HIV necessitates host factors for successful completion of its life cycle. Mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that forms two complexes, mTORC1 and mTORC2. Rapamycin is an allosteric inhibitor of mTOR that selectively inhibits mTORC1. Rapamycin interferes with viral entry of CCR5 (R5)-tropic HIV and with basal transcription of the HIV LTR, potently inhibiting replication of R5 HIV but not CXCR4 (X4)-tropic HIV in primary cells. The recently developed ATP-competitive mTOR kinase inhibitors (TOR-KIs) inhibit both mTORC1 and mTORC2. Using INK128 as a prototype TOR-KI, we demonstrate potent inhibition of both R5 and X4 HIV in primary lymphocytes (EC₅₀ < 50 nM), in the absence of toxicity. INK128 inhibited R5 HIV entry by reducing CCR5 levels. INK128 also inhibited both basal and induced transcription of HIV genes, consistent with inhibition of mTORC2, whose activity is critical for phosphorylation of PKC isoforms and, in turn, induction of NF-κB. INK128 enhanced the antiviral potency of the CCR5 antagonist maraviroc, and had favorable antiviral interactions with HIV inhibitors of reverse transcriptase, integrase and protease. In humanized mice, INK128 decreased plasma HIV RNA by >2 log10 units and partially restored CD4/CD8 cell ratios. Targeting of cellular mTOR with INK128 (and perhaps others TOR-KIs) provides a potential strategy to inhibit HIV, especially in patients with drug resistant HIV strains.

Vicriviroc (VCV) is a chemokine (C-C motif) receptor 5 (CCR5) antagonist with potent anti-HIV activity that currently is being evaluated in phase III clinical trials. In the present study, donor CCR5 density (CCR5 receptors/CD4 lymphocytes) inversely correlated with VCV antiviral activity (Spearman's correlation test;r= 0.746,P= 0.0034). Low doses of the transplant drug rapamycin (RAPA) reduced CCR5 density and enhanced VCV antiviral activity. In drug interaction studies, the RAPA/VCV combination had considerable antiviral synergy (combination indexes of 0.1–0.04) in both multicycle and single-cycle infection of lymphocytes. The synergy between RAPA and VCV translated into dose reduction indexes of 8- to 41-fold reductions for RAPA and 19- to 658-fold reductions for VCV. RAPA enhanced VCV antiviral activity against both B and non-B clade isolates, potently suppressing clade G viruses with reported reduced sensitivities to VCV and to the licensed CCR5 antagonist maraviroc. Importantly, RAPA reduction of CCR5 density in lymphocytes sensitized VCV-resistant strains to VCV, inhibiting virus production by ∼ 90%. We further demonstrated the role of CCR5 density on VCV activity against resistant virus in donor lymphocytes and in cell lines expressing varying CCR5 densities. Together, these results suggest that low doses of RAPA may increase the durability of VCV-containing regimens in patients by enhancing VCV viral suppression, by allowing the use of lower doses of VCV with reduced potential for toxicity, and by controlling emerging VCV-resistant variants.

