Isern_2025_Chembiochem__e202500160

Leishmaniasis, a vector borne neglected tropical disease, caused by the protozoan parasite Leishmania, is a major global public health challenge with millions of new cases annually. Treatment of leishmaniasis is difficult for many reasons including a multiple life-cycle stages, involving both an infective insect vector form, the promastigote, and a disease causing intracellular mammalian host form, the amastigote, and increasing drug-tolerance that are all linked by the interplay between parasite and host. We employed activity-based protein profiling (ABPP) using new cell-permeable fluorophosphonate probes to explore serine hydrolases (SHs) in Leishmania mexicana with subsequent analysis enabled by secondary reaction with an affinity reagent. Importantly, these cell-permeable probes can access all life-cycle stages including the disease-critical intramacrophage amastigote. Probe efficacy is a combination of both target engagement and subsequent accessibility to the affinity agent. Fourteen SHs, including peptidases and lipases, were identified in the L. mexicana proteome with comparative profiling of different parasite life-stages revealing significant changes in SH activity across the life-cycle stages. This intra-cellular ABPP approach provides insights into the host-parasite interactome demonstrating that SHs function as important virulence factors with Z-Pro-Prolinal, a known prolyloligopeptidase inhibitor, being able to reduce parasite infectivity in the macrophage by altering multiple SH targets.