TFA-VAD(OMe)-FMK, TFA-VAD(OMe)-TPH, TFA-VD(OMe)-TPH, TFA- EVD-TPH and TFA-6E8D-TPH inhibit caspase-3/7 activity in Hep G2 cells.
  • Hep G2 cells human hepatocyte carcinoma cell line
  • Inhibitors were added to the indicated concentrations and cells were incubated for 15 minutes at 37°C.
  • Caspase activity was induced by treating cells with 2 ⁇ ⁇ anti-TRAIL R2 agonist antibody for 24 hours at 37°C in a total volume of 60 ⁇ in DMEM + 10% FBS.
  • TFA-VAD(OMe)-FMK, TFA-VAD(OMe)-TPH and TFA-VD(OMe)-TPH inhibit staurosporine-induced apoptosis in Jurkat cells.
  • Jurkat cells (1 x 10 6 cells, 1 ml total volume in RPMI + 10% FBS) were incubated with 5 ⁇ of the indicated caspase inhibitor for 15 minutes prior to stimulation with 1 ⁇
  • annexin V binding buffer 140 mM NaCl, 2.5 mM CaC12, 10 mM HEPES pH 7.4
  • annexin V - Alexa Fluor 488 was added to each sample and cells were incubated in the dark, at room temperature for 15 minutes.
  • 1 ml of annexin V binding buffer was added to each sample followed by 1 ⁇ SYTOX AADvanced (Thermo Scientific).
  • TFA-EVD-TPH and TFA-6E8D-TPH inhibited apoptosis to a lesser extent than TFA-VAD-FMK, TFA-VAD-TPH and TFA-VD-TPH in staurosporine-induced Jurkat cells. ( Figure 8)
  • TFA-VAD(OMe)-FMK, TFA-VAD(OMe)-TPH, TFA-VD(OMe)-TPH, TFA- EVD-TPH and TFA-6E8D-TPH inhibit anti-TRAIL R2 antibody-induced apoptosis in Jurkat cells.
  • Jurkat Cells (1 x 10 6 cells, 1 ml total volume in RPMI + 10% FBS) were incubated with the indicated concentrations of caspase inhibitor for 15 minutes prior to stimulation with 100 ng/ml anti-TRAIL R2 agonist antibody for 24 hours at 37°C.
  • Cells were washed with annexin V binding buffer (140 mM NaCl, 2.5 mM CaC12, 10 mM HEPES pH 7.4) and resuspended in 100 ⁇ annexin V binding buffer. 5 ⁇ Annexin V - Alexa Fluor 488 (Thermo Scientific) was added to each sample and cells were incubated in the dark, at room temperature for 15 minutes.
  • annexin V binding buffer 1 ml was added to each sample followed by 1 ⁇ SYTOX AADvanced (Thermo Scientific). Cells were incubated in the dark, at room temperature for 5 minutes prior to flow cytometry analysis using a Life Technologies Attune NxT acoustic focusing cytometer (488 nm excitation laser, 530 nm/30 nm band pass emission filter for annexin V- Alexa Fluor 488 and 488 nm excitation laser, 695 nm/40 nm band pass emission filter for SYTOX
  • RAW 264.7 cells (mouse macrophage cell line) were plated in 6 well plates at 8 x 10 cells/well in DMEM + 10% FBS and allowed to grow overnight at 37°C. The next day, cells were treated with the indicated concentrations of TFA-FK-TPH, TFA-FR-TPH or E64d (a general cysteine cathepsin and calpain inhibitor) for 1 hour at 37°C followed by the addition of 1 ⁇ BMV109 (a Cy5 labeled activity based probe that binds cathepsin B, L, S and X) for 2 hours at 37°C to label active cathepsins.
  • TFA-FK-TPH TFA-FR-TPH
  • E64d a general cysteine cathepsin and calpain inhibitor
  • Representative compounds of the invention can also be prepared as illustrated below.
  • Intermediate compound 110 can be used to prepare compounds having D-TPH at the carboxy terminus. Similar intermediate compounds can be prepared to provide compounds having the functionality X-J at the carboxy terminus, wherein X is an amino acid or an appropriately protected amino acid and J has any of the values defined herein, by selection of the starting amino acid 101.
  • Solid-phase peptide synthesis can be used to prepare peptide sequences that can be incorporated in to compounds of the invention. Coupling of the carboxy terminus of a peptide sequences 111 with an intermediated of formula 110 followed by optional deprotection and optional functionalization of the N-terminus can provide compounds of the invention as illustrated below. 1. Solid-phase peptide synthesis (SPPS)

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Abstract

L'invention concerne des composés et des compositions qui inhibent une ou plusieurs protéases à cystéine (par exemple des caspases, ou des cathepsines à cystéine, ainsi que des kits comprenant de tels composés et de telles compositions. L'invention concerne également des méthodes thérapeutiques et des procédés de criblage qui utilisent de tels composés et de telles compositions.
PCT/US2016/053539 2015-09-23 2016-09-23 Inhibiteurs de protéases à cystéine WO2017053864A1 (fr)

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WO2020181165A1 (fr) * 2019-03-07 2020-09-10 Conatus Pharmaceuticals Inc. Inhibiteurs de caspase et leurs procédés d'utilisation
EP3766873A4 (fr) * 2018-03-13 2021-12-15 Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Procédé de préparation d'un inhibiteur de caspase
CN114504637A (zh) * 2020-09-04 2022-05-17 复旦大学附属中山医院 一种gsdmd抑制剂在动脉粥样硬化中的应用
WO2022261473A1 (fr) * 2021-06-11 2022-12-15 The Scripps Research Institute Inhibiteurs de la protéase pour le traitement d'infections à coronavirus

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3766873A4 (fr) * 2018-03-13 2021-12-15 Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Procédé de préparation d'un inhibiteur de caspase
WO2020181165A1 (fr) * 2019-03-07 2020-09-10 Conatus Pharmaceuticals Inc. Inhibiteurs de caspase et leurs procédés d'utilisation
US11597703B2 (en) 2019-03-07 2023-03-07 Histogen, Inc. Caspase inhibitors and methods of use thereof
CN110893171A (zh) * 2019-12-30 2020-03-20 济源市万洋华康生物科技有限公司 一种用于抑制伤口瘢痕性愈合的组合物的制备方法
CN110893171B (zh) * 2019-12-30 2021-07-27 河南希百康健康产业有限公司 一种用于抑制伤口瘢痕性愈合的组合物的制备方法
CN114504637A (zh) * 2020-09-04 2022-05-17 复旦大学附属中山医院 一种gsdmd抑制剂在动脉粥样硬化中的应用
CN114504637B (zh) * 2020-09-04 2024-05-14 复旦大学附属中山医院 一种gsdmd抑制剂在动脉粥样硬化中的应用
WO2022261473A1 (fr) * 2021-06-11 2022-12-15 The Scripps Research Institute Inhibiteurs de la protéase pour le traitement d'infections à coronavirus
US11708348B2 (en) 2021-06-11 2023-07-25 The Scripps Research Institute Protease inhibitors for treatment of coronavirus infections

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