Proteases: How naturally occurring inhibitors can facilitate small molecule drug discovery for cysteine proteases

Details of protease inhibitors in clinical use have been reviewed and referenced by Abbenante & Fairlie³ and up-to-date information relating to clinical trials for a wide range of diseases, including those that involve protease inhibitors can be identified using the National Institutes of Health clinical trial database (ClinicalTrials.gov) which currently contains >100,000 clinical trials from 180 countries and receives over 50 million page views per month. Despite the successes in discovering and developing orally administered protease inhibitors, significant challenges still remain with regards to their safety profiles and demonstrable efficacy in clinical trials. Nevertheless, the fact that there are small molecule protease inhibitors undergoing clinical trials confirms the view that the protease target class are tractable for drug discovery⁴. In this article, the roles of synthetic, natural products and endogenous cystatin M/E are discussed, in particular with respect to facilitating cysteine protease small molecule drug discovery.

Synthetic and natural product low molecular mass cysteine protease inhibitors

Most of the biochemical and structural studies carried out on proteases have made use of the model systems such as the serine protease chymotrypsin and cysteine protease papain and these have provided valuable insights into their mechanism of action and specificity characteristics^5-8. The cysteine proteases contain an essential highly reactive thiol group contributed by a cysteine residue which is required for catalytic activity. In addition, they also contain an imidazole group contributed by a histidine residue which is largely responsible for conferring the abnormally low pK_a of the cysteine thiol group (3.4 in the case of papain) rather than the usual pK_a >9 associated with the dissociation of low M_r thiol containing compounds such as 2-mercaptoethanol. The thiol groups in cysteine proteases have an inherent propensity to react with reagents such as iodoacetate⁹ and mercuribenzoate^10,11, however they lack specificity features for cysteine proteases. Specificity for cysteine protease inhibitors can be introduced by the incorporation of features that are complimentary with the binding sites of the enzymes as exemplified by the irreversible substrate derived inhibitors based upon fluoromethyl ketones¹², cyanogen bromide^13-15 and 2,2′-dipyridyl disulphides¹⁶. Many cysteine protease inhibitors also inhibit serine proteases due to their similarities in the catalytic mechanism of action. However, as the catalytic site thiol (of cysteine proteases) has a greater nucleophilicity relative to the hydroxyl of the catalytic serine (of serine proteases), this can allow for selectivity towards cysteine proteases.

As protease enzymes have an inherent propensity to degrade their respective substrates, they are often synthesised in an inactive form (pro-enzyme) in order to prevent aberrant activity. This pro-enzyme subsequently undergoes auto-catalytic processing to release the pro-domain, which thereby results in the generation of mature catalytically competent protease¹⁷. The peptide sequences surrounding the auto-catalytic cleavage sites of proteases are often used to design protease substrates such that they contain similar sequences^18,19. However, proteases usually undergo a conformational change upon autocatalytic processing and the specificity characteristics of the mature protease may not directly resemble that of the pro-enzyme, therefore it is not always the case that a substrate designed on the basis of the auto-catalytic cleavage site will be acted upon by the mature protease.

The mechanisms by which low molecular mass inhibitors act upon cysteine proteases include (a) reaction with their catalytic site thiol group resulting in the formation a product which cannot undergo any further reaction, (b) forming a reactive intermediate that subsequently reacts with the enzyme via a mechanism that is not part of its usual catalytic act or (c) reacting with the enzyme active centre via their usual mechanism and undergoing further reaction at such a slow rate that it is essentially considered to be an irreversible reaction thereby rendering the enzyme catalytically inactive. An extensively characterised low molecular mass cysteine protease inhibitor is the natural product alkylating agent L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) that originates from Aspergillus japonicas. This has been shown to inhibit a variety of plant cysteine proteases (including papain and ficin), human cysteine proteases (cathepsin L²⁰, a protease from human breast-tumour tissue²¹, and the calcium-dependent protease calpain from chicken muscle²², but not to inhibit a variety of serine proteases (trypsin, chymotrypsin, tissue kallikrein, plasmin and pancreatic elastase) or aspartic proteases (pepsin and Paecilomyces acid proteases). X-ray crystallography studies of papain-E64 complex have shown that the epoxide residue of E64 interacts with the papain S₁-subsite and the leucyl residue is bound to the papain S₂-subsite. A variety of E64 derivatives have been synthesised and tested in vitro against cysteine proteases, of which CA-074 has been shown to inhibit cathepsin B with an IC₅₀ in the low nM range with >1,000 fold selectivity against other related cysteine proteases cathepsin L and cathepsin H^23,24. Collectively, these studies suggest that E-64 has been a valuable inhibitor for the study of cysteine proteases.

