A detergent-based assay for the detection of promiscuous inhibitors

• Published in: Nature protocols Vol. 1; no. 2; pp. 550 - 553
• Main Authors: Shoichet, Brian K, Feng, Brian Y
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01.07.2006
• Subjects: Aggregates; beta-Lactamase Inhibitors; Biological assay; Chemical properties; Detergents - chemistry; Detergents - pharmacology; Enzyme inhibitors; Enzyme Inhibitors - analysis; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Enzymes; Experiments; Inhibition; Inhibitors; Ligands; Measurement; Methods; Potassium; Protein Binding - drug effects; Protein Denaturation - drug effects; Protocol; United States
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2006.77

At micromolar concentrations, many small molecules self-associate into colloidal aggregates that non-specifically inhibit enzymes and other proteins. Here we describe a protocol for identifying aggregate-based inhibitors and distinguishing them from small molecules that inhibit via specific mechanisms. As a convenient proxy for promiscuous, aggregate-based inhibition, we monitor inhibition of beta-lactamase in the absence and presence of detergent. Inhibition that is attenuated in the presence of detergent is characteristic of an aggregate-based mechanism. In the 96-well-format assay described here, about 200 molecules can be tested, in duplicate, per hour for detergent-dependent sensitivity. Furthermore, we also describe simple experiments that can offer additional confirmation of aggregate-based inhibition.