P04-41. Kinetics of Antibody Neutralization and Viral Evolution Following Envelope Vaccination in SIV-infected Rhesus Monkeys

Poster presentation. Conclusion: Our results indicate that env vaccination is associated with an accelerated development of autologous neutralizing antibodies. These antibodies were focused at least in part on the V1 region of Env, since there was selective pressure in this region of the envelope for the evolution of mutational changes

The triple combination of tenofovir, emtricitabine and efavirenz shows synergistic anti-HIV-1 activity in vitro: a mechanism of action study

<p>Abstract</p> <p>Background</p> <p>Tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and efavirenz (EFV) are the three components of the once-daily, single tablet regimen (Atripla) for treatment of HIV-1 infection. Previous cell culture studies have demonstrated that the double combination of tenofovir (TFV), the parent drug of TDF, and FTC were additive to synergistic in their anti-HIV activity, which correlated with increased levels of intracellular phosphorylation of both compounds.</p> <p>Results</p> <p>In this study, we demonstrated the combinations of TFV+FTC, TFV+EFV, FTC+EFV, and TFV+FTC+EFV synergistically inhibit HIV replication in cell culture and synergistically inhibit HIV-1 reverse transcriptase (RT) catalyzed DNA synthesis in biochemical assays. Several different methods were applied to define synergy including median-effect analysis, MacSynergy^®II and quantitative isobologram analysis. We demonstrated that the enhanced formation of dead-end complexes (DEC) by HIV-1 RT and TFV-terminated DNA in the presence of FTC-triphosphate (TP) could contribute to the synergy observed for the combination of TFV+FTC, possibly through reduced terminal NRTI excision. Furthermore, we showed that EFV facilitated efficient formation of stable, DEC-like complexes by TFV- or FTC-monophosphate (MP)-terminated DNA and this can contribute to the synergistic inhibition of HIV-1 RT by TFV-diphosphate (DP)+EFV and FTC-TP+EFV combinations.</p> <p>Conclusion</p> <p>This study demonstrated a clear correlation between the synergistic antiviral activities of TFV+FTC, TFV+EFV, FTC+EFV, and TFV+FTC+EFV combinations and synergistic HIV-1 RT inhibition at the enzymatic level. We propose the molecular mechanisms for the TFV+FTC+EFV synergy to be a combination of increased levels of the active metabolites TFV-DP and FTC-TP and enhanced DEC formation by a chain-terminated DNA and HIV-1 RT in the presence of the second and the third drug in the combination. This study furthers the understanding of the longstanding observations of synergistic anti-HIV-1 effects of many NRTI+NNRTI and certain NRTI+NRTI combinations in cell culture, and provides biochemical evidence that combinations of anti-HIV agents can increase the intracellular drug efficacy, without increasing the extracellular drug concentrations.</p

Preclinical Characterization of a Potent, Selective Inhibitor of the Protein Methyltransferase DOT1L for Use in the Treatment of MLL-Rearranged Leukemia.

