Beyond Gatekeeping: Efflux Pumps Remotely Destabilize Cytoplasmic Drug-Target Interactions by Limiting Rebinding.

Bacterial efflux pumps are major contributors to multidrug resistance, classically described as "gatekeepers" that reduce drug entry. Here, we uncover an equally potent post-entry mechanism of efflux pumps, revealing that their function extends deep into intracellular drug-target interactions. Using quantitative live-cell fluorescence imaging, we monitored the activity of major efflux systems in Escherichia coli and Pseudomonas aeruginosa with Hoechst (HCT), a DNA-binding inhibitor. As expected, more HCT entered efflux-deficient cells than efflux-active cells. Unexpectedly, the intracellular HCT in efflux-deficient cells also bound DNA more stably, resulting in higher binding affinity. Statistical-physics-based modeling and experimental testing show that that unlike in dilute in vitro conditions, drug molecules that unbind from their targets in intracellular environments rapidly undergo successive rebinding, prolonging the lifetime of the drug-target complex. However, efflux pumps counteract this effect by suppressing rebinding, thereby kinetically destabilizing drug-target interactions and lowering the binding affinity. This kinetic destabilization acts synergistically with canonical gatekeeping to broaden and amplify drug resistance. Our findings reveal an underappreciated biophysical mechanism of efflux pumps, expanding the classical biochemical view of how efflux pumps confer multidrug resistance.