Synthesis and evaluation of phenoxymethylbenzamide analogues as anti-trypanosomal agents

Mycobacterium tuberculosis glutamine synthetase (GS) is an essential enzyme involved in the pathogenicity of the organism. The screening of a compound library using a robust high-throughput screening (HTS) assay is currently thought to be the most efficient way of getting lead molecules, which are potent inhibitors for this enzyme. The authors have purified the enzyme to a >90% level from the recombinant Escherichia coli strain YMC21E, and it was used for partial characterization as well as standardization experiments. The results indicated that the Kmof the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The Km for ADP, arsenate, and Mn2+ were 2 [.proportional]M, 5 [.proportional]M, and 25 [.proportional]M, respectively. When the components were adjusted according to their Km values, the activity remained constant for at least 3 h at both 25° C and 37° C. The Z′ factor determined in microplate format indicated robustness of the assay. When the signal/noise ratios were determined for different assay volumes, it was observed that the 200-[.proportional]l volume was found to be optimum. The DMSO tolerance of the enzyme was checked up to 10%, with minimal inhibition. The IC50 value determined for L-methionine S-sulfoximine on the enzyme activity was 3 mM. Approximately 18,000 small molecules could be screened per day using this protocol by a Beckman Coulter HTS setup.

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High-Throughput Screening of a 100,000-Compound Library for Inhibitors of Influenza A Virus (H3N2)

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057108323123 ◽

2008 ◽

Vol 13 (9) ◽

pp. 879-887 ◽

Cited By ~ 30

Author(s):

William E. Severson ◽

Michael McDowell ◽

Subramaniam Ananthan ◽

Dong-Hoon Chung ◽

Lynn Rasmussen ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Influenza A ◽

Moderate Activity ◽

Compound Library ◽

Highly Active ◽

Virus Life Cycle ◽

Influenza Activity ◽

Compound Concentration ◽

Viral Cytopathic Effect

Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-µM compound concentration against influenza strain A/Udorn/72 (H3N2). The “hit” rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-µM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI50 = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI50 > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity. ( Journal of Biomolecular Screening 2008:879-887)

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High-Throughput Screening Method of Inhibitors that Block the Interaction between 2 Helical Regions of HIV-1 gp41

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057104269726 ◽

2005 ◽

Vol 10 (1) ◽

pp. 13-19 ◽

Cited By ~ 10

Author(s):

Bong-Suk Jin ◽

Won-Kyu Lee ◽

Kwangseog Ahn ◽

Myung Kyu Lee ◽

Yeon Gyu Yu

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Screening Method ◽

Assay Method ◽

Enzyme Linked Immunosorbent Assay ◽

Compound Library ◽

Elisa Method ◽

Helical Bundle ◽

Fusion Inhibitors ◽

Hiv 1

The HIV-1 envelope glycoprotein transmembrane subunit, gp41, mediates the fusion of viral and target cell membranes. The 2 helical regions in the ectodomain of gp41, the N-helix and the C-helix, form a helical bundle complex that has been suggested as a fusion-active conformation. Previously, an enzyme-linked immunosorbent assay (ELISA) method had been established to measure the interaction of 2 helical regions of gp41. In this study, the ELISA method was modified to apply high-throughput screening (HTS) of an organic compound library. A few compounds had been identified to prevent the interaction between 2 helical regions of gp41, and they were further shown to inhibit the gp41-mediated viral infection. In addition, they specifically quenched the fluorescence of tryptophan in the N-helix region, indicating that these compounds bound to the N-helix rather than the C-helix of gp41. These results suggested that this assay method targeting gp41 could be used for HTS of HIV fusion inhibitors. ( Journal of Biomolecular Screening 2005:13-19)

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Overcoming Problems of Compound Storage in DMSO: Solvent and Process Alternatives

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109335670 ◽

2009 ◽

Vol 14 (6) ◽

pp. 708-715 ◽

Cited By ~ 21

Author(s):

Timothy J. Waybright ◽

John R. Britt ◽

Thomas G. McCloud

Keyword(s):

