**Abstract**

Trypanothione reductase (TR), a flavoprotein oxidoreductase is an important therapeutic target for leishmaniasis. Ligand-based pharmacophore modelling and molecular docking were used to predict selective inhibitors against TR. Homology modelling was employed to generate a three-dimensional structure of *Leishmania major* trypanothione reductase (*Lm*TR). A pharmacophore model used to screen a natural compound library generated 42 hits, which were docked against the *Lm*TR protein. Compounds with lower binding energies were evaluated via *in silico* pharmacological profiling and bioactivity. Four compounds emerged as potential leads comprising Karatavicinol (7-[(2E,6E,10S)-10,11-dihydroxy-3,7,11 trimethyldodeca-2,6-dienoxy]chromen-2-one), Marmin (7-[(E,6R)-6,7-dihydroxy-3,7-dimethyloct-2-enoxy]chromen-2-one), Colladonin (7-[[(4*a*S)-6-hydroxy-5,5,8*a*-trimethyl-2-methylidene-3,4,4*a*,6,7,8-hexahydro-1*H*-naphthalen-1-yl] methoxy]chromen-2-one), and Pectachol (7-[(6-hydroxy-5,5,8*a*-trimethyl-2 methylidene-3,4,4*a*,6,7,8-hexahydro-1*H*-naphthalen-1-yl)methoxy]-6,8-dimethoxychromen-2-one) with good binding energies of 9.4, 9.3, 8.8, and 8.5 kcal/mol, respectively. These compounds bound effectively to the FAD domain of the protein with some critical residues including Asp35, Thr51, Lys61, Tyr198, and Asp327. Furthermore, molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area (MMPBSA) computations corroborated their strong binding. The compounds were also predicted to possess anti-leishmanial activity. The molecules serves as templates for the design of potential drug candidates and can be evaluated *in vitro* with optimistic results in producing plausible attenuating infectivity in macrophages.

**Keywords:** *Leishmania*, trypanothione reductase, oxidative stress, natural product, pharmacophore modeling, virtual screening, molecular dynamics
