<i>In vitro</i> , <i>in silico</i> , and STD-NMR studies of flavonoids from <i>Hypericum helianthemoides</i> (Spach) Boiss. against <i>Leishmania major</i> pteridine reductase 1 ( <i>Lm</i> PTR1)

Apigenin (<b>1</b>) and 3I,8II-biapigenin (<b>2</b>), a dimer of apigenin, were isolated from the aerial parts of <i>Hypericum helianthemoides</i> (Spach) Boiss. (Hypericaceae family). This study aimed to evaluate the <i>in vitro</i> inhibitory effects of flavonoids <b>1</b> and <b>2</b> against <i>Leishmania major</i> pteridine reductase-1 (<i>Lm</i>PTR1), an essential enzyme for the growth of <i>Leishmania</i> parasites and other trypanosomatid protozoa. The second objective was to understand the binding interactions and structural properties of <i>Lm</i>PTR1 inhibition at the atomic level through extensive <i>in silico</i> analyses and Saturation-Transfer Difference (STD)-NMR studies. Anti-<i>Lm</i>PTR1 results showed that the dimeric form (<b>2</b>) was active (IC₅₀ of 34.65 μM), while the monomeric form (<b>1</b>) 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 <i>Lm</i>PTR1 binding site was observed for <b>2</b>. Moreover, several important binding residues in the catalytic triad (Y194 and K198) and the substrate loop (L226, S227, S229, V230, and M233) interacted with <b>2</b>. 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 <i>Lm</i>PTR1 active site. MTT assay results for <b>2</b> against human normal fibroblast cells (BJ cells) exhibited no cytotoxicity at concentrations of 50 and 100 μM. Overall, 3I,8II-biapigenin (<b>2</b>) displayed promise as a candidate for <i>in vivo</i> studies and anti-leishmanial drug development. Further evaluation of the anti-leishmanial and anti-<i>Lm</i>PTR1 activities of bioflavonoid <b>2</b>, along with its analogues, is warranted.