Screening of HIV-1 Protease Using a Combination of an Ultra-High-Throughput Fluorescent-Based Assay and RapidFire Mass Spectrometry
| Autor: | Jay A. Grobler, Paul Zuck, Victor N. Uebele, Jeffrey D. Hermes, Vandna Munshi, Gregory C. Adam, Juncai Meng, Ming-Tain Lai, John A. McCauley, Eric N. Johnson |
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| Rok vydání: | 2015 |
| Předmět: |
medicine.medical_treatment
High-throughput screening Peptide Microbial Sensitivity Tests Mass spectrometry Biochemistry Substrate Specificity Analytical Chemistry HIV-1 protease Drug Discovery Fluorescence Resonance Energy Transfer medicine Humans chemistry.chemical_classification Protease Chromatography Dose-Response Relationship Drug biology Chemistry HIV Protease Inhibitors Molecular biology Fluorescence High-Throughput Screening Assays Kinetics Förster resonance energy transfer biology.protein Molecular Medicine Biotechnology |
| Zdroj: | SLAS Discovery. 20:606-615 |
| ISSN: | 2472-5552 |
| Popis: | HIV-1 protease (PR) represents one of the primary targets for developing antiviral agents for the treatment of HIV-infected patients. To identify novel PR inhibitors, a label-free, high-throughput mass spectrometry (HTMS) assay was developed using the RapidFire platform and applied as an orthogonal assay to confirm hits identified in a fluorescence resonance energy transfer (FRET)-based primary screen of1 million compounds. For substrate selection, a panel of peptide substrates derived from natural processing sites for PR was evaluated on the RapidFire platform. As a result, KVSLNFPIL, a new substrate measured to have a ~ 20- and 60-fold improvement in k cat/K m over the frequently used sequences SQNYPIVQ and SQNYPIV, respectively, was identified for the HTMS screen. About 17% of hits from the FRET-based primary screen were confirmed in the HTMS confirmatory assay including all 304 known PR inhibitors in the set, demonstrating that the HTMS assay is effective at triaging false-positives while capturing true hits. Hence, with a sampling rate of ~7 s per well, the RapidFire HTMS assay enables the high-throughput evaluation of peptide substrates and functions as an efficient tool for hits triage in the discovery of novel PR inhibitors. |
| Databáze: | OpenAIRE |
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