Appraisal of Na+, K+-ATPase and Related Ion Antiporter Functions as Potential Molecular Targets for New Antileishmanial Drug Discovery

Visceral leishmaniasis, caused by infection with a protozoan parasite Leishmania donovani, is a prominent disease in certain tropical regions of the world. Extreme toxicity, increasing resistance of the parasite to currently available drugs and the absence of a suitable vaccine necessitate the discovery of new anti-leishmanial drugs with defined molecular targets for action, better efficacy and safety profiles. Random screening of a library of natural products indicated Na⁺,K⁺-ATPase or related ion transport functions as potential antileishmanial drug targets. The Na⁺-K⁺-ATPases and other related antiporter enzymes are important tools for the maintenance of intracellular ion homeostasis and proton pump functions, which may be essential for survival of Leishmania amastigotes in acidic environment of phagolysomes of mammalian macrophages. To validate this hypothesis, a few selected standard drugs/inhibitors, namely, N-ethylmaleimide, omeprazole, ouabain, amiloride, bufalin, quercetin, oleandrine, digoxin, mansonin, sanguinarine, cinobufagin and procillaridin A, which are known to inhibit Na⁺,K⁺-ATPases or proton pump functions through distinct molecular mechanisms, were evaluated in vitro against different cellular forms of L. donovani namely, promastigotes, axenic amastigotes and intracellular amastigotes growing within terminally differentiated THP1 human acute monocytes cells. The inhibitors were simultaneously tested for cytotoxicity against differentiated THP1 cells. Except omeprazole (inhibitor of H⁺/K⁺-ATPase) and amiloride (selective inhibitor of epithelial sodium channel), all inhibitors showed highly potent and selective anti-leihsmanial effect on intracellular amastigotes, with no significant effect on growth & proliferation of promastigotes and axenic amastigotes. Bufalin, a cardiotonic steroid and inhibitor of Na⁺,K⁺-ATPase, was the most active inhibitor with IC₅₀ value in sub-nanomolar range. Selective action of inhibitors indicate essential role of Na⁺,K⁺-ATPase and related proton transport functions in survival and proliferation of intracellular L. donovani amastigotes. Further analysis of selective susceptibility of intracellular amastigotes, molecular targets for action of specific inhibitors and molecular/functional characteristics of leishmanial Na⁺,K⁺/H⁺-ATPase vis-à-vis their role in leishmania growth and survival would be useful in identification of selective inhibitors, devoid of cardiotonic functions as potential novel antileishmanial drugs. Acknowledgements: The US Army Medical Research & Material Command CDMRP grant No. W81XWH-09 – 2-0093 and the USDA Agricultural Research Service Specific Cooperative Agreement No. 58 – 6408 – 02 – 1-612.