Inhibition of Cysteine Proteases

One of the goals of synthetic medicinal chemistry is to design potent inilibitors of clinically important proteases. Elastase inhibitors may be useful for treatment of emphysema, pancreatitis, and arthritis,a/b while inhibitors of the angiotensinogen-converting enzyme or of renin (Box 22-D) can help control blood pressure. Inhibition of thrombin, factor Xa, or other blood clotting factors (Fig. 12-17) may prevent blood clots and inhibition of the cytosolic tryptase may provide a new treatment for asthma. Inhibition of the cysteine protease cathepsin K may help combat osteoporosis and inhibition of cysteine proteases of corona viruses may fight the common cold. Cysteine proteases of schistosomes are also targets for protease inhibitors.c... 

Shenai, B.R., Lee, B.J., Alvarez-Hernandez, A., Chong, P.Y., Emal, C.D., Neitz, R.J., Roush, W.R. and Rosenthal, P.J. (2003) Structure-activity relationships for inhibition of cysteine protease activity and development... 

Matsumoto K, Mizoue K, Kitamura K, Tse WC, Huber CP, Ishida T (1999) Structural basis of inhibition of cysteine proteases by E-64 and its derivatives. Biopolymers 51 99-107... 

Figure 11 Mechanism-based probes for cysteine protease active site labeling, (a) E-64-mediated inhibition of cysteine proteases via nucleophilic attack of the catalytic cysteine at the epoxy group, (b) E-64-based probe with biotin reporter tag and tyrosine as 125l iodination site for radioactive labeling, (c) Acyloxymethylketone probe for the complementary labeling of cysteine proteases.

Figure 3 Percentage inhibition of cysteine protease activity by array X at 10 pM.

Thompson, S.K., Veber, D.F., Tomaszek, T.A., and Tew, D.G., Preparation of amino acid derivatives for treatment of parasitic diseases by inhibition of cysteine proteases of the papain superfamily, Smithkline Beecham, Int. Patent Appl. WO 9953039, 1999 Chem. Abstr, 131, 299694, 1999. 

The Cystatins are ubiquitous proteinase inhibitors of cysteine proteases that regulate various biolc ical and pathological processes by inhibition of cysteine protease activity. Cystatin is secreted by human mononuclear phagocytes and durii inflammatory processes its expression is down modulated which, in turn contributes to increased cytsteine protease activity. In addition, the study of cystatin in DC maturation and MHC molecule processing si ested that cystatin plays a role in the intracellular control of invariant chain degradation and antigen presentation. ... 

Miyamoto Y, Akaike T. Alam MS et al. Novel functions of human alpha(l)-proteasc inhibitor after S-nitrosylation inhibition of cysteine protease and antibactetial activity. Biochem Biophys Res Commun 200ft 267 918-923. 

Mladenovic M et al (2008) Atomistic insights into the inhibition of cysteine proteases first QM/MM calculations clarifying the stereoselectivity of epoxide-based inhibitors. J Phys ChemB 112 11798-11808... 

More recently, miraziridine A (113) was isolated from a marine sponge related to Theonella mirabilis and shown to inhibit the cysteine protease cathepsin B. It has been shown that the aziridine ring plays a key role in this biological activity and gives rise to irreversible inhibition of cathepsins B and L, presumably through... 

We will synthesize a 6,000-member focused library designed to inhibit the caspase family of cysteine proteases involved in apoptosis. 

Peptide nitriles are reversible inhibitors of cysteine proteases. 1,2 Peptide nitrile reacts with the active site thiol group to form an imidothioate, a dead-end product that does not undergo hydrolysis to an amide.134 This imidothioate derivative has been detected by NMR spectroscopic studies.P 5 The inhibition of papain, a cysteine protease, by a peptide nitrile proved to be reversible in a dialysis experiment. 3 Peptide nitriles are weaker inhibitors of cysteine proteases than the corresponding aldehydes. 61 Most peptide nitriles show poor inhibition toward serine proteases, however those nitriles with proper peptide sequences are potent inhibitors of serine proteases. 7-9 ... 

