Pharmacokinetics Screening, Molecular Docking, and Dynamics Simulations Revealed Novel Antimicrobial Peptide NKLF2 Mutants as Potent Inhibitors of Mycobacterium tuberculosis

The increasing threat of multidrug-resistant Mycobacterium tuberculosis (Mtb) underscores the urgent need for novel therapeutics that can circumvent existing resistance mechanisms. The clinical ineffectiveness of current treatment regimens propelled the exploration for alternative antimicrobials with minimal toxicity and multi-target specificity. This study aimed to design and optimize antimicrobial peptides (AMPs) targeting crucial Mtb enzymes, namely, arabinosyltransferase C, DNA gyrase, 30S ribosomal protein S1, and enoyl-[acyl-carrier-protein] reductase. Curated set of 92 natural peptides, exhibiting high positive charge and reported efficacy against Mtb-H37Rv were screened and evaluated for pharmacokinetic properties. To enhance efficacy and overcome the intrinsic limitations of cationic AMPs, a mutant library was generated. Among them, NKLF2 and its mutants (M16C and M16I) demonstrated improved antibacterial efficacy (~ 5%) and favorable pharmacological profiles with no predicted toxicity. Molecular docking revealed enhanced binding affinities of the mutants across multiple targets. Notably, NKLF2_M16I exhibited enhancements of 9.71% and 7.63% in binding affinities against 4NNI and 5VRL respectively, while NKLF2_M16C achieved increments of 5.4% and 4.37% against 4G3N and 3PTY. Intermolecular interaction profiling identified hydrogen bonds, salt bridges, and hydrophobic interactions with the crucial active site residues of each target. Validations through coarse-grained, molecular, and essential dynamics simulations revealed minimal residue-level fluctuations, stable backbone profile, and minimized energy cluster basins ensuring compactness and stability of the protein-peptide docked complexes. These in silico findings open new avenues for further experimental validations and suggest that NKLF2 mutants in combination with conventional anti-TB drugs could pave the path towards the development of effective therapeutics in combating Mtb.