Conspectus Human immunodeficiency virus (HIV) reverse transcriptase (RT) contains two distinct functional domains: a DNA polymerase (pol) domain and a ribonuclease H (RNase H) domain, both of which ...are required for viral genome replication. Over the last 3 decades, RT has been at the forefront of HIV drug discovery efforts with numerous nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) approved by the FDA. However, all these RT inhibitors target only the pol function, and inhibitors of RT-associated RNase H have yet to enter the development pipeline, which in itself manifests both the opportunity and challenges of targeting RNase H: if developed, RT RNase H inhibitors would represent a mechanistically novel class of HIV drugs that can be particularly valuable in treating HIV strains resistant to current drugs. The challenges include (1) the difficulty in selectively targeting RT RNase H over RT pol due to their close interplay both spatially and temporally and over HIV-1 integrase strand transfer (INST) activity because of their active site similarities; (2) to a larger extent, the inability of active site inhibitors to confer significant antiviral effect, presumably due to a steep substrate barrier by which the pre-existing substrate prevents access of small molecules to the active site. As a result, previously reported RT RNase H inhibitors typically lacked target specificity and significant antiviral potency. Achieving meaningful antiviral activity via active site targeting likely entails selective and ultrapotent RNase H inhibition to allow small molecules to cut into the dominance of substrates. Based on a pharmacophore model informed by prior work, we designed and redesigned a few metal-chelating chemotypes, such as 2-hydroxyisoquinolinedione (HID), hydroxypyridonecarboxylic acid (HPCA), 3-hydroxypyrimidine-2,4-dione (HPD), and N-hydroxythienopyrimidine-2,4-dione (HTPD). Analogues of these chemotypes generally exhibited improved potency and selectivity inhibiting RT RNase H over the best previous compounds and further validated the pharmacophore model. Extended structure–activity relationship (SAR) on the HPD inhibitor type by mainly altering the linkage generated a few subtypes showing exceptional potency (single-digit nanomolar) and excellent selectivity over the inhibition of RT pol and INST. In parallel, a structure-based approach also allowed us to design a unique double-winged HPD subtype to potently and selectively inhibit RT RNase H and effectively compete against the RNA/DNA substrate. Significantly, all potent HPD subtypes consistently inhibited HIV-1 in the cell culture, suggesting that carefully designed active site RNase H inhibitors with ultrapotency could partially overcome the barrier to antiviral phenotype. Overall, in addition to identifying our own inhibitor types, our medicinal chemistry efforts demonstrated the value of pharmacophore and structure-based approaches in designing active side-directed RNase H inhibitors and could provide a viable path to validating RNase H as a novel antiviral target.
4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a nucleoside reverse transcriptase inhibitor (NRTI) with a novel mechanism of action, unique structure, and amongst NRTIs, unparalleled anti-HIV-1 ...activity. We will summarize its structure and function, antiviral activity, resistance profile, and potential as an antiretroviral for use in the treatment and preexposure prophylaxis of HIV-1 infection.
EFdA is active against wild-type (EC50 as low as 50 pmol/l) and most highly NRTI-resistant viruses. The active metabolite, EFdA-triphosphate, has been shown to have a prolonged intracellular half-life in human and rhesus (Rh) blood cells. As a result, single drug doses tested in simian immunodeficiency virus mac251-infected Rh macaques and HIV-1-infected individuals exhibited robust antiviral activity of 7-10 days duration. Preclinical studies of EFdA as preexposure prophylaxis in the Rh macaque/simian/human immunodeficiency virus low-dose intrarectal challenge model have shown complete protection when given in clinically relevant doses.
EFdA is a novel antiretroviral with activity against both wild-type and NRTI-resistant viruses. As a result of the prolonged intracellular half-life of its active moiety, it is amenable to flexibility in dosing of at least daily to weekly and perhaps longer.
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 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 movement of viruses and other large macromolecular cargo through nuclear pore complexes (NPCs) is poorly understood. The human immunodeficiency virus type 1 (HIV-1) provides an attractive model ...to interrogate this process. HIV-1 capsid (CA), the chief structural component of the viral core, is a critical determinant in nuclear transport of the virus. HIV-1 interactions with NPCs are dependent on CA, which makes direct contact with nucleoporins (Nups). Here we identify Nup35, Nup153, and POM121 to coordinately support HIV-1 nuclear entry. For Nup35 and POM121, this dependence was dependent cyclophilin A (CypA) interaction with CA. Mutation of CA or removal of soluble host factors changed the interaction with the NPC. Nup35 and POM121 make direct interactions with HIV-1 CA via regions containing phenylalanine glycine motifs (FG-motifs). Collectively, these findings provide additional evidence that the HIV-1 CA core functions as a macromolecular nuclear transport receptor (NTR) that exploits soluble host factors to modulate NPC requirements during nuclear invasion.
The early steps of HIV-1 infection, such as uncoating, reverse transcription, nuclear import, and transport to integration sites are incompletely understood. Here, we imaged nuclear entry and ...transport of HIV-1 replication complexes in cell lines, primary monocyte-derived macrophages (MDMs) and CD4
T cells. We show that viral replication complexes traffic to and accumulate within nuclear speckles and that these steps precede the completion of viral DNA synthesis. HIV-1 transport to nuclear speckles is dependent on the interaction of the capsid proteins with host cleavage and polyadenylation specificity factor 6 (CPSF6), which is also required to stabilize the association of the viral replication complexes with nuclear speckles. Importantly, integration site analyses reveal a strong preference for HIV-1 to integrate into speckle-associated genomic domains. Collectively, our results demonstrate that nuclear speckles provide an architectural basis for nuclear homing of HIV-1 replication complexes and subsequent integration into associated genomic loci.
Nucleos(t)ide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV) reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable ...long-term side effects. The profound suppression of HBV by the nucleos(t)ide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(t)ide analogs. The HBV ribonuclease H (RNAseH) is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 µM, the best compounds had low micromolar IC(50) values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 µM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug development.
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.
Abstract
The HIV-1 capsid core participates in several replication processes. The mature capsid core is a lattice composed of capsid (CA) monomers thought to assemble first into CA dimers, then into ...∼250 CA hexamers and 12 CA pentamers. CA assembly requires conformational flexibility of each unit, resulting in the presence of unique, solvent-accessible surfaces. Significant advances have improved our understanding of the roles of the capsid core in replication; however, the contributions of individual CA assembly forms remain unclear and there are limited tools available to evaluate these forms in vivo. Here, we have selected aptamers that bind CA lattice tubes. We describe aptamer CA15-2, which selectively binds CA lattice, but not CA monomer or CA hexamer, suggesting that it targets an interface present and accessible only on CA lattice. CA15-2 does not compete with PF74 for binding, indicating that it likely binds a non-overlapping site. Furthermore, CA15-2 inhibits HIV-1 replication when expressed in virus producer cells, but not target cells, suggesting that it binds a biologically-relevant site during virus production that is either not accessible during post-entry replication steps or is accessible but unaltered by aptamer binding. Importantly, CA15-2 represents the first aptamer that specifically recognizes the HIV-1 CA lattice.
PDF