The cystatins: endogenous high molecular mass cysteine protease inhibitors

As described above, proteases are often expressed in vivo in an inactive form (pro-enzyme). Upon cleavage of the pro-domain, it usually dissociates from the mature protease thereby rendering it catalytically functional. The activities of mature cysteine protease enzymes in vivo are regulated by a variety of endogenous protease inhibitors such as cystatins. Additional endogenous protease inhibitors include the serine protease inhibitors (serpins), a noteworthy example of which is the myeloid and erythroid nuclear termination (MENT), a stage-specific protein which has been shown to inhibit the cysteine proteases cathepsin L and cathepsin V²⁵ and the tissue inhibitor of metalloprotease²⁶.

The cystatins are members of a superfamily of evolutionarily-related proteins (each containing >100 amino acid residues) that can be divided into three major families, namely Type-1 cystatins A and B (also known as stefins) which are relatively simple in structure, containing no disulfide bonds or carbohydrate and are found intracellular as well as the cytoplasm of cells as well as body fluids. Type-2 cystatins (C, D, F, G, M/E, S, SN, and SA) containing two disulfide bonds and no carbohydrate which are mainly extracellular secreted polypeptides synthesised with a significantly shorter (19 to 28) residue signal peptide and are broadly distributed and found in most body fluids, and Type-3, also known as kininogens (L- and H-kininogens) which are composed of many domains, disulfide bonds and carbohydrate and these include H-kininogen (high-molecular-mass, IPR002395) and L-kininogen (low-molecular-mass) and are found in a number of species²⁷. The first human cystatin was identified from the sera of autoimmune disease patients and was shown to inhibit the cysteine proteases papain, human cathepsin H and cathespin B²⁸.

In general, cystatins are competitive, reversible, tight binding proteins that inhibit cysteine proteases in a micromolar to picomolar range²⁹. They are capable of rendering their target proteases inactive via a stable complex and preventing any additional proteolysis^30-33. These inhibitors act upon their target proteases that have escaped or upon exogenous proteases of invading microorganisms. The absence of these endogenous inhibitors has been implicated in disease states, for example, cystatin C has been shown to promote atherosclerosis in apolipoprotein E deficient mice³⁴. Each cystatin has a single reactive site and binds to their target cysteine protease in a non-covalent manner. Although the cystatins have many common features, the differences in their structures have a considerable effect upon their abilities to inhibit their target proteases. The chicken egg white cystatin has been purified and extensively characterised with regards to its bio-physical characterisation and kinetics and mechanism of inhibition of a variety of proteases³⁵ and has been shown to be composed of two major forms (Form A and Form B, composed of 108 and 116 amino acid residues respectively and containing two disulfide bonds).

The role of endogenous cystatin M/E as a cysteine protease inhibitor

There is evidence implicating the role of cysteine proteases in the maintenance of epidermal tissues^36,37 as well as being down-regulated in breast cancer³⁸. The most notable example is the characterisation of wild-type cystatin M/E and its N64A and W135E variants against cysteine proteases that led to the identification of key residues of cystatin M/E that are responsible for its inhibition profile. Although wild-type cystatin M/E has been shown to inhibit legumain, cathepsin V and cathepsin L with K_i with values <2 nM, the N64A variant results in a significant decrease in its potency towards legumain (K_i >100 nM) whilst retaining similar activity against cathepsin V and cathepsin L³⁹. In the case of the W135A cystatin M/E mutant, the potency against legumain and cathepsin L is similar to that of wild-type cystatin M/E, however, in the case of cathepsin V a significant decrease in potency was observed (K_i >100 nM). The homology model of cystatin M/E based upon the crystal structure of cystatin D has led to the identification of key regions within the protein that can explain the inhibition profiles cystatin M/E as well its variants³⁹.

