A designed P1 cysteine mimetic for covalent and non-covalent inhibitors of HCV NS3 protease

Narjes, Frank; Koehler, Konrad F.; Koch, Uwe; Gerlach, Benjamin; Colarusso, Stefania; Steinkühler, Christian; Brunetti, Mirko; Altamura, Sergio; Francesco, Raffaele De; Matassa, Victor G.

doi:10.1016/s0960-894x(01)00842-3

Cited by 176 publications

(81 citation statements)

References 24 publications

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“…4). Similarly to compound 1, compound 2 has an alpha carboxylic acid and is a well-characterized product inhibitor (38,39). In compound 3, the P1 carboxylic acid is replaced by an alpha ketoacid, resulting in a covalent inhibitor of the protease (38).…”

Section: Selection Of Replicons Resistant To Compound 1 and Identificmentioning

confidence: 99%

In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

Trozzi

Bartholomew

Ceccacci

et al. 2003

J Virol

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115

136

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The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.Despite the introduction of blood-screening tests ϳ10 years ago, hepatitis C virus (HCV) is still the major cause of bloodborne chronic hepatitis, with nearly 200 million infected people worldwide. HCV infection often evolves into a chronic disease, which can lead to liver dysfunction and hepatocellular carcinoma. Current therapeutic regimens based on alpha interferon (IFN-␣) and the nucleoside analog ribavirin are only partially effective and are limited by the adverse effects of both agents (50). Given the high prevalence of this disease, developing new treatments is a major public health objective. Similarly to human immunodeficiency virus (HIV) research, most efforts to develop antiviral agents for HCV have focused on the inhibition of key viral enzymes, serine protease, helicase, and polymerase (2).The most extensively studied HCV target has been the NS3-4A serine protease, a heterodimeric enzyme comprising the N-terminal domain of the NS3 protein (amino acids 1 to 180) and the small hydrophobic NS4A protein (3). This protease cleaves the viral polyprotein at four junctions (NS3/ NS4A, NS5A/NS5B, NS4A/NS4B, and NS4B/NS5A), and its activity is necessary for viral replication (24). Although the NS3 protease domain possesses enzymatic activity, the 54-amino-acid NS4A protein is required for cleavage at the NS3/ NS4A and NS4B/NS5A sites and increases cleavage efficiency at the NS4A/NS4B and NS5A/NS5B junctions (4, 14, 28, 47). X-ray crystallography (20, 35, 51) and nuclear magnetic resonance (NMR) spectroscopy (1, 36) have shown that the NS3-4A structure is similar to that of other chymotrypsin-like serine proteases, with two domains, both composed of a ␤-barrel and two short ␣-helices. The catalytic triad comprises histidine 57, aspartate 81, and serine 139 and is located between the two domains. The central region of NS4A is an integral part of t...

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Section: Selection Of Replicons Resistant To Compound 1 and Identificmentioning

confidence: 99%

In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

Trozzi

Bartholomew

Ceccacci

et al. 2003

J Virol

Self Cite

115

136

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“…Consequently, the incorporation of fluorine or fluoroalkyl groups has become a daily routine in the design and fine tuning of the biological properties of the drug candidates. The difluoromethyl group (CF 2 H), like its analogue CF 3 group, has attracted increasing interests recently since the difluoromethyl group is known to be bioisostere of carbinol 3 , thiol 4 or hydroxamic acid hydroxyl group 5 . In addition, the difluoromethyl group can act as lipophilic hydrogen bond donor to improve the binding selectivity and cell membrane permeability.…”

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confidence: 99%

Cooperative dual palladium/silver catalyst for direct difluoromethylation of aryl bromides and iodides

2014

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Difluoromethylated arenes are one of the privileged structural motifs that are important for fine tuning the biological properties of drug molecules. No general catalytic method exists for the formation of difluoromethylarenes. Previous methods for the preparation of difluoromethylarenes typically required harsh conditions, multiple steps or stoichiometric amount of catalysts. Here we report a cooperative dual palladium/silver catalyst system for direct difluoromethylation of aryl bromides and iodides under mild conditions. We develop the system by initial preparation of the putative intermediates in the dual-catalytic cycles, followed by studying the elemental steps to demonstrate the viability of the proposed cooperative catalytic cycle. The reaction is compatible with a variety of functional groups such as ester, amide, protected phenoxide, protected ketone, cyclopropyl, bromide and heteroaryl subunits such as pyrrole, benzothiazole, carbazole or pyridine.

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“…The preferred residue in P 1 was the same observed for the substrate, i.e., cysteine, followed in decreasing order of potency by Abu, Val, and Ser. Fluorinated amino acids like trifluoroaminobutyric acid 28 or difluoroaminobutyric acid 29 were later designed as suitable cysteine mimetics, and their use was enabling to synthesize potent "serine-trap" type inhibitors. 28 -31 The second anchor of the product inhibitors resided in the P 6 -P 5 acidic pair (compare peptides 3 and 4, Table I).…”

Section: Optimization Of Product Inhibitorsmentioning

confidence: 99%

“…When used in conjunction with optimization of the peptide moiety, the serine trap concept was successfully exploited by several groups, leading to potent aminoboronic acids, 20,42 ␣-ketoacids, 29,30 (see Table I, no. 19) ␣-ketoesters, 29,38 and ␣-ketoamides.…”

Section: Serine-trap Inhibitorsmentioning

confidence: 99%

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Inhibiting viral proteases: Challenges and opportunities

Bianchi

Pessi

2002

Biopolymers

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Inhibitor design against viral targets must take into account the peculiar characteristics of viral biology-in particular, the plasticity of their replicative machinery. This includes maturational cleavage of the polyprotein, which is mediated by virally encoded proteases. Designing against a movable target is particularly challenging, but at the same time it offers new opportunities. Here we describe our experience with the NS3/4A (NS: nonstructural) serine protease of human hepatitis C virus (HCV). By extensive use of combinatorial peptide libraries, various inhibitor types were generated, including product inhibitors, serine traps, P-P' inhibitors, and prime side inhibitors. The latter represent a first case for a serine protease. A key finding, derived from structural studies utilizing these inhibitors, was that NS3 is an induced-fit protease, requiring both the NS4A cofactor protein and the substrate to fully activate its catalytic machinery. In the absence of cofactor and/or substrate, NS3 exists in solution as a large conformational ensemble, which can be matched by a correspondingly large set of peptide inhibitors, each one stabilizing a given conformer. In the perspective of inhibiting viral proteases in general, we suggest that combinatorial ligand ensembles may be a powerful tool, to contrast the adaptive potential of the viral quasispecies.

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A designed P1 cysteine mimetic for covalent and non-covalent inhibitors of HCV NS3 protease

Cited by 176 publications

References 24 publications

In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

Cooperative dual palladium/silver catalyst for direct difluoromethylation of aryl bromides and iodides

Inhibiting viral proteases: Challenges and opportunities

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