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Rapid LC-MS Based High-Throughput Screening Method, Affording No False Positives or False Negatives, Identifies a New Inhibitor for Carbonic Anhydrase.

ABSTRACT: Developing effective high-throughput screening (HTS) methods is of paramount importance in the early stage of drug discovery. While rugged and robust assays may be easily developed for certain enzymes, HTS assays designed to identify ligands that block protein binding are much more challenging to develop; attenuating the number of false positives and false negatives under high-throughput screening conditions is particularly difficult. We describe an MS-based HTS workflow that addresses these challenges. The assay mitigates false positives by selectively identifying positive hits exclusively when a ligand at the binding site of interest is displaced; it mitigates false negatives by detecting a reporter compound that ionizes well, not by detecting the ligand binder, which may not ionize. The method was validated by detecting known binders of three proteins, pepsin, maltose binding protein (MBP), and carbonic anhydrase (CA) in the presence of hundreds of non-binders. We also identified a novel CA binder, pifithrin-µ, which could not have been identified by any other MS-based assay because of its poor ionization efficiency. This new method addresses many of the challenges that are currently encountered during high-throughput screening.

SUBMITTER: Imaduwage KP

PROVIDER: S-EPMC5583356 | biostudies-other | 2017 Sep

REPOSITORIES: biostudies-other

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Rapid LC-MS Based High-Throughput Screening Method, Affording No False Positives or False Negatives, Identifies a New Inhibitor for Carbonic Anhydrase.

Imaduwage Kasun P KP Lakbub Jude J Go Eden P EP Desaire Heather H

Scientific reports 20170904 1

Developing effective high-throughput screening (HTS) methods is of paramount importance in the early stage of drug discovery. While rugged and robust assays may be easily developed for certain enzymes, HTS assays designed to identify ligands that block protein binding are much more challenging to develop; attenuating the number of false positives and false negatives under high-throughput screening conditions is particularly difficult. We describe an MS-based HTS workflow that addresses these cha ...[more]

PMID: 28871149

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Project description:BackgroundThe international SARS-CoV-2 pandemic has resulted in an urgent need to identify new anti-viral drugs for treatment of COVID-19. The initial step to identifying potential candidates usually involves in vitro screening that includes standard cytotoxicity controls. Under-appreciated is that viable, but stressed or otherwise compromised cells, can also have a reduced capacity to replicate virus. A refinement proposed herein for in vitro drug screening thus includes a simple growth assay to identify drug concentrations that cause cellular stress or "cytomorbidity", as distinct from cytotoxicity or loss of viability.MethodsA simple rapid bioassay is presented for antiviral drug screening using Vero E6 cells and inhibition of SARS-CoV-2 induced cytopathic effects (CPE) measured using crystal violet staining. We use high cell density for cytotoxicity assays, and low cell density for cytomorbidity assays.ResultsThe assay clearly illustrated the anti-viral activity of remdesivir, a drug known to inhibit SARS-CoV-2 replication. In contrast, nitazoxanide, oleuropein, cyclosporine A and ribavirin all showed no ability to inhibit SARS-CoV-2 CPE. Hydroxychloroquine, cyclohexamide, didemnin B, γ-mangostin and linoleic acid were all able to inhibit viral CPE at concentrations that did not induce cytotoxicity. However, these drugs inhibited CPE at concentrations that induced cytomorbidity, indicating non-specific anti-viral activity.ConclusionsWe describe the methodology for a simple in vitro drug screening assay that identifies potential anti-viral drugs via their ability to inhibit SARS-CoV-2-induced CPE. The additional growth assay illustrated how several drugs display anti-viral activity at concentrations that induce cytomorbidity. For instance, hydroxychloroquine showed anti-viral activity at concentrations that slow cell growth, arguing that its purported in vitro anti-viral activity arises from non-specific impairment of cellular activities. The cytomorbidity assay can therefore rapidly exclude potential false positives.

Dataset Information

Rapid LC-MS Based High-Throughput Screening Method, Affording No False Positives or False Negatives, Identifies a New Inhibitor for Carbonic Anhydrase.

Publications

Rapid LC-MS Based High-Throughput Screening Method, Affording No False Positives or False Negatives, Identifies a New Inhibitor for Carbonic Anhydrase.

Similar Datasets

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