Cysteine enzyme inhibitor

A novel concept of using bioadhesive polymers as enzyme inhibitors has been developed [97]. Included are derivatives of poly acrylic acid, polycarbophil, and car-bomer to protect therapeutically important proteins and peptides from proteolytic activity of enzymes, endopeptidases (trypsin and a-chymotrypsin), exopeptidases (carboxypeptidases A and B), and microsomal and cytosolic leucine aminopeptidase. However, cysteine protease (pyroglutamyl aminopeptidase) is not inhibited by polycarbophil and carbomer [97]. 

There are at least three possibile ways in which the inhibitor can bind to the active site (1) formation of a sulfide bond to a cysteine residue, with elimination of hydrogen bromide [Eq. (10)], (2) formation of a thiol ester bond with a cysteine residue at the active site [Eq. (11)], and (3) formation of a salt between the carboxyl group of the inhibitor and some basic side chain of the enzyme [Eq. (12)]. To distinguish between these three possibilities, the mass numbers of the enzyme and enzyme-inhibitor complex were measured with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI). The mass number of the native AMDase was observed as 24766, which is in good agreement with the calculated value, 24734. An aqueous solution of a-bromo-phenylacetic acid was added to the enzyme solution, and the mass spectrum of the complex was measured after 10 minutes. The peak is observed at mass number 24967. If the inhibitor and the enzyme bind to form a sulfide with elimination of HBr, the mass number should be 24868, which is smaller by about one... 

Sharma and Reed, 1976)]. In proteins the coordination number 4 is most common, where the zinc ion is typically coordinated in tetrahedral or distorted tetrahedral fashion. The coordination polyhedron of structural zinc is dominated by cysteine thiolates, and the metal ion is typically sequestered from solvent by its molecular environment the coordination polyhedron of catalytic zinc is dominated by histidine ligands, and the metal ion is exposed to bulk solvent and typically binds a solvent molecule (Vallee and Auld, 1990). The inner-sphere coordination number of catalytic zinc may increase to 5 during the course of enzymatic turnover, and several five-coordinate zinc enzyme—substrate, enzyme product, and enzyme-inhibitor complexes have been studied by high-resolution X-ray crystallographic methods (reviewed by Matthews, 1988 Christianson and Lipscomb, 1989). The coordination polyhedron of zinc in five coordinate examples may tend toward either trigonal bipyramid or octahedral-minus-one geometry. 

Peptide mimetics containing the a-ketoamide moiety are very important because they act as cysteine protease inhibitors. In fact, the a-ketoamide residue forms hemithioacetals with the -SH group of the cysteine residue of the enzyme [32], Nakamura et al. [26b] reported the preparation of a 100-member combinatorial library of a-ketoamides by means of a two-step one-pot synthesis. The first step consisted of the Ugi-4CR between (+/— )lactic acid, amines, isocyanides, and aldehydes leading to the formation of the lactamides 40 which were oxidized to the corresponding pyruvamides 41. This one-pot procedure was performed in THF since the PDC oxidation was incompatible with the presence of methanol. Five a-ketoamides showed an 80% average purity (Scheme 2.17). 

Crystallographic studies of native cysteine proteinases and enzyme-inhibitor complexes have been used to interpret much or the kinetic data for cysteine protemsse-caUlyzed hydrolysis of amide bonds. Analysis of the crystal structures of papain [16]. caricain [38], actinidain [56], etc. shows that these structures are closely related. The active site of all these cysteine proteinases contains the Cys-25 sulfhydryl group in close proximity to the His-159 imidazole ring nitrogens, where the latter can abstract the sulfhydryl proton to facilitate attack on the substrate amide carbonyl group [17]. 

A potent cysteine proteinase inhibitor PCPl 8.3 was isolated from potato tubers. The inhibitor has a broad inhibitor spectrum, including the bromelain enzymes, which am not inhibited by cystatin. PCPI shows a Ki value of 190 nM for stem bromelain, 33 nM for firuit bromelain, 0.06 nM for ananain, and 3.3 nM for papain [78]. ft is proposed (hat the differences of inhibitory spectrum between PCPl and the cystatins may be those of distinct supeifamilies of cysteine proteinase inhibitors. 

Peptide aldehydes constitute a rather general example of protease inhibitors. The electrophilic carbonyl group is attacked reversibly by the cleaving nucleophile, forming a covalent acetal or thioacetal intermediate. With cysteine proteases the preferred inhibitors are strong electrophiles, for example ketones, chloromethyl ketones, epoxides, or vinyl sulfones. Many cysteine protease inhibitors form an enzyme-inhibitor complex irreversibly these are therefore denoted suicide-inhibitors . 

Angiotensin I converting enzyme Acquired immunodeficiency syndrome Aspartic protease inhibitor Bowman Birk protease inhibitor protein Chymotrypsin Cysteine protease inhibitor Endothelin-converting enzyme Elastase... 

As the H+, K+-ATPase inhibition is associated with the modification of mer-capto groups in the enzyme, the disulfide adduct (ESSR) can be considered as a model of the enzyme-inhibitor complex, and the sulfenamide, or possibly the sulfenic acid (C), formed from omeprazole can be considered to be the active inhibitor, which binds covalently to cysteine residues of the H+, K+-ATPase. 

Al. Abrahamson, M., Barrett, A. J., Salvesen, G., and Grubb, A., Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids. J. Biol. Chem. 261(24), 11282—11289 (1986). 

H3. Hall, A., Abrahamson, M., Grubb, A., Trojnar, J., Kania, P., et al., Cystatin C based peptidyl diazomethanes as cysteine proteinase inhibitors Influence of the peptidyl chain length. J. Enzyme Inhib. 6(2), 113-123 (1992). 

Figure 1.16 Left Schematic presentation of the 1,3-diaminopropanone core moiety as cysteine protease-directed and active site-spanning inhibitor principle (top). Upon reaction with the enzyme nucleophile, the ketone is reversibly converted to a hemithioketal (bottom). Right Peptidomimetic cysteine protease inhibitors of subsequent generations are depicted together with their inhibitory activity and primary targets.

Approaches to Enzyme Inhibition Aspartic Proteases and Aspartic Protease Inhibitors Cysteine Proteases and Cysteine Protease Inhibitors Metalloproteinases, Biophysics and Chemistry of Serine Proteases and Serine Protease Inhibitors... 

Figure 4.8. Acid activation of saviprazole to its active principle, the sulphenamide pyridium salt (2), and its L-cysteine (L-Cys) addition product (3) as a model for the enzyme-inhibitor...

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