Abstract Abstract 2379 The enzymatic activity of the protein methyltransferase (PMT) DOT1L has been shown to be a driver of cell proliferation in MLL-rearranged leukemia. Our group has previously reported the design of potent and selective aminonucleoside inhibitors of DOT1L [Daigle et al. (2011) Cancer Cell 20: 53–65; Basavapathruni et al. (2012) Chem. Biol. Drug Design, in press]. Structure-guided design, together with robust biochemical and biological assays, was used to optimize the potency, selectivity and pharmacological features of the aminonucleosides, resulting in the compound EPZ-5676. EPZ-5676 is an S-adenosyl methionine (SAM) competitive inhibitor of DOT11L that displays a Ki value of 80 pM and a drug-target residence time of &gt; 24 hours. The compound is highly selective for DOT1L, demonstrating &gt; 37,000-fold selectivity against all other PMTs tested. Crystallographic studies reveal that the high affinity, durable inhibition of DOT1L by EPZ-5676 has its origin in a conformational adaptation of the protein that attends inhibitor binding, extending the compound binding pocket to include novel recognition elements beyond the SAM binding active site. Treatment of leukemia cells with EPZ-5676 results in concentration- and time-dependent reduction of H3K79 methylation without effect on the methylation status of other histone sites. The reduction of H3K79 methylation leads to inhibition of key MLL target genes and selective, apoptotic cell killing in MLL-rearranged leukemia cells, but has minimal impact on non-rearranged cells. EPZ-5676 is highly soluble in aqueous solution and can thus be formulated for intravenous administration. The effective pharmacokinetic half-life of EPZ-5676 in systemic circulation has been measured to be 0.25 and 1.5 h in rats and dogs, respectively. A nude rat subcutaneous xenograft model of MLL-rearranged leukemia has been established. Continuous intravenous infusion of EPZ-5676 for 21 days in this model leads to dose-dependent anti-tumor activity. At the highest dose, complete tumor regressions are achieved with no regrowth for up to 32 days after the cessation of treatment (Figure 1). Figure 1. EPZ-5676 causes complete and sustained tumor regression in a MV4–11 nude rat xenograft model of MLL-rearranged leukemia. No significant weight loss or obvious toxicity was observed in rats treated with EPZ-5676 during this efficacy study. EPZ-5676 is thus a potent, selective inhibitor of DOT1L that demonstrates strong efficacy in a rat xenograft model of MLL-rearranged leukemia. Details of the preclinical characterization of this compound will be presented. Figure 1. EPZ-5676 causes complete and sustained tumor regression in a MV4–11 nude rat xenograft model of MLL-rearranged leukemia. . / No significant weight loss or obvious toxicity was observed in rats treated with EPZ-5676 during this efficacy study. EPZ-5676 is thus a potent, selective inhibitor of DOT1L that demonstrates strong efficacy in a rat xenograft model of MLL-rearranged leukemia. Details of the preclinical characterization of this compound will be presented. Disclosures: Pollock: Epizyme: Employment, Equity Ownership. Daigle:Epizyme: Employment, Equity Ownership. Therkelsen:Epizyme: Employment, Equity Ownership. Basavapathruni:Epizyme: Employment, Equity Ownership. Jin:Epizyme: Employment, Equity Ownership. Allain:Epizyme: Employment, Equity Ownership. Klaus:Epizyme, Inc.: Employment, Equity Ownership. Raimondi:Epizyme: Employment, Equity Ownership. Porter Scott:Epizyme: Employment, Equity Ownership. Chesworth:Epizyme: Employment, Equity Ownership. Moyer:Epizyme: Employment, Equity Ownership. Copeland:Epizyme Inc.: Employment, Equity Ownership. Richon:Epizyme, Inc.: Employment, Equity Ownership. Olhava:Epizyme: Employment. </jats:sec

Envelope Vaccination Shapes Viral Envelope Evolution following Simian Immunodeficiency Virus Infection in Rhesus Monkeys▿

The evolution of envelope mutations by replicating primate immunodeficiency viruses allows these viruses to escape from the immune pressure mediated by neutralizing antibodies. Vaccine-induced anti-envelope antibody responses may accelerate and/or alter the specificity of the antibodies, thus shaping the evolution of envelope mutations in the replicating virus. To explore this possibility, we studied the neutralizing antibody response and the envelope sequences in rhesus monkeys vaccinated with either gag-pol-nef immunogens or gag-pol-nef immunogens in combination with env and then infected with simian immunodeficiency virus (SIV). Using a pseudovirion neutralization assay, we demonstrate that envelope vaccination primed for an accelerated neutralizing antibody response following virus challenge. To monitor viral envelope evolution in these two cohorts of monkeys, full-length envelopes from plasma virus isolated at weeks 37 and 62 postchallenge were sequenced by single genome amplification to identify sites of envelope mutations. We show that env vaccination was associated with a change in the pattern of envelope mutations. Prevalent mutations in sequences from gag-pol-nef vaccinees included deletions in both variable regions 1 and 4 (V1 and V4), whereas deletions in the env vaccinees occurred only in V1. These data show that env vaccination altered the focus of the antibody-mediated selection pressure on the evolution of envelope following SIV challenge