Water Absorption ◽

High Throughput ◽

High Throughput Screening ◽

Time Course ◽

Molar Concentration ◽

Dmso Solution ◽

Compound Library ◽

Plate Production ◽

The Common ◽

Drug Solution

The common practice of preparing storage libraries of compounds in 100% DMSO solution well in advance of bioassay brings with it difficulties that affect the accuracy of the data obtained. This publication presents a series of studies done on a subset of compounds that are difficult to bioassay because they precipitate from DMSO solution. These compounds are members of a frequently used, diverse compound library of the sort commonly used in the high-throughput screening (HTS) environment. Experiments were performed to determine the concentration of drug in solution above the precipitate, observe the time course and effect of various mixtures of solvents upon precipitation, measure the viscosity of cosolvents to determine compatibility with HTS, determine water absorption rates for various solvent combinations, and investigate resolubilization techniques to ensure proper drug solution for HTS. Recommendations are made on how to best maximize the probability that problem compounds will remain in solution, be accurately transferred during assay plate production, and, as a result, be accurately bioassayed at the specified molar concentration. ( Journal of Biomolecular Screening 2009:708-715)

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Compartmentalization of enhanced biomolecular interactions for high-throughput drug screening in test tubes

10.1101/2020.01.19.911149 ◽

2020 ◽

Author(s):

Min Zhou ◽

Weiping Li ◽

Jian Li ◽

Leiming Xie ◽

Rongbo Wu ◽

...

Keyword(s):

Drug Discovery ◽

High Throughput ◽

Biomedical Research ◽

High Throughput Screening ◽

Biomolecular Interactions ◽

Compound Library ◽

Cellular Processes ◽

High Throughput Drug Screening ◽

Binding Partners ◽

Discovery Method

AbstractModification-dependent and -independent biomolecular interactions (BIs), including protein-protein, protein-DNA/RNA and protein-lipid, play crucial roles in all cellular processes. Dysregulation of BIs or malfunction of the associated enzymes results in various diseases, thus they are attractive targets for therapies. High-throughput screening (HTS) can greatly facilitate the discovery of drugs for these targets. Here we describe a HTS drug discovery method, called compartmentalization of enhanced biomolecular interactions in test tubes (CEBIT). CEBIT uses selective recruitment of biomolecules into phase separated compartments harboring their cognate binding partners as readouts. CEBIT were tailored to detect various BIs and associated modifying enzymes. Using CEBIT-based HTS assays, we successfully identified known inhibitors of the p53/MDM2 interaction and of SUV39H1 from a compound library. CEBIT is simple and versatile, and is likely to become a powerful tool for drug discovery and basic biomedical research.

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Differential Assay for High-Throughput Screening of Antibacterial Compounds

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057107308161 ◽

2007 ◽

Vol 12 (8) ◽

pp. 1102-1108 ◽

Cited By ~ 8

Author(s):

Shaun P. Falk ◽

Andrew T. Ulijasz ◽

Bernard Weisblum

Keyword(s):

Cell Wall ◽

High Throughput ◽

High Throughput Screening ◽

Immediate Release ◽

Cell Wall Synthesis ◽

Compound Library ◽

Fluorogenic Substrate ◽

Cell Wall Disruption ◽

Surfactant Activity ◽

High Throughput Assay

The previously described Bacillus subtilis reporter strain BAU-102 is capable of detecting cell wall synthesis inhibitors that act at all stages of the cell wall synthesis pathway. In addition, this strain is capable of detecting compounds with hydrophobic/ surfactant activity and alternative mechanisms of cell wall disruption. BAU-102 sequesters preformed β-gal in the periplasm, suggesting leakage of β-gal as the means by which this assay detects compound activities. A model is proposed according to which β-gal release by BAU-102 reflects activation of pathways leading to autolysis. The authors also report a simplified high-throughput assay using BAU-102 combined with the fluorogenic substrate N-methylumbelliferyl-β-D-galactoside as a single reagent. Cell wall inhibitors release β-gal consistently only after 60 min of incubation, whereas compounds with surfactant activity show an almost immediate release. A high-throughput screen of a 480-compound library of known bioactives yielded 8 compounds that cause β-gal release. These results validate the BAU-102 assay as an effective tool in antimicrobial drug discovery. ( Journal of Biomolecular Screening 2007:1102-1108)

