The valine at position 82 (Val 82) in the active site of the human immunodeficiency virus (HIV) protease mutates in response to therapy with the protease inhibitor ritonavir. By using the X-ray crystal structure of the complex of HIV protease and ritonavir, the potent protease inhibitor ABT-378, which has a diminished interaction with Val 82, was designed. ABT-378 potently inhibited wild-type and mutant HIV protease (Ki = 1.3 to 3.6 pM), blocked the replication of laboratory and clinical strains of HIV type 1 (50% effective concentration [EC50], 0.006 to 0.017 μM), and maintained high potency against mutant HIV selected by ritonavir in vivo (EC50, ≤0.06 μM). The metabolism of ABT-378 was strongly inhibited by ritonavir in vitro. Consequently, following concomitant oral administration of ABT-378 and ritonavir, the concentrations of ABT-378 in rat, dog, and monkey plasma exceeded the in vitro antiviral EC50 in the presence of human serum by >50-fold after 8 h. In healthy human volunteers, coadministration of a single 400-mg dose of ABT-378 with 50 mg of ritonavir enhanced the area under the concentration curve of ABT-378 in plasma by 77-fold over that observed after dosing with ABT-378 alone, and mean concentrations of ABT-378 exceeded the EC50 for >24 h. These results demonstrate the potential utility of ABT-378 as a therapeutic intervention against AIDS.

BILN 2061 is a novel, specific hepatitis C virus (HCV) NS3 serine protease inhibitor discovered by Boehringer Ingelheim that has shown potent activity against HCV replicons in tissue culture and is currently under clinical investigation for the treatment of HCV infection. The poor fidelity of the HCV RNA-dependent RNA polymerase will likely lead to the development of drug-resistant viruses in treated patients. The development of resistance to BILN 2061 was studied by the in vitro passage of HCV genotype 1b replicon cells in the presence of a fixed concentration of the drug. Three weeks posttreatment, four colonies were expanded for genotypic and phenotypic characterization. The 50% inhibitory concentrations of BILN 2061 for these colonies were 72-to 1,228-fold higher than that for the wild-type replicon. Sequencing of the individual colonies identified several mutations in the NS3 serine protease gene. Molecular clones containing the single amino acid substitution A156T, R155Q, or D168V resulted in 357-fold, 24-fold, and 144-fold reductions in susceptibility to BILN 2061, respectively, compared to the level of susceptibility shown by the wild-type replicon. Modeling studies indicate that all three of these residues are located in close proximity to the inhibitor binding site. These findings, in addition to the three-dimensional structure analysis of the NS3/NS4A serine protease inhibitor complex, provide a strategic guide for the development of next-generation inhibitors of HCV NS3/NS4A serine protease.Hepatitis C virus (HCV) infection is believed to be the leading cause of chronic hepatitis, end-stage cirrhosis, and hepatocellular carcinoma, affecting over 4 million Americans and about 170 million people worldwide. Currently, the most effective treatment of HCV infection involves a combination of the nucleoside analog ribavirin with alpha interferon (IFN-␣). However, the regimen is prolonged and not well tolerated, and only approximately half of the genotype 1 HCV-infected individuals have a sustained virological response, although the response rate improves significantly (ϳ80%) when genotypes 2 and 3 are treated (7,29). An oral agent that offers promise as an efficacious alternative to IFN or that may be used in IFNcontaining regimens and that improves efficacy and/or the side effect profile is in great demand.The HCV genome is a 9.6-kb single-stranded RNA of positive polarity encoding a large polyprotein that is posttranslationally cleaved into structural and nonstructural proteins (3,12,23). The N-terminal domain (approximately 180 amino acids) of NS3 and the small hydrophobic NS4A protein compose a heterodimeric enzyme catalyzing the posttranslational processing of the HCV nonstructural proteins (1, 2, 23). Its structure has been extensively studied by X-ray crystallography (9, 30, 31) and nuclear magnetic resonance spectroscopy (1, 6). The proteolytic activity of NS3/NS4A serine protease is known to be essential for viral RNA replication (10,14). A recent study indicated that the NS3/NS4A serine protease a...

