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Rapid identification of antifungal compounds against Exserohilum rostratum using high throughput drug repurposing screens.Sun W, Park YD, Sugui JA, Fothergill A, Southall N, Shinn P, McKew JC, Kwon-Chung KJ, Zheng W, Williamson PRPLoS ONE , (8), e70506, 2013. Article Pubmed A recent large outbreak of fungal infections by Exserohilum rostratum from contaminated compounding solutions has highlighted the need to rapidly screen available pharmaceuticals that could be useful in therapy. The present study utilized two newly-developed high throughput assays to screen approved drugs and pharmaceutically active compounds for identification of potential antifungal agents. Several known drugs were found that have potent effects against E. rostratum including the triazole antifungal posaconazole. Posaconazole is likely to be effective against infections involving septic joints and may provide an alternative for refractory central nervous system infections. The anti-E. rostratum activities of several other drugs including bithionol (an anti-parasitic drug), tacrolimus (an immunosuppressive agent) and floxuridine (an antimetabolite) were also identified from the drug repurposing screens. In addition, activities of other potential antifungal agents against E. rostratum were excluded, which may avoid unnecessary therapeutic trials and reveals the limited therapeutic alternatives for this outbreak. In summary, this study has demonstrated that drug repurposing screens can be quickly conducted within a useful time-frame. This would allow clinical implementation of identified alternative therapeutics and should be considered as part of the initial public health response to new outbreaks or rapidly-emerging microbial pathogens.
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A phenotypic compound screening assay for lysosomal storage diseases.Xu M, Liu K, Swaroop M, Sun W, Dehdashti SJ, McKew JC, Zheng WJ Biomol Screen , (19), 168-75, 2014. Article Pubmed The lysosome is a vital cellular organelle that primarily functions as a recycling center for breaking down unwanted macromolecules through a series of hydrolases. Functional deficiencies in lysosomal proteins due to genetic mutations have been found in more than 50 lysosomal storage diseases that exhibit characteristic lipid/macromolecule accumulation and enlarged lysosomes. Recently, the lysosome has emerged as a new therapeutic target for drug development for the treatment of lysosomal storage diseases. However, a suitable assay for compound screening against the diseased lysosomes is currently unavailable. We have developed a Lysotracker staining assay that measures the enlarged lysosomes in patient-derived cells using both fluorescence intensity readout and fluorescence microscopic measurement. This phenotypic assay has been tested in patient cells obtained from several lysosomal storage diseases and validated using a known compound, methyl-β-cyclodextrin, in primary fibroblast cells derived from Niemann Pick C disease patients. The results demonstrate that the Lysotracker assay can be used in compound screening for the identification of lead compounds that are capable of reducing enlarged lysosomes for drug development.
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A high-throughput screening assay for determining cellular levels of total tau protein.Dehdashti SJ, Zheng W, Gever JR, Wilhelm R, Nguyen T, Sittampalam G, McKew JC, Austin C, Prusiner SBCurr Alzheimer Res , (10), 679-87, 2013. Pubmed The microtubule-associated protein (MAP) tau has been implicated in the pathology of numerous neurodegenerative diseases. In the past decade, the hyperphosphorylated and aggregated states of tau protein have been important targets in the drug discovery field for the potential treatment of Alzheimer's disease. Although several compounds have been reported to reduce the hyperphosphorylated state of tau or impact the stabilization of tau, their therapeutic activities are remain to be validated. Recently, reduction of total cellular tau protein has emerged as an alternate intervention point for drug development and a potential treatment of tauopathies. We have developed and optimized homogenous assays, using the AlphaLISA and HTRF assay technologies, for the quantification of total cellular tau protein levels in the SH-SY5Y neuroblastoma cell line. The signal-to-basal ratios were 375 and 5.3, and the Z' factors were 0.67 and 0.60 for the AlphaLISA and HTRF tau assays, respectively. The clear advantages of these homogeneous tau assays over conventional total tau assays, such as ELISA and Western blot, are the elimination of plate wash steps and miniaturization of the assay into 1536-well plate format for the ultra-high-throughput screening of large compound libraries.
