Antiprotozoal activity of 6-aminonicotinamide against Leishmania infantum promastigotes

Background Leishmaniasis (L) is one of the most important vector-borne human diseases, transmitted via infected female sandfly bite. Leishmania (L) protozoan parasites transmission is zoonotic, with dogs as main reservoir, but anthroponotic in the most hit Asia regions. In vertebrates L lives in macrophages. Diverse L species mainly cause visceral, cutaneous and mucocutaneous L,L. infantum visceral L in the Mediterranean region and in Latin America, (called there L. chagasi). Chemotherapy is fundamental since vaccines are available only for dogs, with limited efficacy. Drugs are lipid-associated amphotericin B, antimonials, pentamidine, paramomycin, miltefosine and few other compounds like ketoconazole for cutaneous L. Except for liposomal amphotericin B, combination therapy is recommended to avoid drug-resistance, as happened in India for antimonials, and to reduce side toxic effects presented by all these drugs. New compounds or drug repurposing are welcome. 6-aminonicotinamide (6AN) is an antimetabolite shown in some cell types to inhibit pentose phosphate pathway (PPP). It was initially proposed for cancer chemotherapy and psoriasis. Materials/methods L. infantum strain MO1 was cultured at 25 °C in RPMI containing 15% FBS and penicillin/streptomycin. 6AN (Sigma) solved in DMSO, aliquoted at -20°C, was diluted in RPMI as other compounds before use. Viable cells were counted by either hemocytometer or Alamar Blue fluorescence. For oxidative stress resistance evaluation, 6AN treated and control cells were diluted to 6 x 104 cells/mL and challenged 45 min with H2O2. Surviving parasites were counted using trypan blue and hemocytometer. 6AN treated and control cells were washed with PBS and lysed in 50 mM Tris, pH 7.5, 100 mM NaCl, 1% Triton X100, 5 mM EDTA and other protease inhibitors by freeze-thaw and sonication. 6-phosphogluconate dehydrogenase (6PGD) was assayed at 340 nm in presence of 6PG and NADP by microplate reader and normalized for the cells number. Treatments were in triplicate and in several independent experiments. Results • 6AN at 100 M inhibits cells growth of 40% after 3 days-treatment and of 33% after 2 days at 200 M, similarly to 30 M antimony potassium tartrate. In the same experiment miltefosine, used as control at 50 M for 2 days, was highly toxic completely killing the cells. • Oxidative challenge with 100 M H2O2 reduced of 10% control cells number while cells survived to 6AN treatment, decreased of a further 40% (graph). • Cells treated with 6AN for 3 days, showed a reduction of 75% (compared to control cells) in activity of 6PGD, the 3rd enzyme of PPP already shown to be the main target of 6AN in human cells. Conclusion 6AN, a drug already used in clinics for other purposes, is able to kill L. infantum promastigotes, by decreasing its redox power supply, fundamental to fight oxidative stress to which the parasite is exposed.