Immobilized α-amylase magnetic beads for ligand fishing: Proof of concept and identification of α-amylase inhibitors in Ginkgo biloba

Diabetes mellitus is a widespread metabolic disorder that affects millions of people around the world. The disease is a major burden on both economic and social levels, and there is a need for improved drugs with fewer side effects in the management of the disease. Current methods for isolation of anti-diabetic lead compounds from complex mixtures suffer from low resolution and sensitivity, and there is a need for improved alternatives. In this work, magnetic ligand fishing combined with high-performance liquid chromatography – photodiode-array detection – high-resolution mass spectrometry – solid-phase extraction – nuclear magnetic resonance spectroscopy (HPLC-PDA-HRMS-SPE-NMR) was developed and validated, with the aim of accelerating discovery of natural products targeting α-amylase. The enzyme was successfully immobilized onto magnetic beads and retained its catalytic activity for a period of 75 days, and the specificity of this method was successfully validated by testing the N-terminus coupled α-amylase immobilized magnetic beads on an artificial mixture. A proof of concept experiment, using a crude ethyl acetate extract of Ginkgo biloba leaves, proved that it was possible to fish out four α-amylase ligands. HPLC-PDA-HRMS-SPE-NMR analysis confirmed the presence of bilobetin, isoginkgetin, ginkgetin and sciadopitysin in the solutions resulting from α-amylase ligand fishing with Ginkgo biloba. IC50 curves revealed a reversed relationship between concentration of sciadopitysin and inhibition of α-amylase activity, suggesting that this compound activated the enzyme instead of inhibiting it. α-Amylase, a target for lowering blood glucose in diabetic patients, were immobilized on magnetic beads, and shown to effectively fish out binders from crude Ginkgo biloba ethyl acetate extract. [Display omitted] •Magnetic ligand fishing with N-terminus-coupled α-amylase was developed and validated.•Storage and reuse of N-terminus-coupled α-amylase for magnetic ligand fishing investigated.•α-Amylase binder fished out in proof-of-concept study with artificial mixture.•Four α-amylase binders fished out in proof-of-concept study with Ginkgo biloba extract.