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A high-throughput screening assay for fungicidal compounds against Cryptococcus neoformans.Rabjohns JL, Park YD, Dehdashti J, Sun W, Henderson C, Zelazny A, Metallo SJ, Zheng W, Williamson PRJ Biomol Screen , (19), 270-7, 2014. Article Pubmed Cryptococcus neoformans is a pathogenic fungus that causes meningitis worldwide, particularly in human immunodeficiency virus (HIV)-infected individuals. Although amphotericin B is the "gold standard" treatment for cryptococcal meningitis, the toxicity and inconvenience of intravenous injection emphasize a need for development of new anticryptocccal drugs. Recent data from humans and animal studies suggested that a nutrient-deprived host environment may exist in cryptococcal meningitis. Thus, a screening assay for identifying fungicidal compounds under nutrient-deprived conditions may provide an alternative strategy to develop new anticryptococcal drugs for this disease. A high-throughput fungicidal assay was developed using a profluorescent dye, alamarBlue, to detect residual metabolic activity of C. neoformans under nutrient-limiting conditions. Screening the Library of Pharmacologically Active Compounds (LOPAC) with this assay identified a potential chemical scaffold, 10058-F4, that exhibited fungicidal activity in the low micromolar range. These results thus demonstrate the feasibility of this alamarBlue-based assay for high-throughput screening of fungicidal compounds under nutrient-limiting conditions for new anticryptococcal drug development.
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Phenotypic screens as a renewed approach for drug discovery.Zheng W, Thorne N, McKew JCDrug Discov. Today , (18), 1067-73, 2013. Article Pubmed The significant reduction in the number of newly approved drugs in the past decade has been partially attributed to failures in discovery and validation of new targets. Evaluation of recently approved new drugs has revealed that the number of approved drugs discovered through phenotypic screens, an original drug screening paradigm, has exceeded those discovered through the molecular target-based approach. Phenotypic screening is thus gaining new momentum in drug discovery with the hope that this approach may revitalize drug discovery and improve the success rate of drug approval through the discovery of viable lead compounds and identification of novel drug targets.
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A high-throughput screening assay for assessing the viability of Cryptococcus neoformans under nutrient starvation conditions.Dehdashti SJ, Abbott J, Nguyen T, McKew JC, Williamson PR, Zheng WAnal Bioanal Chem , (405), 6823-9, 2013. Article Pubmed Cryptococcus neoformans causes an estimated 600,000 AIDS-related deaths annually that occur primarily in resource-limited countries. Fluconazole and amphotericin B are currently available for the treatment of cryptococcal-related infections. However, fluconazole has limited clinical efficacy and amphotericin B requires intravenous infusion and is associated with high renal toxicity. Therefore, there is an unmet need for a new orally administrable anti-cryptococcal drug. We have developed a high-throughput screening assay for the measurement of C. neoformans viability in 1,536-well plate format. The signal-to-basal ratio of the ATP content assay was 21.9 fold with a coefficient of variation and Z' factor of 7.1% and 0.76, respectively. A pilot screen of 1,280 known compounds against the wild-type C. neoformans (strain H99) led to the identification of four active compounds including niclosamide, malonoben, 6-bromoindirubin-3'-oxime, and 5-[(4-ethylphenyl)methylene]-2-thioxo-4-thiazolidinone. These compounds were further tested against nine clinical isolates of C. neoformans, and their fungicidal activities were confirmed. The results demonstrate that this miniaturized C. neoformans assay is advantageous for the high-throughput screening of large compound collections to identify lead compounds for new anti-cryptococcal drug development.
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Induction and reversal of myotonic dystrophy type 1 pre-mRNA splicing defects by small molecules.Childs-Disney JL, Stepniak-Konieczna E, Tran T, Yildirim I, Park H, Chen C, Hoskins J, Southall N, Marugan J, Patnaik S, Zheng W, Austin C, Schatz GC, Sobczak K, Thornton CA, Disney MDNat Commun , (4), 2044, 2013. Article Pubmed The ability to control pre-mRNA splicing with small molecules could facilitate the development of therapeutics or cell-based circuits that control gene function. Myotonic dystrophy type 1 is caused by the dysregulation of alternative pre-mRNA splicing due to sequestration of muscleblind-like 1 protein (MBNL1) by expanded, non-coding r(CUG) repeats (r(CUG)(exp)). Here we report two small molecules that induce or ameliorate alternative splicing dysregulation. A thiophene-containing small molecule (1) inhibits the interaction of MBNL1 with its natural pre-mRNA substrates. Compound (2), a substituted naphthyridine, binds r(CUG)(exp) and displaces MBNL1. Structural models show that 1 binds MBNL1 in the Zn-finger domain and that 2 interacts with UU loops in r(CUG)(exp). This study provides a structural framework for small molecules that target MBNL1 by mimicking r(CUG)(exp) and shows that targeting MBNL1 causes dysregulation of alternative splicing, suggesting that MBNL1 is thus not a suitable therapeutic target for the treatment of myotonic dystrophy type 1.
