Abstract
MAPK3 has been identified as a potential drug target due to its role in the Leishmania life cycle. This study focused on two species prevalent in Thailand: Leishmania martiniquensis and Leishmania orientalis. Using an in silico strategies, we screened an in-house library of 8000 tripeptides to identify potential MAPK3 inhibitors. A multi-level computational workflow was employed, including homology modeling, molecular docking study, and molecular dynamics (MD) simulations. Promising candidates were further validated by an in vitro kinase assay. The three-dimensional (3D) structures of MAPK3 from L.martiniquensis and L. orientalis were constructed by using the Swiss Model. Structure-based virtual screening identified nine promising tripeptides, with WSY demonstrated strong binding affinity, MD simulation and MM-GBSA binding free energy calculation confirmed its stable interaction with key regions in the MAPK3 active site, including the DFG motif, hinge region, αC-helix through hydrogen bonds, hydrophobic interaction, and π-π stacking. In vitro kinase assays demonstrated that the peptide WSY was significantly more effective at inhibiting MAPK3 than WHW, with IC(50) values of 0.9 ± 11.3 and 26.9 ± 6.7 μM, respectively. These findings suggest that WSY is a promising anti-leishmanial drug candidate targeting LmqMAPK3 and LoMAPK3. Our hybrid computational-experimental approach provides a solid framework for identification of novel inhibitors and can be extended to other compound libraries to help the discovery of new anti-leishmania therapies.