Major depression, a prevalent mental disorder, severely affects the psychosocial functioning and life quality of approximately 280 million people worldwide (according to Global Health Data Exchange at https://vizhub.healthdata.org/gbd-results/) [1]. A major and common hypothesis associates depression with the deficiency of monoamine neurotransmitters in synaptic cleft, such as serotonin, dopamine and norepinephrine [2]. Currently, the primary treatment approach predominantly revolves around modulating monoamine neurotransmitters [3]. Monoamine oxidase A (MAOA), a vital enzyme involved in monoamine metabolism, plays a crucial role in regulating the levels of these neurotransmitters [4,5]. Selective MAOA inhibitors (MAOAIs) [6], like moclobemide and toloxatone, represent an important class of pharmacological therapy for depression. However, due to the reported side effects of these drugs, such as anticholinergic effects, postural hypotension, and impaired sexual function [7,8], there is an urgent need to discover safer and more potent novel MAOAIs.

Serotonin, one of the most important monoamine neurotransmitters, is synthesized from tryptophan and primarily catabolized by MAOA in human body, playing a key role in the regulation of emotion [9,10]. In nature, various of tryptophan-derived alkaloids share significant structural similarities with serotonin in the central nervous system. Previous studies have shown that tryptophan-derived alkaloids like β-carbolines and monoterpene indoles possess significant inhibitory effects against MAOA [11]. Passos et al. [12] discovered four monoterpene indole alkaloids from Psychotria with IC50 values against MAOA ranging from 0.85 to 2.14 µM. Herraiz et al. [13] isolated four β-carbolines from Peganum harmala and observed their significant inhibitory effects against MAOA with IC50 values ranging from 8.7 to 352 nM. Hunteria zeylanica, a member of the Apocynaceae family, is well-known for its abundance of monoterpene indole alkaloids [14,15]. An in vitro study revealed that the alkaloid extract of H. zeylanica has good inhibitory effect against MAOA (IC50= 62.8 ± 18.7 μg/mL). However, the active ingredients responsible for this effect remain unclear, making it a promising candidate for discovering novel MAOAIs.

Magnetic beads (MBs)-based ligand fishing, exploiting the specific affinity between ligands and immobilized enzymes, enables rapid isolation of "targets" from complex systems [16,17]. Compared to conventional inhibitor screening techniques, MBs-based ligand fishing is radically easier, faster and more cost-effective [18], [19], [20], [21]. Previously, we established a MAOAIs screening method based on amine-terminated MBs modified with MAOA (MBs@NH2@MAOA) [22]. However, our initial results showed that the enzyme immobilization efficiency and biocatalytic activity of MBs@NH2@MAOA were unsatisfactory, which led to a low-efficiency ligand fishing from the alkaloid extract of H. zeylanica. Previous studies have demonstrated that the surface chemistry of solid supports significantly affects the amount and activity of the immobilized enzyme [23,24]. Amine-terminated MBs performed better than MBs terminated with other moieties for the immobilization of xanthine oxidase [25] and Lathyrus cicera amine oxidase [26], while carboxyl-terminated mesoporous silica provides higher loading efficiency and better activity for organophosphorus hydrolase than the corresponding amine-terminated support [27]. To improve the ligand fishing efficiency of MAOA immobilized MBs, it is necessary to study the effect of the surface moieties of MBs on the immobilization of MAOA.

In this work, we aimed to develop a successful MBs-based ligand fishing approach for rapid discovery of monoterpene indoles as MAOAIs from H. zeylanica. The immobilization efficiency of two commonly available MBs, i.e. amine- and carboxyl-terminated MBs, were comparatively evaluated based on the activity and amount of the immobilized MAOA. The specific binding and non-specific adsorption on these MBs were also investigated using a model mixture. The more effective approach, i.e. carboxyl-terminated MBs with immobilized MAOA (MBs@COOH@MAOA), was applied for screening potential MAOAIs in the alkaloid extract of H. zeylanica. The chemical structures of the ligands fished out from the extract were then identified using HPLC-Obitrap-MS/MS, while their IC50 values were determined using the Spark 10 M microplate reader. To gain further insights into the interactions between these ligands and MAOA, molecular docking studies were also carried out. The workflow for the established ligand fishing approach is illustrated in Fig. 1 which summarizes the different stages of the research process.