A simple assay for detection of small-molecule redox activity

Area of Science:

• Medicinal Chemistry
• Biochemistry
• Drug Discovery

Background:

• High-throughput screening (HTS) campaigns can yield drug candidates with undesirable mechanisms, hindering development.
• Common promiscuous mechanisms include target alkylation/acylation and compound aggregation.
• Oxidation of enzyme targets by small molecules is an under-documented but problematic mechanism.

Purpose of the Study:

• To provide direct evidence of small-molecule-induced oxidation of cysteine proteases.
• To develop a simple assay for predicting compound oxidizing behavior.
• To validate the assay's utility in distinguishing problematic redox compounds from well-behaved inhibitors in drug discovery projects.

Main Methods:

• Liquid chromatography/tandem mass spectrometry (LC/MS/MS) was used to demonstrate direct oxidation of a cysteine protease by a small molecule.
• A predictive assay was developed to identify compounds with oxidizing potential.
• The assay's performance was evaluated using historical drug discovery data from GlaxoSmithKline.

Main Results:

• Direct proof of small-molecule oxidation of a cysteine protease was established using LC/MS/MS.
• A straightforward assay was successfully developed to predict compound-mediated oxidation.
• The assay effectively differentiated nuisance redox compounds from desirable inhibitors in past drug discovery efforts.

Conclusions:

• Compound oxidation represents a significant, yet under-recognized, mechanism of undesirable activity in drug discovery.
• The developed assay provides a valuable tool for early identification and mitigation of redox-active compounds.
• Implementing this assay can enhance the specificity and developability of drug candidates, improving the success rate of drug discovery programs.