title: Screening of HIV-1 Protease Using a Combination of an Ultra-High-Throughput Fluorescent-Based Assay and RapidFire Mass Spectrometry.
abstract: 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 of > 1 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.
authors: Screening and Protein Sciences, Merck Research Labs, North Wales, PA, USA juncai_meng@merck.com gregory_adam@merck.com.
authors: Juncai
authors: J
authors: Meng
authors: Department of Infectious Disease, Merck Research Labs, West Point, PA, USA.
authors: Ming-Tain
authors: MT
authors: Lai
authors: Department of Infectious Disease, Merck Research Labs, West Point, PA, USA.
authors: Vandna
authors: V
authors: Munshi
authors: Department of Infectious Disease, Merck Research Labs, West Point, PA, USA.
authors: Jay
authors: J
authors: Grobler
authors: Medicinal Chemistry, Merck Research Labs, West Point, PA, USA.
authors: John
authors: J
authors: McCauley
authors: Screening and Protein Sciences, Merck Research Labs, North Wales, PA, USA.
authors: Paul
authors: P
authors: Zuck
authors: Screening and Protein Sciences, Merck Research Labs, North Wales, PA, USA Wuxi Apptech.
authors: Eric N
authors: EN
authors: Johnson
authors: Screening and Protein Sciences, Merck Research Labs, North Wales, PA, USA.
authors: Victor N
authors: VN
authors: Uebele
authors: Screening and Protein Sciences, Merck Research Labs, North Wales, PA, USA.
authors: Jeffrey D
authors: JD
authors: Hermes
authors: Screening and Protein Sciences, Merck Research Labs, North Wales, PA, USA.
authors: Gregory C
authors: GC
authors: Adam
mesh_terms: D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D013379:Substrate Specificity
chemical_list: D017320:HIV Protease Inhibitors
keywords: FRET assay; HIV-1 protease; RapidFire MS; high-throughput screening