Activity and selectivity of compounds that induce HIV-1 transcription.

<p>Activity and selectivity of compounds that induce HIV-1 transcription.</p

HDAC inhibition assay.

<p>Hela nuclear extract (NE) or purified HDACs were assayed for deacetylation activity in the presence of Compound <b>1</b>, Compound <b>2</b>, or known HDAC inhibitors. Data are plotted as mean ± SEM of two independent replicates and is normalized to vehicle-treated sample. Dotted line indicates 100% HDAC activity (0% inhibition). SAHA, suberanilohydroxamic acid; TSA, trichostatin A; VA, valproic acid; NaBu, sodium butyrate.</p

Compounds 1 and 2 induce HIV-1 transcription in ACH-2 and U1.

<p>Cells were treated with 30 μM of Cmpd 1 and Cmpd 2 for 24 h. RNA was prepared and HIV expression was monitored by qRT-PCR. Data are presented as the log fold induction over DMSO treated controls. Each bar represents treatments performed in triplicate. Error bars represent the standard error. These data are from an individual experiment that is representative of 3 independent experiments.</p

Induction of HIV-1 expressing cells following treatment with latency reversing agents.

<p>ACH-2 cells were not treated or treated with a final concentration of 30 μM Cmpd 1 or Cmpd 2, 10 μM SAHA, 1 mM JQ1 or 10 ng/ml PMA for 16 h. Cells were stained using anti-HIV-Gag-PE and analyzed by flow cytometry. Numbers within the profiles represent the percentage of positive cells. These profiles are from a single experiment. B) Data from four independent experiments. Y-axis is mean of % p24 positive cells. * Cmpd 1 p-value >0.005 compared to other treatments using a two-tailed t-test.</p

In vitro profiling properties of HIV-1 latency antagonists.

<p>In vitro profiling properties of HIV-1 latency antagonists.</p

Mouse pharmacokinetics of benzazole compounds (10 mg/kg, PO).

<p>Mouse pharmacokinetics of benzazole compounds (10 mg/kg, PO).</p

Structures of compounds that induce HIV transcription.

<p>Compound <b>1</b> was identified through high throughput screening utilizing 24STNLSG cells. A series of analogs (Compounds <b>2–6</b>) were synthesized as a preliminary investigation of structure-activity relationship.</p

Compounds induce HIV-1 transcription in cellular models.

<p>Compound <b>1</b> (A), TNF-α (B), and Compound <b>2</b> (C) were evaluated in the 24STNLSG cell model by measuring increase in SEAP reporter activity 72 hours post addition (closed symbols, left axis). Cellular ATP was quantified in parallel as a measure of cytotoxicity (open symbols, right axis). Data is presented as mean and standard deviation of 2 replicates per compound concentration. Experiment is representative of at least five independent experiments for each compound.</p

Compounds 1 and 2 do not activate latent gamma herpesviruses.

<p>BCP-1 (A) or Raji (B) cells were treated with Compound <b>1</b>, Compound <b>2</b>, or phorbol 12-myristate 13-acetate (PMA) for 24 hours and total RNA was purified. ORF50 (panel A) or BLZF1 (panel B) viral RNAs were quantitated by qRT-PCR and normalized to cellular GADPH on a per-well basis. Data are displayed as mean ± SEM fold increase compared to vehicle-treated control cells. Data point represents two independent replicates, each evaluated by qRT-PCR in duplicate.</p

Evaluation of compounds in cytokine and transcription assays.

<p>(A) PBMCs were exposed to test compounds for 24 hours and IL-2 was quantitated by ELISA. (B) Jurkat cells expressing an NF-κB-responsive Fluc reporter were exposed to compounds and luciferase activity was measured 4 hours post addition. Results are expressed as change from vehicle-treated control. (C) HeLa cells transfected with an HIV LTR-driven Rluc reporter were treated with compounds for 24 hours. Rluc was measured and normalized to vehicle-treated control. For all panels, data is presented as the mean and standard deviation of two replicates and is representative of at least two independent experiments.</p