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J Biomol Screen. 2015 Jun;20(5):606-15. doi: 10.1177/1087057115570838. Epub 2015 Feb 13.

Screening of HIV-1 Protease Using a Combination of an Ultra-High-Throughput Fluorescent-Based Assay and RapidFire Mass Spectrometry.

Author information

1
Screening and Protein Sciences, Merck Research Labs, North Wales, PA, USA juncai_meng@merck.com gregory_adam@merck.com.
2
Department of Infectious Disease, Merck Research Labs, West Point, PA, USA.
3
Medicinal Chemistry, Merck Research Labs, West Point, PA, USA.
4
Screening and Protein Sciences, Merck Research Labs, North Wales, PA, USA.
5
Screening and Protein Sciences, Merck Research Labs, North Wales, PA, USA Wuxi Apptech.

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.

KEYWORDS:

FRET assay; HIV-1 protease; RapidFire MS; high-throughput screening

PMID:
25681434
DOI:
10.1177/1087057115570838
[Indexed for MEDLINE]

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