Conserved mode of peptidomimetic inhibition and substrate recognition of human cytomegalovirus protease

Tong, Liang; Qian, Chungeng; Massariol, Marie-Josée; Déziel, Robert; Yoakim, Christiane; Lagacé, Lisette

doi:10.1038/1860

Cited by 53 publications

(90 citation statements)

References 30 publications

(49 reference statements)

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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.…”

supporting

confidence: 89%

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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supporting

confidence: 89%

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

et al. 2002

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“…This loop is poorly ordered in the crystal structures and changes its conformation upon substrate binding for CMV protease [31]. In the EBV protease structure the loop is involved in the crystal contact between the dimers.…”

Section: Discussionmentioning

confidence: 99%

“…The introduction of hydrophobic groups on the C-terminal side of the cleavage site has also been used to increase the affinity of peptide-based inhibitors containing an a-keto-amide moiety between residues P1 and P1 0 which were reported for the CMV protease [16,17,31]. Such inhibitors form a reversible covalent complex through the serine residue of the catalytic triad.…”

mentioning

confidence: 99%

“…Such inhibitors form a reversible covalent complex through the serine residue of the catalytic triad. The covalent complexes of two of them (IC 0 50 s of <0.05 lM and 0.3 lM) with the CMV protease have been studied crystallographically [17,31]. Like the EBV enzyme, the hepatitis C protease NS3-4A has a relatively open catalytic site which sparked a great interest in the development of a-ketoamide based inhibitors [34,35].…”

mentioning

confidence: 99%

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Kinetics, inhibition and oligomerization of Epstein‐Barr virus protease

Buisson

Rivail

Hernandez³

et al. 2006

FEBS Letters

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Epstein-Barr virus (EBV) is an omnipresent human virus causing infectious mononucleosis and EBV associated cancers. Its protease is a possible target for antiviral therapy. We studied its dimerization and enzyme kinetics with two enzyme assays based either on the release of paranitroaniline or 7-amino-4-methylcoumarin from labeled pentapeptide (Ac-KLVQA) substrates. The protease is in a monomer-dimer equilibrium where only dimers are active. In absence of citrate the K d is 20 lM and drops to 0.2 lM in presence of 0.5 M citrate. Citrate increases additionally the activity of the catalytic sites. The inhibitory constants of different substrate derived peptides and a-keto-amide based inhibitors, which have at best a K i of 4 lM, have also been evaluated.

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“…Fig. 6a compares the calculated B-factors for the dimeric form of assemblin complexed noncovalently to a peptidyl-activated carbonyl inhibitor, [94] very similar structurally to BILC 821, with the experimental B-factors for the covalent complex between BILC 821 and the dimer of assemblin [95]. Figs.…”

Section: Molecular Dynamics Studies On the Human Cytomegalovirus Protmentioning

confidence: 99%

On the application of simple explicit water models to the simulations of biomolecules

Guimarães¹,

Barreiro²,

Oliveira³

et al. 2004

Braz. J. Phys.

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Computer simulations of biomolecular systems have achieved a significant importance in science as they provide information regarding structure, dynamics, and energetics of biomolecules that are inaccessible to experimental measuring techniques. In this work, some important aspects of the simulation of biomolecular systems are described. An overview of the most popular protein force fields, simple explicit water models for the simulation of liquid water, and different approaches to treat the boundaries of the system is presented. Also, studies conducted in our group illustrating successful simulations of three biomolecules (thrombin, L-type calcium channel and human Cytomegalovirus protease) through the application of simple explicit water models combined with protein force fields are discussed.

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Conserved mode of peptidomimetic inhibition and substrate recognition of human cytomegalovirus protease

Cited by 53 publications

References 30 publications

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

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

Kinetics, inhibition and oligomerization of Epstein‐Barr virus protease

On the application of simple explicit water models to the simulations of biomolecules

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