5′-Methylthioadenosine phosphorylase from Schistosoma mansoni ( Sm MTAP) is a promising target within the parasite's purine salvage pathway. In this study, Sm MTAP was covalently immobilized onto amine-functionalized cobalt ferrite magnetic nanoparticles (CoFe 2 O 4 ), generating a reusable bioreactor (CoFe 2 O 4 @ Sm MTAP) 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, R 2 = 0.9999; LOD 0.005 μmol/L; LOQ 0.01 μmol/L). The immobilized enzyme displayed Michaelis-Menten behavior (apparent K M = 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 Sm MTAP inhibitors (IC 50 = 8.29 ± 0.94 μg/mL). Affinity selection–mass spectrometry (AS-MS) with CoFe 2 O 4 @ Sm MTAP 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 Sm MTAP inhibitors in complex natural libraries. • Sm MTAP was covalently immobilized onto CoFe 2 O 4 nanoparticles. • An LC-DAD method enabled direct monitoring of Sm MTAP via adenine quantification in a 3.5 min analysis. • H. stigonocarpa stem extract was identified as a potential source of Sm MTAP inhibitors (IC 50 = 8.29 μg/mL). • Affinity selection–MS revealed six Sm MTAP ligands from the crude extract. • Integration of AS-MS and microfractionation enabled prioritization of active fractions.
Amaral et al. (Fri,) studied this question.