HIV reverse transcriptase (RT) is an enzyme that plays a major role in the replication cycle of HIV and has been a key target of anti-HIV drug development efforts. Because of the high genetic ...diversity of the virus, mutations in RT can impart resistance to various RT inhibitors. As the prevalence of drug resistance mutations is on the rise, it is necessary to design strategies that will lead to drugs less susceptible to resistance. Here we provide an in-depth review of HIV reverse transcriptase, current RT inhibitors, novel RT inhibitors, and mechanisms of drug resistance. We also present novel strategies that can be useful to overcome RT’s ability to escape therapies through drug resistance. While resistance may not be completely avoidable, designing drugs based on the strategies and principles discussed in this review could decrease the prevalence of drug resistance.
The detailed molecular interactions between native HIV-1 capsid protein (CA) hexamers that shield the viral genome and proteins have been elusive. We report crystal structures describing interactions ...between CA monomers related by sixfold symmetry within hexamers (intrahexamer) and threefold and twofold symmetry between neighboring hexamers (interhexamer). The structures describe how CA builds hexagonal lattices, the foundation of mature capsids. Lattice structure depends on an adaptable hydration layer modulating interactions among CA molecules. Disruption of this layer alters interhexamer interfaces, highlighting an inherent structural variability. A CA-targeting antiviral affects capsid stability by binding across CA molecules and subtly altering interhexamer interfaces remote to the ligand-binding site. Inherent structural plasticity, hydration layer rearrangement, and effector binding affect capsid stability and have functional implications for the retroviral life cycle.
HIV-1 capsid protein (CA) is the molecular target of the recently FDA-approved long acting injectable (LAI) drug lenacapavir (GS-6207). The quick emergence of CA mutations resistant to GS-6207 ...necessitates the design and synthesis of novel sub-chemotypes. We have conducted the structure-based design of two new sub-chemotypes combining the scaffold of GS-6207 and the N-terminal cap of PF74 analogs, the other important CA-targeting chemotype. The design was validated via induced-fit molecular docking. More importantly, we have worked out a general synthetic route to allow the modular synthesis of novel GS-6207 subtypes. Significantly, the desired stereochemistry of the skeleton C
was confirmed via an X-ray crystal structure of the key synthetic intermediate
. Although the newly synthesized analogs did not show significant potency, our efforts herein will facilitate the future design and synthesis of novel subtypes with improved potency.
4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is the most potent nucleoside analog inhibitor of HIV reverse transcriptase (RT). It retains a 3′-OH yet acts as a chain-terminating agent by diminishing ...translocation from the pretranslocation nucleotide-binding site (N site) to the posttranslocation primer-binding site (P site). Also, facile misincorporation of EFdA-monophosphate (MP) results in difficult-to-extend mismatched primers. To understand the high potency and unusual inhibition mechanism of EFdA, we solved RT crystal structures (resolutions from 2.4 to 2.9 Å) that include inhibition intermediates (i) before inhibitor incorporation (catalytic complex, RT/DNA/EFdA-triphosphate), (ii) after incorporation of EFdA-MP followed by dT-MP (RT/DNAEFdA-MPP•dT-MPN
), or (iii) after incorporation of two EFdA-MPs (RT/DNAEFdA-MPP•EFdA-MPN
); (iv) the latter was also solved with EFdA-MP mismatched at the N site (RT/DNAEFdA-MPP•EFdA-MP*N
). We report that the inhibition mechanism and potency of EFdA stem from interactions of its 4′-ethynyl at a previously unexploited conserved hydrophobic pocket in the polymerase active site. The high resolution of the catalytic complex structure revealed a network of ordered water molecules at the polymerase active site that stabilize enzyme interactions with nucleotide and DNA substrates. Finally, decreased translocation results from favorable interactions of primer-terminating EFdA-MP at the pretranslocation site and unfavorable posttranslocation interactions that lead to observed localized primer distortions.
The capsid core of HIV-1 is a large macromolecular assembly that surrounds the viral genome and is an essential component of the infectious virus. In addition to its multiple roles throughout the ...viral life cycle, the capsid interacts with multiple host factors. Owing to its indispensable nature, the HIV-1 capsid has been the target of numerous antiretrovirals, though most capsid-targeting molecules have not had clinical success until recently. Lenacapavir, a long-acting drug that targets the HIV-1 capsid, is currently undergoing phase 2/3 clinical trials, making it the most successful capsid inhibitor to-date. In this review, we detail the role of the HIV-1 capsid protein in the virus life cycle, categorize antiviral compounds based on their targeting of five sites within the HIV-1 capsid, and discuss their molecular interactions and mechanisms of action. The diverse range of inhibition mechanisms provides insight into possible new strategies for designing novel HIV-1 drugs and furthers our understanding of HIV-1 biology.
Targeting the clinically unvalidated reverse transcriptase (RT) associated ribonuclease H (RNase H) for human immunodeficiency virus (HIV) drug discovery generally entails chemotypes capable of ...chelating two divalent metal ions in the RNase H active site. The hydroxypyridonecarboxylic acid scaffold has been implicated in inhibiting homologous HIV integrase (IN) and influenza endonuclease via metal chelation. We report herein the design, synthesis, and biological evaluations of a novel variant of the hydroxypyridonecarboxylic acid scaffold featuring a crucial N-1 benzyl or biarylmethyl moiety. Biochemical studies show that most analogues consistently inhibited HIV RT-associated RNase H in the low micromolar range in the absence of significant inhibition of RT polymerase or IN. One compound showed reasonable cell-based antiviral activity (EC50 = 10 μM). Docking and crystallographic studies corroborate favorable binding to the active site of HIV RNase H, providing a basis for the design of more potent analogues.
