Hepatitis Central

Evasive Maneuvers By Hepatitits C Virus

Brett D. Lindenbach, Ph.D.
Charles M. Rice, Ph.D.
Center for the Study of Hepatitis C, Laboratory for Virology and Infectious Disease, The Rockefeller University, New York, NY

Abstract

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.

Comments

Due to their compact genome size, efficient replication, and high mutation rates, RNA viruses like hepatitis C virus (HCV) evolve at a rapid pace. For such viruses, measured error frequencies teeter just below the limit for reliable genome propagation.¹ Indeed, HCV exhibits such sequence diversity that the term “quasispecies” is used to describe the swarm of viral sequences within an infected patient.² This term was originally developed by Eigen and Schuster to describe the distribution of mutant RNA populations related to one or more master sequences during evolution of primordial life forms.³ The high evolutionary capacity of RNA viruses endows them with the ability to quickly adapt to selective pressures, including antiviral drugs. The recent paper by Trozzi et al. at the Istituto di Ricerche di Biologia Molecolare (IRBM)—Merck Research Laboratories in Italy, is the first demonstration of HCV adapting to a specific antiviral drug, and underscores the urgency to develop new therapeutic strategies to defeat this foe.

Currently, the most effective treatment for chronic HCV infection involves the combined use of pegylated interferon alfa and ribavirin.⁴ Both of these agents work in nonspecific ways to inhibit HCV. Interferon alfa induces cellular antiviral pathways that shut down virus production and prevent infection of new cells, as well as to stimulate the specific immune response.⁵ Pegylation is a recent modification that sustains interferon's half-life in vivo.⁶ Ribavirin is a nucleoside analog with pleiotropic effects,⁷ including mutagenic potential that may degrade the ability of HCV to sustain reliable replication.⁸ Despite the improved efficacy of combined drug therapy, around half of patients infected with genotype 1 HCV fail to show a sustained virologic response and remain chronically infected.⁴ Moreover, these treatments are expensive, prolonged, and not well tolerated. Clearly, better therapies for chronic HCV are needed.

Biochemical and genetic studies have revealed new targets for the development of drugs that could potentially inhibit specific steps in the virus life cycle. Despite an inability to efficiently propagate HCV in culture, much has been learned about viral proteins by using heterologous expression systems. The HCV genome encodes one large open reading frame that is translated as a polyprotein and proteolytically processed to yield the viral structural and nonstructural (NS) proteins.⁹ Two virally encoded proteases participate in polyprotein processing, the NS2-3 autoprotease (which cleaves in cis at the NS2-3 junction) and the NS3-4A serine protease (which cleaves at 4 downstream NS protein junctions). Other known enzymatic functions of the NS proteins include an RNA helicase activity in NS3 and an RNA polymerase activity in NS5B. In addition to these biochemical studies, genetic tools for dissecting HCV molecular biology have been developed. Using an HCV infectious clone that can initiate productive infection in chimpanzees, Kolykhalov et al. showed that all of the HCV enzyme activities are required for virus viability and are therefore key targets for antiviral design.¹⁰ Furthermore, Lohmann et al. showed stable replication of HCV “replicon” RNAs following transfection into the human hepatoma line Huh-7.¹¹ Initiation of this process was very inefficient, and the original replicon constructs contained dominant selectable marker genes to select rare cells that stably supported HCV RNA replication. It was subsequently found that selected cell populations contained replicons bearing cell culture-adaptive mutations. When these mutations were re-engineered back into new replicons, the rate of replication initiation could be improved by up to 20,000-fold.^12-15 These results illustrated the remarkable ability of HCV to adapt to selective pressure, dramatically improved the ability to genetically dissect this virus, and provided a convenient cell culture system for the evaluation of potential antiviral drugs.

The most extensively studied HCV enzyme is the NS3-4A serine protease, which cleaves at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions.⁹ These cleavage sites are highly conserved and correspond to the consensus (Asp/Glu)XXX(Cys/Thr)Ser/Ala). By convention, the C-terminal residue generated by protease cleavage is termed P1, while the nascent N-terminal residue is termed P1´, and residues are numbered progressively from the cleavage site. Crystal structures for the serine protease domain of NS3 revealed a chymotrypsin-like fold, containing two domains, each containing two short -helices and a -barrel.^16-19 The NS4A cofactor peptide contributes a strand to one of these barrel structures. As for similar proteases, the active site residues lie within a cleft between these domains. Interestingly, the substrate binding surface was found to be remarkably smooth and shallow, unlike any other serine protease. NS3-4A is therefore thought to bind its substrate via an extensive network of weak interactions, spanning over 10 substrate residues.^20,21 These observations posed a challenge to the development of traditional competitive substrate inhibitors. 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.

