HIV-1 reverse transcriptase (RT) catalytically incorporates individual nucleotides into a viral DNA strand complementing an RNA or DNA template strand; the polymerase active site of RT adopts multiple conformational and structural states while performing this task. The states associated are dNTP binding at the N site, catalytic incorporation of a nucleotide, release of a pyrophosphate, and translocation of the primer 3′-end to the P site. Structural characterization of each of these states may help in understanding the molecular mechanisms of drug activity and resistance and in developing new RT inhibitors. Using a 38-mer DNA template-primer aptamer as the substrate mimic, we crystallized an RT/dsDNA complex that is catalytically active, yet translocation-incompetent in crystals. The ability of RT to perform dNTP binding and incorporation in crystals permitted obtaining a series of structures: (I) RT/DNA (P-site), (II) RT/DNA/AZTTP ternary, (III) RT/AZT-terminated DNA (N-site), and (IV) RT/AZT-terminated DNA (N-site)/foscarnet complexes. The stable N-site complex permitted the binding of foscarnet as a pyrophosphate mimic. The Mg2+ ions dissociated after catalytic addition of AZTMP in the pretranslocated structure III, whereas ions A and B had re-entered the active site to bind foscarnet in structure IV. The binding of foscarnet involves chelation with the Mg2+ (B) ion and interactions with K65 and R72. The analysis of interactions of foscarnet and the recently discovered nucleotide-competing RT inhibitor (NcRTI) α-T-CNP in two different conformational states of the enzyme provides insights for developing new classes of polymerase active site RT inhibitors.

Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry has emerged as a powerful tool for analyzing the conformational dynamics of protein–ligand and protein–protein interactions. Recent advances in instrumentation and methodology have expanded the utility of HDX for the analysis of large and complex proteins; however, asymmetric dimers with shared amino acid sequence present a unique challenge for HDX because assignment of peptides with identical sequence to their subunit of origin remains ambiguous. Here we report the use of differential isotopic labeling to facilitate HDX analysis of multimers using HIV-1 reverse transcriptase (RT) as a model. RT is an asymmetric heterodimer of 51 kDa (p51) and 66 kDa (p66) subunits. The first 440 residues of p51 and p66 are identical. In this study differentially labeled RT was reconstituted from isotopically enriched (15N-labeled) p51 and unlabeled p66. To enable detection of 15N-deuterated RT peptides, the software HDX Workbench was modified to follow a 100% 15N model. Our results demonstrated that 15N enrichment of p51 did not affect its conformational dynamics compared to unlabeled p51, but 15N-labeled p51 did show different conformational dynamics than p66 in the RT heterodimer. Differential HDX-MS of isotopically labeled RT in the presence of the non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV) showed subunit-specific perturbation in the rate of HDX consistent with previously published results and the RT-EFV cocrystal structure.

HIV-1 reverse transcriptase (RT) undergoes a series of conformational changes during viral replication and is a central target for antiretroviral therapy. The intrinsic flexibility of RT can provide novel allosteric sites for inhibition. Crystals of RT that diffract X-rays to better than 2 Å resolution facilitated the probing of RT for new druggable sites using fragment screening by X-ray crystallography. A total of 775 fragments were grouped into 143 cocktails, which were soaked into crystals of RT in complex with the non-nucleoside drug rilpivirine (TMC278). Seven new sites were discovered, including the Incoming Nucleotide Binding, Knuckles, NNRTI Adjacent, and 399 sites, located in the polymerase region of RT, and the 428, RNase H Primer Grip Adjacent, and 507 sites, located in the RNase H region. Three of these sites (Knuckles, NNRTI Adjacent, and Incoming Nucleotide Binding) are inhibitory and provide opportunities for discovery of new anti-AIDS drugs.

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.

