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

Sonali Das, Anindyajit Banerjee, Mohd Kamran, Sarfaraz Ahmad Ejazi, Mohammad Asad, Nahid Ali, Saikat Chakrabarti
Journal of Biological Chemistry 2020 May 29
Emergence of resistance to available anti-leishmanial drugs advocates identification of new drug targets and their inhibitors for visceral leishmaniasis. Here, we identified heat shock protein 78 in Leishmania donovani (LdHSP78), a putative ClpB 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 L. donovani ( LdHSP78+/- ) and L. mexicana ( LmxHSP78+/- ) were generated using flanking untranslated region (UTR) based multi-fragment ligation strategy and CRISPR-Cas9 technique, respectively to investigate the significance of HSP78 for disease manifestation. LdHSP78+/- parasite burden was dramatically reduced in both murine bone marrow-derived macrophages and hamsters, associated with enrichment of pro-inflammatory cytokines and nitric oxide (NO). This finding implies that LdHSP78+/- parasites cannot suppress immune activation and escape NO-mediated toxicity in macrophages. Further, phosphorylation of the mitogen-activated protein kinase (MAPK) p38 was enhanced, and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) was decreased in cells infected with LdHSP78+/- compared to wildtype (WT) infection. Virulence of the LdHSP78+/- strain was restored by episomal addition of LdHSP78 gene. Finally, using high-throughput virtual screening, we identified P1,P5-di(adenosine-5')-penta-phosphate ammonium salt (Ap5A) as an LdHSP78 inhibitor. It selectively induced amastigote death at doses similar to amphotericin B (AmB) dosing, while exhibiting much less cytotoxicity toward healthy macrophages than AmB. 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 anti-leishmanial agents.

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