Inhibitors of human heart chymase based on a peptide library.

Bastos, Maria Lourdes; Maeji, N. Joe; Abeles, Robert H.

doi:10.1073/pnas.92.15.6738

Cited by 60 publications

(52 citation statements)

References 29 publications

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“…The relative intolerance of P3 for aromatic residues in our study contrasts somewhat with the study by Powers et al (29), who found suc-L-Phe-Pro-Phe-4-NA to be a good substrate, although not as good as otherwise identical substrates with a sampling of neutral/aliphatic, hydrophilic, or acidic residue at P3. In our study, the P3 preference for Glu and intolerance of Pro agrees with a study by Bastos et al (40) of non-cleavable chymase inhibitors. Differences between our study and those of Powers et al in part may be because of differences in the length of the peptide or in the identity of the leaving group.…”

Section: P1 Subsite Preferences Predicted By the Combinatorial Substrsupporting

confidence: 93%

Albumin Is a Substrate of Human Chymase

Raymond

Ruggles

Craik

et al. 2003

Journal of Biological Chemistry

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Human chymase is a chymotryptic serine peptidase stored and secreted by mast cells. Compared with other chymotryptic enzymes, such as cathepsin G and chymotrypsin, it is much more slowly inhibited by serum serpins. Although chymase hydrolyzes several peptides and proteins in vitro, its target repertoire is limited compared with chymotrypsin because of selective interactions in an extended substrate-binding site. The bestknown natural substrate, angiotensin I, is cleaved to generate vasoactive angiotensin II. Selectivity of angiotensin cleavage depends in major part on interactions involving substrate residues on the carboxyl-terminal (P1-P2) side of the cleaved bond. To identify new targets based on interactions with residues on the aminoterminal (P4 -P1) side of the site of hydrolysis, we profiled substrate preferences of recombinant human chymase using a combinatorial, fluorogenic peptide substrate library. Data base queries using the peptide (Arg-Glu-Thr-Tyr-X) generated from the most preferred amino acid at each subsite identify albumin as the sole, soluble, human extracellular protein containing this sequence. We validate the prediction that this site is chymase-susceptible by showing that chymase hydrolyzes albumin uniquely at the predicted location, with the resulting fragments remaining disulfide-linked. The site of hydrolysis is highly conserved in vertebrate albumins and is near predicted sites of metal cation binding, but nicking by chymase does not alter binding of Cu 2؉ or Zn 2؉ . A synthetic peptidic inhibitor, diphenyl N ␣ -benzoxycarbonyl-L-Arg-Glu-Thr-Phe P -phosphonate, was designed from the preferred P4 -P1 substrate sequence. This inhibitor is highly potent (IC 50 3.8 nM) and 2,700-and 1,300-fold selective for chymase over cathepsin G and chymotrypsin, respectively. In summary, these findings reveal albumin to be a substrate for chymase and identify a potentially useful new chymase inhibitor.

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Section: P1 Subsite Preferences Predicted By the Combinatorial Substrsupporting

confidence: 93%

Albumin Is a Substrate of Human Chymase

Raymond

Ruggles

Craik

et al. 2003

Journal of Biological Chemistry

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“…In this respect, they were thus similar to previously reported examples of serine protease inhibitors spanning both enzyme subsites (4,(24)(25)(26)(27)(28)(29)(30)(31). Here we report on further work, which led to the development of peptidic and nonpeptidic inhibitors that bind exclusiVely to the prime site of NS3/4A.…”

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confidence: 90%

Prime Site Binding Inhibitors of a Serine Protease: NS3/4A of Hepatitis C Virus

et al. 2002

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Serine proteases are the most studied class of proteolytic enzymes and a primary target for drug discovery. Despite the large number of inhibitors developed so far, very few make contact with the prime site of the enzyme, which constitutes an almost untapped opportunity for drug design. In the course of our studies on the serine protease NS3/4A of hepatitis C virus (HCV), we found that this enzyme is an excellent example of both the opportunities and the challenges of such design. We had previously reported on two classes of peptide inhibitors of the enzyme: (a) product inhibitors, which include the P 6 -P 1 region of the substrate and derive much of their binding energy from binding of their C-terminal carboxylate in the active site, and (b) decapeptide inhibitors, which span the S 6 -S 4 ′ subsites of the enzyme, whose P 2 ′-P 4 ′ tripeptide fragment crucially contributes to potency. Here we report on further work, which combined the key binding elements of the two series and led to the development of inhibitors binding exclusiVely to the prime site of NS3/4A. We prepared a small combinatorial library of tripeptides, capped with a variety of constrained and unconstrained diacids. The SAR was derived from multiple analogues of the initial micromolar lead. Binding of the inhibitor(s) to the enzyme was further characterized by circular dichroism, site-directed mutagenesis, a probe displacement assay, and NMR to unequivocally prove that, according to our design, the bound inhibitor(s) occupies (occupy) the S′ subsite and the active site of the protease. In addition, on the basis of the information collected, the tripeptide series was evolved toward reduced peptide character, reduced molecular weight, and higher potency. Beyond their interest as HCV antivirals, these compounds represent the first example of prime site inhibitors of a serine protease. We further suggest that the design of an inhibitor with an analogous binding mode may be possible for other serine proteases.Serine proteases are perhaps the most studied class of proteolytic enzymes and a primary target for drug discovery. Despite the large number of inhibitors developed so far, very few make contact with the prime site of the enzyme. 1 Given that the starting point for inhibitor design is generally the substrate, this is not surprising, since for this class of enzymes the key determinants of substrate specificity reside in the P-region (1, 2-5). Therefore, developing inhibitors of serine proteases, which specifically target the prime site, is an almost untapped opportunity for drug design. In the course of our studies on the serine protease NS3/4A of hepatitis C virus (HCV), we found that this enzyme is an excellent example of both the opportunities and the challenges of such design.The target of our work is a key virally encoded enzyme, whose inhibition holds much promise for an effective anti-HCV therapy. Infection by hepatitis C virus (HCV) is widely recognized today as a huge public health concern, with more than 170 million people infe...

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“…The determination of distinct functional activities for ACE and chymases has been possible in some cases by the use of selective inhibitors (42) and substrates (43). The development of the compound Z-Ile-Glu-Pro-Phe-CO2H (CH5450), a peptide inhibitor of human heart chymase (42), provided an important pharmacological tool to investigate the enzymes responsible for tissue Ang II formation (41).…”

Section: Alternative Pathways For Ang II Generationmentioning

confidence: 99%