Integrative computational and experimental approaches for identifying potent antimalarials by targeting falcipain-2 of <i>Plasmodium falciparum</i>

Malaria remains a critical global health issue, particularly in tropical and subtropical regions. Understanding its biology and epidemiology is vital for developing effective prevention and control strategies. Falcipain-2 (FP-2), a cysteine protease in <i>Plasmodium falciparum</i>, the deadliest malaria parasite, is essential for parasite survival in red blood cells, making it an attractive drug target. Inhibiting FP-2 disrupts key metabolic processes, leading to parasite death, offering a promising antimalarial drug development avenue. This study utilized computational approaches to design novel antimalarials. We prepared large fragment libraries, Enamine (∼220,174 fragments) and ChemDiv (∼18,713 fragments), for virtual screening against FP-2 (PDB ID: 6JW9). Fragments with binding free energies ≤ −4.0 kcal/mol were selected and evolved into drug-like ligands. SP and XP docking-based screenings prioritized these ligands, refined further using MM-GBSA calculations. Promising ligands underwent 100 ns molecular dynamics simulations to assess conformational changes and complex stability. This study identified two viable inhibitors, <b>T4</b> and <b>A3</b>, with high affinity, stability, and selectivity towards FP-2 compared to E64.Following computational studies, compound <b>A3</b> and its ten analogues (<b>KA-series</b>) were synthesized and characterized by using multi-spectroscopic techniques with high purity confirmed by LC-MS. Biological testing against <i>P. falciparum</i> 3D7 strain revealed <b>KA-5</b> as the most potent compound with an IC₅₀ value of <b>3.0 μM</b>. Future work will involve synthesizing additional analogue series for an extensive structure-activity relationship (SAR) study. Further experimental validation is essential to develop these compounds as effective therapeutic agents for malaria treatment.