Proteolytic activity of NS3 serine proteinase of hepatitis C virus efficiently expressed inescherichia coli

Shoji, Ikuo; Suzuki, Tetsuro; Chieda, Shinya; Sato, Mitsuru; Harada, Takashi; Chiba, Tsutomu; Matsuura, Yoshiharu; Miyamura, Tatsuo

doi:10.1002/hep.1840220606

Cited by 17 publications

(3 citation statements)

References 48 publications

(10 reference statements)

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“…Initially, the substrate specificity of NS3 has been qualitatively investigated using transient transfection (31,32), in vitro translation (33), or intracellular processing of fusion proteins in Escherichia coli (34). More recently, efficient heterologous expression and purification of the enzymatically active protease domain have been described (24,(35)(36)(37)(38)(39)(40)(41)(42)(43), and optimized conditions for the determination of protease activity have been established (43)(44)(45). This has allowed a systematic study of NS3 substrate specificity using synthetic peptides derived from the NS4A-NS4B (46) or NS5A-NS5B cleavage sites (47,48).…”

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confidence: 99%

Potent Peptide Inhibitors of Human Hepatitis C Virus NS3 Protease Are Obtained by Optimizing the Cleavage Products

et al. 1998

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In the absence of a broadly effective cure for hepatitis caused by hepatitis C virus (HCV), much effort is currently devoted to the search for inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is required for the maturation of the viral polyprotein, cleaving it at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites. In the course of our studies on the substrate specificity of NS3, we found that the products of cleavage corresponding to the P6-P1 region of the substrates act as competitive inhibitors of the enzyme, with IC50s ranging from 360 to 1 microM. A detailed study of product inhibition by the natural NS3 substrates is described in the preceding paper [Steinkühler, C., et al. (1997) Biochemistry 37, 8899-8905]. Here we report the results of a study of the structure-activity relationship of the NS3 product inhibitors, which suggest that the mode of binding of the P region-derived products is similar to the ground-state binding of the corresponding substrates, with additional binding energy provided by the C-terminal carboxylate. Optimal binding requires a dual anchor: an "acid anchor" at the N terminus and a "P1 anchor" at the C-terminal part of the molecule. We have then optimized the sequence of the product inhibitors by using single mutations and combinatorial peptide libraries based on the most potent natural product, Ac-Asp-Glu-Met-Glu-Glu-Cys-OH (Ki = 0.6 microM), derived from cleavage at the NS4A-NS4B junction. By sequentially optimizing positions P2, P4, P3, and P5, we obtained several nanomolar inhibitors of the enzyme. These compounds are useful both as a starting point for the development of peptidomimetic drugs and as structural probes for investigating the substrate binding site of NS3 by modeling, NMR, and crystallography.

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confidence: 99%

Potent Peptide Inhibitors of Human Hepatitis C Virus NS3 Protease Are Obtained by Optimizing the Cleavage Products

et al. 1998

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“…Availability of quantitative data using peptidic substrates has been so far hampered by difficulties in expressing and purifying sufficient amounts of enzymatically active recombinant NS3 protease. We (17,26) and others (15,20,21,(27)(28)(29)(30)(31)(32)(33) have recently described efficient heterologous expression and purification of the enzyme and were able to define optimized conditions for the determination of protease activity (26).…”

