High-throughput screening identifies idarubicin as a preferential inhibitor of smooth muscle versus endothelial cell proliferation.

Intimal hyperplasia is the cause of the recurrent occlusive vascular disease (restenosis). Drugs currently used to treat restenosis effectively inhibit smooth muscle cell (SMC) proliferation, but also inhibit the growth of the protective luminal endothelial cell (EC) lining, leading to thrombosis. To identify compounds that selectively inhibit SMC versus EC proliferation, we have developed a high-throughput screening (HTS) format using human cells and have employed this to screen a multiple compound collection (NIH Clinical Collection). We developed an automated, accurate proliferation assay in 96-well plates using human aortic SMCs and ECs. Using this HTS format we screened a 447-drug NIH Clinical Library. We identified 11 compounds that inhibited SMC proliferation greater than 50%, among which idarubicin exhibited a unique feature of preferentially inhibiting SMC versus EC proliferation. Concentration-response analysis revealed this differential effect most evident over an ∼10 nM-5 µM window. In vivo testing of idarubicin in a rat carotid injury model at 14 days revealed an 80% reduction of intimal hyperplasia and a 45% increase of lumen size with no significant effect on re-endothelialization. Taken together, we have established a HTS assay of human vascular cell proliferation, and identified idarubicin as a selective inhibitor of SMC versus EC proliferation both in vitro and in vivo. Screening of larger and more diverse compound libraries may lead to the discovery of next-generation therapeutics that can inhibit intima hyperplasia without impairing re-endothelialization

High Throughput Small Molecule Screen for Reactivation of <i>FMR1</i> in Fragile X Syndrome Human Neural Cells

Fragile X syndrome (FXS) is the most common inherited cause of autism and intellectual disability. The majority of FXS cases are caused by transcriptional repression of the FMR1 gene due to epigenetic changes that are not recapitulated in current animal disease models. FXS patient induced pluripotent stem cell (iPSC)-derived gene edited reporter cell lines enable novel strategies to discover reactivators of FMR1 expression in human cells on a much larger scale than previously possible. Here, we describe the workflow using FXS iPSC-derived neural cell lines to conduct a massive, unbiased screen for small molecule activators of the FMR1 gene. The proof-of-principle methodology demonstrates the utility of human stem-cell-based methodology for the untargeted discovery of reactivators of the human FMR1 gene that can be applied to other diseases

Bacillimidazoles A&minus;F, Imidazolium-Containing Compounds Isolated from a Marine Bacillus

Chemical investigations of a marine sponge-associated Bacillus revealed six new imidazolium-containing compounds, bacillimidazoles A&ndash;F (1&ndash;6). Previous reports of related imidazolium-containing natural products are rare. Initially unveiled by timsTOF (trapped ion mobility spectrometry) MS data, extensive HRMS and 1D and 2D NMR analyses enabled the structural elucidation of 1&ndash;6. In addition, a plausible biosynthetic pathway to bacillimidazoles is proposed based on isotopic labeling experiments and invokes the highly reactive glycolytic adduct 2,3-butanedione. Combined, the results of structure elucidation efforts, isotopic labeling studies and bioinformatics suggest that 1&ndash;6 result from a fascinating intersection of primary and secondary metabolic pathways in Bacillus sp. WMMC1349. Antimicrobial assays revealed that, of 1&ndash;6, only compound six displayed discernible antibacterial activity, despite the close structural similarities shared by all six natural products

Dose-responses of HuAoSMCs and HuAoECs to idarubicin treatment.

<p>Proliferation of SMCs or ECs in the presence of various concentrations of idarubicin or resveratrol was assayed in a 96-well plate and handled by the same robotic system as described in Materials and Methods. Each data point is a mean ± SD of triplicates, *P<0.05.</p

Inhibitory effect of idarubicin on intimal hyperplasia in balloon-injured rat carotid arteries.

<p>Following balloon angioplasty, idarubicin was applied locally around the injured arteries. Morphometric analysis was performed on the sections of carotid arteries collected on day 14 post angioplasty, as described in detail in Materials and Methods. Shown in A and B are representative H&E-stained sections from the arteries treated with vehicle (DMSO) and idarubicin, respectively. Arrow heads point to IEL. Statistics of the area ratio of intima versus media (C), residual lumen (the ratio of lumen area versus IEL area) (D), and EEL length (E) were calculated with the data pooled from 5 rats in each treatment group. Each bar represents a mean ± SEM (*P<0.05).</p

HTS against the NIH Clinical Collection for HuAoSMC proliferation.

<p>Assays were performed with SMCs using the automated assay system as described in detail in Materials and Methods. DMSO (blue, final 0.05% in each of 8 wells) and resveratrol (red, final 50 µM in each of 8 wells) served as negative control and positive control, respectively, on each of six 96-well plates. Total 447 compounds in the NIH Clinical Collection (yellow) were tested at a final concentration of 5 µM (1 well for each drug). <i>For confirmation of the hits with a different method (Cell Titer Glo), please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089349#pone.0089349.s002" target="_blank">Figure S2</a>. </i><b>A</b>). Percent Alamar Blue fluorescence reading. The dashed line marks 50% inhibition of SMC proliferation. <b>B</b>). Consistency of HTS assay on each of six 96-well plates.</p

Test of reproducibility of the automated 96-well proliferation assay format.

<p>Experiments were performed as described in detail in Materials and Methods. DMSO (40 wells) and resveratrol (40 wells) were used as negative control and positive control, respectively. Data are presented either as Alamar Blue fluorescence reading from individual wells (A), or a mean ± SD (standard deviation) of 40 wells (B) (***P<0.001).</p