Objective: The M184V mutation in the HIV-1 reverse transcriptase gene is frequent (>50%) in patients, both in resource-rich and resource-limited countries, conferring high-level resistance (>100-fold) to the cytosine analog reverse transcriptase inhibitors lamivudine and emtricitabine. The reverse transcriptase enzyme of M184V HIV-1 mutants has reduced processivity, resulting in reduced viral replication, particularly at low deoxynucleotide (dNTP) levels. We hypothesized that lowering intracellular dNTPs with resveratrol, a dietary supplement, could interfere with replication of M184V HIV-1 mutants. Design and methods: Evaluation of the activity of resveratrol on infection of primary peripheral blood lymphocytes by wild-type and M184V mutant HIV-1. We assayed both molecular clones and primary isolates of HIV-1, containing M184V alone and in combination with other reverse transcriptase mutations. Viral infection was quantified by p24 ELISA and by quantitative real-time PCR analysis. Cell viability was measured by colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays. Results: In virus-infectivity assays, resveratrol did not inhibit replication of wild-type NL4-3 (resveratrol EC₅₀ > 10 μmol/l), but it inhibited NL4-3 184V mutant (resveratrol EC₅₀ = 5.8 μmol/l). These results were confirmed by real-time PCR analysis of early and late products of reverse transcription. Resveratrol inhibited molecular clones and primary isolates carrying M184V, alone or in combination with other reverse transcriptase mutations (resveratrol EC₅₀ values ranging from 2.5 to 7.7 μmol/l). Conclusions: Resveratrol inhibits HIV-1 strains carrying the M184V mutation in reverse transcriptase. We propose resveratrol as a potential adjuvant in HIV-1 therapy, particularly in resource-limited settings, to help control emtricitabine-resistant M184V HIV-1mutants.

Successful propagation of HIV in the human host requires entry into a permissive cell, reverse transcription of viral RNA, integration into the human genome, transcription of the integrated provirus, and assembly/release of new virus particles. Currently, there are antiretrovirals against each of these viral steps, except for provirus transcription. An inhibitor of HIV transcription could both increase potency of treatment and suppress drug-resistant strains. Cellular cyclin-dependent kinase 9 (CDK9) serves as a cofactor for the HIV Tat protein and is required for effective transcription of the provirus. Previous studies have shown that the CDK9 inhibitor Indirubin 3’-monoxime (IM) inhibits HIV transcription in vitro and in short-term in vivo studies of HIV acute infection in humanized mice (PBMC-NSG model), suggesting a therapeutic potential. The objective of this study is to evaluate the toxicity, pharmacokinetics and long-term antiviral activity of IM during chronic HIV infection in humanized mice (HSC-NSG model). We show that IM concentrations above EC₅₀ values are rapidly achieved and sustained for > 3 h in plasma, and that non-toxic concentrations durably reduce HIV RNA levels. In addition, IM enhanced the antiviral activity of antiretrovirals from the reverse transcriptase, protease and integrase inhibitor classes in in vitro infectivity assays. In summary, IM may enhance current antiretroviral treatments and could help achieve a “functional cure” in HIV patients by preventing expression of proviruses.

Sustained inhibition of HIV-1, the goal of antiretroviral therapy, is often impeded by the emergence of viral drug resistance. For patients infected with HIV-1 resistant to conventional drugs from the viral reverse transcriptase and protease inhibitor classes, the recently approved entry and integration inhibitors effectively suppress HIV-1 and offer additional therapeutic options. Entry inhibitors are particularly attractive because, unlike conventional antiretrovirals, they target HIV-1 extracellularly, thereby sparing cells from both viral- and drug-induced toxicities. The fusion inhibitor enfuvirtide and the CCR5 antagonist maraviroc are the first entry inhibitors licensed for patients with drug-resistant HIV-1, with maraviroc restricted to those infected with CCR5-tropic HIV-1 (R5 HIV-1) only. Vicriviroc (another CCR5 antagonist) is in Phase III clinical trials, whereas the CCR5 antibodies PRO 140 and HGS 004 are in early stages of clinical development. Potent antiviral synergy between maraviroc and CCR5 antibodies, coupled with distinct patterns of resistance, suggest their combinations might be particularly effective in patients. In addition, given that oral administration of maraviroc achieves high drug levels in cervicovaginal fluid, combinations of maraviroc and other CCR5 inhibitors could be effective in preventing HIV-1 transmission. Moreover, since CCR5 antagonists prevent rejection of transplanted organs, maraviroc could both suppress HIV-1 and prolong organ survival for the growing number of HIV-1 patients with kidney or liver failure necessitating organ transplantation. Thus, maraviroc offers an important treatment option for patients with drug-resistant R5 HIV-1, who presently account for >50% of drug-resistance cases.