5 research outputs found
Secondary screening of neuroprotective compounds.
<p>Compounds identified through high-throughput screening were administered at 0.1 (white), 1 (light gray), 10 (dark gray), and 100 µM (black) at reoxygenation after 2 hours OGD. Twenty-four hours later, cells survival was measured by TUNEL assay and compared to DMSO vehicle-treated neurons. Compounds providing at least 2-fold increased neuroprotection over vehicle were further investigated. MIA, mianserine hydrochloride, ISO, isoxsuprine hydrochloride, MER, meropenem, MEC, meclofenamic acid, ETI, etilifrine hydrochloride, HAL, haloperidol, MOX, moxonidine, CHL, chlorphenesin carbamate, PRO, prothionamide, EPI, epitiostanol.</p
Dose optimization and time course of administration of neuroprotective compounds.
<p>A. Dose response of compounds administered at reoxygenation after 2(ISO) and etilifrine hydrochloride (ETI) and 200 µM for chlorphenesin carbamate (CHL). Isoxsuprine was significantly more protective at 1 nM compared to 0.01, 0.1, and 100 nM (*, <i>p</i><0.01). Etilifrine was significantly more protective at 1 nM compared to 0.01 and 0.1 nM (*, <i>p</i><0.05). Chlorphenesin carbamate was significantly more neuroprotective at 200 µM compared to all other doses (*, <i>p</i><0.05). B. Time course of administration of compounds at the optimal dose at 0, 15, 30, and 60 minutes after reoxygenation onset. Isoxsuprine and chlorphenesin carbamate demonstrated no decrease in neuroprotection when administered up to 60 minutes after reoxygenation onset. Neuroprotection by etilifrine significantly decreased when administered at 60 minutes versus time 0 (*, <i>p</i><0.01).</p
Neuroprotection by isoxsuprine hydrochloride in an animal stroke model.
<p>A. Representative TTC-stained brain sections showing differences in infarction between animals receiving vehicle or isoxsuprine hydrochloride. B. Effect of isoxsuprine hydrochloride on infarct volume. Isoxsuprine hydrochloride (1 mg/kg, IV) given at the onset of reperfusion after a 90-minute MCAO significantly reduced infarct volume compared to vehicle (137±18 mm<sup>3</sup> versus 279±25 mm<sup>3</sup>), <i>p</i><0.001. Closed circles, vehicle, closed squares, isoxsuprine hydrochloride, n = 7 animals for each group.</p
Neuroprotective compounds identified through high-throughput screening of the Prestwick Chemical Library.
1<p>Compounds previously investigated in models of cerebral ischemia are italicized.</p
Identification of a Small Molecule Yeast TORC1 Inhibitor with a Multiplex Screen Based on Flow Cytometry
TOR (target of rapamycin) is a serine/threonine kinase,
evolutionarily
conserved from yeast to human, which functions as a fundamental controller
of cell growth. The moderate clinical benefit of rapamycin in mTOR-based
therapy of many cancers favors the development of new TOR inhibitors.
Here we report a high-throughput flow cytometry multiplexed screen
using five GFP-tagged yeast clones that represent the readouts of
four branches of the TORC1 signaling pathway in budding yeast. Each
GFP-tagged clone was differentially color-coded, and the GFP signal
of each clone was measured simultaneously by flow cytometry, which
allows rapid prioritization of compounds that likely act through direct
modulation of TORC1 or proximal signaling components. A total of 255
compounds were confirmed in dose–response analysis to alter
GFP expression in one or more clones. To validate the concept of the
high-throughput screen, we have characterized CID 3528206, a small
molecule most likely to act on TORC1 as it alters GFP expression in
all five GFP clones in a manner analogous to that of rapamycin. We
have shown that CID 3528206 inhibited yeast cell growth and that CID
3528206 inhibited TORC1 activity both <i>in vitro</i> and <i>in vivo</i> with EC<sub>50</sub>'s of 150 nM and 3.9 μM,
respectively. The results of microarray analysis and yeast GFP collection
screen further support the notion that CID 3528206 and rapamycin modulate
similar cellular pathways. Together, these results indicate that the
HTS has identified a potentially useful small molecule for further
development of TOR inhibitors