Maxwell D. Leslie

Biochemical Mechanism of HIV-1 Resistance to Rilpivirine

Background: Reverse transcriptase mutations E138K and M184I emerged most frequently in HIV-1 patients who failed rilpivirine/emtricitabine/tenofovir combination therapy. Results: M184I reduces polymerase efficiency, and E138K restores it. E138K also reduces rilpivirine binding affinity mainly by increasing its dissociation rate. Conclusion: E138K abrogates the polymerase defect of M184I and increases rilpivirine dissociation. Significance: Our results provide a biochemical explanation for the selection of E138K/M184I in patients who failed combination therapy. Rilpivirine (RPV) is a second generation nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) that efficiently inhibits HIV-1 resistant to first generation NNRTIs. Virological failure during therapy with RPV and emtricitabine is associated with the appearance of E138K and M184I mutations in RT. Here we investigate the biochemical mechanism of RT inhibition and resistance to RPV. We used two transient kinetics approaches (quench-flow and stopped-flow) to determine how subunit-specific mutations in RT p66 or p51 affect association and dissociation of RPV to RT as well as their impact on binding of dNTP and DNA and the catalytic incorporation of nucleotide. We compared WT with four subunit-specific RT mutants, p66M184I/p51WT, p66E138K/p51E138K, p66E138K/M184I/p51E138K, and p66M184I/p51E138K. Ile-184 in p66 (p66184I) decreased the catalytic efficiency of RT (kpol/Kd.dNTP), primarily through a decrease in dNTP binding (Kd.dNTP). Lys-138 either in both subunits or in p51 alone abrogated the negative effect of p66184I by restoring dNTP binding. Furthermore, p51138K reduced RPV susceptibility by altering the ratio of RPV dissociation to RPV association, resulting in a net reduction in RPV equilibrium binding affinity (Kd.RPV = koff.RPV/kon.RPV). Quantum mechanics/molecular mechanics hybrid molecular modeling revealed that p51E138K affects access to the RPV binding site by disrupting the salt bridge between p51E138 and p66K101. p66184I caused repositioning of the Tyr-183 active site residue and decreased the efficiency of RT, whereas the addition of p51138K restored Tyr-183 to a WT-like conformation, thus abrogating the Ile-184-induced functional defects.

Structural and Inhibition Studies of the RNase H Function of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase

ABSTRACT RNase H inhibitors (RNHIs) have gained attention as potential HIV-1 therapeutics. Although several RNHIs have been studied in the context of HIV-1 reverse transcriptase (RT) RNase H, there is no information on inhibitors that might affect the RNase H activity of other RTs. We performed biochemical, virological, crystallographic, and molecular modeling studies to compare the RNase H function and inhibition profiles of the gammaretroviral xenotropic murine leukemia virus-related virus (XMRV) and Moloney murine leukemia virus (MoMLV) RTs to those of HIV-1 RT. The RNase H activity of XMRV RT is significantly lower than that of HIV-1 RT and comparable to that of MoMLV RT. XMRV and MoMLV, but not HIV-1 RT, had optimal RNase H activities in the presence of Mn2+ and not Mg2+. Using hydroxyl-radical footprinting assays, we demonstrated that the distance between the polymerase and RNase H domains in the MoMLV and XMRV RTs is longer than that in the HIV-1 RT by ∼3.4 Å. We identified one naphthyridinone and one hydroxyisoquinolinedione as potent inhibitors of HIV-1 and XMRV RT RNases H with 50% inhibitory concentrations ranging from ∼0.8 to 0.02 μM. Two acylhydrazones effective against HIV-1 RT RNase H were less potent against the XMRV enzyme. We also solved the crystal structure of an XMRV RNase H fragment at high resolution (1.5 Å) and determined the molecular details of the XMRV RNase H active site, thus providing a framework that would be useful for the design of antivirals that target RNase H.

Inhibitors of Foot and Mouth Disease Virus Targeting a Novel Pocket of the RNA-Dependent RNA Polymerase

Background Foot-and-Mouth Disease Virus (FMDV) is a picornavirus that infects cloven-hoofed animals and leads to severe losses in livestock production. In the case of an FMD outbreak, emergency vaccination requires at least 7 days to trigger an effective immune response. There are currently no approved inhibitors for the treatment or prevention of FMDV infections. Methodology/Principal Findings Using a luciferase-based assay we screened a library of compounds and identified seven novel inhibitors of 3Dpol, the RNA-dependent RNA polymerase of FMDV. The compounds inhibited specifically 3Dpol (IC50s from 2-17 µM) and not other viral or bacterial polymerases. Enzyme kinetic studies on the inhibition mechanism by compounds 5D9 and 7F8 showed that they are non-competitive inhibitors with respect to NTP and nucleic acid substrates. Molecular modeling and docking studies into the 3Dpol structure revealed an inhibitor binding pocket proximal to, but distinct from the 3Dpol catalytic site. Residues surrounding this pocket are conserved among all 60 FMDV subtypes. Site directed mutagenesis of two residues located at either side of the pocket caused distinct resistance to the compounds, demonstrating that they indeed bind at this site. Several compounds inhibited viral replication with 5D9 suppressing virus production in FMDV-infected cells with EC50 = 12 µM and EC90 = 20 µM). Significance We identified several non-competitive inhibitors of FMDV 3Dpol that target a novel binding pocket, which can be used for future structure-based drug design studies. Such studies can lead to the discovery of even more potent antivirals that could provide alternative or supplementary options to contain future outbreaks of FMD.

