Skip to Content
Merck

Proteome-wide covalent ligand discovery in native biological systems.

Nature (2016-06-17)
Keriann M Backus, Bruno E Correia, Kenneth M Lum, Stefano Forli, Benjamin D Horning, Gonzalo E González-Páez, Sandip Chatterjee, Bryan R Lanning, John R Teijaro, Arthur J Olson, Dennis W Wolan, Benjamin F Cravatt
ABSTRACT

Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered 'undruggable'. Fragment-based ligand discovery can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes, including those that can access regions of proteins that are difficult to target through binding affinity alone. Here we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins in human proteomes and cells. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and -10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, 1 mg/mL, clone M2, affinity isolated antibody, buffered aqueous solution (50% glycerol, 10 mM sodium phosphate, and 150 mM NaCl, pH 7.4)
Sigma-Aldrich
N-(4-Bromophenyl)-N-phenylacrylamide, ≥95%
Sigma-Aldrich
BMK-alkyne
Sigma-Aldrich
MST-alkyne, ≥95%
Sigma-Aldrich
BrBT-alkyne, ≥95%
Sigma-Aldrich
MI-alkyne, ≥95%
Sigma-Aldrich
AlkFAA-alkyne, ≥95%
Sigma-Aldrich
ArVSA-alkyne, ≥95%
Sigma-Aldrich
MSBT-alkyne, ≥95%
Sigma-Aldrich
2-Chloro-1-(6-methoxy-1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-one, ≥95%
Sigma-Aldrich
EBX1-alkyne, ≥95.0%
Sigma-Aldrich
PFPSA-alkyne, ≥95%
Sigma-Aldrich
AlkVSA-alkyne, ≥95%
Sigma-Aldrich
EBX2-alkyne, ≥95%
Sigma-Aldrich
MSOD-alkyne, ≥95%
Sigma-Aldrich
CA-alkyne, ≥95%
Sigma-Aldrich
CA-nitrile, ≥95%
Sigma-Aldrich
ArVS-alkyne, ≥95%