Several 3-hydroxyquinolin-2(1H)-ones derivatives were synthesized and evaluated as inhibitors of 2009 pandemic H1N1 influenza A endonuclease. All five of the monobrominated 3-hydroxyquinolin(1H)-2-ones derivatives were synthesized. Suzuki-coupling of p-fluorophenylboronic acid with each of these brominated derivatives provided the respective p-fluorophenyl 3-hydroxyquinolin(1H)-2-ones. In addition to 3-hydroxyquinolin-2(1H)-one, its 4-methyl, 4-phenyl, 4-methyl-7-(p-fluorophenyl), and 4-phenyl-7-(p-fluorophenyl) derivatives were also synthesized. Comparative studies on their relative activity revealed that both 6- and 7-(p-fluorophenyl)-3-hydroxyquinolin-2(1H)-one are among the more potent inhibitors of H1N1 influenza A endonuclease. An X-ray crystal structure of 7-(p-fluorophenyl)-3-hydroxyquinolin-2(1H)-one complexed to the influenza endonuclease revealed that this molecule chelates to two metal ions at the active site of the enzyme.Keywords: 3-hydroxyquinolin-2-ones; antiviral; endonuclease; influenza A; quinolinones;

tert-Butyldimethylsilyl-spiroaminooxathioledioxide (TSAO) compounds have an embedded thymidine-analogue backbone; however, TSAO compounds invoke non-nucleoside RT inhibitor (NNRTI) resistance mutations. Our crystal structure of RT:7 (TSAO-T) complex shows that 7 binds inside the NNRTI-binding pocket, assuming a “dragon” shape, and interacts extensively with almost all the pocket residues. The structure also explains the structure−activity relationships and resistance data for TSAO compounds. The binding of 7 causes hyper-expansion of the pocket and significant rearrangement of RT subdomains. This nonoptimal complex formation is apparently responsible (1) for the lower stability of a RT (p66/p51) dimer and (2) for the lower potency of 7 despite of its extensive interactions with RT. However, the HIV-1 RT:7 structure reveals novel design features such as (1) interactions with the conserved Tyr183 from the YMDD-motif and (2) a possible way for an NNRTI to reach the polymerase active site that may be exploited in designing new NNRTIs.

Clustering of 99 available X-ray crystal structures of HIV-1 reverse transcriptase (RT) at the flexible non-nucleoside inhibitor binding pocket (NNIBP) provides information about features of the conformational landscape for binding non-nucleoside inhibitors (NNRTIs), including effects of mutation and crystal forms. The ensemble of NNIBP conformations is separated into eight discrete clusters based primarily on the position of the functionally important primer grip, the displacement of which is believed to be one of the mechanisms of inhibition of RT. Two of these clusters are populated by structures in which the primer grip exhibits novel conformations that differ from the predominant cluster by over 4 Å and are induced by the unique inhibitors capravirine and rilpivirine/TMC278. This work identifies a new conformation of the NNIBP that may be used to design NNRTIs. It can also be used to guide more complete exploration of the NNIBP free energy landscape using advanced sampling techniques.

The non-nucleoside reverse transcriptase inhibitor (NNRTI) TMC278/rilpivirine is an anti-AIDS therapeutic agent with high oral bioavailability despite its high hydrophobicity. Previous studies established a correlation between ability of the drug molecule to form stable, homogeneous populations of spherical nanoparticles (∼100−120 nm in diameter) at low pH in surfactant-independent fashion and good oral bioavailability. Here, we hypothesize that the drug is able to assume surfactant-like properties under physiologically relevant conditions, thus facilitating formation of nanostructures in the absence of other surfactants. The results of all-atom molecular dynamics simulations indeed show that protonated drug molecules behave as surfactants at the water/aggregate interface while neutral drug molecules assist aggregate packing via conformational variability. Our simulation results suggest that amphiphilic behavior at low pH and intrinsic flexibility influence drug aggregation and are believed to play critical roles in the favorable oral bioavailability of hydrophobic drugs.

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 (∼25–75 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 high-resolution 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 ∼1.5 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.

