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Bruce A Malcolm

Publications and source records attributed to Bruce A Malcolm.

9 recordsLinked to original sources
PubMed2006

Impact of naturally occurring variants of HCV protease on the binding of different classes of protease inhibitors.

HCV drug discovery efforts have largely focused on genotype 1 virus due to its prevalence and relatively poor response to current therapy. However, patients infected with genotype 2 and 3 viruses account for a significant number of cases and would also benefit from new therapies. In vitro studies using two chemically distinct protease inhibitors with clinical potential showed that one, VX-950, was equally active on proteases from all three genotypes, whereas the other, BILN 2061, was significantly less active on genotype 2 and 3 proteases. Naturally occurring variation near the inhibitor binding site was identified based on sequence alignment of the protease region from genotype 1-3 sequences. Substitution of amino acids in genotype 1 based on genotype 2 and 3 has revealed residues which impact binding of BILN 2061. Substitution of residues 78-80, together with 122 and 132, accounted for most of the reduced sensitivity of genotype 2. The most critical position affecting inhibitor binding to genotype 3 protease was 168. Substitution of residues at positions 168, 123, and 132 fully accounted for the reduced sensitivity of genotype 3. Comparative studies of BILN 2061 and a closely related nonmacrocycle inhibitor suggested that the rigidity of BILN 2061, while conferring greater potency against genotype 1, rendered it more sensitive to variations near the binding site. Free energy perturbation analysis confirmed the experimental observations. The identification of naturally occurring variations which can affect inhibitor binding is an important step in the design of broad-spectrum, second generation protease inhibitors.

Binding Sites↗
PubMed2006

Identification and analysis of fitness of resistance mutations against the HCV protease inhibitor SCH 503034.

HCV NS3 protease variants resistant to the protease inhibitor SCH 503034 were selected. Three mutations, T54A, V170A and A156S mutations conferred low to moderate levels of resistance (<20-fold). Longer exposure (>10 passages) or selection with higher levels of compound led to the selection of a more resistant variant, A156T (>100-fold). [Lin, C., Lin, K., Luong, Y.P., Rao, B.G., Wei, Y.Y., Brennan, D.L., Fulghum, J.R., Hsiao, H.M., Ma, S., Maxwell, J.P., Cottrell, K.M., Perni, R.B., Gates, C.A., Kwong, A.D., 2004. In vitro resistance studies of hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061: structural analysis indicates different resistance mechanisms. J. Biol. Chem. 279(17), 17508-17514; Lu, L., Pilot-Matias, T.J., Stewart, K.D., Randolph, J.T., Pithawalla, R., He, W., Huang, P.P., Klein, L.L., Mo, H., Molla, A., 2004. Mutations conferring resistance to a potent hepatitis C virus serine protease inhibitor in vitro. Antimicrob. Agents Chemother. 48(6), 2260-2266.] Combination with IFN-alpha drastically reduced the number of emergent colonies. Resistant colonies showed no change in sensitivity to IFN-alpha. Although the A156T mutation conferred the highest level of resistance to SCH 503034, it significantly reduced the colony formation efficiency (CFE) of the mutant replicon RNA, and rendered replicon cells less fit than those bearing wild-type replicons. Replicon cells bearing mutation A156S were less fit than wild-type in co-culture growth competition assays but showed no impact on CFE. The V170A mutation, on the other hand, did not affect replicon fitness in either assay, which was consistent with its emergence as the dominant mutant after 12 months of continuous selection. The reduced fitness of the most resistant variant suggests that it may be rare in naïve patients and that development of high-level resistance may be slow. Combination therapy with IFN-alpha should also greatly reduce the potential emergence of resistance.

Blotting, Western↗
PubMed2005

Mutations conferring resistance to SCH6, a novel hepatitis C virus NS3/4A protease inhibitor. Reduced RNA replication fitness and partial rescue by second-site mutations.

