Dipiperazine‐Phenyl Derivatives Based on Convergent Molecular Platforms Can Reverse Multidrug Resistance in Gram‐Negative Bacteria by Inhibiting Efflux and Permeabilizing Cell Membranes

By integrating a convergent molecular platform strategy, this study designed a novel dual‐target C5 to combat multidrug‐resistant Gram‐negative bacteria. C5 synergistically enhances antibiotic efficacy by inhibiting efflux pumps and increasing bacterial membrane permeability. This innovative “efflux pump‐membrane dual blockade” mechanism offers a new therapeutic paradigm for addressing the growing problem of bacterial resistance. Abstract With the global surge of infections caused by multidrug‐resistant (MDR) Gram‐negative bacteria, there is an urgent need for breakthrough therapeutic approaches. To overcome the intrinsic resistance mechanisms of bacteria, End‐alkyl‐modified dipiperazine‐phenyl derivatives are designed via convergent molecular platforms (CMPs)‐guided multi‐target directed ligand (MTDL) strategy. These dual‐functional compounds not only inhibit the AcrB‐TolC efflux pump system but also enhance bacterial membrane permeability and display a distinctive activity profile across a broad concentration range. Through integrated evaluation combining in vitro activity screening and computational ADMET (absorption, distribution, metabolism, excretion, toxicity) profiling, compound C5 is identified as a promising lead candidate. This compound achieved three notable breakthroughs. First, it reduces biofilm formation by 80% at 1/64 minimum inhibitory concentration (MIC) when combined with antibiotics. Second, unlike conventional antibiotic adjuvants that typically display potentiation within a narrow concentration window (1/4 MIC), C5 maintained robust and consistent synergistic activity across a broad range from 1/64 MIC to 1/4 MIC. Third, C5 markedly enhanced the therapeutic efficacy of antibiotics such as minocycline by over 1000‐fold in in vivo infection models, without causing detectable acute toxicity or cytotoxicity. The established MTDL‐CMPs integrated platform pioneers a novel “pump‐membrane dual blockade” therapeutic paradigm against MDR Enterobacteriaceae infections. By integrating a convergent molecular platform strategy, this study designed a novel dual-target C5 to combat multidrug-resistant Gram-negative bacteria. C5 synergistically enhances antibiotic efficacy by inhibiting efflux pumps and increasing bacterial membrane permeability. This innovative “efflux pump-membrane dual blockade” mechanism offers a new therapeutic paradigm for addressing the growing problem of bacterial resistance. Abstract With the global surge of infections caused by multidrug-resistant (MDR) Gram-negative bacteria, there is an urgent need for breakthrough therapeutic approaches. To overcome the intrinsic resistance mechanisms of bacteria, End-alkyl-modified dipiperazine-phenyl derivatives are designed via convergent molecular platforms (CMPs)-guided multi-target directed ligand (MTDL) strategy. These dual-functional compounds not only inhibit the AcrB-TolC efflux pump system but also enhance bacterial membrane permeability and display a distinctive activity profile across a broad concentration range. Through integrated evaluation combining in vitro activity screening and computational ADMET (absorption, distribution, metabolism, excretion, toxicity) profiling, compound C5 is identified as a promising lead candidate. This compound achieved three notable breakthroughs. First, it reduces biofilm formation by 80% at 1/64 minimum inhibitory concentration (MIC) when combined with antibiotics. Second, unlike conventional antibiotic adjuvants that typically display potentiation within a narrow concentration window (1/4 MIC), C5 maintained robust and consistent synergistic activity across a broad range from 1/64 MIC to 1/4 MIC. Third, C5 markedly enhanced the therapeutic efficacy of antibiotics such as minocycline by over 1000-fold in in vivo infection models, without causing detectable acute toxicity or cytotoxicity. The established MTDL-CMPs integrated platform pioneers a novel “pump-membrane dual blockade” therapeutic paradigm against MDR Enterobacteriaceae infections. Advanced Science, EarlyView.