Phenothiazine-Bile Acid Conjugates: A Novel Approach to Combat Gram-Negative Bacterial Resistance

Gram-negative bacterial resistance continues to pose a major therapeutic challenge, largely due to outer membrane impermeability and biofilm-associated tolerance mechanisms. In this study, we report the rational design, synthesis, and mechanistic characterization of bile acid-phenothiazine (BA-PTZ) conjugates as membrane-targeted antibacterial hybrids. Triazole and Schiff base linkers were strategically incorporated to systematically tune electronic properties and optimize amphiphilic balance. The synthesized conjugates exhibited markedly enhanced antibacterial activity compared to the parent phenothiazine (PTZ). Notably, cholic acid triazole-phenothiazine (CTPTZ) and cholyhydrazide Schiff base-phenothiazine (ChHSB-PTZ) displayed IC₅₀ values of 32.34 ± 3.7 and 34.76 ± 3.5 μM, respectively, against Serratia marcescens (S. marcescens), along with improved potency against Escherichia coli (E. coli). Quantitative antibiofilm assays demonstrated strain-dependent efficacy, with ChHSB-PTZ effectively inhibiting E. coli biofilms (IC₅₀ = 62.49 ± 1.3 μM) and CTPTZ exhibiting superior activity against S. marcescens biofilms (IC₅₀ = 79.74 ± 2.2 μM). Cytotoxicity profiling in mammalian cells revealed tunable selectivity, particularly for triazole-linked derivatives. Density functional theory calculations and global reactivity descriptor analysis established correlations between electronic softness, electrophilicity, and antibacterial performance. Complementary docking studies indicated enhanced binding affinity of the conjugates toward Gram-negative membrane-associated proteins relative to PTZ alone. Intracellular ROS assessment excluded oxidative stress-mediated cytotoxicity, supporting a membrane-associated mechanism modulated by electronic properties. Collectively, these results validate BA-PTZ conjugation as a rational design strategy to overcome Gram-negative permeability barriers and provide structure-guided principles for the optimization of membrane-targeted antimicrobial bioconjugates.