Background: 4Ј-Ethynyl-2-fluoro-2Ј-deoxyadenosine (EFdA) is a highly potent nucleoside analog reverse transcriptase (RT) inhibitor with a 3Ј-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.

Technical limitations in simultaneous microscopic visualization of RNA, DNA, and proteins of HIV have curtailed progress in this field. To address this need we develop a microscopy approach, multiplex immunofluorescent cell-based detection of DNA, RNA and Protein (MICDDRP), which is based on branched DNA in situ hybridization technology. MICDDRP enables simultaneous single-cell visualization of HIV (a) spliced and unspliced RNA, (b) cytoplasmic and nuclear DNA, and (c) Gag. We use MICDDRP to visualize incoming capsid cores containing RNA and/or nascent DNA and follow reverse transcription kinetics. We also report transcriptional “bursts” of nascent RNA from integrated proviral DNA, and concomitant HIV-1, HIV-2 transcription in co-infected cells. MICDDRP can be used to simultaneously detect multiple viral nucleic acid intermediates, characterize the effects of host factors or drugs on steps of the HIV life cycle, or its reactivation from the latent state, thus facilitating the development of antivirals and latency reactivating agents.

BackgroundThe K65R substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is the major resistance mutation selected in patients treated with first-line antiretroviral tenofovir disoproxil fumarate (TDF). 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), is the most potent nucleoside analog RT inhibitor (NRTI) that unlike all approved NRTIs retains a 3'-hydroxyl group and has remarkable potency against wild-type (WT) and drug-resistant HIVs. EFdA acts primarily as a chain terminator by blocking translocation following its incorporation into the nascent DNA chain. EFdA is in preclinical development and its effect on clinically relevant drug resistant HIV strains is critically important for the design of optimal regimens prior to initiation of clinical trials.ResultsHere we report that the K65R RT mutation causes hypersusceptibility to EFdA. Specifically, in single replication cycle experiments we found that EFdA blocks WT HIV ten times more efficiently than TDF. Under the same conditions K65R HIV was inhibited over 70 times more efficiently by EFdA than TDF. We determined the molecular mechanism of this hypersensitivity using enzymatic studies with WT and K65R RT. This substitution causes minor changes in the efficiency of EFdA incorporation with respect to the natural dATP substrate and also in the efficiency of RT translocation following incorporation of the inhibitor into the nascent DNA. However, a significant decrease in the excision efficiency of EFdA-MP from the 3’ primer terminus appears to be the primary cause of increased susceptibility to the inhibitor. Notably, the effects of the mutation are DNA-sequence dependent.ConclusionWe have elucidated the mechanism of K65R HIV hypersusceptibility to EFdA. Our findings highlight the potential of EFdA to improve combination strategies against TDF-resistant HIV-1 strains.

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e 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. Human immunodeficiency virus type 1 (HIV-1) replicates primarily in activated CD4 ϩ T cells, while showing poor reproductive capacity in monocytes, macrophages, dendritic cells, and resting CD4 ϩ T cells (1-10). Sterile alpha motif-and histidine/ aspartic acid domain-containing protein 1 (SAMHD1) is responsible for blocking HIV-1 replication in such cells (5,(11)(12)(13), reportedly by acting as a dGTP-stimulated deoxynucleotide triphosphohydrolase that hydrolyzes deoxynucleoside triphosphates (dNTPs), thus decreasing the amounts of dNTPs available for reverse transcription (3,4,(14)(15)(16)(17)(18)(19).Nucleoside reverse transcriptase inhibitors (NRTIs) are nucleoside analogs and key components of antiretroviral therapies (20-26). They generally lack a 3=-OH group and thus act as chain terminators upon incorporation into viral DNA by reverse transcriptase (RT) (26-29). However, 4=-ethynyl-2-fluoro-2=-deoxyadenosine (EFdA) retains a 3=-OH group, acts primarily by blocking RT translocation following incorporation of EFdA monophosphate (MP) into the template-primer, and has picomolar antiviral potency (30-37). NRTIs are administered as nucleosides and are phosphorylated to their active forms by cellular kinases (38). Hence, they compete with dNTPs for activation by cellular kinases, and their incorporation by RT is influenced by the cellular concentrations of dNTPs, which compete with NRTI triphosphates (TPs) at the RT active site (39,40). Amie et al. (19) recently reported that SAMHD1 does not significantly hydrolyze dideoxynucleoside triphosphates (ddNTPs) or zidovudine (AZT)-TP and that depletion of SAMHD1 in monocytic THP-1 cells decreased the potency of these NRTIs in a pseudotype-based assay. Strong evidence that the decreased potency of these NRTIs was due to increased amounts of competing dNTPs was presented. Our parallel independent study confirmed their data, extended the number of NRTIs studied, validated the results with fully infectious HIV-1, and found an unexpected disparity in the effects of SAMHD1 on the deoxyribosylthymine (dT) analogs AZT and stavudine (d4T). We demonstrate that this is due to differences in the activation of AZT and d4T, highlighting the importance of distinct metabolic pathways in NRTI activation, in addition to competition with dNTPs.We tested purified Escherichia coli-produced recombinant SAMHD1 for dGTP-regulated NRTI-TP hydrolysis (using dNTPs as a re...

