Identification of ZINC database-derived antitrypanosomal candidates targeting Trypanosoma congolense lysosomal-α-mannosidase using computational, biochemical and in vivo methods.

Animal African Trypanosomiasis (AAT), primarily caused by Trypanosoma congolense, continues to threaten livestock productivity across sub-Saharan Africa. The persistence of the disease is largely driven by antigenic variation of Variant Surface Glycoproteins (VSGs), which enable immune evasion. Lysosomal α-D-mannosidase (LDM) suggestively plays a key role in VSG catabolism, and its inhibition may impair parasite survival. This study employed computational and biochemical approaches to characterize T. congolense lysosomal α-D-mannosidase (TcLDM) and identify potential inhibitory compounds. A high-quality TcLDM homology model was constructed and validated, followed by molecular docking of 38 structural analogues of known α-mannosidase inhibitors retrieved from the ZINC database which led to 4 hits with good selectivity ratio toward the parasite orthologues (ZINC000003795857, ZINC000043929526, ZINC000257502749, ZINC000257502751). Molecular dynamics simulations (100 ns) using GROMACS 2022 confirmed the structural stability of the 4 top ligands, particularly kifunensine (ZINC000003795857) and its analogue ZINC000257502749, which exhibited persistent interactions with key catalytic residues. Experimentally, TcLDM was isolated and partially purified using a combination of ion exchange chromatography and gel filtration to a purification fold of 1.62, revealing a molecular weight of ∼66 kDa, optimal activity at pH 5.0, and KM ≈ 0.503 mM. Kifunensine showed mixed-type inhibition with IC50 = 52.97 μM, significantly reducing catalytic efficiency. In vivo drug incubation infectivity assay showed that kifunensine suppressed parasitemia in a dose-dependent manner, achieving near clearance at higher concentrations. These findings collectively highlight TcLDM as a viable enzyme target and suggest kifunensine and ZINC000257502749 as promising scaffolds for anti-trypanosomal drug design.