Influenza A viruses are responsible for seasonal epidemics and high mortality pandemics. A major function of the viral NS1A protein, a virulence factor, is the inhibition of the production of IFN-β mRNA and other antiviral mRNAs. The NS1A protein of the human influenza A/Udorn/72 (Ud) virus inhibits the production of these antiviral mRNAs by binding the cellular 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), which is required for the 3′ end processing of all cellular pre-mRNAs. Here we report the 1.95-Å resolution X-ray crystal structure of the complex formed between the second and third zinc finger domain (F2F3) of CPSF30 and the C-terminal domain of the Ud NS1A protein. The complex is a tetramer, in which each of two F2F3 molecules wraps around two NS1A effector domains that interact with each other head-to-head. This structure identifies a CPSF30 binding pocket on NS1A comprised of amino acid residues that are highly conserved among human influenza A viruses. Single amino acid changes within this binding pocket eliminate CPSF30 binding, and a recombinant Ud virus expressing an NS1A protein with such a substitution is attenuated and does not inhibit IFN-β pre-mRNA processing. This binding pocket is a potential target for antiviral drug development. The crystal structure also reveals that two amino acids outside of this pocket, F103 and M106, which are highly conserved (>99%) among influenza A viruses isolated from humans, participate in key hydrophobic interactions with F2F3 that stabilize the complex.

Tenofovir, also known as PMPA, R-9-(2-(phosphonomethoxypropyl)adenine, is a nucleotide reverse transcriptase (RT) inhibitor. We have determined the crystal structures of two related complexes of HIV-1 RT with template primer and tenofovir: (i) a ternary complex at a resolution of 3.0 Å of RT crosslinked to a dideoxy-terminated DNA with tenofovir-diphosphate bound as the incoming substrate; and (ii) a RT−DNA complex at a resolution of 3.1 Å with tenofovir at the 3' primer terminus. The tenofovir nucleotide in the tenofovir-terminated structure seems to adopt multiple conformations. Some nucleoside reverse transcriptase inhibitors, including 3TC and AZT, have elements ('handles') that project beyond the corresponding elements on normal dNTPs (the 'substrate envelope'). HIV-1 RT resistance mechanisms to AZT and 3TC take advantage of these handles; tenofovir's structure lacks handles that could protrude through the substrate envelope to cause resistance.

X-ray crystallography has been an under-appreciated screening tool for fragment-based drug discovery due to the perception of low throughput and technical difficulty. Investigators in industry and academia have overcome these challenges by taking advantage of key factors that contribute to a successful crystallographic screening campaign. Efficient cocktail design and soaking methodologies have evolved to maximize throughput while minimizing false positives/negatives. In addition, technical improvements at synchrotron beamlines have dramatically increased data collection rates thus enabling screening on a timescale comparable to other techniques. The combination of available resources and efficient experimental design has resulted in many successful crystallographic screening campaigns. The three-dimensional crystal structure of the bound fragment complexed to its target, a direct result of the screening effort, enables structure-based drug design while revealing insights regarding protein dynamics and function not readily obtained through other experimental approaches. Furthermore, this “chemical interrogation” of the target protein crystals can lead to the identification of useful reagents for improving diffraction resolution or compound solubility.

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, such as RNH, should 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 noncompetitive kinetics and suggest that it forms a tropylium ion that interacts favorably with RT and the RNA:DNA substrate.