Drug resistance is a major issue in the development and use of specific antiviral therapies. Here we report the isolation and characterization of hepatitis C virus RNA replicons resistant to a novel ketoamide inhibitor of the NS3/4A protease, SCH6 (originally SCH446211). Resistant replicon RNAs were generated by G418 selection in the presence of SCH6 in a dose-dependent fashion, with the emergence of resistance reduced at higher SCH6 concentrations. Sequencing demonstrated remarkable consistency in the mutations conferring SCH6 resistance in genotype 1b replicons derived from two different strains of hepatitis C virus, A156T/A156V and R109K. R109K, a novel mutation not reported previously to cause resistance to NS3/4A inhibitors, conferred moderate resistance only to SCH6. Structural analysis indicated that this reflects unique interactions of SCH6 with P'-side residues in the protease active site. In contrast, A156T conferred high level resistance to SCH6 and a related ketoamide, SCH503034, as well as BILN 2061 and VX-950. Unlike R109K, which had minimal impact on NS3/4A enzymatic function, A156T significantly reduced NS3/4A catalytic efficiency, polyprotein processing, and replicon fitness. However, three separate second-site mutations, P89L, Q86R, and G162R, were capable of partially reversing A156T-associated defects in polyprotein processing and/or replicon fitness, without significantly reducing resistance to the protease inhibitor.

Binding Sites↗
PubMed2005

Trans-complementation of HCV replication by non-structural protein 5A.

Although establishment of the subgenomic replicon system has considerably facilitated genetic analysis of HCV replication, many details remain largely unknown. To initially test whether HCV replication could be affected in trans, complementation studies were conducted in which defective replicon RNAs carrying a luciferase reporter were introduced into stable cells bearing functional replicons. The NS3 protease and the NS5B viral polymerase genes on the transfected replicons were rendered null by active site mutations and shown not to be complemented in trans by functional proteins expressed from the endogenous replicons. A new strategy was also developed to examine whether adapted copies of NS4B and NS5A could enhance the replication of transfected replicons carrying non-adapted genes. The replication efficiency of a replicon carrying two adaptive mutations in NS3 (E1202G and T1280I) had previously been shown to be greatly enhanced by the presence of a third mutation in either NS4B (K1846T) or NS5A (S2197P). A partially adapted luciferase replicon carrying only the two NS3 mutations was used to transfect cells containing replicons bearing the adapted NS4B or NS5A. Using this approach, NS5A, but not NS4B, was found to trans-complement. In a final confirmatory study, ectopically expressed NS5A also complemented the HCV replicon genome bearing the non-adapted NS5A. These studies strongly suggest that HCV non-structural proteins, with the exception of NS5A, can only act in cis on the RNA from which they were translated.

Adaptation, Biological↗
PubMed2004

A continuous nonradioactive assay for RNA-dependent RNA polymerase activity.

Current assays for the activity of viral RNA-dependent RNA polymerases (RdRps) are inherently end-point measurements, often requiring the use of radiolabeled or chemically modified nucleotides to detect reaction products. In an effort to improve the characterization of polymerases that are essential to the life cycle of RNA viruses and develop antiviral therapies that target these enzymes, a continuous nonradioactive assay was developed to monitor the activity of RdRps by measuring the release of pyrophosphate (PP(i)) generated during nascent strand synthesis. A coupled-enzyme assay method based on the chemiluminescent detection of PP(i), using ATP sulfurylase and firefly luciferase, was adapted to monitor poliovirus 3D polymerase (3D(pol)) and the hepatitis C virus nonstructural protein 5B (NS5B) RdRp reactions. Light production was dependent on RdRp and sensitive to the concentration of oligonucleotide primer directing RNA synthesis. The assay system was found to be amenable to sensitive kinetic studies of RdRps, requiring only 6nM 3D(pol) to obtain a reliable estimate of the initial velocity in as little as 4 min. The assay can immediately accommodate the use of both homopolymer and heteropolymer RNA templates lacking uridylates and can be adapted to RNA templates containing uridine by substituting alpha-thio ATP for ATP. The low background signal produced by other NTPs can be corrected from no enzyme (RdRp) controls. The effect of RdRp/RNA template preincubation was assessed using NS5B and a homopolymer RNA template and a time-dependent increase of RdRp activity was observed. Progress curves for a chain terminator (3(')-deoxyguanosine 5(')-triphosphate) and an allosteric NS5B inhibitor demonstrated the predicted time- and dose-dependent reductions in signal. This assay should facilitate detailed kinetic studies of RdRps and their potential inhibitors using either standard or single-nucleotide approaches.

