Probing the conserved catalytic mechanism of ThiL protein in pathogenic Leptospira species: An in silico strategy for inhibitor discovery to combat leptospirosis

Leptospirosis is a zoonotic bacterial disease caused by Leptospira spirochetes, with limited therapeutic options, symptoms ranging from mild flu-like illness to severe organ failure and death. It presents a broad clinical spectrum, complicating diagnosis and treatment. This study targets Leptospira thiamine monophosphate kinase (ThiL), an essential enzyme conserved across the pathogenic species of Leptospira that is crucial for bacterial survival, with no known human homolog, making it a promising and selective therapeutic candidate for drug development. This study aims to discover effective inhibitors of Leptospira ThiL using Structure-Based Virtual Screening (SBVS). Potential hits were evaluated for drug-like properties, followed by Density Functional Theory (DFT) calculations to assess electronic structure properties. Further molecular dynamics simulations and binding free energy calculations were performed using the MM/PBSA approach to confirm the stability and affinity of the inhibitor. High-throughput Virtual Screening (HTVS) of phytochemicals revealed five promising candidates, namely, IMPHY004345, IMPHY005869, IMPHY006284, IMPHY002964, and IMPHY005688, exhibiting better docking scores (-12.36 to -10.54 kcal/mol) and strong MM/GBSA binding energies (-47.26 to -40.72 kcal/mol), along with optimal pharmacokinetic profiles. DFT analysis assessed the electronic properties of these compounds, providing insights into their chemical reactivity. MD simulations demonstrated stable binding and persistent hydrogen-bond interactions in the ThiL-ligand complexes. The conformational stability was monitored through MD-based distance plot analysis, revealing sustained interactions with catalytically significant residues (Glu9, Gln23, Asp39, Arg140, Thr209 Lys218) across all pathogenic Leptospira species, underscoring ThiL's evolutionary and functional importance. MM/PBSA calculations also support the high-affinity binding, with key residues emerging as crucial for maintaining complex stability and contributing to energy. This study establishes ThiL as a structurally stable, evolutionarily conserved, and highly druggable target in Leptospira. The identified leads, IMPHY006284 and IMPHY004345, emerged as the most potent broad-spectrum ThiL inhibitors, exhibiting multi-target inhibition across pathogenic species. This offers a promising strategy to overcome strain-specific variability and deliver broad-spectrum therapeutics for leptospirosis management.