Product Inhibition of the Hepatitis C Virus NS3 Protease

Steinkühler, Christian; Biasiol, Gabriella; Brunetti, Mirko; Urbani, Andrea; Koch, Uwe; Cortese, Riccardo; Pessi, and Antonello; Francesco, Raffaele De

doi:10.1021/bi980313v

Cited by 222 publications

(202 citation statements)

References 45 publications

(62 reference statements)

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“…A breakthrough in this arena was the unusual observation that N-terminal cleavage products (i.e., the nonprime side) bind NS3-4A and competitively block substrate recognition. 22,23 Combinatorial chemistry was then used to further optimize and derive increasingly potent inhibitors, such as compound 1, which was used in this study.…”

Section: Commentsmentioning

confidence: 99%

Evasive maneuvers by hepatitits C virus

Lindenbach

Rice

2003

Hepatology

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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 of 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.

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Section: Commentsmentioning

confidence: 99%

Evasive maneuvers by hepatitits C virus

Lindenbach

Rice

2003

Hepatology

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“…Early drug design efforts were hampered by the relatively shallow, featureless architecture of the protease active site. The eventual observation of N-terminal product inhibition served as a stepping stone for the discovery of more potent peptidomimetic inhibitors (9,10). Over the past decade, pharmaceutical companies have further developed these lead compounds.…”

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confidence: 99%

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Romano

Ali

Royer

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

182

245

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Hepatitis C virus infects an estimated 180 million people worldwide, prompting enormous efforts to develop inhibitors targeting the essential NS3/4A protease. Resistance against the most promising protease inhibitors, telaprevir, boceprevir, and ITMN-191, has emerged in clinical trials. In this study, crystal structures of the NS3/4A protease domain reveal that viral substrates bind to the protease active site in a conserved manner defining a consensus volume, or substrate envelope. Mutations that confer the most severe resistance in the clinic occur where the inhibitors protrude from the substrate envelope, as these changes selectively weaken inhibitor binding without compromising the binding of substrates. These findings suggest a general model for predicting the susceptibility of protease inhibitors to resistance: drugs designed to fit within the substrate envelope will be less susceptible to resistance, as mutations affecting inhibitor binding would simultaneously interfere with the recognition of viral substrates.drug design | hepatitis C | substrate envelope D rug resistance is a major obstacle in the treatment of quickly evolving diseases. Hepatitis C virus (HCV) is a genetically diverse Hepacivirus of the Flaviviridae family infecting an estimated 180 million people worldwide (1). The viral RNA genome is translated as a single polyprotein and subsequently processed by host-cell and viral proteases into structural (C, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (2). The viral RNA-dependent RNA polymerase, NS5B, is inherently inaccurate and misincorporation of bases accounts for a very high mutation rate (3). While some mutations are neutral, others will alter the viability of the virus and propagate with varying efficiencies in each patient. Thus HCV infected individuals will develop a heterogeneous population of virus variants known as quasispecies (4). As patients begin treatment, the selective pressures of antiviral drugs will favor drug resistant variants (5). Therefore, an inhibitor must not only recognize one protein variant, but an ensemble of related enzymes. A detailed understanding of the atomic mechanisms of resistance is essential to effectively combat drug resistance against HCV antivirals.The essential HCV NS3/4A protease is an attractive therapeutic target responsible for cleaving at least four sites along the viral polyprotein. These sites share little sequence homology except for an acid at position P6, Cys or Thr at P1, and Ser or Ala at P1′ (Table S1). The first cleavage event at the 3-4A junction occurs in cis as a unimolecular process, while the remaining substrates are processed bimolecularly in trans. The NS3/4A protease also cleaves the human cellular targets TRIF and MAVS, which confounds the innate immune response to viral infection (6-8).Early drug design efforts were hampered by the relatively shallow, featureless architecture of the protease active site. The eventual observation of N-terminal product inhibition served as a stepping stone f...

