α-Methylene-β-Lactone Probe for Measuring Live-Cell Reactions of Small Molecules

13 April 2020, Version 2
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

The novel use of the α-methylene-β-lactone (MeLac) moiety as a warhead of multiple electrophilic sites is reported. In this study, we demonstrate that a MeLac-alkyne is a competent covalent probe and reacts with diverse proteins in live cells. Proteomics analysis of affinity-enriched samples identifies probe-reacted proteins, resolves their modified peptides/residues, and thus characterizes probe-protein reactions. Unique methods are developed to evaluate confidence in the identification of the reacted proteins and modified peptides. Tandem mass spectra of the peptides reveal that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael addition or acyl addition. A peptide-centric proteomics platform, using MeLac-alkyne as the measurement probe, successfully analyzes the Orlistat selectivity in live HT-29 cells. MeLac is a versatile warhead demonstrating enormous potential to expedite the development of covalent probes and inhibitors in interrogating protein (re)activity. MeLac-empowered platforms in chemical proteomics are widely adaptable for measuring the live-cell action of reactive molecules.

Keywords

α-methylene-β-lactone
warhead
covalent probe
covalent inhibitor
covalent drug
chemical proteomics
peptide-centric competitive activity-based protein profiling
tagging triplication
cysteinome
Orlistat
glutathione S-transferase

Supplementary materials

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MeLac UConn XYao Appn 2020Apr
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MeLac UConn XYao preprint 2020Apr ver2
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MeLac UConn XYao preprint SI 2020Apr ver2
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