Allosteric Regulation↗
PubMed2004

Conserved C-terminal threonine of hepatitis C virus NS3 regulates autoproteolysis and prevents product inhibition.

Inspection of over 250 hepatitis C virus (HCV) genome sequences shows that a threonine is strictly conserved at the P1 position in the NS3-NS4A (NS3-4A) autoproteolysis junction, while a cysteine is maintained as the P1 residue in all of the putative trans cleavage sites (NS4A-4B, NS4B-5A, and NS5A-5B). To understand why T631 is conserved at the NS3-4A junction of HCV, a series of in vitro transcription-translation studies were carried out using wild-type and mutant (T631C) NS3-4A constructs bearing native, truncated, and mutant NS4A segments. The autocleavage of the wild-type junction was found to be dependent on the presence of the central cofactor domain of NS4A (residues 21 to 34). In contrast, all NS3-4A T631C mutant proteins underwent self-cleavage even in the absence of the cofactor. Subgenomic replicons derived from the Con1 strain of HCV and bearing the T631C mutation showed reduced levels of colony formation in transfection studies. Similarly, replicons derived from a second genotype 1b virus, HCV-N, demonstrated a comparable reduction in replication efficiency in transient-transfection assays. These data suggest that the threonine is conserved at position 631 because it serves two functions: (i) to slow processing at the NS3-4A cleavage site, ensuring proper intercalation of the NS4A cofactor with NS3 prior to polyprotein scission, and (ii) to prevent subsequent product inhibition by the NS3 C terminus.

Gene Expression Regulation, Viral↗
PubMed2003

The effect of ribavirin and IMPDH inhibitors on hepatitis C virus subgenomic replicon RNA.

The recent development of in vitro hepatitis C virus (HCV) RNA replication systems has provided useful tools for studying the intracellular anti-HCV activity of ribavirin. Ribavirin has been shown to: (1) induce "error catastrophe" in poliovirus, Proc. Natl. Acad. Sci. USA 98, 6895-6900), (2) be a pseudo-substrate of the HCV RNA-dependent RNA polymerase (RdRp) in vitro, J. Biol. Chem. 276, 46094-46098), and (3) increase mutations in HCV RNA in the binary T7 polymerase/HCV cDNA replication system, J. Virol. 76, 8505-8517). These findings have led to the hypothesis that ribavirin may also induce error catastrophe in HCV. However, the functional relevance of ribavirin-induced HCV RNA mutagenesis is unclear. By use of a colony formation assay, in which RNA is isolated from the HCV subgenomic replicon system following treatment, the impact of ribavirin, inosine-5'-monophosphate dehydrogenase (IMPDH) inhibitors, and the combination was assessed. Ribavirin reduced HCV replicon colony-forming efficiency (CFE) in a dose-dependent fashion, suggesting that ribavirin may be misincorporated into replicon RNA and result in an anti-replicon effect analogous to error catastrophe. This effect was markedly suppressed by addition of exogenous guanosine. Combination treatment with ribavirin and mycophenolic acid (MPA) or VX-497, both potent, nonnucleoside IMPDH inhibitors, led to a greatly enhanced anti-replicon effect. This enhancement was reversed by inclusion of guanosine with the treatment. In contrast, MPA or VX-497 alone had only marginal effects on both the quantity and quality (CFE) of replicon RNA, suggesting that although IMPDH inhibition is an important contributing factor to the overall ribavirin anti-HCV replicon activity, IMPDH inhibition by itself is not sufficient to exert an anti-HCV effect. Sequencing data targeting the neo gene segment of the HCV replicon indicated that ribavirin together with MPA or VX-497 increased the replicon error rate by about two-fold. Taken together these results further suggest that lethal mutagenesis may be an effective anti-HCV strategy. The colony formation assay provides a useful tool for evaluating mutagenic nucleoside analogs for HCV therapy. Finally, the data from combination treatment indicate potential therapeutic value for an enhanced anti-HCV effect when using ribavirin in combination with IMPDH inhibition.

Antimetabolites↗