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Identification and optimization of small-molecule agonists of the human relaxin hormone receptor RXFP1.Xiao J, Huang Z, Chen C, Agoulnik IU, Southall N, Hu X, Jones RE, Ferrer-Alegre M, Zheng W, Agoulnik AI, Marugan JNat Commun , (4), 1953, 2013. Article Pubmed The anti-fibrotic, vasodilatory and pro-angiogenic therapeutic properties of recombinant relaxin peptide hormone have been investigated in several diseases, and recent clinical trial data has shown benefit in treating acute heart failure. However, the remodelling capacity of these peptide hormones is difficult to study in chronic settings because of their short half-life and the need for intravenous administration. Here we present the first small-molecule series of human relaxin/insulin-like family peptide receptor 1 agonists. These molecules display similar efficacy as the natural hormone in several functional assays. Mutagenesis studies indicate that the small molecules activate relaxin receptor through an allosteric site. These compounds have excellent physical and in vivo pharmacokinetic properties to support further investigation of relaxin biology and animal efficacy studies of the therapeutic benefits of relaxin/insulin-like family peptide receptor 1 activation.
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A novel brain penetrant NPS receptor antagonist, NCGC00185684, blocks alcohol-induced ERK-phosphorylation in the central amygdala and decreases operant alcohol self-administration in rats.Thorsell A, Tapocik JD, Liu K, Zook M, Bell L, Flanigan M, Patnaik S, Marugan J, Damadzic R, Dehdashti SJ, Schwandt ML, Southall N, Austin C, Eskay R, Ciccocioppo R, Zheng W, Heilig MJ. Neurosci. , (33), 10132-42, 2013. Article Pubmed The Neuropeptide S receptor, a Gs/Gq-coupled GPCR expressed in brain regions involved in mediating drug reward, has recently emerged as a candidate therapeutic target in addictive disorders. Here, we describe the in vitro and in vivo pharmacology of a novel, selective and brain penetrant NPSR antagonist with nanomolar affinity for the NPSR, NCGC00185684. In vitro, NCGC00185684 shows biased antagonist properties, and preferentially blocks ERK-phosphorylation over intracellular cAMP or calcium responses to NPS. In vivo, systemic NCGC00185684 blocks alcohol-induced ERK-phosphorylation in the rat central amygdala, a region involved in regulation of alcohol intake. NCGC00185684 also decreases operant alcohol self-administration, and lowers motivation for alcohol reward as measured using progressive ratio responding. These effects are behaviorally specific, in that they are observed at doses that do not influence locomotor activity or reinstatement responding following extinction. Together, these data provide an initial validation of the NPSR as a therapeutic target in alcoholism.
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An alternative direct compound dispensing method using the HP D300 digital dispenser.Jones RE, Zheng W, McKew JC, Chen CJ Lab Autom , (18), 367-74, 2013. Article Pubmed Evaluation of compound activity in vitro is crucial to drug discovery efforts and require that the compounds be accurately and reliably titrated and dispensed to the assay wells. The HP D300 dispenser uses inkjet technology to achieve small-volume dispensing that allows concentration-response testing using the direct dilution paradigm. Although inkjet technology has been long in existence, it is new to the field of screening and drug development. We have evaluated the D300 dispenser in a biochemical assay, a cell-based reporter gene assay, and a cytotoxicity assay. The software for this instrument is user friendly, and the compound-dispensing process is streamlined. However, a limitation is that this dispenser is currently applicable to only 96-well and 384-well plate formats and not to 1536-well high-density plates. Our results indicate that the D300 generates clean and reproducible results that correlate with those produced with more commonly used instruments such as the pin tool. We found that the instrument is useful and can improve the throughput of compound dispensing in 96-well and 384-well plates.
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