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Compounds A-782759 (an N-1-aza-4-hydroxyquinolone benzothiadiazine) and BILN-2061 are specific antihepatitis C virus (HCV) agents that inhibit the RNA-dependent RNA polymerase and the NS3 serine protease, respectively. Both compounds display potent activity against HCV replicons in tissue culture. In order to characterize the development of resistance to these anti-HCV agents, HCV subgenomic 1b-N replicon cells were cultured with A-782759 alone or in combination with BILN-2061 at concentrations 10 times above their corresponding 50% inhibitory concentrations in the presence of neomycin. Single substitutions in the NS5B polymerase gene (H95Q, N411S, M414L, M414T, or Y448H) resulted in substantial decreases in susceptibility to A-782759. Similarly, replicons containing mutations in the NS5B polymerase gene (M414L or M414T), together with single mutations in the NS3 protease gene (A156V or D168V), conferred high levels of resistance to both A-782759 and BILN-2061. However, the A-782759-resistant mutants remained susceptible to nucleoside and two other classes of nonnucleoside NS5B polymerase inhibitors, as well as interferon. In addition, we found that the frequency of replicons resistant to both compounds was significantly lower than the frequency of resistance to the single compound. Furthermore, the dually resistant mutants displayed significantly reduced replication capacities compared to the wild-type replicon. These findings provide strategic guidance for the future treatment of HCV infection.Hepatitis C virus (HCV) is a leading cause of chronic liver disease, affecting over 4 million Americans and about 170 million people worldwide. The current standard of care for chronic HCV infection involves extended dosing with alpha interferon (IFN-␣) and ribavirin (10,14,18). However, these regimens have limited clinical benefit due to poor tolerability and limited efficacy (only approximately half of genotype 1 HCV-infected individuals have a sustained virological response, whereas the response rate improves significantly [ϳ80%] when genotypes 2 and 3 are treated) (9,11,35). Therefore, development of inhibitors against virally encoded targets is urgently needed.The HCV genome is a 9.6-kb single-stranded RNA of positive polarity encoding a large polyprotein, which is the precursor of at least 10 mature viral proteins: C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (2). The HCV polymerase encoded by nonstructural protein 5B (NS5B) is responsible for HCV RNA-dependent RNA polymerase (RdRp) and terminal transferase activities (4,31,32). The N-terminal domain (approximately 180 amino acids) of NS3 and the small hydrophobic NS4A protein compose a heterodimeric enzyme catalyzing the posttranslational processing of the HCV NS proteins (1, 21). Both NS5B RdRp and NS3 serine protease are believed to be components of the HCV replication complex, responsible for viral RNA replication, and have been shown to be indispensable for HCV replication in chimpanzees (26).To date, a number of distinct classes of NS5B RdRp inhibitors...

Combinatorial and structure-based medicinal chemistry strategies were used together to advance a lead compound with an activity of K(i) = 58 microM via a potency enhancement of >70 000-fold to an analogue with an activity of K(i) = 0.8 nM against influenza neuraminidase (A/Tokyo/67). Lead optimization was initiated using molecular modeling and combinatorial chemistry. Protein crystal structures revealed that inconsistent structure-activity relationship (SAR) data resulted from different binding orientations of the inhibitor core five-membered rings from one series to another. Binding modes for a series of compounds showed up to a 180 degrees variation in orientation of the five-membered ring within the active site. Potent analogues were only achieved with chemical series that were observed to bind in the same orientation and yielded consistent SAR. In one series, consistent binding was obtained by an unprecedented occupation of a negatively charged binding pocket by a neutral methyl ester unit. The structural rationale for this novel SAR variation, based on protein crystallographic data, is given.