4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) Inhibits HIV-1 Reverse Transcriptase with Multiple Mechanisms

Background: 4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a highly potent nucleoside analog reverse transcriptase (RT) inhibitor with a 3′-OH. Results: EFdA inhibits RT as an immediate or delayed chain terminator depending on the DNA substrate sequence. RT efficiently misincorporates EFdA, producing non-extendable mismatched primers protected from excision. Conclusion: EFdA blocks RT by multiple mechanisms. Significance: Understanding the EFdA inhibition mechanism will help develop better antivirals. 4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a nucleoside analog that, unlike approved anti-human immunodeficiency virus type 1 (HIV-1) nucleoside reverse transcriptase inhibitors, has a 3′-OH and exhibits remarkable potency against wild-type and drug-resistant HIVs. EFdA triphosphate (EFdA-TP) is unique among nucleoside reverse transcriptase inhibitors because it inhibits HIV-1 reverse transcriptase (RT) with multiple mechanisms. (a) EFdA-TP can block RT as a translocation-defective RT inhibitor that dramatically slows DNA synthesis, acting as a de facto immediate chain terminator. Although non-translocated EFdA-MP-terminated primers can be unblocked, they can be efficiently converted back to the EFdA-MP-terminated form. (b) EFdA-TP can function as a delayed chain terminator, allowing incorporation of an additional dNTP before blocking DNA synthesis. In such cases, EFdA-MP-terminated primers are protected from excision. (c) EFdA-MP can be efficiently misincorporated by RT, leading to mismatched primers that are extremely hard to extend and are also protected from excision. The context of template sequence defines the relative contribution of each mechanism and affects the affinity of EFdA-MP for potential incorporation sites, explaining in part the lack of antagonism between EFdA and tenofovir. Changes in the type of nucleotide before EFdA-MP incorporation can alter its mechanism of inhibition from delayed chain terminator to immediate chain terminator. The versatility of EFdA in inhibiting HIV replication by multiple mechanisms may explain why resistance to EFdA is more difficult to emerge.

SAMHD1 Has Differential Impact on the Efficacies of HIV Nucleoside Reverse Transcriptase Inhibitors

ABSTRACT Sterile alpha motif- and histidine/aspartic acid domain-containing protein 1 (SAMHD1) limits HIV-1 replication by hydrolyzing deoxynucleoside triphosphates (dNTPs) necessary for reverse transcription. Nucleoside reverse transcriptase inhibitors (NRTIs) are components of anti-HIV therapies. We report here that SAMHD1 cleaves NRTI triphosphates (TPs) at significantly lower rates than dNTPs and that SAMHD1 depletion from monocytic cells affects the susceptibility of HIV-1 infections to NRTIs in complex ways that depend not only on the relative changes in dNTP and NRTI-TP concentrations but also on the NRTI activation pathways.

Effects of Substitutions at the 4′ and 2 Positions on the Bioactivity of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine

ABSTRACT Nucleos(t)ide reverse transcriptase inhibitors (NRTIs) form the backbone of most anti-HIV therapies. We have shown that 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a highly effective NRTI; however, the reasons for the potent antiviral activity of EFdA are not well understood. Here, we use a combination of structural, computational, and biochemical approaches to examine how substitutions in the sugar or adenine rings affect the incorporation of dA-based NRTIs like EFdA into DNA by HIV RT and their susceptibility to deamination by adenosine deaminase (ADA). Nuclear magnetic resonance (NMR) spectroscopy studies of 4′-substituted NRTIs show that ethynyl or cyano groups stabilize the sugar ring in the C-2′-exo/C-3′-endo (north) conformation. Steady-state kinetic analysis of the incorporation of 4′-substituted NRTIs by RT reveals a correlation between the north conformation of the NRTI sugar ring and efficiency of incorporation into the nascent DNA strand. Structural analysis and the kinetics of deamination by ADA demonstrate that 4′-ethynyl and cyano substitutions decrease the susceptibility of adenosine-based compounds to ADA through steric interactions at the active site. However, the major determinant for decreased susceptibility to ADA is the 2-halo substitution, which alters the pKa of N1 on the adenine base. These results provide insight into how NRTI structural attributes affect their antiviral activities through their interactions with the RT and ADA active sites.

Evaluation of Combinations of 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine with Clinically Used Antiretroviral Drugs

ABSTRACT Drug combination studies of 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) with FDA-approved drugs were evaluated by two different methods, MacSynergy II and CalcuSyn. Most of the combinations, including the combination of the two adenosine analogs EFdA and tenofovir, were essentially additive, without substantial antagonism or synergism. The combination of EFdA and rilpivirine showed apparent synergism. These studies provide information that may be useful for the design of EFdA combination regimens for initial and salvage therapy assessment.

3-hydroxypyrimidine-2,4-diones as novel hepatitis B virus antivirals targeting the viral ribonuclease H

ABSTRACT Hepatitis B virus (HBV) RNase H (RNH) is an appealing therapeutic target due to its essential role in viral replication. RNH inhibitors (RNHIs) could help to more effectively control HBV infections. Here, we report 3-hydroxypyrimidine-2,4-diones as novel HBV RNHIs with antiviral activity. We synthesized and tested 52 analogs and found 4 that inhibit HBV RNH activity in infected cells. Importantly, 2 of these compounds inhibited HBV replication in the low micromolar range.

Biochemical mechanism of clinical resistance to rilpivirine

Background The introduction of HAART has significantly prolonged the life span of HIV-infected patients. However, the error-prone nature of HIV-1 reverse transcriptase (HIV-1 RT) results in the emergence of drug-resistant viruses and threatens the effectiveness of HAART. HIV-1 RT is a primary target of two classes of drugs: nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). Recent phase III clinical trials have shown that two HIV-1 RT mutations, E138K and M184I, were the most frequent mutations found in patients that experienced virological failure during therapy that included rilpivirine (RPV), emtricitabine (FTC), and tenofovir (TDF).