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Data-driven approaches used for compound library design, hit triage and bioactivity modeling in high-throughput screening

Briefings in Bioinformatics ◽

10.1093/bib/bbw105 ◽

2016 ◽

pp. bbw105 ◽

Cited By ~ 7

Author(s):

Shardul Paricharak ◽

Oscar Méndez-Lucio ◽

Aakash Chavan Ravindranath ◽

Andreas Bender ◽

Adriaan P. IJzerman ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Data Driven ◽

Library Design ◽

Compound Library

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High-Throughput Hit Screening Cascade to Identify Respiratory Syncytial Virus (RSV) Inhibitors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057115569428 ◽

2015 ◽

Vol 20 (5) ◽

pp. 597-605 ◽

Cited By ~ 9

Author(s):

Helen Plant ◽

Clare Stacey ◽

Choi-Lai Tiong-Yip ◽

Jarrod Walsh ◽

Qin Yu ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

High Throughput ◽

High Throughput Screening ◽

Compound Library ◽

Viral Inhibition ◽

The United Kingdom ◽

Physiological Relevance ◽

Syncytial Virus ◽

Infant Hospitalization ◽

Screening Cascade

Respiratory syncytial virus (RSV) infects 99% of children by age 2 years and is a leading cause of serious lower respiratory tract infection (LRTI) and infant hospitalization in the United Kingdom. Identification of efficacious RSV therapeutics has been hindered by the lack of a robust and appropriate primary assay for high-throughput screening (HTS). Here we report an HTS cascade that identified inhibitors of RSV replication using a robust RSV replicon luminescence-reporter assay for the primary campaign. The performance of the assay was consistent and reliable at scale, with Z′ of 0.55 ± 0.08 across 150 assay plates and signal-to-background ratios >40. The HTS assay was used to screen the AstraZeneca compound library of 1 million compounds at a single concentration of 10 µM. Hits specifically targeting the RSV replicon were determined using a series of hit generation assays. Compounds nonspecifically causing cell toxicity were removed, and hits were confirmed in live viral inhibition assays exhibiting greater physiological relevance than the primary assay. In summary, we developed a robust screening cascade that identified hit molecules that specifically targeted RSV replication.

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A High-throughput Screening of a Chemical Compound Library in Ovarian Cancer Stem Cells

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207321666180124093406 ◽

2018 ◽

Vol 21 (1) ◽

pp. 50-56 ◽

Cited By ~ 2

Author(s):

F. Ricci ◽

L. Carrassa ◽

M. S. Christodoulou ◽

D. Passarella ◽

B. Michel ◽

...

Keyword(s):

Ovarian Cancer ◽

Stem Cells ◽

Cancer Stem Cells ◽

High Throughput ◽

Chemical Compound ◽

High Throughput Screening ◽

Response To Therapy ◽

Compound Library ◽

Ovarian Cancer Stem Cells ◽

Differentiated Cells

Background: Epithelial ovarian cancer has a poor prognosis, mostly due to its late diagnosis and the development of drug resistance after a first platinum-based regimen. The presence of a specific population of “cancer stem cells” could be responsible of the relapse of the tumor and the development of resistance to therapy. For this reason, it would be important to specifically target this subpopulation of tumor cells in order to increase the response to therapy. Method: We screened a chemical compound library assembled during the COST CM1106 action to search for compound classes active in targeting ovarian stem cells. We here report the results of the high-throughput screening assay in two ovarian cancer stem cells and the differentiated cells derived from them. Results and Conclusion: Interestingly, there were compounds active only on stem cells, only on differentiated cells, and compounds active on both cell populations. Even if these data need to be validated in ad hoc dose response cytotoxic experiments, the ongoing analysis of the compound structures will open up to mechanistic drug studies to select compounds able to improve the prognosis of ovarian cancer patients.

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