Dataset Information

Computational study to discover potent phytochemical inhibitors against drug target, squalene synthase from Leishmania donovani.

ABSTRACT:

Aims

The parasite, Leishmania donovani is responsible for lethal visceral leishmaniasis (VL) in humans. There is a need to investigate novel medicines as antileishmanial drugs, as medication currently introduced for leishmaniasis may cause resistance, serious side-effects, chemical instability and high cost. Therefore, this computational study was designed to explore potential phytochemical inhibitors against Leishmania donovani squalene synthase (LdSQS) enzyme, a drug target.

Main methods

Multiple sequence alignment was carried to detect conserved regions across squalene synthases from different Leishmania spp. Their evolutionary relationships were studied by generating phylogenetic tree. Homology modeling method was used to build a three dimensional model of the protein. The validated model was explored by docking simulation with the phytochemicals of interest to identify the most potent inhibitors. Two reported inhibitors were used as references in the virtual screening. The top hit compounds (binding energy less than -9 kcal/mol) were further subjected to intermolecular interaction analysis, pharmacophore modeling, pharmacokinetic and toxicity prediction.

Key findings

Seven phytochemicals displayed binding energies less than -9 kcal/mol hence demonstrating ability to be strongly bound to the active site of LdSQS to inhibit the enzymatic activity. Ancistrotanzanine B demonstrated the lowest binding affinity of -9.83 kcal/mol superior to reported inhibitors in literature. Conserved two aspartate rich regions and two signatory motifs were found in the L. donovani squalene synthase by multiple sequence alignment. In addition, study of pharmacophore modeling confirmed that top hit phytochemicals and the reported inhibitor (E5700) share common chemical features for their biochemical interaction with LdSQS. Among seven phytochemicals, 3-O-methyldiplacol showed admissible physicochemical, pharmacokinetic and toxicity predictions compared to the reported inhibitors. All seven phytochemicals satisfied in silico prediction criteria for oral bioavailability.

Significance

Based on the current study, these hits can be further structurally optimized and validated under laboratory conditions to develop antileishmanial drugs.

SUBMITTER: Wadanambi PM

PROVIDER: S-EPMC8188062 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundGene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo.Methodology/principal findingsTwo parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.Conclusion/significanceOur study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target.