The Benzopyrone Biochanin-A as a reversible, competitive, and selective monoamine oxidase B inhibitor

As part of our search for safe, natural MAO-B inhibitors, and guided by selecting benzopyrone structures, we focused our efforts to investigate compounds that could be responsible for the plant PCS selective MAO-B inhibition. Furthermore, we characterized the action of the most potent and selective inhibitor. In the current investigation, we tested PCS as well as six of its benzopyrone constituents for their hMAO-A and hMAO-B inhibitions. The PCS isoflavone constituent BIO-A was found to be the most potent and selective hMAO-B inhibitor in this study. BIO-A was also more potent and selective than PCSEE. The presence of BIO-A in PCSEE was verified, and their inhibition of MAOs did not involve H₂O₂ scavenging activity. Furthermore, our results indicated that BIO-A is a reversible and competitive hMAOI with an SI to inhibit hMAO-B. In molecular docking experiments, BIO-A selective activity was accompanied with two reversible H-bonds and three hydrophobic interactions with the human MAO-B active site, which were more than the reversible interactions formed with the MAO-A active site.

The results obtained showed that BIO-A (5, 7-dihydroxy-4′-methoxyisoflavone) is the most potent hMAO-A and hMAO-B inhibitor with the highest MAO-B selectivity out of the tested benzopyrones and PCSEE (Figs. 2 and 3). The obtained PCSEE results are consistent with our previous reports on the potency and selectivity using spectrophotometric and fluorescence assays with different extraction methods [15, 16]. We verified the presence of BIO-A in our used PCSEE to confirm previously reported 0.063?±?0.003% w/w of BIO-A in the dried PCS [24]. BIO-A IC₅₀ and RS results on both isozymes were consistent with the more reflective parameter of its binding affinity, K_i; that was higher in hMAO-B than hMAO-A (Fig. 5). Notably, BIO-A at a very high concentration becomes less selective due to the structure similarities of both isozymes (Figs. 2 and 3). Similarly, standards pirlindole, DEP, and RAS become non-selective when their concentrations are highly increased.

The obtained results indicate the non-selectivity of the studied coumarins as MAOIs. These results are supported by previous reports which indicate PCS total furocoumarins had antidepressant effects in mice and showed in vitro rat MAOs inhibitory effects [29], and another study reported PS and IPS inhibitory effects of rat MAOs [30]. Additionally, we used luminescence assay for confirmation of spectrophotometric assay isozymes effective inhibitions; the luminescence assay is independent of measuring H₂O₂ with a lower representation of the biological reactions. The varied inhibitory potencies may differ with the enzyme source and methods used as it was previously reported with other MAO-BIs [31].

BIO-A structure and the tested benzopyrones are absent of any reactive terminal amino groups as in DEP or phenelzine that can covalently bind at the MAO active site. Our docking evaluation indicated that the non-covalent molecular interactions were underlying BIO-A competitiveness for hMAO-A and hMAO-B (Fig. 5), and thus, supported our biochemical data. In the human MAO-B, BIO-A contained two H-bonds comprising the acceptor and the donor of the C7-OH and the C4’-OCH₃ groups, respectively (Fig. 1). Previous docking studies indicated that flavonoids H-bond donors or acceptors complemented with lipophilic interactions are candidates to modulate both rat MAOs activities [32]. Additionally, two predicted H-bonds at hMAO-B in our previous reports of flavonoids bavachinin and genistein were accompanied with competitive hMAO-B inhibitory effects with higher hMAO-B affinity than MAO-A [17, 33]. Thus, the two H-bond acceptors with the three hydrophobic groups in BIO-A held the best features of the MAO-BI flavonoid pharmacophore more than MAO-A. These consistent results point out H-bonds as a possible critical factor for the human MAO-B affinity stabilization and consequently a better flavonoid inhibitory activity. In MAO-A however, the prediction of a single but crucial H-bond with FAD may explain BIO-A lower affinity, potency, and higher competitiveness to inhibit MAO-A than B.

In addition to our results in hMAO-A and hMAO-B inhibitions, BIO-A was reported to have several multiple pharmacological functions. BIO-A was reported to be neuroprotective in in-vitro and in vivo studies through its multimechanistic antiinflammatory, antioxidant, and phytoestrogenic properties. BIO-A inhibited lipopolysaccharide (LPS)-induced dopaminergic cell damage in rats [34], and LPS-induced activation of microglia [35], protected from the glutamate- and A?- induced cytotoxicity in neuropathological rat models [36, 37], and the glutamate-induced cytotoxicity in human cortical neurons [38].

More importantly, BIO-A ameliorated age- and drug-induced cognitive and behavioral dysfunctions in mice model [39], improved the neuronal viability, vascular functions, and memory [40], and promoted the recovery of peripheral nerve injuries [41] in rat models. It also reduced acetylcholinesterase in dementia mouse [40] and rat [39] models. Such reports indicate that BIO-A can provide neuroprotective effects in different animal models and provide evidence that BIO-A can cross the blood-brain barrier. From another perspective, BIO-A was reported of being metabolized by 4’-O-demethylation by P450 isoforms mainly CYP1A2 to genistein [42, 43]. Moreover, genistein was also reported to be metabolized back to BIO-A by 4’-O-methylation by rat liver enzymes [44]. Furthermore, in our recent investigations, genistein was found to be an active MAOI [33]. BIO-A with its metabolite genistein shared hepatoprotective [45], antimicrobial [46], and anticancer activities [44]. Having BIO-A and genistein to be human MAOIs, the two analogs mutual presence is highly possible in the blood, and hence, their possible activity in the brain against neurodegeneration.

The present kinetics studies indicate that BIO-A can be a safe and effective MAO-BI. The relatively superior selectivity of BIO-A can eliminate oxidative stress produced by both H₂O₂ and toxic aldehydes, which are generated by hMAO-B. The reversibility and competitiveness of BIO-A for hMAOs inhibitions can also eliminate the classical side effects related to MAOIs irreversibilities such as the cheese effect, psychosis, withdrawal and possible drug interactions. Consequently, BIO-A may safely reduce the aging brain oxidative stress by introducing a new mechanism for reversibly inhibiting both human MAOs with MAO-B inhibitory selectivity. There are other natural products with MAO-B inhibition proposed for neuroprotection [47–49], and there is a need to disclose their value in PD and AD patients.