In silico screening, molecular dynamic simulations, and in vitro activity of selected natural compounds as an inhibitor of Leishmania donovani 3-mercaptopyruvate sulfurtransferase

Abstract

In Leishmania sp., the enzymes of de novo cysteine biosynthesis pathway require sulfide. Other organisms utilize sulfide through the sulfide reduction pathway, but Leishmania lacks the gene that encodes these enzymes. Hence, the major source of sulfide for Leishmania is believed to be from the action of 3-mercaptopyruvate sulfurtransferase (3MST) on 3-mercapto-pyruvate (3MP). There has been no effort reported in the past to screen inhibitors against L. donovani 3MST (Ld3MST). As a result, this study examines natural compounds that are potent against Ld3MST and validates it by in vitro activity and cytotoxicity tests. Initially, a library of ~ 5000 natural compounds was subjected to molecular docking approach for screening, and the best hit was validated using a long-term molecular dynamic simulation (MD). Among the docking results, quercetine-3-rutinoside (Rutin) was deemed the best hit. The results of the MD indicated that Rutin was highly capable of interacting with the varied active site segments, possibly blocking substrate access. Additionally, promastigotes and amastigotes were tested for Rutin activity and the IC50 was found to be 40.95 and 90.09 μM, respectively. Similarly, the cytotoxicity assay revealed that Rutin was not toxic even at a concentration of 819.00 μM to THP-1 cell lines. Additionally, the Ld3MST was cloned, purified, and evaluated for enzyme activity in the presence of Rutin. Reduction in the enzyme activity (~ 85%) was observed in the presence of ~ 40 μM Rutin. Thus, this study suggests that Rutin may act as a potent inhibitor of Ld3MST. With further in vivo investigations, Rutin could be a small molecule of choice for combating leishmaniasis.

Keywords: 3MST; Drug target; IC50; Inhibitor; Leishmania; Rutin.

Fig. 1

Cysteine biosynthesis pathway in Leishmania sp

Fig. 2

Structure of Ld3MST. Pink: 3-MP; blue: active site helix; green: active site sheet; brown: active site loops; and yellow: active site center residue cysteine 253

Fig. 3

Comparison of residues of Ld3MST interacting with A 3MP and B Rutin. Dotted lines: H-bond interactions. Spiked half circle: Hydrophobic interactions

Fig. 4

RMSD analysis of 100 ns trajectory. A Backbone RMSD of all systems. A1 RMSD density plot of unbound 3MST. A2 RMSD density plot of 3MST-3MP complex. A3 RMSD density plot of 3MST–Rutin complex. B All atoms RMSD of ligands. C RMSF of all residues of all the systems

Fig. 5

Radius of gyration (Rg) analysis of 100 ns trajectory. A Rg of all protein atoms. A1 Rg density plot of unbound 3MST. A2 Rg density plot of 3MST–3MP complex. A3 Rg density plot of 3MST–Rutin complex

Fig. 6

Solvent accessible surface area (SASA) analysis of 100 ns trajectory. A SASA of all protein atoms; A1 SASA density plot of unbound 3MST; A2 SASA density plot of 3MST–3MP complex. A3 SASA density plot of 3MST–Rutin complex. B SASA of active site. B1 SASA density plot of active site of unbound 3MST. B2 SASA density plot of active site of 3MST–3MP complex. B3 SASA density plot of active site of 3MST–Rutin complex

Fig. 7

A Distance of active site of Ld3MST with 3MP and Rutin. Number of hydrogen bonds of Ld3MST with B 3MP and C Rutin

Fig. 8

Antileishmanial activity and cytotoxicity of Rutin. A Cell viability MTT assay on promastigotes. B Cytotoxicity assay on THP-1. C Cell viability MTT assay on amastigotes. D Activity assay of recombinant Ld3MST on substrate, sodium thiosulfate. * p < 0.05; ** p < 0.01; *** p < 0.001; NS—non-significant; AmB—AmBisome as positive control; and NC—phosphate buffer solution as negative control; C—observation of cell morphology under microscope (× 40); C1—control untreated promastigote cells with PBS; C2—control untreated THP-1 cells; C3—control untreated amastigote cells with PBS; C4—enzyme activity of Ld3MST in the absence of Rutin. E1—control untreated; E2—treated with 40.95 μM Rutin; E3—treated with 327.6 μM Rutin