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A Detergent-based Assay for the Detection of Promiscuous Inhibitors

Overview
Journal Nat Protoc
Specialties Biology
Pathology
Science
Date 2006 Dec 28
PMID 17191086
Citations 197
Authors
Brian Y Feng
Brian K Shoichet
Affiliations
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Abstract

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.

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