Thianthrene is a novel inhibitor of Leishmania donovani pteridine reductase 1 (PTR1)

Pteridine reductase 1 (PTR1) from Leishmania donovani is a short chain reductase that catalyses the NADPH-dependent reduction of folates and pterins. It has gained attention as a therapeutic target because it acts as a metabolic bypass for dihydrofolate reductase (DHFR) targeting drugs and is thought to be responsible for the failure of conventional therapies against the trypanosomatids. In the present study, we report the identification of thianthrene as a potent inhibitor of L. donovani PTR1 (LdPTR1) based on both structure-based virtual screening and experimental verification. Thianthrene displayed uncompetitive mixed type inhibition in a recombinant enzyme inhibition assay. In addition, cell based assays and flow cytometry showed that the intracellular amastigotes were inhibited by thianthrene in vitro. The results of our study could be considered for the development of novel therapeutics based on PTR1 inhibition

Degradation of pteridine reductase 1 (PTR1) enzyme during growth phase in the protozoan parasite Leishmania donovani

Pteridine reductase 1 (PTR1) is an essential enzyme of pterin and folate metabolism in the protozoan parasite Leishmania. The present work is focused on the degradation of PTR1 during growth phase in Leishmania donovani. Western blot analysis with PTR1-GFP transfected promastigotes revealed that PTR1 protein was degraded in the stationary phase of growth at the time when the parasites were undergoing metacyclogenesis. Fluorescence microscopy revealed cytoplasmic localization of GFP tagged protein extending to the flagellum in these stationary phase promastigotes, implying that degradation of the protein was not by the usual multivesicular tubule lysosome (MVT) pathway. A probable destruction box of nine amino acids Q63ADLSNVAK71 and possible lysine residue K156 was identified in L. donovani PTR1 to be the site for ubiquitin conjugation. This suggests that PTR1 degradation during the stationary phase of growth is mediated by the proteasome. This leads to lower levels of H4-biopterin, which favors metacyclogenesis, and subsequently results in a highly infective stage of the parasite. Therefore, this finding has importance to identify new target molecule like the proteasome for therapeutic intervention

Novel mechanism of drug resistance in kala azar field isolates

Clinical resistance to pentavalent antimonial drugs in the form of sodium antimony gluconate (SAG) has become a major problem in the treatment of kala azar (visceral leishmaniasis) in India. The mechanism of resistance is unclear in these clinical isolates, although work has been conducted with Leishmania species mutants selected in vitro by stepwise increase of drug concentration, using antimony-related metal arsenic and, more recently, SAG. In the present study, we investigated the molecular aspect of drug resistance in clinically confirmed SAG-resistant field isolates. Our results show that the mechanisms of resistance postulated for laboratory mutants of Leishmania species are not operating in field isolates of Leishmania donovani. Instead, we identified a novel gene amplified in these drug-resistant parasites whose locus is on chromosome 9. The significant finding was that this isolated fragment confers antimony resistance to wild-type Leishmania species after transfection. We speculate that protein phosphorylation may play a role in signal transduction pathway in the parasite after exposure to drug-conferring resistance

Refractoriness to the treatment of sodium stibogluconate in Indian kala-azar field isolates persist in in vitro and in vivo experimental models

Ever since their discovery about 60 years ago as therapeutic agent for visceral leishmaniasis (VL) or kala-azar, pentavalent antimonials (Sbv) have remained the first line treatment of choice all over the world including India. But recently, the number of kala-azar patients unresponsive to sodium stibogluconate (SSG) therapy, is steadily increasing in India. In this study, three clinical isolates, of which two were from SSG unresponsive and one from SSG responsive patients were evaluated for their infectivity and for their chemotherapeutic responses in vitro (macrophage–amastigote system) and in vivo (in hamsters). Persistence of SSG resistance was also checked by repeated passages in vitro as well as in vivo. The drug resistant strains (2039 and 2041) did not respond to SSG therapy both in vitro as well as in vivo but strains 2001 and Dd8 showed full sensitivity to SSG treatment. All the four strains responded well to amphotericin B and miltefosine treatment both in macrophages and in hamsters. The specific chemotherapeutic responses of all the strains to SSG were consistently persistent after repeated passages in cultures and in vivo, which indicates that these isolates are truly refractory to SSG treatment in field conditions. Two isolates were also transfected with green fluorescent protein (GFP) for the development of in vitro assay for studying antileishmanial activities of new and reference drugs in macrophages by flow cytometry

Molecular docking, structure-activity relationship and biological evaluation of the anticancer drug monastrol as a pteridine reductase inhibitor in a clinical isolate of Leishmania donovani

Objectives: Using the pteridine reductase (PTR1) enzyme of Leishmania as the target, the objective of our study was to find a drug candidate that can enter the clinical development process after being evaluated for safety and efficacy in animals. Methods: Monastrol (R) and (S) enantiomers were docked using the QUANTUM program into the active site of a Leishmania donovani PTR1 (LdPTR1) homology model. A structure-activity relationship based on a homology model of a recombinant enzyme was substantiated by a recombinant enzyme inhibition assay. We adapted an L. donovani (transfected with green fluorescent protein) intramacrophage amastigote screening assay as a cellular model for leishmaniasis. Furthermore, since the clinicopathological features and immunopathological mechanisms of visceral leishmaniasis (VL) in a hamster model are remarkably similar to those of human disease, systemic infection of hamsters with L. donovani was utilized to collect in vivo data for monastrol. Results: Both monastrol (R) and (S) enantiomers fit well in the ligand-binding pocket of LdPTR1. Monastrol exhibits a Ki value of 0.428 μM in the recombinant enzyme inhibition assay. We confirm monastrol as a potent inhibitor of PTR1 in Leishmania; it inhibits proliferation of amastigotes with an IC50 (50% inhibitory concentration) of 10 μM in macrophage cultures infected with an L. donovani clinical isolate, with no host cytotoxicity. We also show that in experimental animals, oral administration of a 5 mg/kg dose of monastrol on two alternate days inhibits 50% of parasite growth, giving therapeutic backing to the use of monastrol as a potent antileishmanial in human VL cases. Conclusions: To our knowledge, this is the first report presenting monastrol as a potent oral antileishmanial