**3.15 Exploring possible implications and structure similarities of predicted leads**

Karatavicinol and Marmin had lower binding energies of 9.4 and 9.3 kcal/ mol, respectively, as compared to Colladonin (8.5 kcal/mol) and Pectachol (8.5 kcal/mol). These binding energies are closer to that of FAD (9.0 kcal/mol) for which can possibly compete in binding at the FAD domain. These compounds were concluded to have drug-likeness by satisfying Lipinski's rule of 5. They also do not pass the blood–brain barrier which is good. Also, Marmin and Karatavicinol checked false for p-glycoprotein substrate. This gives the compounds an advantage to maintain their concentrations in cellular level to maximize efficacy. Pectachol and Colladonin however were implicated as P-gp substrates. These predicted preferable properties can favor their lead likeness and chances of going a long way in experimental studies. The four lead compounds were predicted as antileishmanial compounds. The four leads are confirmed not to be already existing antileishmanial drugs by structural similarity searches in www.DrugBank.ca but rather observed to be analogues of chrome 2-one. In regard to this, studies over the years have however shown some novel compounds such as 7-{[(2R\*)-3,3-dimethyloxiran-2-yl] methoxy}-8-[(2R\*,3R\*)-3-isopropenyloxiran-2-yl]-2H-chromen-2-one and 7 methoxy-8-(4-methyl-3-furyl)-2H-chromen-2-one against *Leishmania donovani* with EC50 of 9.9 and 10.5 μg/mL, respectively [70]. These tested compounds with antileishmanial effect tend to be analogues of chromen-2-one. We emphasize that Karatavicinol is not a unique lead compound since it has already been experimented on other *Leishmania* species excluding *L. major* [57]. But the study identified it via these computational processes and therefore would report it as a potential compound against *L. major*. This augments the fact that the computational drug discovery pipeline has an optimistic potential of yielding good candidates for experimental work. Colladonin on the other hand is an enantiomer of Feselol for which Feselol is experimented as an antileishmanial agent [57]. Marmin also holds a very good potential of being an anti-ulcerative agent [71]. This favors it being a good


#### **Table 2.**

*The energy terms obtained after MM-PBSA analysis of the protein–ligand complexes.*

compound for the treatment of cutaneous leishmaniasis. These compounds classified are coumarins and more other studies have reported good antileishmanial activities from this class of compounds [72, 73]. This work supports the fact that Karatavicinol, Marmin, Pectachol and Colladonin may possibly exhibit good antileishmanial activity if tested *in vitro* (**Table A6**).

Further in this study, the interaction of the active site residues with all four lead compounds showed hydrogen bonding with Val34, Thr51, Lys60, Thr160, Ala159, Arg287, Thr293, Asp327, Asn330, Thr335, and Gly376 (**Table 1**). Superimposition of the docked 2JK6 and co-crystallized revealed common residues such as Ser14, Gly15, Arg287, and Thr335 (**Figure A1**). These residues can be observed to be unique to the FAD domain of *Lm*TR in anchoring the FAD molecule. Comparing these residues to the hydrogen bonding residues from the four leads shows that possible interruption of any of these residues can cause conformational changes which might not favor the selective binding of FAD at its domain. Baiocco et al. in 2009 identified Thr335 of trypanothione reductase at the FAD catalytic site of *L. infantum* [74]. They proposed that the FAD molecule binds tightly to the protein and orients itself towards the hydride transfer region of the active site by hydrogen bonding with specific residues Lys60, Thr335, and His461. Having observed this, interrupting one of these residues can potentially inhibit the reduction of T[S]2 by interfering with the hydride transfer. These compounds can potentially covey a competitive mode for binding to Thr355 which can affect the hydride transfer reaction in the active site preventing direct inactivation of trypanothione reductase. Other studies with quinone derivatives also have identified Thr355 and Ser14 as unique to the FAD domain of TR [75, 76].
