A chemical inhibitor of heat shock protein 78 (HSP78) from Leishmania donovani represents a potential antileishmanial drug candidate

Abstract

The emergence of resistance to available antileishmanial drugs advocates identification of new drug targets and their inhibitors for visceral leishmaniasis. Here, we identified <i>Leishmania donovani</i> heat shock protein 78 (LdHSP78), a putative caseinolytic protease, as important for parasite infection of host macrophages and a potential therapeutic target. Enrichment of LdHSP78 in infected humans, hamsters, and parasite amastigotes suggested its importance for disease persistence. Heterozygous knockouts of <i>L. donovani</i> HSP78 (<i>LdHSP78</i>+/-) and <i>Leishmania mexicana</i> HSP78 (<i>LmxHSP78</i>+/-) were generated using a flanking UTR-based multifragment ligation strategy and the CRISPR-Cas9 technique, respectively to investigate the significance of HSP78 for disease manifestation. The <i>LdHSP78</i>+/- parasite burden was dramatically reduced in both murine bone marrow-derived macrophages and hamsters, in association with enrichment of proinflammatory cytokines and NO. This finding implies that <i>LdHSP78</i>+/- parasites cannot suppress immune activation and escape NO-mediated toxicity in macrophages. Furthermore, phosphorylation of the mitogen-activated protein kinase p38 was enhanced and phosphorylation of extracellular signal-regulated kinase 1/2 was decreased in cells infected with <i>LdHSP78</i>+/- parasites, compared with WT parasites. Virulence of the <i>LdHSP78</i>+/- strain was restored by episomal addition of the <i>LdHSP78</i> gene. Finally, using high-throughput virtual screening, we identified <i>P</i>¹,<i>P</i>⁵-di(adenosine-5')-pentaphosphate (Ap5A) ammonium salt as an LdHSP78 inhibitor. It selectively induced amastigote death at doses similar to amphotericin B doses, while exhibiting much less cytotoxicity to healthy macrophages than amphotericin B. In summary, using both a genetic knockout approach and pharmacological inhibition, we establish LdHSP78 as a drug target and Ap5A as a potential lead for improved antileishmanial agents.