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“…Thus, La-HCV IRES interaction could be a potential target for drug design. In fact, previously, Izumi et al (16) also reported a short peptide, LAP (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28), derived from the La motif, which could selectively inhibit IRES-mediated translation of HCV and PV. Our results are consistent with those of the study by Izumi et al (16) that show a single-point mutation in the La motif of the full-length La protein can completely abrogate the RNA binding activity.…”

Section: Discussionmentioning

confidence: 99%

“…Design of the peptidomimetic inhibitor BILIN-2061 against HCV protease was possible because it is based on structural relationship studies, availability of the crystal structure of the protease, replacement of the natural amino acids, etc. (27,32). Availability of the structure of this small peptide will be helpful in developing more stable peptidomimetic structures with higher affinity for HCV IRES, so that it could effectively inhibit IRES-dependent translation at much lower concentrations while increasing the bioavailability and solving the stability issues.…”

Section: Discussionmentioning

confidence: 99%

Structural Determinant of Human La Protein Critical for Internal Initiation of Translation of Hepatitis C Virus RNA

Mondal

Ray

Manna

et al. 2008

J Virol

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Human La protein has been implicated in facilitating internal ribosome entry site (IRES)-mediated translation of hepatitis C virus (HCV).The mechanism of internal initiation of translation of hepatitis C virus (HCV) RNA is unique and fundamentally different from the cap-dependent translation of host cell mRNAs, and thus, the HCV internal ribosome entry site (IRES) offers a potential target for developing novel antiviral therapeutics (6).The ribosome assembly at the HCV IRES has been shown to be prokaryotic-like and requires a minimal number of initiation factors (23). The HCV IRES has been shown to be capable of binding directly to 40S ribosomal subunit with the help of the ribosomal protein S5 (10, 18). Although, HCV IRES binds to the 40S ribosomal subunit specifically and stably even in the absence of any initiation factors, efficient translation requires some of the canonical initiation factors and noncanonical trans-acting factors, possibly to facilitate ribosome binding and to ensure proper positioning of the initiator AUG (iAUG) codon in the P site (13). Several cellular trans-acting factors have been reported to be critically required for HCV IRES-mediated translation, which includes human La autoantigen (2).Human La protein was originally identified in the sera from patients with systemic lupus erythematosus and Sjögren's syndrome (28). Earlier, it was reported in the literature that La protein contains three RNA recognition motifs (RRMs), which were putatively located between residues 1 and 100 (previously named RRM1), 101 and 208 (previously, RRM2), and 209 and 300 (previously, RRM3) (12, 24). However, according to the recent nomenclature, the first structured domain in human La is termed "La motif" located between residues 16 and 75 ], followed by two RRMs, RRM (112-184) and RRM (230-300) (22,29). Although, based on the structure determinations, the precise boundaries of the structured cores were found to be slightly different , RRM (229-327)], they largely encompass the above regions (1, 17). La becomes associated with the 3Ј termini of many newly synthesized small RNAs made by RNA polymerase III, as well as certain small RNAs synthesized by other RNA polymerases (20,31). The N terminal 80 amino acid (aa) residues termed the La motif and the adjacent RRM part have been shown to be required for high-affinity binding with polymerase III transcripts, where the La motif helps in specific recognition for the polyuridylate (UUU OH ) sequence at the 3Ј end of the RNA. The C-terminal RRM (230-300) has been shown to have a beta sheet comprising five strands and a long C-terminal helix that binds to the putative RNA binding site (17). The central RRM (112-184) has been shown to possess a classical RRMtype fold containing four stranded beta sheets backed by two alpha helices. It also has an additional beta strand inserted between alpha 2 and beta 4. The helix alpha 3 sheet of RRM (112-184) is predominantly hydrophilic and protrudes away

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Product Inhibition of the Hepatitis C Virus NS3 Protease

Cited by 222 publications

References 45 publications

Evasive maneuvers by hepatitits C virus

Evasive maneuvers by hepatitits C virus

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Structural Determinant of Human La Protein Critical for Internal Initiation of Translation of Hepatitis C Virus RNA

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