TMC278 is a diarylpyrimidine (DAPY) nonnucleoside reverse transcriptase inhibitor (NNRTI) that is highly effective in treating wild-type and drug-resistant HIV-1 infections in clinical trials at relatively low doses (ϳ25-75 mg/day). We have determined the structure of wild-type HIV-1 RT complexed with TMC278 at 1.8 Å resolution, using an RT crystal form engineered by systematic RT mutagenesis. This highresolution structure reveals that the cyanovinyl group of TMC278 is positioned in a hydrophobic tunnel connecting the NNRTI-binding pocket to the nucleic acid-binding cleft. The crystal structures of TMC278 in complexes with the double mutant K103N/Y181C (2.1 Å) and L100I/K103N HIV-1 RTs (2.9 Å) demonstrated that TMC278 adapts to bind mutant RTs. In the K103N/Y181C RT/TMC278 structure, loss of the aromatic ring interaction caused by the Y181C mutation is counterbalanced by interactions between the cyanovinyl group of TMC278 and the aromatic side chain of Y183, which is facilitated by an ϳ1.5 Å shift of the conserved Y183MDD motif. In the L100I/K103N RT/ TMC278 structure, the binding mode of TMC278 is significantly altered so that the drug conforms to changes in the binding pocket primarily caused by the L100I mutation. The flexible binding pocket acts as a molecular ''shrink wrap'' that makes a shape complementary to the optimized TMC278 in wild-type and drug-resistant forms of HIV-1 RT. The crystal structures provide a better understanding of how the flexibility of an inhibitor can compensate for drug-resistance mutations.drug design ͉ drug resistance ͉ nonnucleoside reverse transcriptase inhibitor ͉ polymerase ͉ x-ray crystallography C ombinations of drugs have been used to successfully treat HIV-1 infections, permitting dramatic reduction in viral loads and restoration of the immune system. However, drug treatments do not eliminate the infection and treatment must be life-long. There are problems with drug toxicity, and prolonged drug exposure can lead to the emergence of drug-resistant mutant viruses. Because toxicity and drug resistance complicate treatment strategies, the development of new anti-AIDS drugs remains essential., also referred to as rilpivirine and R278474, is a diarylpyrimidine (DAPY) nonnucleoside reverse transcriptase inhibitor (NNRTI) that was developed in a multidisciplinary structure-based design effort (3, 4) intended to discover drugs that can inhibit a wide range of the known drug-resistant HIV-1 strains. In cell-based assays, TMC278 inhibits a broad spectrum of HIV-1 variants (Table 1), including strains that are partially or completely resistant to the existing NNRTI drugs (3). Phase I and II clinical trials have shown that TMC278 effectively reduces viral load and maintains low viral load levels with no virus rebound in patients even after 48 weeks of treatment (5, 6).Crystal structures of HIV-1 RT/NNRTI complexes were solved in the course of developing TMC278 (7, 8) along a path that originated with the first-generation TIBO compounds (9). Most of those structures were obtained at ϳ...

Combinations of nucleoside and nonnucleoside inhibitors (NNRTIs) of HIV-1 reverse transcriptase (RT) are widely used in anti-AIDS therapies. Five NNRTIs including nevirapine are clinical drugs; however, the molecular mechanism of inhibition by NNRTIs is not clear. We determined the crystal structures of RT–DNA–nevirapine, RT–DNA, and RT–DNA–AZT-triphosphate complexes at 2.85, 2.70, and 2.80 Å, respectively. The RT–DNA complex in the crystal could bind nevirapine or AZT-triphosphate; however, not both. Binding of nevirapine led to opening of the NNRTI-binding pocket. The pocket formation caused shifting of the 3’-end of DNA primer by ~5.5 Å away from its polymerase active site position. Nucleic acid interactions with fingers and palm subdomains were reduced, the dNTP-binding pocket was distorted, and the thumb opened up. The structures elucidate complementary roles of nucleoside and nonnucleoside inhibitors in inhibiting RT.

Summary Novel inhibitors are needed to counteract the rapid emergence of drug-resistant HIV variants. HIV-1 reverse transcriptase (RT) has both DNA polymerase and RNase H (RNH) enzymatic activities, but approved drugs that inhibit RT target the polymerase. Inhibitors that act against new targets, like RNH, would be effective against all of the current drug-resistant variants. Here, we present 2.80 Å and 2.04 Å resolution crystal structures of an RNH inhibitor, β-thujaplicinol, bound at the RNH active site of both HIV-1 RT and an isolated RNH domain. β-thujaplicinol chelates two divalent metal ions at the RNH active site. We provide biochemical evidence that β-thujaplicinol is a slow-binding RNH inhibitor with non-competitive kinetics and suggest that it forms a tropylium ion that interacts favorably with RT and the RNA:DNA substrate.

Seasonal and pandemic influenza viruses continue to be a leading global health concern. Emerging resistance to the current drugs and the variable efficacy of vaccines underscore the need for developing new flu drugs that will be broadly effective against wild-type and drug-resistant influenza strains. Here, we report the discovery and development of a class of inhibitors targeting the cap-snatching endonuclease activity of the viral polymerase. A high-resolution crystal form of pandemic 2009 H1N1 influenza polymerase acidic protein N-terminal endonuclease domain (PAN) was engineered and used for fragment screening leading to the identification of new chemical scaffolds binding to the PAN active site cleft. During the course of screening, binding of a third metal ion that is potentially relevant to endonuclease activity, was detected in the active site cleft of PAN in the presence of a fragment. Using structure-based optimization, we developed a highly potent hydroxypyridinone series of compounds from a fragment hit that defines a new mode of chelation to the active site metal ions. A compound from the series demonstrating promising enzymatic inhibition in a fluorescence-based enzyme assay with an IC50 value of 11 nM was found to have an antiviral activity (EC50) of 11 μM against PR8 H1N1 influenza A in MDCK cells.