The human cytochrome P450 (CYP) enzymes CYP3A4 and CYP3A5 metabolize most drugs and have high similarities in their structure and substrate preference. Whereas CYP3A4 is predominantly expressed in the liver, CYP3A5 is upregulated in cancer, contributing to drug resistance. Selective inhibitors of CYP3A5 are, therefore, critical to validating it as a therapeutic target. Here we report clobetasol propionate (clobetasol) as a potent and selective CYP3A5 inhibitor identified by high-throughput screening using enzymatic and cell-based assays. Molecular dynamics simulations suggest a close proximity of clobetasol to the heme in CYP3A5 but not in CYP3A4. UV–visible spectroscopy and electron paramagnetic resonance analyses confirmed the formation of an inhibitory type I heme–clobetasol complex in CYP3A5 but not in CYP3A4, thus explaining the CYP3A5 selectivity of clobetasol. Our results provide a structural basis for selective CYP3A5 inhibition, along with mechanistic insights, and highlight clobetasol as an important chemical tool for target validation.

Despite the availability of effective vaccines and treatments, HBV remains a significant global health concern, with more than 240 million individuals chronically infected. Current treatments are highly effective at controlling viral replication and disease progression but rarely cure infections. Therefore, much emphasis is being placed on finding therapeutics with new drug targets, such as viral gene expression, covalently closed circular DNA formation and stability, capsid formation, and host immune modulators, with the ultimate goal of an HBV cure. Understanding the mechanisms by which novel antiviral agents act will be imperative for the development of curative HBV therapies.

The RNase H (RNH) function of HIV-1 reverse transcriptase (RT) plays an essential part in the viral life cycle. We report the characterization of YLC2-155, a 2-hydroxyisoquinoline-1,3-dione (HID)-based active-site RNH inhibitor. YLC2-155 inhibits both polymerase (50% inhibitory concentration [IC 50 ] ϭ 2.6 M) and RNH functions (IC 50 ϭ 0.65 M) of RT but is more effective against RNH. X-ray crystallography, nuclear magnetic resonance (NMR) analysis, and molecular modeling were used to show that YLC2-155 binds at the RNH-active site in multiple conformations.KEYWORDS RNase H, human immunodeficiency virus, inhibitor, reverse transcriptase H IV-1 reverse transcriptase (RT) plays a critical role in virus replication. It has multiple functions, including RNA-dependent DNA synthesis, RNase H (RNH) activity, and DNA-dependent DNA synthesis to convert the viral single-stranded RNA genome into double-stranded DNA for downstream incorporation into the host cell genome (1). At least one component of highly active antiretroviral therapy (HAART) administered to patients includes RT polymerase inhibitors, either a nucleoside RT inhibitor(s) or a nonnucleoside RT inhibitor, or both. Prolonged use of antivirals can lead to side effects or drug resistance (2, 3). Hence, new antivirals that act by novel mechanisms of action are needed. No currently approved therapeutics target the RNH function of HIV-1 RT. Hence, RNH is an attractive target for future antiviral therapies.RNH inhibitors of several different chemotypes have been identified and characterized for their effectiveness against HIV-1 (4). These include acylhydrazones (5-7), diketo acids (8, 9), ␣-hydroxytropolones (10), vinylogous ureas (11), naphthyridinones (12), pyridopyrimidinones (13,14), pyrimidinol carboxylic acids (15), hydroxypyridonecarboxylic acids (16), 3-hydroxypyrimidine-2,4-diones (17, 18), and 2-hydroxyisoquinoline-1,3-diones (HIDs) (19). Notably, many 2-hydroxyisoquinoline-1,3-diones inhibit both RT polymerase and RNH functions of RT (19). In order to further understand the mechanism of RT inhibition by HIDs, we further characterized YLC2-155, which is substituted at the C7 position of the 2-hydroxyisoquinoline-1,3-dione with a furan ring (Fig. 1).