Profiling Serine Hydrolases in the Leishmania Host–Pathogen Interactome Using Cell‐Permeable Activity‐Based Fluorophosphonate Probes

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 multiple lifecycle 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. Activity‐based protein profiling (ABPP) was employed 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 are capable of accessing all lifecycle 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 lifecycle stages. This intracellular ABPP approach provides insights into the host–parasite interactome demonstrating that SHs function as important virulence factors with Z‐Pro‐Prolinal, a known prolyl‐oligopeptidase inhibitor, being able to reduce parasite infectivity in the macrophage by altering multiple SH targets.