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Bioactive small-molecule inhibitors represent a treasure chest for future drugs. In vitro high-throughput screening is a common approach to identify the small-molecule inhibitors that bind tightly to purified targets. Here, we investigate the inhibitor-target binding/unbinding kinetics in E. coli cells using a benzimidazole-derivative DNA inhibitor as a model system. We find that its unbinding rate is not constant but depends on cell growth rate. This dependence is mediated by the cellular activity, forming a feedback loop with the inhibitor’s activity. In accordance with this feedback, we find cell-to-cell heterogeneity in inhibitor-target interaction, leading to co-existence of two distinct subpopulations: actively growing cells that dissociate the inhibitors from the targets and non-growing cells that do not. We find similar heterogeneity for other clinical DNA inhibitors. Our studies reveal a mechanism that couples inhibitor-target kinetics to cell physiology and demonstrate the significant effect of this coupling on drug efficacy.

Background Serum levels of pyridinoline, measured by high-performance liquid chromatography, and pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen (ICTP), measured by a newly developed radioimmunoassay kit, were compared and analyzed to determine whether ICTP can replace pyridinoline as a marker of bone resorption in hemodialysis patients.