Structure of the complex of proteinase K with a substrate analogue hexapeptide inhibitor at 2.2-A resolution

Betzel, Christian; Singh, T.P.; Visanji, Marcia; Peters, Κ.; Fittkau, S.; Saenger, Wolfram; Wilson, K.S.

doi:10.1016/s0021-9258(18)82332-8

Cited by 40 publications

(8 citation statements)

References 19 publications

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“…While proving that prime site binding can be a component of a high-affinity interaction, our inhibitors did not bind to the prime site in the absence of contacts with the nonprime region of the enzyme. 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.…”

supporting

confidence: 88%

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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confidence: 88%

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

et al. 2002

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“…Three unusual product complexes are shown for proteinase K. Two of these complexes are of hydrolyzed peptidic inhibitors, AcPAPF- d A-A-NH 2 and AcPAPF- d A-AAA-NH 2 (Figure k; gray and green ribbons, respectively). , The complexes are noncovalent but stabilized by an extensive hydrogen-bonding network, with their inhibition most likely imparted by the presence of the d -alanine at position P1‘. These transition-state complexes do show each product in an isolated extended conformation .…”

Section: 2 Protease-bound Product Structuresmentioning

confidence: 99%

Proteases Universally Recognize Beta Strands In Their Active Sites

2005

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“…This argument is also supported by our recent report with this ligand in its octaprotonated form, showing an encapsulated chloride which was stabilized by only two bridgehead protons without the involvement of secondary protons. 21 As compared with normal or bifurcated hydrogen bonds, the trifurcated H-bonds are less common in synthetic complexes, 15 however they exist in many natural systems, for example, in enzyme-peptide interactions 22 and protein a-helices. 23 The structure, reported herein, represents the first crystallographic evidence of triply bridged anions as [NH(NO 3 ) 3 HN] + in a synthetic macrocycle, showing a surprising corollary 24 to a dinuclear metal complex [MA 3 M] z+ , where the bridgehead protons in the cryptand play the topological role of two transition metal ions in a classical Werner type coordination complex bridging three anions.…”

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confidence: 99%