73 research outputs found
Surface topology of Minibody by selective chemical modifications and mass spectrometry
No full textThe surface topology of the Minibody, a small de novo-designed beta-protein, has been probed by a strategy that combines selective chemical modification with a variety of reagents and mass spectrometric analysis of the modified fragments. Under appropriate conditions, the susceptibility of individual residues primarily depends on their surface accessibility so that their relative reactivities can be correlated with their position in the tertiary structure of the protein. Moreover, this approach provides information on interacting residues, since intramolecular interactions might greatly affect the reactivity of individual side chains by altering their pKa values. The results of this study indicate that, while overall the Minibody model is correct, the beta-sheet formed by the N- and C-terminal segments is most likely distorted. This is also in agreement with previous results that were obtained using a similar approach where mass spectrometry was used to identify Minibody fragments from limited proteolysis (Zappacosta F, Pessi A, Bianchi E, Venturini S, Sollazzo M, Tramontano A, Ma?ino G, Pucci P. 1996. Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: The case of Minibody. Protein Sci 5:802-813). The chemical modification approach, in combination with limited proteolysis procedures, can provide useful, albeit partial, structural information to complement simulation techniques. This is especially valuable when, as in the Minibody case, an NMR and/or X-ray structure cannot be obtained due to insufficient solubility of the molecule
Structural investigations on human erythrocyte acylpeptide hydrolase by mass spectrometric procedures.
No full textStructural studies of peptide inhibitors bound to hepatitis C virus protease yield insights into mechanism of action of the enzyme
No full textStructural investigations on human erythrocyte acylpeptide hydrolase by mass spectrometric procedures.
No full textStructural investigations on human erythrocyte acylpeptide hydrolase by mass spectrometric procedures.
No full text
Prime Site-binding Inhibitors of a Serine Protease: NS3/4A of Hepatitis C Virus
No full textSerine 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
The effect of prime-site occupancy on the hepatitis C virus NS3 protease structure
No full textWe recently reported a new class of inhibitors of the chymotrypsin-like serine protease NS3 of the hepatitis C virus. These inhibitors exploit the binding potential of the S′ site of the protease, which is not generally used by the natural substrates. The effect of prime-site occupancy was analyzed by circular dichroism spectroscopy and limited proteolysis-mass spectrometry. Generally, nonprime inhibitors cause a structural change in NS3. Binding in the S′ site produces additional conformational changes with different binding modes, even in the case of the NS3/4A cofactor complex. Notably, inhibitor binding either in the S or S′ site also has profound effects on the stabilization of the protease. In addition, the stabilization propagates to regions not in direct contact with the inhibitor. In particular, the N-terminal region, which according to structural studies is endowed with low structural stability and is not stabilized by nonprime inhibitors, was now fully protected from proteolytic degradation. From the perspective of drug design, P-P′ inhibitors take advantage of binding pockets, which are not exploited by the natural HCV substrates; hence, they are an entry point for a novel class of NS3/4A inhibitors. Here we show that binding of each inhibitor is associated with a specific structural rearrangement. The development of a range of inhibitors belonging to different classes and an understanding of their interactions with the protease are required to address the issue of the most likely outcome of viral protease inhibitor therapy, that is, viral resistanc
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