Mianserin, an antidepressant kills Leishmania donovani by depleting ergosterol levels.

In the present study, we have investigated the antileishmanial potential of mianserin, an antidepressant. Mianserin was found to inhibit both the promastigote and amastigote forms of the parasite in a dose dependant manner. The IC50 values for promastigotes and amastigotes were 21 µM and 46 µM respectively. Interestingly, mianserin failed to inhibit THP-1 differentiated macrophages up to 100 µM concentration thus, exhibiting parasite selectivity. When mianserin was incubated with recombinant Leishmania donovani 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme, it exhibited an IC50 value of 19.8 µM. Inhibition kinetics revealed competitive mode of enzyme inhibition as the Km increased with no change in Vmax. Further structural investigation of enzyme-inhibitor interaction revealed quenching of HMGR tryptophan intrinsic fluorescence with a K(sv) value of 3.025±0.37 M(-1) and an apparent binding constant of 0.0954 mM. We further estimated ergosterol levels which is a major component of Leishmania cell membrane. It is synthesized by HMGR enzyme, the first rate limiting enzyme of the sterol biosynthetic pathway. Analysis of ergosterol levels by HPLC revealed ∼2.5-fold depletion in mianserin treated promastigotes with respect to untreated parasites. This data was further validated by exogenous supplementation of mianserin treated cells with ergosterol and cholesterol. Reversal of growth inhibition was observed only upon ergosterol addition though it was refractory to cholesterol supplementation. Overall, our results demonstrate the possibility of repositioning of an antidepressant for the treatment of Visceral Leishmaniasis.

AMP-acetyl CoA synthetase from Leishmania donovani: identification and functional analysis of 'PX4GK' motif.

An adenosine monophosphate forming acetyl CoA synthetase (AceCS) which is the key enzyme involved in the conversion of acetate to acetyl CoA has been identified from Leishmania donovani for the first time. Sequence analysis of L. donovani AceCS (LdAceCS) revealed the presence of a 'PX4GK' motif which is highly conserved throughout organisms with higher sequence identity (96%) to lower sequence identity (38%). A ∼ 77 kDa heterologous protein with C-terminal 6X His-tag was expressed in Escherichia coli. Expression of LdAceCS in promastigotes was confirmed by western blot and RT-PCR analysis. Immunolocalization studies revealed that it is a cytosolic protein. We also report the kinetic characterization of recombinant LdAceCS with acetate, adenosine 5'-triphosphate, coenzyme A and propionate as substrates. Site directed mutagenesis of residues in conserved PX4GK motif of LdAceCS was performed to gain insight into its potential role in substrate binding, catalysis and its role in maintaining structural integrity of the protein. P646A, G651A and K652R exhibited more than 90% loss in activity signifying its indispensible role in the enzyme activity. Substitution of other residues in this motif resulted in altered substrate specificity and catalysis. However, none of them had any role in modulation of the secondary structure of the protein except G651A mutant.