In the current scenario, the canine visceral Leishmaniosis that causes approximately 20,000 deaths, annual incidence of 300,000 new cases and 1 billion of people at risk of infection drives the major concern around the glove for control of this disease
(Hailu et al., 2016). Although various chemotherapeutics have been trailed against this disease, but all compounds cause serious side effects, such as renal, pancreatic and hepatic toxicity, teratogenicity, cardiac as well as gastrointestinal problems
(Copeland and Aronson, 2015). Further, a single FDI approved drug miltefosine which has been used since 2014 against this disease but it has been facing lot of difficulties in its implementation due to problems of affordability and accessibility
(Sunyoto et al., 2018). It has also been reported that the trypanothione synthetase has vital role in the survival and growth of protozoa as this enzyme mediates the final step of tryptophan synthesis. Moreover, it was reported that humans do not have tryptophan synthase, so this enzyme was explored as a potential drug target against
leishmania infantum (Chaudhary and Roos, 2005). Although the binding interaction studies, targeting different enzymes of
leishmania infantum such as dihydrofolate reductase, thymidylate synthase
(Vadloori et al. 2018) Glutamyl cysteine Synthetase
(Agnihotri et al., 2017), Inositol phosphoryl ceramide synthase
(Norcliffe et al., 2018) with natural inhibitors have been reported in previous finding, but here authors focused on the discovery of potential natural compounds against thetryptophan synthase enzyme. As the crystal structure of
leishmania infantum trypanothione synthetase (LiTs) was not avaible, the primary sequence analysis resulted a conserved domain
i.
e CHAP domain present between 109
th to 257
th amino acid which mediates the utilization of catalytic cysteine residue in a nucleophilic-attack mechanism during tryptophan synthesis
(Bateman et al., 2003). To its support, the avaible crystal structure of
leishmania major was retrieved and utilized for generation of protein models.
The model structure of
Leishmania infantum trypanothione synthetase (LiTS) was generated using trypanothione synthetase of
Leishmania major (PDB ID: 2VPM chain: A) as template. Out of ten models, Model-9 with lowest DOPE and GA341 score of -19960.97 and 0.02942 was considered for best model shown in Table 1 and performed for further structural validation studies. Further the structural superimposition of LdTS model with template 2VPM-A chain both before and after energy minimization (Fig 1) revealed a Root Mean Square Deviation (RMSD) score of 0.112 Å. The RAMPAGE analysis of this model showed (88.5%, 7.1%, 4.3%) amino acid residues in the favored, allowed in the outlier region (Fig 2A). The ProSA-web analysis revealed a Z score of -5.19 (Fig 2B). The ProQ analysis resulted Levitt-Gerstein (LG) and Max sub score of 2.86 and 0.73.Further the ERRAT programme showed the overall quality value for LiTS model of
Leishmania infantum was 61.83% (Fig 2C). In addition, Verify3D plot of the modelled protein (Fig 2D) showed PASS and the 3D environment profile resulted 81.93% of the residues have averaged 3D-1D score~*0.2. The structural validation study suggested our model is best in terms of quality and stability.
Due to lack of crystal structure, the secondary structure of
Leishmania infantum trypanothione synthetase (LiTS) protein was predicted from its primary sequence using PSIPRED web server which showed 1 long, 3 medium, 2 short helical regions and 3 medium, 11 short β-sheets within the structure of LiTS protein (Fig 3).
Few drugs such as pentavalent antimonial derivatives, sodium stibogluconate, paromomycin, pentamidine, miltefosine and amphotericin-B showed promising effect against this protozoa, but all these drugs showed potent toxic with serious side effects and also exhibit drug resistance
(Oh et al., 2014). These drugs also cause severe adverse reactions such as reversible peripheral neuropathy; pancreatitis, nephrotoxicity cardiotoxicity, pancytopenia, myalgia and bone pain
(Croft et al., 2006).
This study has been able to find the binding efficiency of selected phytochemicals (10-hydroxycamptothecin, Theaflavin, Hecogenin acetate, glycyrrhizic acid, convallatoxin, tubocurarine, cafestol, mundulone, pomiferin, catechin) against tryptophan synthase of the protozoa. The separate binding interaction study between selected natural inhibitors and antiprotozoal drug with LiTS protein was done to assay the better therapeutic agent against
L.
infantum based upon their free binding energy and the inhibition constant of each binding complex which was reported in Table 2.
The result showed that among all selected inhibitor, Glycyrrhetic acid (-7.34 kcal/mol; KI: 4.18uM), Theaflavin (-6.95 kcal/mol; KI: 8.04uM) showed best binding efficiency with LiTS protein. The binding interaction between LiTS with glycyrrhetic acid and Theaflavin are shown in surface and ribbon structure presented in Fig 4 A, B, C and D respectively.
In this study it was showed that among natural inhibitor, glycyrrhetic acid and Theaflavin showed highest binding affinity against LiTs which is a good agreement with the findings of
Venkatesan et al., 2011 and It has been reported that Glycyrrhetic acid which is a pentacyclic triterpenoid aglycone, a product derived from the plant
Glycyrrhiza glabra showed tremendous antiparasitic activity by activation through nitric oxide (NO) upregulation, proinflammatory cytokine expression and NF-κB activation through p38 kinase
(Gupta et al., 2015). Similarly theaflavin is a class of natural flavonoids derived from the dried leaves of the plant
Camellia sinensis (tea) and related plants with potent antioxidant properties. The antiprozoal activity of theaflavin might be due to inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway previously reported by
Hui et al., 2016.
