*2.5.3 Docking validation via superimposition and alignment*

Superimposition of the crystallographic ligand and re-docking poses was used as a means of validating docking. The five crystallographic ligands in complex with *Leishmania* trypanothione reductase were removed from the co-crystallized complex and re-docked. The pdb files of the re-docked complex were uploaded into PyMOL together with the solved complex from the Protein Data Bank. LigAlign [41] was then used to calculate the root mean square deviation between the superimposed re-docked and co-crystalized ligands. Superimposition also allowed the identification of critical overlapping residues via LigPlot<sup>+</sup> . The FAD molecule from the template selected for modeling (PDB ID: 2JK6) was also extracted and docked in *Lm*TR. A comparative study of the original FAD-2JK6 complex and FAD-*Lm*TR complex was done using LigPlot<sup>+</sup> .

## **2.6 Identification of lead compounds**

To identify lead compounds the binding energy, molecular bond interactions, pharmacological, and physiochemical properties were considered. This step helped to filter generalized hit compounds. These compounds in SMILES format were submitted to SwissADME [42], which calculates the corresponding ADME (absorption, distribution, metabolism, and excretion) properties of the compounds. Hydrogen bond interactions of the ligand–protein complexes were studied using LigPlot<sup>+</sup> and PyMOL.

The hit compounds were physiochemically profiled to identify their druglikeness and solubility in water. Lipinski's rule of 5 was used as a metric to narrow down druggable compounds [43]. Pharmacokinetic properties of predicted compounds were determined *in silico*. This included cytochrome inhibition, P-glycoprotein (P-gp) substrates, gastrointestinal (GI) absorption, and the blood– brain barrier (BBB) permeant.

## **2.7 Prediction of activity spectra for substances (PASS) for leads**

PASS assesses the probability that a compound has a suspected biological activity [44]. It has been well known that each substance has a wide spectrum of biological activities as evident from some new uses of many old drugs. The SMILES format of the leads were submitted to Way2Drug.com [45] to predict possible biological activity.
