Active site cathepsin

When the target enzyme is difficult to obtain, related enzymes could be used to provide insights in the design of novel ligands. For example, papain was used to design a class of potent cathepsin K inhibitors [33] spanning both sides of the papain active site. However, fine-tuning these inhibitors to produce more potent ones required the use of the crystal structure of cathepsin K itself [34],... 

Fig. 6.14. Quenched activity based probe for the imaging of cathepsins. Upon covalent binding of the sensor to histidine and serine residues in the active site of the enzyme, the quencher is released and increased fluorescence indicates the now covalently labeled enzyme of interest. 

Cathepsin K (Cat K) is a member of the CA1 family of lysosomal cysteine proteases. This family is comprised of 11 human members (cathepsins B, C, F, H, K, L, O, S, V, W, Z) which share a common papain-like structural fold and a conserved active site Cys-Asn-His triad of residues [1-3]. These enzymes are synthesized as pre-pro-enzymes and are converted from the catalytically inactive zymogen into the active form in acidic lysosomal environment. In some cases, cathepsins are also secreted in the active form from cells. The sequence identity of... 

The most convenient way of categorizing the classes of cathepsin inhibitors is based on the nature of the electrophilic warhead that interacts with the sulfhydryl group of the active site cysteine residue. Since a large portion of the binding energy of a cysteine protease inhibitor comes from the covalent interaction with this thiol, the properties of the resulting molecules are largely derived from the electrophile. In broad terms, these inhibitors can be broken down into ketone and nitrile-based reversible covalent inhibitors, or the more recent non-covalent inhibitors based on an aminoaniline template. 

Metcalf, P., and Fusek, M. (1993). Two crystal structure for cathepsin D The lysosomal targeting signal and active site. EMBO J. 12, 1293-1302. 

Figure 6. An example of inter-family target hopping between human and viral aspartyl proteases. The aspartyl protease active site is located at a homodimer interface in HIV and within a single domain in Cathepsin D, so sequence and structure alignments between these proteins cannot be constructed. By using an approach independent of sequence or structure homology to directly align the sites, SiteSorter finds that the HIV protease and Cathepsin D substrate sites are highly similar (identical chemical groups within 1 A are colored dark blue). It has been verified experimentally that Cathepsin D is susceptible to inhibition by HIV-protease inhibitors. ...

The cysteinyl proteases include papain calpains I and II cathepsins , H, and L proline endopeptidase and interleukin-converting enzyme (ICE) and its homologs. The most well-studied cysteinyl protease is likely papain, and the first x-ray crystallographic structures of papain [193] and a peptide chloromethylketone inhibitor-papain complex [194] provided the first high resolution molecular maps of the active site. Pioneering studies in the discovery of papain substrate peptide-based inhibitors having P, electrophilic moieties such as aldehydes [195], ketones (e.g., fluoromethylketone, which has been determined [196] to exhibit selectivity for cysteinyl proteases versus serinyl proteases), semicarbazones, and nitriles are noteworthy since 13C-NMR spectro-... 

Most of the lysosomal proteases called cathepsins are small 20- to 40-kDa glycoproteins found in all animal tissues.313 Most are cysteine proteases which function best and are most stable in the low pH reducing environment of lysosomes. They resemble papain in size, amino acid sequence, and active site structures. Papain is nonspecific but most cathepsins have definite substrate preferences. Cathepsin B is the most abundant. There are smaller amounts of related cathepsins H (an aminopeptidase)314 and L315 and still less of cathepsins C, K, and others. Cathepsin B is both an endopep-tidase and an exopeptidase.316 It acts on peptides with arginine at either Pj or P2 but also accepts bulky hydro-phobic residues in Pj and prefers tyrosine at P3.317 Cathepsin S is less stable at higher pH than other cathepsins and has a more limited tissue distribution, being especially active in the immune system.318 319... 

Zymogens of cysteine proteases usually have a long terminal extension which is removed, sometimes by autoactivation. Propapain has a 107-residue extension.343 The 322-residue cathepsin B carries an unusually short 62-residue extension in its proenzyme form.315 343 344 In every case the N-terminal extension folds into a domain, one of whose functions is to block the active site cleft. 

Epoxy groups, such as that of E-64, a compound isolated from the culture medium of a species of Aspergillus, react irreversibly with the active site thiolate group of cysteine proteases.1/U/V Related epoxides may become useful medications against abnormal cathepsin levels. 

Like antithrombin, heparin cofactor II inhibits proteases by forming a I I stoichiometric complex with the enzyme. The protease attacks the reactive site of heparin cofactor II located on the C-terminus, resulting in the formation of a covalent bond. Heparin cofactor II has higher protease specificity than antithrombin. Of the coagulation enzymes, heparin cofactor II is known only to inhibit thrombin (92). Additionally heparin cofactor II has been shown to inhibit chymotrypsin (93) and leukocyte cathepsin G (94), This protease specificity appears to be due to the active site bond present in heparin cofactor II. Whereas antithrombin contains an Arg-Ser bond as its active site, heparin cofactor II is unique in containing a Leu-Ser bond. This suggests than another portion of the heparin cofactor II molecular may be required for protease binding,... 

Sulfonyl Fluorides. Sulfonyl fluorides inhibit serine proteases by reacting with the active-site serine residue. Previously we investigated the rates of inhibition of human leukocyte elastase and cathepsin G by a variety of sulfonyl fluorides and found relatively little selectivity or reactivity (38). However, we have discovered recently that the introduction of fluoroacyl groups into the sulfonyl fluoride structure gives considerable reactivity and selectivity for elastase (39). 

Lecaille, F. et al. 2003. Probing cathepsin K activity with a selective substrate spanning its active site. Biochem. J. 375, 307-312. 

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