Ketanserin, an antidepressant, exerts its antileishmanial action via inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme of Leishmania donovani

Abstract

Leishmaniasis is one of the major health problems existing globally. The current chemotherapy for leishmaniasis presents several drawbacks like toxicity and increased resistance to existing drugs, and hence, there is a necessity to look out for the novel drug targets and new chemical entities. Current trend in drug discovery arena is the "repurposing" of old drugs for the treatment of diseases. In the present study, an antidepressant, ketanserin, was found lethal to both Leishmania donovani promastigotes and intracellular amastigotes with no apparent toxicity to the cells. Ketanserin killed promastigotes and amastigotes with an IC50 value of 37 μM and 28 μM respectively, in a dose-dependent manner. Ketanserin was found to inhibit L. donovani recombinant 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme with an IC50 value of 43 μM. Ketanserin treated promastigotes were exogenously supplemented with sterols like ergosterol and cholesterol to rescue cell death. Ergosterol could recover the inhibition partially, whereas cholesterol supplementation completely failed to rescue the inhibited parasites. Further, HMGR-overexpressing parasites were generated by transfecting Leishmania promastigotes with an episomal pspα hygroα-HMGR construct. Wild-type and HMGR overexpressors of L. donovani were used to study the effect and mode of action of this inhibitor. The HMGR overexpressors showed twofold resistance to ketanserin. These observations suggest that the lethal effect of ketanserin is due to inhibition of HMGR, the rate-limiting enzyme of the ergosterol biosynthetic pathway. Since targeting of the sterol biosynthetic pathway enzymes may be useful therapeutically, the present study may have implications in treatment of leishmaniasis.

Antileishmanial effect of mevastatin is due to interference with sterol metabolism

“…The medium was maintained at pH 7.2. Promastigotes, transfected with pspα hygroα shuttle vector and pspα hygroα-LdHMGR episomal overexpression construct, were routinely maintained in 60 μg/mL hygromycin antibiotic as reported earlier (Singh et al 2014). These mutant parasites were used in all subsequent experiments.…”

“…Briefly, the reaction mixture contained 50 mM KH 2 PO 4 , 50 mM KCl, 5 mM DTT, 1 mM EDTA, 0.27 mM NADPH, 0.27 mM HMG-CoA, and recombinant enzyme in a final volume of 200 μL at pH 7.2. The HMG-CoA-dependent oxidation of NADPH was monitored at 340 nm (Singh et al 2014).…”

“…Neutral lipids were isolated and separated by high-pressure liquid chromatography (HPLC) as previously described (Singh et al 2014). Approximately 1 × 10 6 L. donovani promastigotes were incubated at 24°C for 48 h and treated with mevastatin for 48 h. Promastigotes were harvested, washed with phosphate-buffered saline (PBS), and resuspended in a mixture of methanol/chloroform/water and incubated on ice for 1 h. After centrifugation, the supernatant was collected and a mixture of water/chloroform (1:1 v/v) was added.…”

“…To determine the specificity of LdHMGR towards mevastatin, we had evaluated its effect on HMGR-overexpressing transgenic parasites which were generated by transfecting the pspα hygroα-LdHMGR construct into promastigotes as reported previously (Singh et al 2014). Wild-type promastigotes were taken as control.…”

“…However, recent studies on statins have been widely investigated for their clinical benefits in various diseases like colon, lung, kidney, bone cancer, and central nervous system, as well as diabetes (Liao 2005) apart from exhibiting immunomodulatory effects (Danesh et al 2003). We had previously demonstrated the antileishmanial potential of inhibitors belonging to statin class of compounds like atorvastatin, simvastatin, and antidepressants like ketanserin and mianserin via targeting HMGR enzyme (Dinesh et al 2014a, b;Singh et al 2014).…”