c-di-AMP analogs as allosteric inhibitors against tuberculosis: A 500 ns molecular dynamics study

The emergence of multidrug- and extensively drug-resistant tuberculosis remains a major global health challenge. Current therapies are limited by long treatment durations, toxicity, and limited efficacy, highlighting an urgent need for novel drug design strategies targeting alternative pathways such as allosteric modulation. Recombinase A (RecA) is a bacterial stress response protein that plays multi-functional roles in homologous recombination, DNA maintenance, and repair mechanisms, making it a promising therapeutic target. Recent studies have indicated the binding potential of cyclic di-adenylate monophosphate (c-di-AMP) to an allosteric site on RecA, offering a novel route for modulation. The current study focuses on designing and computationally evaluating novel c-di-AMP analogs as potential allosteric inhibitors of MtRecA. Over 100 c-di-AMP analogs were designed by employing non-allergenic, non-toxic, and well-characterized conventional functional groups such as amines, amides, hydroxyls, iodine, bromine, and fluorine and screened using molecular docking. Top candidates were evaluated using ADMET prediction, 500 ns molecular dynamics (MD) simulations, and MM/GBSA binding free energy calculations. The top three analogs, Analog-1, Analog-2, and Analog-3, with respective CDOCKER energies of −77.98 kcal/mol, −56.43 kcal/mol, and −49.97 kcal/mol, were found to have favorable binding in the allosteric binding site of MtRecA. The MD simulation studies revealed the stability of the RecA-analog complexes (Analog-1: 0.627 nm; Analog-2: 0.731 nm; Analog-3: 0.700 nm) in comparison to the native c-di-AMP (1.126 nm). The MM/GBSA binding free energy analysis was favorable for the RecA-analog-2 complex with a ΔG value of −10.21 kcal/mol. Reduced RMSF, compact radius of gyration, and lower SASA further supported enhanced structural stability of analog-bound complexes. Among the designed analogs based on pharmacophore modeling, Analog-2 was found as a promising allosteric modulator of MtRecA. These results are computational and hypothesis-generating; experimental validation will be essential to confirm their potential as allosteric modulators of RecA and adjunctive anti-tubercular candidates.