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

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

Hypoxia and oxidative stress in breast cancer: Tumour hypoxia – therapeutic considerations

Conclusive research has shown that regions of acute/chronic hypoxia, which exist within the majority of solid tumours, have a profound influence on the therapeutic outcome of cancer chemotherapy and radiotherapy and are a strong prognostic factor of disease progression and survival. A strong argument therefore exists for assessing the hypoxic fraction of tumours, prior to patient treatment, and to tailor this treatment accordingly. Tumour hypoxia also provides a powerful physiological stimulus that can be exploited as a tumour-specific condition, allowing for the rationale design of hypoxia-activated anticancer drugs or novel hypoxia-regulated gene therapy strategies

Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression

Variation in the CYP3A enzymes, which act in drug metabolism, influences circulating steroid levels and responses to half of all oxidatively metabolized drugs. CYP3A activity is the sum activity of the family of CYP3A genes, including CYP3A5, which is polymorphically expressed at high levels in a minority of Americans of European descent and Europeans (hereafter collectively referred to as 'Caucasians'). Only people with at least one CYP3A5*1 allele express large amounts of CYP3A5. Our findings show that single-nucleotide polymorphisms (SNPs) in CYP3A5*3 and CYP3A5*6 that cause alternative splicing and protein truncation result in the absence of CYP3A5 from tissues of some people. CYP3A5 was more frequently expressed in livers of African Americans (60%) than in those of Caucasians (33%). Because CYP3A5 represents at least 50% of the total hepatic CYP3A content in people polymorphically expressing CYP3A5, CYP3A5 may be the most important genetic contributor to interindividual and interracial differences in CYP3A-dependent drug clearance and in responses to many medicines

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

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

Bezout matrices, Subresultants and Parameters

The problem of computing the greatest common divisor of two univariate polynomials in D[x], with D a field or an integral domain, is one of the cornerstones in Computer Algebra. When D is an integral domain we shall talk about a greatest common divisor as a polynomial in D[x] which is a greatest common divisor of th