16 research outputs found

    High-throughput screening in larval zebrafish identifies novel potent sedative-hypnotics

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    BACKGROUND: Many general anesthetics were discovered empirically, but primary screens to find new sedative-hypnotics in drug libraries have not used animals, limiting the types of drugs discovered. The authors hypothesized that a sedative-hypnotic screening approach using zebrafish larvae responses to sensory stimuli would perform comparably to standard assays, and efficiently identify new active compounds. METHODS: The authors developed a binary outcome photomotor response assay for zebrafish larvae using a computerized system that tracked individual motions of up to 96 animals simultaneously. The assay was validated against tadpole loss of righting reflexes, using sedative-hypnotics of widely varying potencies that affect various molecular targets. A total of 374 representative compounds from a larger library were screened in zebrafish larvae for hypnotic activity at 10 µM. Molecular mechanisms of hits were explored in anesthetic-sensitive ion channels using electrophysiology, or in zebrafish using a specific reversal agent. RESULTS: Zebrafish larvae assays required far less drug, time, and effort than tadpoles. In validation experiments, zebrafish and tadpole screening for hypnotic activity agreed 100% (n = 11; P = 0.002), and potencies were very similar (Pearson correlation, r > 0.999). Two reversible and potent sedative-hypnotics were discovered in the library subset. CMLD003237 (EC50, ~11 µM) weakly modulated γ-aminobutyric acid type A receptors and inhibited neuronal nicotinic receptors. CMLD006025 (EC50, ~13 µM) inhibited both N-methyl-D-aspartate and neuronal nicotinic receptors. CONCLUSIONS: Photomotor response assays in zebrafish larvae are a mechanism-independent platform for high-throughput screening to identify novel sedative-hypnotics. The variety of chemotypes producing hypnosis is likely much larger than currently known.This work was supported by grants from Shanghai Jiaotong University School of Medicine, Shanghai, China, and the Chinese Medical Association, Beijing, China (both to Dr. Yang). The Department of Anesthesia, Critical Care and Pain Medicine of Massachusetts General Hospital, Boston, Massachusetts, supported this work through a Research Scholars Award and an Innovation Grant (both to Dr. Forman). Contributions to this research from the Boston University Center for Molecular Discovery, Boston, Massachusetts (to Drs. Porco, Brown, Schaus, and Xu, and to Mr. Trilles), were supported by a grant from the National Institutes of Health, Bethesda, Maryland (grant No. R24 GM111625). (Shanghai Jiaotong University School of Medicine, Shanghai, China; Chinese Medical Association, Beijing, China; Department of Anesthesia, Critical Care and Pain Medicine of Massachusetts General Hospital, Boston, Massachusetts; R24 GM111625 - National Institutes of Health, Bethesda, Maryland)Accepted manuscript2019-09-0

    Human Telomerase Reverse Transcriptase Promoter-Driven Oncolytic Adenovirus with E1B-19 kDa and E1B-55 kDa Gene Deletions

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    We constructed an oncolytic adenovirus, Adeno-hTERT-E1A, with deletions of the viral E1B, E3A, and E3B regions and insertion of a human telomerase reverse transcriptase (hTERT) promoter-driven early viral 1A (E1A) cassette that confers high transcriptional activity in multiple human tumor cell lines. The oncolytic potential of Adeno-hTERT-E1A was characterized in comparison with that of the E1B-55kDa- and E3B-region-deleted oncolytic adenovirus ONYX-015. Tumor cells infected with Adeno-hTERT-E1A expressed dramatically higher levels of E1A oncoprotein, underwent enhanced lysis, and displayed an earlier and higher apoptotic index than cells infected with ONYX-015. Despite the increase in virus-induced apoptotic death, Adeno-hTERT-E1A replicated and produced functional progeny leading to viral spread, but with reduced efficiency compared with ONYX-015, in particular in A549 cells. Virus-induced E1A expression, host cell apoptosis, viral hexon protein production, and DNA synthesis were markedly reduced in primary human hepatocytes after infection with Adeno-hTERT-E1A as compared with ONYX-015. The strong oncolytic activity of Adeno-hTERT-E1A in tumor cell culture translated into superior antitumor activity in vivo in an MDA-MB-231 solid tumor xenograft model. Adeno-hTERT-E1A thus has strong therapeutic potential and an improved safety profile compared with ONYX-015, which may lead to reduced toxicity in the clinic

    The binding isotherm of the agonist [<sup>3</sup>H]muscimol to the α4β3N-Flag-δ GABA<sub>A</sub> receptor in native membranes and reconstituted into CHAPS/lipid micelles.

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    <p>Binding curves of [<sup>3</sup>H]muscimol to α4β3N–Flag–δ GABA<sub>Α</sub>Rs, both, in cell membranes (pmol/mg membrane protein) and after purification and reconstitution into micelles of 5 mM CHAPS and 200 μM DOPC:DOPS:Cholesterol in mole ratio 52:15:33 (pmol/mL). Displaceable binding was determined as the difference between binding in the presence and absence of 1 mM GABA using a filtration assay in triplicate. The displaceable binding and its standard deviation was determined by subtracting these two values and propagating errors at each total muscimol concentration. The curves were fitted by nonlinear least squares with weighting by standard deviation. These yielded apparent dissociation constants of 9.2 ± 0.6 and 35 ± 12 nM, respectively. The B<sub>max</sub> of the membranes was 22.6 ± 0.5 pmol/mg and for reconstituted receptors in micelles was 19 ± 3 pmol/mL. The Hill coefficients differed little from one (1.07 ± 0.3 and 0.90 ± 0.05 respectively).</p

    DS2 enhances agonist binding in δ-subunit containing receptors.

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    <p>The δ-subunit specific modulator, DS2, modulates [<sup>3</sup>H]muscimol binding (2 nM) in α4β3N-Flag-δ GABA<sub>A</sub>Rs. For the membranes, the data are the mean and standard deviation of two experiments, and for micelle reconstituted receptors for a single experiment in triplicate. The curves were fitted by nonlinear least squares to the Hill equation. The EC<sub>50</sub> (μM), Hill coefficient and maximum modulation were: for membranes, 2.0 ± 0.7 μM, 0.9 ± 0.2, 139 ± 4%; for reconstituted 2.3 ± 0.8 μM, 1.2 ± 0.5, 132 ± 4%.</p

    The gating properties of α4β3N–Flag–δ GABA<sub>A</sub>Rs.

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    <p>(<b>A</b>) GABA concentration-response curve for α4β3N–Flag–δ receptor mediated currents. Currents were elicited by application of varying concentrations of GABA (0.1–10 mM). Peak current amplitudes in each cell were normalized to that obtained with 10 mM GABA. (<b>B</b>) Representative current trace obtained by application of an 8.5 ms pulse of 100 μM GABA to measure the deactivation rate. (<b>C</b>) α4β3N–Flag–δ receptors are spontaneously open. (Left panel) Representative trace of outward currents observed by application of 2 mM Picrotoxin to inhibit the spontaneously open receptors. (Right panel) The estimate of the maximum inward currents obtained by co-application of 10 mM GABA with 10 μM Etomidate to gate all available receptors. The gray lines are drawn by eye to represent the baseline. At least three cells per concentration were used throughout experiments.</p
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