Compound 1 inhibited cellular activation of GTPases.

<p>Rab7 (A), Cdc42 (B), and Ras (C) in response to growth factor stimulation. HeLa cells were treated with sequential starvation, compound addition and EGF stimulation. Active GTPases in the cell lysates were quantified using effector linked glutathione beads, probed with fluorescent antibody and analyzed on flow cytometer. Results are given as (sample MCF—negative control MCF)/positive control MCF where the negative control is the MCF reading obtained using lysates from unstimulated cells, and the positive control is the MCF reading obtained from the stimulated cells treated with DMSO.</p

A Pan-GTPase Inhibitor as a Molecular Probe

<div><p>Overactive GTPases have often been linked to human diseases. The available inhibitors are limited and have not progressed far in clinical trials. We report here a first-in-class small molecule pan-GTPase inhibitor discovered from a high throughput screening campaign. The compound CID1067700 inhibits multiple GTPases in biochemical, cellular protein and protein interaction, as well as cellular functional assays. In the biochemical and protein interaction assays, representative GTPases from Rho, Ras, and Rab, the three most generic subfamilies of the GTPases, were probed, while in the functional assays, physiological processes regulated by each of the three subfamilies of the GTPases were examined. The chemical functionalities essential for the activity of the compound were identified through structural derivatization. The compound is validated as a useful molecular probe upon which GTPase-targeting inhibitors with drug potentials might be developed.</p></div

Compound 1 inhibited EGFR degradation in SCC-12F cells.

<p>A. Immunoblots of EGFR at different time points after treatment with <b>1</b> at different concentrations or DMSO. Actin served as a loading control. Each experiment included duplicate samples and was repeated in triplicate. B. Time course of EGFR degradation. The ratio of EGFR to actin was quantified at different time points and compared to time zero when ligand EGF was added.</p

Lack of covalent bond formation between GTPases and compound 1.

<p>Rab7 (A), Cdc42 (B) or Ras (C) was incubated with or without compound <b>1</b> overnight followed by extensive washing. Binding of BODIPY^-FL GTP to GTPases was indistinguishable whether the enzymes had been treated with compound <b>1</b> or not.</p

Compound 1 inhibited cellular activation of GTPases.

<p>Rab7 (A), Cdc42 (B), and Ras (C) in response to growth factor stimulation. HeLa cells were treated with sequential starvation, compound addition and EGF stimulation. Active GTPases in the cell lysates were quantified using effector linked glutathione beads, probed with fluorescent antibody and analyzed on flow cytometer. Results are given as (sample MCF—negative control MCF)/positive control MCF where the negative control is the MCF reading obtained using lysates from unstimulated cells, and the positive control is the MCF reading obtained from the stimulated cells treated with DMSO.</p

Compound 1 structure and dose-response curves.

<p>(A) Structure of <b>1</b> and sites of functionalization. Shaded areas are modified regions for SAR. (B) General structure of compound <b>1</b> and analogues. (C) The concentration dependence of compound <b>1</b> on BODIPY^- FL GTP binding to eight GTPases. (D) The concentration dependence of compound <b>1</b> on BODIPY^-FL GDP binding to eight GTPases. Data shown in (C) are representative of at least three independent sets of measurements with each set conducted in duplicate. All the data points from each set were used to fit the sigmoidal dose-response equation. Percent response was calculated as the ratio of (sample MCF–negative control MCF) / (positive control MCF–negative control MCF). For the positive control, DMSO instead of the compound was added; for the negative control, the compound was replaced with GTP at a concentration 5000 fold greater than BODIPY^-FL GTP. MCF stands for median channel fluorescence.</p

Development of (<i>E</i>)‑2-((1,4-Dimethylpiperazin-2-ylidene)amino)-5-nitro‑<i>N</i>‑phenylbenzamide, ML336: Novel 2‑Amidinophenylbenzamides as Potent Inhibitors of Venezuelan Equine Encephalitis Virus

Venezuelan equine encephalitis virus (VEEV) is an emerging pathogenic alphavirus that can cause significant disease in humans. Given the absence of therapeutic options available and the significance of VEEV as a weaponized agent, an optimization effort was initiated around a quinazolinone screening hit <b>1</b> with promising cellular antiviral activity (EC₅₀ = 0.8 μM), limited cytotoxic liability (CC₅₀ > 50 μM), and modest in vitro efficacy in reducing viral progeny (63-fold at 5 μM). Scaffold optimization revealed a novel rearrangement affording amidines, specifically compound <b>45</b>, which was found to potently inhibit several VEEV strains in the low nanomolar range without cytotoxicity (EC₅₀ = 0.02–0.04 μM, CC₅₀ > 50 μM) while limiting in vitro viral replication (EC₉₀ = 0.17 μM). Brain exposure was observed in mice with <b>45</b>. Significant protection was observed in VEEV-infected mice at 5 mg kg^–1 day^–1 and viral replication appeared to be inhibited through interference of viral nonstructural proteins

Effects of compound 1 on the Ras-dependent H358 cells.

<p>The U937 FPRΔST cell line was used as control since it had no known Ras-dependency. (A) Compound <b>1</b> decreased the viability of H358 cells at 48 h, compared to the control. (B) Apoptosis analysis showed increase response of H358 cells over time compared to control cells. (C) Treatment of compound <b>1</b> decreased the phosphorylation level of ERK 1/2 in H358 cells. (-) 0.1% DMSO treated controls; (+) 10 μM compound <b>1</b> treated samples. Cells were either grown in complete medium (lane 1 and 2) or stimulated after starvation (lane 3 and 4). The experiment was conducted three times. A representative immunoblot is shown. Immunoblots from all three experiments were quantified by densitometry using exposures in the linear range. The relative ratios of phospho Erk1/2 to total Erk1/2 are plotted. <i>p</i> = 0.0142, calculated with an unpaired two-tailed t-test using GraphPad Prism.</p

Correlation between EC50 values and the concentration of BODIPY^-FL GTP.

<p>The EC50 values of compound <b>1</b> and its analogues increased almost linearly (<i>R</i>² > 0.7) when the concentration of BODIPY^-FL GTP increased. A: compound <b>1</b>; B: compound <b>3</b>; C: compound <b>9</b>. The linear changes suggest a competitive inhibition mechanism [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134317#pone.0134317.ref037" target="_blank">37</a>]. The experiments were carried out in triplicate.</p

Compound 1 inhibited the integrin VLA-4 activation.

<p><b>A.</b> LDV-FITC binds to VLA-4 in the resting state, while the <i>N</i>-fMLFF addition led to high-affinity binding that was inhibited by <b>1</b>. Note that the different curves of fluorescence reading versus time are graphed in a staggered manner for ease of viewing. B. Percent Inhibition for compounds <b>1</b> and <b>3</b>. The percent inhibition was calculated according to Equation 1 in Supporting Information.</p