Clotrimazole causes membrane depolarization and induces sub G0 cell cycle arrest in Leishmania donovani.

Clotrimazole is an FDA approved drug and is widely used as an antifungal agent. An extensive body of research is available about its mechanism of action on various cell types but its mode of killing of Leishmania donovani parasites is unknown. L. donovani causes Visceral Leishmaniasis which is a public health problem with limited treatment options. Its present chemotherapy is expensive, has adverse effects and is plagued with drug resistance issues. In this study we have explored the possibility of repurposing clotrimazole as an antileishmanial drug. We have assessed its efficacy on the parasites and attempted to understand its mode of action. We found that it has a half-maximal inhibitory concentration (IC50) of 35.75 ± 1.06 µM, 12.75 ± 0.35 µM and 73 ± 1.41 µM in promastigotes, intracellular amastigotes and macrophages, respectively. Clotrimazole is 5.73 times more selective for the intracellular amastigotes as compared to the mammalian cell. Effect of clotrimazole was reduced by ergosterol supplementation. It leads to impaired parasite morphology. It alters plasma membrane permeability and disrupts plasma membrane potential. Mitochondrial function is compromised as is evident from increased ROS generation, depolarized mitochondrial membrane and decreased ATP levels. Cell cycle analysis of clotrimazole treated parasites shows arrest at sub-G0 phase suggesting apoptotic mode of cell death.

Pyridoxal kinase gene deletion leads to impaired growth, deranged redox metabolism and cell cycle arrest in Leishmania donovani.

Pyridoxal kinase (PdxK) is a vitamin B6 salvage pathway enzyme which produces pyridoxal phosphate. We have investigated the impact of PdxK deletion in Leishmania donovani on parasite survivability, infectivity and cellular metabolism. LdPdxK mutants were generated by gene replacement strategy. All mutants showed significant reduction in growth in comparison to wild type. For PdxK mediated biochemical perturbations, only heterozygous mutants and complementation mutants were used as the growth of null mutants were compromised. Heterozygous mutant showed reduction invitro infectivity and higher cytosolic and mitochondrial ROS levels. Glutathione levels decreased significantly in heterozygous mutant indicating its involvement in cellular oxidative metabolism. Pyridoxal kinase gene deletion resulted in reduced ATP levels in parasites and arrest at G0/G1 phase of cell cycle. All these perturbations were rescued by PdxK gene complementation. This is the first report to confirm that LdPdxK plays an indispensable role in cell survival, pathogenicity, redox metabolism and cell cycle progression of L. donovani parasites. These results provide substantial evidence supporting PdxK as a therapeutic target for the development of specific antileishmanial drug candidates.

Mechanistic and structural insights into vitamin B2 metabolizing enzyme riboflavin kinase from Leishmania donovani.

Leishmania donovani relies on specific vitamins and cofactors crucial for its survival and pathogenesis. Tailoring therapies to disrupt these pathways offers a promising strategy for the treatment of Visceral Leishmaniasis. Current treatment regimens are limited due to drug resistance and high costs. The dependency of Leishmania parasites on Vitamin B2 and its metabolic products is not known. In this study, we have biochemically and biophysically characterized a Vitamin B2 metabolism enzyme, riboflavin kinase from L. donovani (LdRFK) which converts riboflavin (vitamin B2) into flavin mononucleotide (FMN). Sequence comparison with human counterpart reflects 31.58 % identity only, thus opening up the possibility of exploring it as drug target. The rfk gene was cloned, expressed and the recombinant protein was purified. Kinetic parameters of LdRFK were evaluated with riboflavin and ATP as substrates which showed differential binding affinity when compared with the human RFK enzyme. Thermal and denaturant stability of the enzyme was evaluated. The rfk gene was overexpressed in the parasites and its role in growth and cell cycle was evaluated. In the absence of crystal structure, homology modelling and molecular dynamic simulation studies were performed to predict LdRFK structure. The data shows differences in substrate binding between human and parasite enzyme. This raises the possibility of exploring LdRFK for specific designing of antileishmanial molecules. Gene disruption studies can further validate its candidature as antileishmanial target.