A target-directed platform for identification of ligands of Schistosoma mansoni methylthioadenosine phosphorylase in natural product extracts.

5'-Methylthioadenosine phosphorylase from Schistosoma mansoni (SmMTAP) is a promising target within the parasite's purine salvage pathway. In this study, SmMTAP was covalently immobilized onto amine-functionalized cobalt ferrite magnetic nanoparticles (CoFe2O4), generating a reusable bioreactor (CoFe2O4@SmMTAP) with an immobilization efficiency of 66.3 ± 6.6%. Enzymatic activity was monitored by HPLC-DAD through the direct quantification of adenine using a validated analytical method (linearity 1-100 μmol/L, R2 = 0.9999; LOD 0.005 μmol/L; LOQ 0.01 μmol/L). The immobilized enzyme displayed Michaelis-Menten behavior (apparent KM = 29.45 ± 7.34 μmol/L), retained more than 60% residual activity over two weeks, and preserved more than 50% activity after seven reaction cycles. The platform was applied to the screening of eight natural product extracts, identifying the ethanolic stem extract from Hymenaea stigonocarpa as a potential source of SmMTAP inhibitors (IC50 = 8.29 ± 0.94 μg/mL). Affinity selection-mass spectrometry (AS-MS) with CoFe2O4@SmMTAP revealed six putative ligands (affinity ratios 1.45-2.89) in the crude extract. Analytical-scale microfractionation was conducted, specifically targeting the retention time windows of the ligands previously detected by AS-MS. This strategy successfully localized the main bioactive region to the 30.75-33.0 min window, which overlaps with the retention times of two ligands, including a compound tentatively annotated as asperphenamate. Complementary UHPLC-HRMS dereplication enabled the putative annotation of 23 compounds. Overall, the integrated workflow provided a selective, material-efficient, and reusable bioanalytical platform for screening SmMTAP inhibitors in complex natural libraries.