The studies of Grzonka et al⁴⁰ involved the characterisation of the potential of various cystatins to inhibit papain and cathepsins B, H, L and S and identified the key residues that are responsible for the inhibition profiles against a range of plant cysteine proteases (papain, ficin, actinidin and cathepsin B).

Studies have shown that cystatin A, cystatin B and cystatin C inhibit the cysteine proteases cathepsin B, cathepsin H and cathepsin L with K_i in the double digit nanomolar range. Many of these enzymes have been implicated in tissue degradation and excessive proteolytic activity, leading to diseases such as arthritis, stroke, Alzheimer’s and cataracts. The structural basis for the inhibition of the cysteine protease papain by chicken white cystatin has been determined  and shown to interact with the S₁-S₃ subsite of papain and hairpin loops interacting with the S₁‘-S₂‘ subsite.

The use of cystatins to facilitate small molecule drug discovery for cysteine proteases

Considerable progress has been made in relation to the understanding of the roles cysteine proteases play in diseases. The existence of a variety of endogenous protease inhibitors, notably the cystatins, have been relatively under-exploited for the discovery of inhibitors of proteases despite the extensive kinetic characterisation of their mechanism of inhibition of their respective protease target. As a variety of natural cysteine protease inhibitors have been identified with a range of potencies, some which are relatively potent and elucidation of their mechanisms of action, identification of key binding interactions and kinetics of inhibition can be used to facilitate drug discovery. A comprehensive list of small molecule cysteine protease inhibitors can be found in the review of Otto and Schirmeiter⁴¹. Recent examples of proteases against which inhibitors have been developed, shown to be efficacious in clinical trials and approved by the Food and Drug Administration (FDA), include Sitagliptin which inhibits the serine protease Dipeptidyl Peptidase 4⁴². Although the results from these extensive studies can be exploited in order to identify key interactions for drug discovery purposes, it has remained a considerable challenge to develop suitable compounds with appropriate potency and selectivity.

