Antimicrobial Potential of A Theobromine-Based Hydrazone Derivative Against Acinetobacter baumannii : In Vitro and In Silico Investigation

Background: Nosocomial Acinetobacter baumannii infections in immunocompromised cancer patients represent an urgent challenge, and drug repositioning of established pharmacological scaffolds offers an efficient strategy to address this burden. T-1-NBHPA [2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)-N-(3-(1-(2-(4-nitrobenzoyl)hydrazono)ethyl)phenyl)acetamide] was previously identified as a potent anticancer VEGFR-2 inhibitor with sub-micromolar activity against HepG2 and MCF-7 cell lines. Objectives: This study aimed to investigate, for the first time, the antibacterial and antibiofilm activities of T-1-NBHPA against four A. baumannii strains and to elucidate its potential mechanism of antibiofilm action through molecular docking and molecular dynamics (MD) simulation targeting the biofilm master regulator BfmR. Methods: Antibacterial activity was assessed by agar well diffusion and broth microdilution against A. baumannii J26, Au299, 339, and S10 (colistin as reference). Antibiofilm activity was evaluated at 0.5×MIC by crystal violet staining and confirmed by scanning electron microscopy (SEM). Time-kill kinetics were conducted against A. baumannii Au299 over 24 h at MIC, 2×MIC, and 4×MIC, and a checkerboard assay assessed the interaction between T-1-NBHPA and ceftriaxone. Molecular docking and a 300×ns MD simulation against the AlphaFold-predicted BfmR structure were performed to explore the compound's binding mode. Results: T-1-NBHPA exhibited inhibition zone diameters of 11–22 mm and MIC values of 64–256 µg/mL across all tested strains. At 0.5×MIC, biofilm inhibition ranged from 33.75±1.1% to 52.65–3.0%, and SEM confirmed extensive disruption of biofilm ultrastructure. Time-kill analysis demonstrated concentration-dependent bactericidal activity, with complete growth elimination at 2× and 4×MIC within 24 h. The checkerboard assay showed a synergistic interaction with ceftriaxone (FICi = 0.375), with eight- and four-fold decreases in MIC values for ceftriaxone and T-1-NBHPA, respectively. Molecular docking predicted a binding affinity of ΔG = -8.0 kcal/mol against BfmR, and a 300-ns MD simulation supported the stability of the predicted complex. Conclusion: T-1-NBHPA demonstrates antibacterial and potent antibiofilm activity against A. baumannii, including a biofilm-disruption-mediated synergy with ceftriaxone, with computational data providing a plausible, though not yet experimentally confirmed, BfmR-mediated mechanistic hypothesis. Combined with its previously reported anticancer activity, these findings position T-1-NBHPA as a promising dualactivity theobromine scaffold warranting further mechanistic and in vivo validation, particularly for oncology patients at heightened risk of nosocomial A. baumannii infection.