PAINS in the Assay: Chemical Mechanisms of Assay Interference and Promiscuous Enzymatic Inhibition Observed during a Sulfhydryl-Scavenging HTS

Significant resources in early drug
discovery are spent unknowingly pursuing artifacts and promiscuous
bioactive compounds, while understanding the chemical basis for these
adverse behaviors often goes unexplored in pursuit of lead compounds.
Nearly all the hits from our recent sulfhydryl-scavenging high-throughput
screen (HTS) targeting the histone acetyltransferase Rtt109 were such
compounds. Herein, we characterize the chemical basis for assay interference
and promiscuous enzymatic inhibition for several prominent chemotypes
identified by this HTS, including some pan-assay interference compounds
(PAINS). Protein mass spectrometry and ALARM NMR confirmed these compounds
react covalently with cysteines on multiple proteins. Unfortunately,
compounds containing these chemotypes have been published as screening
actives in reputable journals and even touted as chemical probes or
preclinical candidates. Our detailed characterization and identification
of such thiol-reactive chemotypes should accelerate triage of nuisance
compounds, guide screening library design, and prevent follow-up on
undesirable chemical matter.