Lys103Asn and Tyr181Cys are the two mutations frequently observed in patients exposed to various non-nucleoside reverse transcriptase inhibitor drugs (NNRTIs). Human immunodeficiency virus (HIV) strains containing both reverse transcriptase (RT) mutations are resistant to all of the approved NNRTI drugs. We have determined crystal structures of Lys103Asn/Tyr181Cys mutant HIV-1 RT with and without a bound non-nucleoside inhibitor (HBY 097, (S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthio-methyl)-3,4-dihydroquinoxalin-2(1H)-thione) at 3.0 Å and 2.5 Å resolution, respectively. The structure of the double mutant RT/HBY 097 complex shows a rearrangement of the isopropoxycarbonyl group of HBY 097 compared to its binding with wild-type RT. HBY 097 makes a hydrogen bond with the thiol group of Cys181 that helps the drug retain potency against the Tyr181Cys mutation. The structure of the unliganded double mutant HIV-1 RT showed that Lys103Asn mutation facilitates coordination of a sodium ion with Lys101 O, Asn103 N and Oδ1, Tyr188 Oη, and two water molecules. The formation of the binding pocket requires the removal of the sodium ion. Although the RT alone and the RT/HBY 097 complex were crystallized in the presence of ATP, only the RT has an ATP coordinated with two Mn2+ at the polymerase active site. The metal coordination mimics a reaction intermediate state in which complete octahedral coordination was observed for both metal ions. Asp186 coordinates at an axial position whereas the carboxylates of Asp110 and Asp185 are in the planes of coordination of both metal ions. The structures provide evidence that NNRTIs restrict the flexibility of the YMDD loop and prevent the catalytic aspartate residues from adopting their metal-binding conformations.

The rapid replication of HIV-1 and the errors made during viral replication cause the virus to evolve rapidly in patients, making the problems of vaccine development and drug therapy particularly challenging. In the absence of an effective vaccine, drugs are the only useful treatment. Anti-HIV drugs work; so far drug therapy has saved more than three million years of life. Unfortunately, HIV-1 develops resistance to all of the available drugs. Although a number of useful anti-HIV drugs have been approved for use in patients, the problems associated with drug toxicity and the development of resistance means that the search for new drugs is an ongoing process. The three viral enzymes, reverse transcriptase (RT), integrase (IN), and protease (PR) are all good drug targets. Two distinct types of RT inhibitors, both of which block the polymerase activity of RT, have been approved to treat HIV-1 infections, nucleoside analogs (NRTIs) and nonnucleosides (NNRTIs), and there are promising leads for compounds that either block the RNase H activity or block the polymerase in other ways. A better understanding of the structure and function(s) of RT and of the mechanism(s) of inhibition can be used to generate better drugs; in particular, drugs that are effective against the current drug-resistant strains of HIV-1.

•Both the DNA polymerase and RNase H activities of RT are vital for HIV viability.•Only the DNA polymerase activity of this enzyme has been targeted by anti-AIDS drugs.•F3284-8495 is a micromolar inhibitor of HIV RNase H that is chemically modifiable.•A 1.71-Å crystal structure is presented of F3284-8495 bound to the RNase H active site and analysis of more active analogs.•Our analysis establishes proof of concept that the F3284-8495 scaffold can be the basis for development of more potent HIV RNase H inhibitors.Human immunodeficiency virus (HIV) encodes four essential enzymes: protease, integrase, reverse transcriptase (RT)-associated DNA polymerase, and RT-associated ribonuclease H (RNase H). Current clinically approved anti-AIDS drugs target all HIV enzymatic activities except RNase H, which has proven to be a very difficult target for HIV drug discovery. Our high-throughput screening activities identified the dihydroxycoumarin compound F3284-8495 as a specific inhibitor of RT RNase H, with low micromolar potency in vitro. Optimization of inhibitory potency can be facilitated by structural information about inhibitor–target binding. Here, we report the crystal structure of F3284-8495 bound to the active site of an isolated RNase H domain of HIV-1 RT at a resolution limit of 1.71 Å. From predictions based on this structure, compounds were obtained that showed improved inhibitory activity. Computational analysis suggested structural alterations that could provide additional interactions with RT and thus improve inhibitory potency. These studies established proof of concept that F3284-8495 could be used as a favorable chemical scaffold for development of HIV RNase H inhibitors.Download high-res image (77KB)Download full-size image

In this issue of Structure, Lu et al. (2008) report results of structural and functional analysis of rotavirus RNA-dependent RNA polymerase, VP1. Based on their analyses of VP1 in RNA free and bound forms, the authors propose a mechanism for coordinated genome packaging and replication.