Exploring transmembrane allostery in the MexB: DB08385 variant as a promising inhibitor-like candidate against antibiotic resistance: a computational study.

, a formidable pathogen renowned for its antimicrobial resistance, poses a significant threat to immunocompromised individuals. In this regard, the MexAB-OprM efflux pump acts as a pivotal line of defense by extruding antimicrobials from bacterial cells. The inner membrane homotrimeric protein MexB captures antibiotics and translocates them into the outer membrane OprM channel protein connected through the MexA adaptor protein. Despite extensive efforts, competitive inhibitors targeting the tight (T) protomer of the MexB protein have not received FDA approval for medical use. Over the past few years, allosteric inhibitors have become popular as alternatives to the classical competitive inhibitor-based approach because of their higher specificity, lower dosage, and reduced toxicological effects. Hence, in this study, we unveiled the existence of a transmembrane allosteric binding pocket of MexB inspired by the recent discovery of an important allosteric inhibitor, BDM88855, for the homolog AcrB protein. While repurposing BDM88855 proved ineffective in controlling the MexB loose (L) protomer, our investigation identified a promising alternative: a chlorine-containing variant of DB08385 (2-Cl DB08385 or Variant 1). Molecular dynamics simulations, including binding free energy estimation coupled with heterogeneous dielectric implicit membrane model (implicit-membrane MM/PBSA), interaction entropy (IE) analysis and potential of mean force (PMF) calculation, demonstrated Variant 1's superior binding affinity to the transmembrane pocket, displaying the highest energy barrier in the ligand unbinding process. To elucidate the allosteric crosstalk between the transmembrane and porter domain of MexB, we employed the 'eigenvector centrality' measure in the linear mutual information obtained from the protein correlation network. Notably, this study confirmed the presence of an allosteric transmembrane site in the MexB L protomer. In addition to this, Variant 1 emerged as a potent regulator of allosteric crosstalk, inducing an 'O-L intermediate state' in the MexB L protomer. This induced state might hold the potential to diminish substrate intake into the access pocket, leading to the ineffective efflux of antibiotics.