Thiol protease inhibitor

Yamamoto, A., Tomoo, K., Doi, M, Ohishi, H., Inoue, M., Ishida, T., Yamamoto, D., Tsuboi, S., Okamoto, H., Okada, Y. (1992). Crystal structure of papain-succinyl-Gbi-Val-Val-Ala-Ala-p-nitroaniUde complex at 1.7-A resolution noncovalent binding mode of a common sequence of endogenous thiol protease inhibitors. Biochemistry 31,11305-11309. 

Shaw E, Angliker H, Rauber P et al (1986) Peptidyl fluoromethyl ketones as thiol protease inhibitors. Biomed Biochim Acta 45 1397-1403... 

Hamada K, Tamai M, Yamagishi M, Ohmura S, Sawada J, Tanaka I (1978) Isolation and characterization of E-64, a new thiol protease inhibitor. Agric Biol Chem 42 523-528... 

Rev-Eyes daplprazole. reviparin sodium [ban, inn] is a (parenteral) ANTICOAGULANT that is chemically a low-molecular weight form of heparin. It can be used therapeutically in the treatment of deep vein thrombosis, rexostatine (proteinase inhibitor E 64 E-64) is a microbial product isolated from AspergillusJaponicus. It is a (thiol) PROTEASE INHIBITOR, and can be used to sensitize lung and colon carcinomas to ANTICANCER chemotherapy. 

Various protease inhibitors were incubated with the enzyme to determine what class of protease this enzyme might be. The serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), the thiol protease inhibitor iodoacetamide (lAA) and the metalloprotease inhibitor EGTA were incubated with the enzyme and compared to a control with no reagent. Results shown in Table 3 indicated that the enzyme is neither a thiol nor metalloprotease. However, the enzyme was inhibited significantly by PMSF, strongly suggesting that it is similar to a serine protease. 

Peptide antibiotic. Prod, by Chromelosporium fulvum. Thiol protease inhibitor. Powder -f 2H2O. [a] -I-14.6 (c, 0.5 in H2O). Related to Estatin A, E-01609 and Rexostatine, E-01608. 

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. 

A straightforward approach is to hunt for short polypeptides that meet the specificity requirement of an enzyme but which, because of peculiarities of the sequence, are acted upon very slowly. Such a peptide may contain unusual or chemically modified amino acids. For example, the peptide Thr-Pro-nVal-NMeLeu-Tyr-Thr (nVal=norvaline NMeLeu = N-methylleucine) is a very slow elastase substrate whose binding can be studied by X-ray diffraction and NMR spectroscopy.6 Thiol proteases are inhibited by succinyl-Gln-Val-Val-Ala-Ala-p-nitroanilide, which includes a sequence common to a number of naturally occurring peptide inhibitors called cystatins.f They are found in various animal tissues where they inhibit cysteine proteases. 

Protease Classification. In order to rationally design an inhibitor for a protease it is first necessary to place it into one of four families of proteases (see Table V). For a new enzyme, a study of its inhibition profile with a series of general protease inhibitors is sufficient to classify it into one of the four families. The inhibitors usually used are diiso-propylphosphofluoridate (DFP) or phenylmethane sulfonyl fluoride (PMSF) for serine proteases, 1,10-phenanthroline for metalloproteases, thiol reagents such as iodoacetate or N-ethylmaleimide for thiol proteases, and pepstatin or diazo compounds such as diazoacetyl-norleucine methyl ester for carboxyl proteases. 

Inhibitors of Thiol and Carboxyl Proteases. Thiol proteases are inactivated by peptide chloromethyl ketones (30) and other alkylating agents. Peptide diazomethyl ketones are much more selective reagents since they do not react with serine proteases as do chloromethyl ketones. Diazoketones have been applied to papain and cathepsin B (48) thus far and it appears that they should be applicable to most thiol proteases. Specificity should be obtainable by changing the peptide sequence of the inhibitor to match that of the enzyme being studied. 

Inhibition. Since papain, ficin, and bromelain are all enzymes whose activity depends on a free SH group, it is to be expected that all thiol reagents act as inhibitors. Thus, a-halogen acids or amides and N-ethyl-maleimide irreversibly inhibit the thiol proteases. Heavy metal ions and organic mercurial salts inhibit in a fashion that can be reversed by low molecular weight thiols, particularly in the presence of EDTA which... 

One of the key intermediates shown in this reaction scheme is the formation of a tetrahedral adduct during acylation and deacylation (84). Additional support for the formation of a tetrahedral intermedite comes from the observation already referred to— that aldehydes may act as potent inhibitors of papain. Westerik and Wolfenden (65) attribute the inhibitory eflFect of aldehydes to the formation of a stable thiol adduct (thiohemiacetal) analogous to the tetrahedral intermediate produced when papain acts on a substrate. This relationship is depicted in Figure 14. When the complete picture for the mechanism of catalysis by the thiol proteases finally emerges, it will no doubt be similar to the mechanism of action of the serine proteinases. 

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