High content analysis of macrophage‐targeting ehpib‐compounds against cutaneous and visceral leishmania species

An immunostimulatory glycolipid molecule from the intestinal protozoan parasite Enta-moeba histolytica (Eh) and its synthetic analogs derived from its phosphatidylinositol‐b‐anchor (EhPIb) previously showed considerable immunotherapeutic effects against Leishmania major infection in vitro and in vivo. Here, we describe a high content screening assay, based on primary murine macrophages. Parasites detection is based on a 90 kDA heat shock protein‐specific staining, enabling the detection of several Leishmania species. We validated the assay using L. major, L. braziliensis, L. donovani, and L. infantum as well as investigated the anti‐leishmanial activity of six immunostimulatory EhPIb‐compounds (Eh‐1 to Eh‐6). Macrophages infected with dermotropic species were more sensitive towards treatment with the compounds as their viability showed a stronger reduction compared to macrophages infected with viscerotropic species. Most compounds caused a significant reduction of the infection rates and the parasite burdens depending on the infecting species. Only compound Eh‐6 was found to have activity against all Leishmania species. Considering the challenges in anti‐leishmanial drug discovery, we developed a multi‐species screening assay capable of utilizing non‐recombinant parasite strains, and demonstrated its usefulness by screening macrophage‐targeting EhPIb‐compounds showing their potential for the treatment of cutaneous and visceral leishmaniasis.