In vitro, in silico, and STD-NMR studies of flavonoids from Hypericum helianthemoides (Spach) Boiss. against Leishmania major pteridine reductase 1 (LmPTR1).

Apigenin (1) and 3I,8II-biapigenin (2), a dimer of apigenin, were isolated from the aerial parts of Hypericum helianthemoides (Spach) Boiss. (Hypericaceae family). This study aimed to evaluate the in vitro inhibitory effects of flavonoids 1 and 2 against Leishmania major pteridine reductase-1 (LmPTR1), an essential enzyme for the growth of Leishmania parasites and other trypanosomatid protozoa. The second objective was to understand the binding interactions and structural properties of LmPTR1 inhibition at the atomic level through extensive in silico analyses and Saturation-Transfer Difference (STD)-NMR studies. Anti-LmPTR1 results showed that the dimeric form (2) was active (IC₅₀ of 34.65 μM), while the monomeric form (1) was inactive. Computational analyses yielded a grid score of -52.14 kcal/mol and a free energy binding score of -38.23 kcal/mol. A stable ligand-receptor complex at the LmPTR1 binding site was observed for 2. Moreover, several important binding residues in the catalytic triad (Y194 and K198) and the substrate loop (L226, S227, S229, V230, and M233) interacted with 2. The STD-NMR results corroborated the computational simulations, indicating that H-6I and H-6II of the conjugated ring system on the biapigenin structure showed the highest interaction with the LmPTR1 active site. MTT assay results for 2 against human normal fibroblast cells (BJ cells) exhibited no cytotoxicity at concentrations of 50 and 100 μM. Overall, 3I,8II-biapigenin (2) displayed promise as a candidate for in vivo studies and anti-leishmanial drug development. Further evaluation of the anti-leishmanial and anti-LmPTR1 activities of bioflavonoid 2, along with its analogues, is warranted.