References

1. Meyer−Hoffert, U. (2009) Reddish, scaly, and itchy: how proteases and their inhibitors contribute to inflammatory skin diseases Arch Immunol Ther Exp 57:345-354
2. Turk B (2006) Targeting proteases: successes, failures and future prospects. Nat Rev Drug Discov 5:785-799
3. Abbenante G, Fairlie DP (2005) Protease inhibitors in the clinic. Med Chem 1:71-104
4. Overington, JP, Al-Lazikani, B. Hopkins, AL (2006) Nat Rev Drug Discov 5:993-996
5. Wharton CW (1997) A Mechanistic Reference. In: Sinnot ML (ed) Comprehensive Biological Catalysis. vol I. Academic Press, London, 345
6. Brocklehurst K, Willenbrock F, Salih, E (1987) Cysteine proteinases. In: Neuberger A, Brocklehurst K (eds) Hydrolytic enzymes 16:39-158
7. Barrett AJ, Rawlings ND, Woessner JF. (2004). Handbook of Proteolytic Enzymes, Elsevier, London
8. Brocklehurst K, Gul S, Pickersgill RW (2009) Substrate recognition. In: Hughes AB (ed) Amino acids, peptides and proteins in organic chemistry, vol 2. Wiley-VCH publishers, Germany, pp 473-504
9. Cigic B, Pain RH (1999) Location of the binding site for chloride ion activation of cathepsin C. Eur J Biochem 264:944-951
10. Satoh M, Yokosawa H, Ishii S (1989) Characterization of cysteine proteases functioning in degradation of dynorphin in neuroblastoma cells: evidence for the presence of a novel enzyme with strict specificity toward paired basic residues. J Neurochem 52:61-68
11. Ødum L, Yding Andersen C, Jessen TE (2002) Characterization of the coupling activity for the binding of inter-a-trypsin inhibitor to hyaluronan in human and bovine follicular fluid. Reproduction 124:249-257
12. Marzo I, Péréz-Galan P, Giraldo P, Rubio-Félix D, Anel A, Naval J (2001) Cladribine induces apoptosis in human leukaemia cells by caspase dependent and -independent pathways acting on mitochondria. Biochem J 359:537-546
13. Robertus JD, Alden RA, Birktoft JJ, Kraut J, Powers JC, Wilcox PE (1972) An x-ray crystallographic study of the binding of peptide chloromethyl ketone inhibitors to subtilisin BPN’. Biochemistry 11:2439-2449
14. Poulos TL, Alden RA, Freer ST, Birktoft JJ, Kraut J (1976) Polypeptide halomethyl ketones bind to serine proteases as analogs of the tetrahedral intermediate. X-ray crystallographic comparison of lysine- and phenylalanine-polypeptide chloromethyl ketone-inhibited subtilisin. J Biol Chem 251:1097-1103
15. Machleidt W, Thiele U, Laber B, Assfalg-Machleidt I, Esterl A, Wiegand G. Kos J, Turk V, Bode W (1989) Mechanism of inhibition of papain by chicken egg white cystatin: Inhibition constants of N-terminally truncated forms and cyanogens bromide fragments of the inhibitor. FEBS Letters 243:234-238
16. Brocklehurst K, Little G (1973) Reactions of papain and of low-molecular-weight thiols with some aromatic disulphides. 2,2′-Dipyridyl disulphide as a convenient active-site titrant for papain even in the presence of other thiols. Biochem J 133:67-80
17. Hsu MF, Kuo CJ, Chang KT, Chang HC, Chou CC, Ko TP, Shr HL, Chang GG, Wang AH, Liang PH (2005) Mechanism of the maturation process of SARS-CoV 3CL protease. J Biol Chem 280:31257-31266
18. Harris JL, Backes BJ, Leonetti F, Mahrus S, Ellman JA, Craik CS (2000) Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proc Natl Acad Sci USA 97:7754-7759
19. Thomas DA, Francis P, Smith C, Ratcliffe S, Ede NJ, Kay C, Wayne G, Martin SL, Moore K, Amour A, Hooper NM (2006) A broad-spectrum fluorescence-based peptide library for the rapid identification of protease substrates. Proteomics 6:2112-2120
20. Barrett AJ, Kembhavi AA, Brown MA, Kirschke H, Knight CG, Tamai M, Hanada, K (1982) L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem J 201:189-198
21. Mort JS, Recklies AD, Poole AR (1980) Characterization of a thiol proteinase secreted by malignant human breast tumours. Biochim Biophys Acta 614:134-143
22. Sugita H, Ishiura S, Suzuki K, Imahori K (1980) Ca-activated neutral protease and its inhibitors: in vitro effect on intact myofibrils. Muscle Nerve 3:335-339
23. Murata M, Miyashita S, Yokoo C, Tamai M, Hanada K, Hatayama K, Towatari T, Nikawa T, Katunuma N (1991) Novel epoxysuccinyl peptides. Selective inhibitors of cathepsin B, in vitro. FEBS Lett 280:307-310
24. Steverding D (2011) The cathepsin B-selective inhibitors CA-074 and CA-074Me inactivate cathepsin L under reducing conditions. The Open Enzyme Inhibition Journal 4:11-16