Abstract
Background
In vitro selection experiments identified viruses resistant to integrase strand transfer inhibitors (INSTIs) carrying mutations in the G-tract (six guanosines) of the ...3′-polypurine tract (3′-PPT). A clinical study also reported that mutations in the 3′-PPT were observed in a patient receiving dolutegravir monotherapy. However, recombinant viruses with the 3′-PPT mutations that were found in the clinical study were recently shown to be susceptible to INSTIs.
Objectives
To identify the specific mutation(s) in the G-tract of the 3′-PPT for acquiring INSTI resistance, we constructed infectious clones bearing single or multiple mutations and systematically characterized the susceptibility of these clones to both first- and second-generation INSTIs.
Methods
The infectious clones were tested for their infectivity and susceptibility to INSTIs in a single-cycle assay using TZM-bl cells.
Results
A single mutation of thymidine (T) at the fifth position (GGG GTG) in the G-tract of the 3′-PPT had no effect on INSTI resistance. A double mutation, cytidine (C) or ‘T’ at the second position and ‘T’ at the fifth position (GCG GTG and GTG GTG), increased resistance to INSTIs, with the appearance of a plateau in the maximal percentage inhibition (MPI) of the dose–response curves, consistent with a non-competitive mechanism of inhibition.
Conclusions
Mutations at the second and fifth positions in the G-tract of the 3′-PPT may result in complex resistance mechanism(s), rather than simply affecting INSTI binding at the IN active site.
Coronavirus Disease 2019 (COVID-19) is a deadly emerging infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Because SARS-CoV-2 is easily transmitted through ...the air and has a relatively long incubation time, COVID-19 has rapidly developed into a global pandemic. As there are no antiviral agents for the prevention and treatment of this severe pathogen except for remdesivir, development of antiviral therapies to treat infected individuals remains highly urgent. Here, we showed that baicalein and baicalin exhibited significant antiviral activity against SARS-CoV-2, the causative agent of COVID-19 through in vitro studies. Our data through cell-based and biochemical studies showed that both compounds act as SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibitors directly and inhibit the activity of the SARS-CoV-2 RdRp, but baicalein was more potent. We also showed specific binding of baicalein to the SARS-CoV-2 RdRp, making it a potential candidate for further studies towards therapeutic development for COVID-19 as a selective non-nucleoside polymerase inhibitor.
Genetic mutations in the phosphatase PTPN11 (SHP2) are associated with childhood leukemias. These mutations cause hyperactivation of SHP2 due to the disruption of the autoinhibitory conformation. By ...targeting the activation-associated protein conformational change, we have identified an SHP2 inhibitor (E)-1-(1-(5-(3-(2,4-dichlorophenyl)acryloyl)-2-ethoxy-4-hydroxybenzyl)-1,2,5,6-tetrahydropyridin-3-yl)-1H-benzodimidazol-2(3H)-one (LY6, 1) using computer-aided drug design database screening combined with cell-based assays. This compound inhibited SHP2 with an IC50 value of 9.8 μM, 7-fold more selective for SHP2 than the highly related SHP1. Fluorescence titration, thermal shift, and microscale thermophoresis quantitative binding assays confirmed its direct binding to SHP2. This compound was further verified to effectively inhibit SHP2-mediated cell signaling and proliferation. Furthermore, mouse and patient leukemia cells with PTPN11 activating mutations were more sensitive to this inhibitor than wild-type cells. This small molecule SHP2 inhibitor has a potential to serve as a lead compound for further optimization studies to develop novel anti-SHP2 therapeutic agents.
Reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not targeted by current chemotherapy against human immunodeficiency virus (HIV). ...Although numerous chemotypes have been reported to inhibit HIV RNase H biochemically, few show significant antiviral activity against HIV. We report herein the design, synthesis, and biological evaluations of a novel variant of 2-hydroxyisoquinoline-1,3-dione (HID) scaffold featuring a crucial C-6 benzyl or biarylmethyl moiety. The synthesis involved a recently reported metal-free direct benzylation between tosylhydrazone and boronic acid, which allowed the generation of structural diversity for the hydrophobic aromatic region. Biochemical studies showed that the C-6 benzyl and biarylmethyl HID analogues, previously unknown chemotypes, consistently inhibited HIV RT-associated RNase H and polymerase with IC50s in low to submicromolar range. The observed dual inhibitory activity remained uncompromised against RT mutants resistant to non-nucleoside RT inhibitors (NNRTIs), suggesting the involvement of binding site(s) other than the NNRTI binding pocket. Intriguingly, these same compounds inhibited the polymerase, but not the RNase H function of Moloney Murine Leukemia Virus (MoMLV) RT and also inhibited Escherichia coli RNase H. Additional biochemical testing revealed a substantially reduced level of inhibition against HIV integrase. Molecular docking corroborates favorable binding of these analogues to the active site of HIV RNase H. Finally, a number of these analogues also demonstrated antiviral activity at low micromolar concentrations.
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