E64 [trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane] analogues as inhibitors of cysteine proteinases: investigation of S2 subsite interactions

Abstract

A number of epoxysuccinyl amino acid benzyl esters (HO-Eps-AA-OBzl) and benzyl amides (HO-Eps-AA-NHBzl) (where AA represents amino acid) were synthesized as analogues of E64, a naturally occurring inhibitor of cysteine proteinases. These inhibitors were designed to evaluate if selectivity for cathepsin B could be achieved by varying the amino acid on the basis of known substrate specificity. Contrary to the situation with substrates, it was found that variation of the amino acid in the E64 analogues does not lead to major changes in the kinetic parameter kinac./Ki and that the specificity of these analogues does not parallel that observed for substrates. This is particularly true in the case of the benzyl ester derivatives where the deviation from substrate-like behaviour is more important than with the benzyl amide derivatives. The results suggest that the amide proton of the benzyl amide group in HO-Eps-AA-NHBzl interacts in the S2 subsite in both cathepsin B and papain and contributes to increase the potency of these inhibitors. The kinetic data also suggest that differences in the orientation of the C alpha-C beta bond of the side chain in the S2 subsite of the enzyme might explain the differences between substrate and E64 analogue specificities. This hypothesis is supported by the fact that the order of inactivation rates with chloromethane inhibitors (which are believed to be good models of enzyme-substrate interactions) is indeed very similar to that observed with the corresponding amidomethylcoumarin substrates. In conclusion, the information available from S2-P2 interactions with substrates cannot be used to enhance the selectivity of the E64 analogues in a rational manner.

Cysteine cathepsins: From structure, function and regulation to new frontiers

“…The predicted binding geometry of the doubleheaded inhibitors has been confirmed by the crystal structures of papain-CLIK complexes as the model for cathepsin L [107] and the cathepsin B-NS-134 complex [78]. There are several, more detailed, papers on this subject worthy of further reading [75,108,109].…”

The Apaf-1-binding protein Aven is cleaved by Cathepsin D to unleash its anti-apoptotic potential

“…To test whether a protease was responsible for generating the 30-kDa Aven fragment, we treated MCF-7 cells with various protease inhibitors ( Figure 2c). While incubation with the cysteine protease inhibitor E64 22 and the caspase inhibitor z-VAD 23 showed no influence on Aven processing, the serine protease inhibitor aprotinin 24 and the serine and cysteine protease inhibitor pAPMSF 25 CathD is a prominent member of the subfamily of lysosomal aspartic proteases, and its enzymatic function is not restricted solely to the acidic milieu of lysosomes (for a review see Liaudet-Coopman et al 27 and Masson et al 28 ). CathD has been implicated in positive and negative regulation of apoptosis, and its overexpression has been reported to promote tumorigenesis and metastasis of breast cancer (for a review see Masson et al 28 and Garcia et al 29 ).…”

Identification of a silicatein(-related) protease in the giant spicules of the deep-sea hexactinellidMonorhaphis chuni

“…Next, using this technique we determined whether the proteolytic activity in the S. domuncula spicule extract can be affected by the inhibitors E-64 and cystatin. E-64, a well-established irreversible cysteine proteinase inhibitor (Barrett et al, 1982;Gour-Salin et al, 1994), interacts with the S 2 subsite (binding pocket) of the enzyme and blocks its binding to bulky hydrophobic or aromatic residues of the inhibitor, e.g. to Phe in the P 2 position (Gour-Salin et al, 1994).…”

“…E-64, a well-established irreversible cysteine proteinase inhibitor (Barrett et al, 1982;Gour-Salin et al, 1994), interacts with the S 2 subsite (binding pocket) of the enzyme and blocks its binding to bulky hydrophobic or aromatic residues of the inhibitor, e.g. to Phe in the P 2 position (Gour-Salin et al, 1994). Of particular interest are residues 133 and 157 (referring to papain numbering) in the enzyme, which form part of the binding pocket, and residue 205, which closes the end of the pocket (Brömme et al, 1994).…”