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

2009 ◽  
Vol 14 (6) ◽  
pp. 708-715 ◽  
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)

scholarly journals Development of a Simple Assay Protocol for High-Throughput Screening of Mycobacterium tuberculosis Glutamine Synthetase for the Identification of Novel Inhibitors

2005 ◽  
Vol 10 (7) ◽  
pp. 725-729 ◽  
Author(s):  
Upasana Singh ◽  
Vinita Panchanadikar ◽  
Dhiman Sarkar
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Glutamine Synthetase ◽  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Coli Strain ◽  
Compound Library ◽  
Essential Enzyme ◽  
Assay Protocol

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.

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

2008 ◽  
Vol 13 (9) ◽  
pp. 879-887 ◽  
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)

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

2005 ◽  
Vol 10 (1) ◽  
pp. 13-19 ◽  
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)

scholarly journals High-Throughput Quantitative Intrinsic Thiol Reactivity Evaluation Using a Fluorescence-Based Competitive Endpoint Assay

2017 ◽  
Vol 22 (9) ◽  
pp. 1168-1174 ◽  
Author(s):  
Tomoya Sameshima ◽  
Ikuo Miyahisa ◽  
Seiji Yamasaki ◽  
Mika Gotou ◽  
Toshitake Kobayashi ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Time Course ◽  
Reaction Rate ◽  
Evaluation Method ◽  
Chemical Reactivity ◽  
Competitive Reaction ◽  
Quantitative Calculation ◽  
Thiol Reactivity ◽  
Endpoint Assay

In a high-throughput screening (HTS) process, the chemical reactivity of test samples should be carefully examined because such reactive compounds may lead to false-positive results and adverse effects in vivo. Among all natural amino acids, the thiol side chain in cysteine has the highest nucleophilicity; thus, assessment of intrinsic thiol group reactivity in the HTS processes is expected to accelerate drug discovery. In general, kchem (M−1s−1), the secondary reaction rate constant of a compound to thiol, can be evaluated via time course measurements of thiol-compound adducts using liquid chromatography–mass spectroscopy; this requires time-consuming and labor-intensive procedures. To overcome this issue, we developed a fluorescence-based competitive endpoint assay that allows quantitative calculation of the reaction rate of test compounds in an HTS format. Our assay is based on the competitive reaction for a free thiol (e.g., glutathione) between the test compounds and a fluorescent probe, o-maleimide BODIPY. Our assay provides robust data with a satisfactory throughput at an affordable cost. Our kchem evaluation method has advantages over previous assays in terms of higher throughput and quantitativeness. Thus, it contributes to early elimination of reactive compounds as well as quantitative evaluation of the kchem values of covalent inhibitors.

Synthesis and evaluation of phenoxymethylbenzamide analogues as anti-trypanosomal agents

MedChemComm ◽  
2015 ◽  
Vol 6 (3) ◽  
pp. 403-406 ◽  
Author(s):  
Alexandra Manos-Turvey ◽  
Emma E. Watson ◽  
Melissa L. Sykes ◽  
Amy J. Jones ◽  
Jonathan B. Baell ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Compound Library

The synthesis of a compound library based on a high throughput screening hit led to the discovery of several potent anti-trypanosomal agents.

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

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.

Differential Assay for High-Throughput Screening of Antibacterial Compounds

2007 ◽  
Vol 12 (8) ◽  
pp. 1102-1108 ◽  
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)

scholarly journals Validation of FLIPR Membrane Potential Dye for High Throughput Screening of Potassium Channel Modulators

2001 ◽  
Vol 6 (5) ◽  
pp. 305-312 ◽  
Author(s):  
Kristi L. Whiteaker ◽  
Sujatha M. Gopalakrishnan ◽  
Duncan Groebe ◽  
Char-Chang Shieh ◽  
Usha Warrior ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Potassium Channel ◽  
High Throughput ◽  
High Throughput Screening ◽  
Time Course ◽  
Functional Characterization ◽  
K Channel ◽  
Potassium Channel Modulators ◽  
Alternative Choice ◽  
Fluorescent Compounds

A fluorescence-based assay using the FLIPR Membrane Potential Assay Kit (FMP) was evaluated for functional characterization and high throughput screening (HTS) of potassium channel (ATP-sensitive Ki channel; KATP) modulators. The FMP dye permits a more sensitive evaluation of changes in membrane potential with a more rapid response time relative to DiBAC4(3). The time course of responses is comparable to ligand-evoked activation of the channel measured by patch-clamp studies. The pharmacological profile of the K+ channel evaluated by using reference KATP channel openers is in good agreement with that derived previously by DiBAC4(3)-based FLIPR assays. Improved sensitivity of responses together with the diminished susceptibility to artifacts such as those evoked by fluorescent compounds or quenching agents makes the FMP dye an alternative choice for HTS screening of potassium channel modulators.

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

2016 ◽  
pp. bbw105 ◽  
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

High-Throughput Mechanism of Inhibition

2021 ◽  
Vol 26 (2) ◽  
pp. 248-256
Author(s):  
Gareth Davies ◽  
Hannah Semple ◽  
Megan McCandless ◽  
Jonathan Cairns ◽  
Geoffrey A. Holdgate
Keyword(s):  
High Throughput ◽  
Substrate Concentration ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Significant Proportion ◽  
Mechanism Of Inhibition ◽  
The Common ◽  
High Throughput Screens ◽  
The Impact ◽  
Druggable Genome

Enzymes represent a significant proportion of the druggable genome and constitute a rich source of drug targets. Delivery of a successful program for developing a modulator of enzyme activity requires an understanding of the enzyme’s mechanism and the mode of interaction of compounds. This allows an understanding of how physiological conditions in disease-relevant cells will affect inhibitor potency. As a result, there is increasing interest in evaluating hit compounds from high-throughput screens to determine their mode of interaction with the target. This work revisits the common inhibition modalities and illustrates the impact of substrate concentration relative to Km upon the pattern of changes in IC50 that are expected for increasing substrate concentration. It proposes a new, high-throughput approach for assessing mode of inhibition, incorporating analyses based on a minimal descriptive model, to deliver a workflow that allows rapid and earlier compound classification immediately after high-throughput screening.

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