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confidence: 99%

Substrate Specificity of the Hepatitis C Virus Serine Protease NS3

Urbani¹,

Bianchi²,

Narjes³

et al. 1997

Journal of Biological Chemistry

110

122

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The substrate specificity of a purified protein encompassing the hepatitis C virus NS3 serine protease domain was investigated by introducing systematic modifications, including non-natural amino acids, into substrate peptides derived from the NS4A/NS4B cleavage site. Kinetic parameters were determined in the absence and presence of a peptide mimicking the protease co-factor NS4A (Pep4A). Based on this study we draw the following conclusions: (i) the NS3 protease domain has an absolute requirement for a small residue in the P1 position of substrates, thereby confirming previous modelling predictions. (ii) Optimization of the P1 binding site occupancy primarily influences transition state binding, whereas the occupancy of distal binding sites is a determinant for both ground state and transition state binding. (iii) Optimized contacts at distal binding sites may contribute synergistically to cleavage efficiency.The N-terminal third of the hepatitis C virus (HCV) 1 NS3 protein contains a trypsin-like serine protease that accomplishes four out of the five processing events that take place during maturation of the nonstructural portion of the HCV polyprotein, performing cleavages at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions (1-6). It has been shown that cleavage between NS3 and NS4A is an intramolecular event, whereas all remaining junctions are processed in trans.In vivo, NS3 appears to form a heterodimer whereby the protease domain associates with the viral protein NS4A. The latter is a 54-residue protein that has been shown to bind to the N-terminal region of the protease via a central hydrophobic domain spanning residues 21-34 (7-13). NS4A acts as a cofactor of the protease enhancing cleavage at all sites and being an absolute requirement for processing of the NS4B/NS5A junction ex vivo (7). Several studies have shown that a peptide encompassing the central hydrophobic domain of NS4A is sufficient for eliciting activation of the protease (12, 14 -17).Serine proteases contact the P1 residue of their substrates through characteristic specificity pockets. The residues flanking the specificity pocket are important determinants of substrate recognition. Homology modelling of the S1 specificity pocket of the NS3 protease has predicted the presence of a phenylalanine as a prominent feature, thus rendering the pocket rather small and hydrophobic (18). These characteristics have led to the prediction of the preference for small, hydrophobic residues, ideally cysteine residues, in the P1 position of NS3 substrates. Radiosequencing of the single cleavage products has subsequently confirmed these predictions, yielding the consensus sequence (D/E)XXXXC(A/S) for all trans cleavage sites, with X being any amino acid and the scissile bond being located between Cys and Ala or Ser (2, 18). The homology model has been used to successfully redesign the enzyme's specificity, thereby increasing its validity. Very recentyl the three-dimensional structure of the protease has been solved by two different groups (20, 21...

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“… 14 , 19 Expression of the NS3 1–181 domain alone in Escherichia coli yielded greater quantities of protein with better solubility characteristics, but the resultant enzyme still displayed weakened proteolytic activity. 20 Improved proteolytic activity was achieved by using a synthetic 12-amino acid NS4A peptide (amino acids 22–33) in trans to complex with the NS3 protease. 21 However, the overall catalytic efficiency of this enzyme remained 10-fold lower than that of the full-length NS3 1–631 /4A 1–54.…”

Section: Biochemistry and Molecular Virology: Translating Science Intmentioning

confidence: 99%

The Discovery and Development of Boceprevir: A Novel, First-generation Inhibitor of the Hepatitis C Virus NS3/4A Serine Protease

2013

JCTH

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An estimated 2–3% of the world's population is infected with hepatitis C virus (HCV), making it a major global health problem. Consequently, over the past 15 years, there has been a concerted effort to understand the pathophysiology of HCV infection and the molecular virology of replication, and to utilize this knowledge for the development of more effective treatments. The virally encoded non-structural serine protease (NS3) is required to process the HCV polyprotein and release the individual proteins that form the viral RNA replication machinery. Given its critical role in the replication of HCV, the NS3 protease has been recognized as a potential drug target for the development of selective HCV therapies. In this review, we describe the key scientific discoveries that led to the approval of boceprevir, a first-generation, selective, small molecule inhibitor of the NS3 protease. We highlight the early studies that reported the crystal structure of the NS3 protease, its role in the processing of the HCV polyprotein, and the structural requirements critical for substrate cleavage. We also consider the novel attributes of the NS3 protease-binding pocket that challenged development of small molecule inhibitors, and the studies that ultimately yielded milligram quantities of this enzyme in a soluble, tractable form suitable for inhibitor screening programs. Finally, we describe the discovery of boceprevir, from the early chemistry studies, through the development of high-throughput assays, to the phase III clinical development program that ultimately provided the basis for approval of this drug. This latest phase in the development of boceprevir represents the culmination of a major global effort to understand the pathophysiology of HCV and develop small molecule inhibitors for the NS3 protease.

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Proteolytic activity of NS3 serine proteinase of hepatitis C virus efficiently expressed inescherichia coli

Cited by 17 publications

References 48 publications

Potent Peptide Inhibitors of Human Hepatitis C Virus NS3 Protease Are Obtained by Optimizing the Cleavage Products

Potent Peptide Inhibitors of Human Hepatitis C Virus NS3 Protease Are Obtained by Optimizing the Cleavage Products

Substrate Specificity of the Hepatitis C Virus Serine Protease NS3

The Discovery and Development of Boceprevir: A Novel, First-generation Inhibitor of the Hepatitis C Virus NS3/4A Serine Protease

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