25. Irving JA, Shushanov SS, Pike RN, Popova EY, Brömme D, Coetzer TH, Bottomley SP, Boulynko IA, Grigoryev SA, Whisstock JC (2002) Inhibitory activity of a heterochromatin-associated serpin (MENT) against papain-like cysteine proteinases affects chromatin structure and blocks cell proliferation. J Biol Chem 277:13192-13201
26. Murphy G (2011) Tissue inhibitors of metalloproteinases. Genome Biol 12:233
27. Ochieng J, Chaudhuri G (2010) Cystatin superfamily. J Health Care Poor Underserved 21:51-70
28. Brzin J, Popovic T, Turk V, Borchart U, Machleidt W (1984) Human cystatin, a new protein inhibitor of cysteine proteinases. Biochem Biophys Res Commun 118:103-109
29. Brömme D, Rinne R, Kirschke H (1991) Tight-binding inhibition of cathepsin S by cystatins. Biomed Biochim Acta 50:631-635
30. Barrett AJ (1986) The cystatins: a diverse superfamily of cysteine peptidase inhibitors. Biomed Biochim Acta 45:1363-1374
31. Bode W, Huber R (2000) Structural basis of the endoproteinase-protein inhibitor interaction. Biochim Biophys Acta 1477:241-252
32. Turk V, Bode W (1991) The cystatins: protein inhibitors of cysteine proteinases. FEBS Lett  285:213-219
33. Turk B, Turk D, Salvesen GS (2002) Regulating cysteine protease activity: essential role of protease inhibitors as guardians and regulators. Curr Pharm Des 8:1623-1637
34. Sukhova GK, Wang B, Libby P, Pan J-H, Zhang Y, Grubb A, Fang K, Chapman HA, Shi G-P (2005) Cystatin C deficiency increases elastic lamina degradation and aortic dilatation in apolipoprotein E-null mice. Circulation Res 96:368-375
35. Auerswald EA, Nägler DK, Gross S., Assfalg-Machleidt I., Stubbs MT, Eckerskorn C, Machleidt W and Fritz H (1996) Hybrids of chicken cystatin with human kininogen domain 2 sequences exhibit novel inhibition of calpain, improved inhibition of actinidin and impaired inhibition of papain, cathepsin L and cathepsin B. Eur J Biochem 235:534-542
36. Zeeuwen PL, van Vlijmen-Willems IM, Olthuis D, Johansen HT, Hitomi K, Hara-Nishimura I, Powers JC, James KE, op den Camp HJ, Lemmens R, Schalkwijk J (2004) Evidence that unrestricted legumain activity is involved in disturbed epidermal cornification in cystatin M/E deficient mice. Hum Mol Genet 13:1069-1079
37. Zeeuwen PL, van Vlijmen-Willems IM, Cheng T, Rodijk-Olthuis D, Hitomi K, Hara-Nishimura I, John S, Smyth N, Reinheckel T, Hendriks WJ, Schalkwijk J (2010) The cystatin M/E-cathepsin L balance is essential for tissue homeostasis in epidermis, hair follicles, and cornea. FASEB J 10:3744-3755
38. Sotiropoulou G, Anisowicz A, Sager R (1997) Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer. J Biol Chem 272:903-910
39. Cheng T, Hitomi K, van Vlijmen-Willems IM, de Jongh GJ, Yamamoto K, Nishi K, Watts C, Reinheckel T, Schalkwijk J, Zeeuwen PL (2006) Cystatin M/E is a high affinity inhibitor of cathepsin V and cathepsin L by a reactive site that is distinct from the legumain-binding site. A novel clue for the role of cystatin M/E in epidermal cornification. J Biol Chem 281:15893-15899
40. Grzonka Z, Jankowska E, Kasprzykowski F, Kasprzykowska R, Lankiewicz L, Wiczk W, Wieczerzak E, Ciarkowski J, Drabik P, Janowski R, Kozak M, Jaskólski M, Grubb A (2001) Structural studies of cysteine proteases and their inhibitors. Acta Biochim Pol 48:1-20
41. Otto HH, Schirmeiter T (1997) Cysteine proteases and their inhibitors. Chem Rev 97:133-171
42. Augeri DJ, Robl JA, Betebenner DA, Magnin DR, Khanna A, Robertson JG, Wang A, Simpkins LM, Taunk P, Huang Q, Han SP, Abboa-Offei B, Cap M, Xin L, Tao L, Tozzo E, Welzel GE, Egan DM, Marcinkeviciene J, Chang SY, Biller SA, Kirby MS, Parker RA, Hamann LG (2005) Discovery and preclinical profile of Saxagliptin (BMS-477118): a highly potent, long-acting, orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. J Med Chem 48:5025-5037

Author biography

Sheraz Gul ([email protected]) is Head of Biology at European ScreeningPort, Hamburg, Germany where he manages the assay development and screening of academic targets. Prior to this, he worked for GlaxoSmithKline for seven years where he developed biochemical and cellular assays for High Throughput Screening as well as hit characterisation. In addition, he has worked in academia for five years on proteases and kinases. He is the co-authored of the Enzyme Assays: Essential Data Handbook. He is involved in many European Initiatives involving government, the pharmaceutical industry and academia (e.g. EU Framework 7 and IMI). His research interests are directed towards maximising the impact of HTS for drug discovery.