Compound 1 is a tripeptide containing several modified side chains. This compound is an extremely effective competitive protease inhibitor, functioning at nanomolar concentrations in vitro. When added to replicon-bearing cell cultures in the low micromolar range, HCV RNA levels were dramatically decreased, as were the kinetics of polyprotein processing. Drug-resistant HCV replicons were obtained by selecting for HCV replicon-bearing cells in the presence of compound 1. Sequence analysis revealed that the resistant clones contained cell-culture adaptive mutations, as previously observed. In addition, 3 independent drug-resistant clones each contained a distinct substitution at codon 168 in the substrate binding region of NS3, changing an Asp residue to residues with uncharged side chains. These mutations were engineered back into the HCV replicon and NS3-4A enzyme and shown to confer resistance to compound 1. Further analysis confirmed that these mutations were not detrimental to normal serine protease activity in vitro or in cell culture, although processing at the NS4B-5A site was slightly impaired. Additional peptide inhibitors containing different side chains were tested and found to inhibit both wild-type and mutant NS3-4A. Based on these results, a model for compound 1 binding to NS3-4A was proposed. Interestingly, Asp 168 is not thought to directly contact the inhibitor, but rather participates in a local charge relay with nearby basic residues. Neutralization of this site presumably increases interaction of the protein surface with water, and thereby raises the energy required to form hydrophobic interactions with the P2 position of compound 1, which contains a bulky, planar flap. This interpretation was subsequently reinforced by the X-ray structure analysis of the NS3-4A protease in complex with a macrocyclic analogue of compound 1, described by scientists at Boehringer Ingelheim.²⁴ Indeed, binding of the macrocyclic inhibitor induces a rearrangement of the side chains of Asp 168 and Arg 155 leading to the formation of a salt bridge between these residues and facilitating the interaction of Arg 155 with the P2 substituent of the inhibitor. These results show the ability of HCV to adapt to a protease inhibitor, suggesting that drug-resistant viruses are also likely to arise in vivo. However, this remains to be seen, and it should be noted that some cell culture adaptive mutations selected in replicons are not tolerated in full length HCV genomes following intrahepatic inoculation into chimpanzees.²⁵ Moreover, these results contain lessons to guide further optimization of protease inhibitors.

Development of effective HCV serine protease inhibitors remains a challenge. Many product-based inhibitors currently under investigation by Merck, Boehringer Ingelheim, and Bristol-Meyers Squibb contain planar hydrophobic moieties at the P2 position that help to increase the binding affinity for peptides that are too short to include electrostatic interactions at the P5/P6 position.²⁶ It would be very interesting to know whether mutations at Asp 168 will confer resistance to these other inhibitors as well. If so, this would indicate that additional interactions would be required to retain product-based inhibitor binding to mutant proteases. BILN-2061, a particularly promising product-based inhibitor developed at Boehringer-Ingelheim and currently in phase II trials, gains additional binding energy by cyclization of the P1 and P3 positions into a protease-bound conformation. In addition to non-prime-side product-based peptide inhibitors, peptides that mimic prime-side protease products, as well as a number of substrate-based inhibitor strategies are also currently being pursued.²⁶

The ability of HCV to develop drug resistance will place limits on the effectiveness of inhibitors for specific pathways of viral replication. We might imagine that the virus is like a crucible for testing the yield of our efforts. Yet given the inherent genetic plasticity of RNA viruses, resistance should almost be expected and does not necessarily preclude clinical usefulness. An informative model for this interplay has been the continued development of better anti-retroviral drugs to treat human immunodeficiency virus, followed by the subsequent emergence of drug-resistant viruses. For that virus, the use of combination drug therapies, careful monitoring for drug resistance, and the use of stuttered high-dose regimens have led to significant improvements in the overall outlook for patients. It is hoped that with the further development of HCV inhibitors and optimization of combined drug regimens, we may see continued improvement in our abilities to cure HCV.

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Publishing and Reprint Information

Trozzi C, Bartholomew L, Ceccacci A, Biasiol G, Pacini L, Altamura S, Narjes F, Muraglia E, Paonessa G, Koch U, De Francesco R, Steinkuhler C, Migliaccio G. In vitro selection and characterization of hepatitis C virus serine protease variants resistant to an active-site peptide inhibitor. J Virol 2003;77:3669-3679. (Reprinted with permission.)