Solanum (potato) Kunitz PEPs inhibit the aspartic protease cathepsin D as well as trypsin [125-134] and potato cysteine protease inhibitor (PCPI) inhibits a variety of cysteine proteases [185-188]. The crystal structures of soybean trypsin inhibitor (STI) [362, 368] and of Erythrina trypsin inhibitor (ETI) [350] have been determined. The structure of this type of plant Kunitz serine PIP involves a [3-barrel formed by 6 loop-linked antiparallel [3-strands with a lid formed by 6 further loop-linked antiparallel [3-strands. The scissile bond is located within a loop that extends out from the surface of the [3-barrel [350, 362, 368]. 

In addition to being an inhibitor of papain-like cysteine proteases, cystatin C has recently been shown be an efficient inhibitor of some of the cysteine proteases of another family of cysteine proteases, called the peptidase family C13, with human legumain as a typical enzyme (C6). Human legumain has, like cathepsin S, been proposed to be involved in the class n MHC presentation of antigens (M3). It has also been shown that the cystatin C inhibitory site for mammalian legumain does not overlap with the cystatin C inhibitory site for papain-like cysteine proteases (Fig. 1) and that the same cystatin C molecule therefore is able to simultaneously inhibit one cysteine protease of each type (A 10). 

TIMPs inhibit matrix metaiioproteases (MMPs) via a two-step mechanism in a manner somewhat similar to that of cystatins (Fig. 3). While the N-terminal residues of cystatins bind to the nonprime side of cysteine proteases, TIMPs N-termini bind in the P1-P3 pockets of the protease, coordinate the catalytic... 

Figure 8 Irreversible inhibitors of proteases. Serine and cysteine proteases can be acylated by aza-peptides, which release an alcohol, but cannot be deacylated due to the relative unreactivity of the (thio) acyl-enzyme intermediate. Reactive carbons, such as the epoxide of E64, can alkylate the thiol of cysteine proteases. Phosphonate inhibitors form covalent bonds with the active site serine of serine proteases. Phosphonates are specific for serine proteases as a result of the rigid and well-defined oxyanion hole of the protease, which can stabilize the resulting negative charge. Mechanism-based inhibitors make two covalent bonds with their target protease. The cephalosporin above inhibits elastase [23]. After an initial acylation event that opens the p-lactam ring, there are a number of isomerization steps that eventually lead to a Michael addition to His57. Therefore, even if the serine is deacylated, the enzyme is completely inactive.

Inhibitors of cathepsin K illustrate the principles developed to inhibit this class of enzyme. This enzyme sequence was detected in 1994 by sequencing of human DNA for the human genome project (126).Cathepsin K was found to be inhibited by leupeptin (63) and by compound (64), which surprisingly binds "backwards" to the active site (Fig. 15.30). A hypothesis to develop symmetrical inhibitors of cathepsin K derived from the superposition of both aldehydes on the carbonyl carbon this led to the diamino ketone TSA (65). The diamino ketone moiety seems to work in several classes of cysteine proteases (127). 

Bromelain is a mixture of cysteine proteases obtained from pineapple stems (Ananas comosus, Bromeliaceae) that has been used therapeutically for the treatment of inflammation and trauma [119]. 7n vitro, it has varied stimulatory effects on leukocyte populations, increases CD2-mediated T cell activation, enhances Ag-independent binding to monocytes, etc. The effects of bromelain have previously been attributed to its degradative action at cell surfaces. However, it also acts independent of the removal of cell surface molecules [120]. In order to investigate the possible hormonelike effects of bromelain on intracellular signalling, its effects on TCR7CD3 signalling and IL-2 production were studied. It was observed that bromelain inhibits ERK-2 activation in ThO cells stimulated via the TCR, or with combined TPA plus calcium ionophore. In addtion, bromelain decreased IL-2, IFN-y, and IL-4 mRNA accumulation in ThO cells stimulated with TPA plus calcium ionophore, while the cytokine mRNA accumulation in cells stimulated via TCR was not affected. It seems that bromelain does not act on ERK-2 directly but also inhibits p2r activation, an effector molecule upstream from ERK-2 in the Raf-1/MEKl/ERK kinase cascade. Since p21 is an effector for multiple MAPK pathways, it is likely that bromelain affects other MAPK signalling cascades, such as the INK pathway or p38 MAPK pathway [121],... 

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