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

Posted: 20 August 2013 | | No comments yet

Cysteine proteases are expressed ubiquitously in the animal and plant kingdom and are thought to play key roles in maintaining homeostasis. The aberrant function of cysteine proteases in humans are known to lead to a variety of epidermal disease states such as inflammatory skin disease. In marked contrast, the serine proteases have been most widely implicated in disease states, including hypertension, periodontisis, AIDS, thrombosis, respiratory disease, pancreatitis and cancer, and a number of their inhibitors have been approved for clinical use.


Cysteine proteases are expressed ubiquitously in the animal and plant kingdom and are thought to play key roles in maintaining homeostasis. The aberrant function of cysteine proteases in humans are known to lead to a variety of epidermal disease states such as inflammatory skin disease1. In marked contrast, the serine proteases have been most widely implicated in disease states, including hypertension, periodontisis, AIDS, thrombosis, respiratory disease, pancreatitis and cancer2, and a number of their inhibitors have been approved for clinical use.

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 ( 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.

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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 characteristics5-8. 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 pKa of the cysteine thiol group (3.4 in the case of papain) rather than the usual pKa >9 associated with the dissociation of low Mr 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 mercuribenzoate10,11, 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 bromide13-15 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 sequences18,19. 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 H23,24. 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 proteolysis30-33. 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 tissues36,37 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 Ki with values <2 nM, the N64A variant results in a significant decrease in its potency towards legumain (Ki >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 (Ki >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 Ki 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.


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Author biography

Sheraz GulSheraz 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.

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