Among elderly patients, the most prevalent disease of the modern age is Alzheimer’s disease (AD). It is comprised of amyloid plaques consisting of β-amyloid (Aβ) peptides and neurofibrillary tangles of hyperphosphorylated tau protein which results in neuronal cell loss with dementia like symptom [1
]. Of the two most prevalent current hypotheses, the amyloid cascade and cholinergic hypotheses, the amyloid cascade hypothesis illustrates Aβ as the pathological causative agent for dementia [2
]. β-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the β-secretase enzyme required for the production of neurotoxic Aβ together with γ-secretase. The formation of Aβ is a sequential proteolytic process beginning with cleavage of APP by the β-secretase enzyme. Next, the remaining C99 is further cleaved by γ-secretase to release Aβ. Various studies have thus explored the production of BACE1 inhibitors to slow down the formation of Aβ [3
]. Similarly, the cholinergic hypothesis is widely accepted and has been the focus of many AD investigations. The cholinergic hypothesis of AD states that cholinergic dysfunction may not cause cognitive impairment directly, but instead interferes with attentional processing, thereby causing dementia [4
]. The recovery of cholinergic transmitter levels via acetyl- and butyrylcholinesterase (AChE/BChE) inhibitors has been proposed as the most effective target for AD treatment [5
Oxidation is responsible for the pathogenesis of various age-related degenerative diseases such as cancer, diabetes, macular degeneration, AD, and Parkinson’s disease, as reactive pro-oxidant species can damage proteins, lipids, carbohydrates, and nucleic acids over time [6
]. In addition, various studies have suggested that oxidation induces and activates multiple cell signalling pathways that contribute to lesion formation of toxic substances, finally potentiating AD. In fact, oxidative stress resulting from an increase in reactive oxygen species (ROS) and reactive nitrogen species (RNS) has also been thought to play a malicious role in AD progression [7
]. It is thus a salient feature of neurodegeneration linked to the aging process that exacerbates AD [8
]. However, there are doubts as to whether antioxidants play a beneficial role in mitigating AD, while there is ample evidence suggesting the beneficial effects of antioxidants [9
]. As a result, our study aims to provide additional evidence regarding antioxidant use.
Despite the availability of cholinesterase (ChE) inhibitors like donepezil, galantamine, and rivastigmine for AD, adverse effects still outweigh the benefit of commercial drugs over natural products. The most common side effects related to cholinergic stimulation in the brain and peripheral tissues include, gastrointestinal, cardiorespiratory, extrapyramidal, genitourinary, and musculoskeletal symptoms, as well as sleep disturbance [10
]. Different generations of BACE1 inhibitors are also available, such as E2609 and verubecestat; however, side effects including liver toxicity, low oral bioavailability, and low efficacy have limited their use [11
]. The significance of natural products in healthcare was supported by a report that 80% of the global population still relies on plant derived medicines to address their health care needs. It was also reported that 50% of all drugs in clinical use are natural products, and 74% of the most important drugs consist of plant-derived active ingredients [12
]. Thus, our interest in plant-based drugs has guided us to study the anti-Alzheimer’s disease potential of two major Pueraria lobata
-derived antioxidant compounds.
Coumestrol is a phytoestrogen occurring in plants in the coumestan family of compounds; it shares a common structure with isoflavones and estradiol and exhibits estrogenic and antiestrogenic activity based on estrogen levels in the body [13
]. Research on such potential phytoestrogens has been ongoing since ancient times. It was first isolated from ladino clover in 1956; it is widely distributed in plants like clover, soya beans, peas, and strawberries [14
]. In a successive research paper, Bickoff et al. [15
] elaborated on 13 years of research on coumestrol, which highlighted its pharmacological importance. Coumestrol is able to pass through cell membranes due to its low molecular weight and stable structure and was reported to exhibit a neuroprotective effect via cerebral ischemia prevention [16
]. Foodstuffs containing coumestrol exert beneficial effects in cancer, menopause, osteoporosis, atherosclerosis, and cardiovascular disease [17
]. In addition, it has been reported to exhibit anti-aging [18
], neuroprotective [19
], anti-adipogenic [20
], and depigmenting activity [21
]. With such diverse potential, further investigation of its neuroprotective properties is necessary. On the other hand, the coumestan derivative puerarol which has a common structure with coumestrol is a compound with high potential that has been overlooked. Thus, our study aimed to elucidate the nature of puerarol and to shed more light on such potentially interesting compounds. Another interesting fact about these two compounds is they share a common structure differing only by an aliphatic side chain in puerarol.
Our previous work on P. lobata
-derived compounds illustrated obvious anti-AD [22
], anti-diabetic [23
], and hepatoprotective potential [24
]. In the current study, we sought to explore the potential of two similar isoflavonoids in the prevention of AD by assimilation of two approaches: antioxidant therapy and amyloid cascade/cholinergic pathways. These approaches would enable protection against AD via a multifaceted mechanism while also illuminating structure-activity relationships. Furthermore, the antioxidant potential of coumestrol and puerarol was visualized by western blot analysis for inhibition of peroxynitrite (ONOO−
)-mediated nitrotyrosine formation, while chemical kinetics and molecular docking studies cleared the mechanism of AD (Figure 1
A myriad of approaches to AD treatment are available. ROS generation is thought to be the most common target, and several antioxidant therapies have been used to address it. Oxidative and nitrosative stress are associated with the formation and accumulation of Aβ and BACE1, which several studies have affirmed [25
]. Nitric oxide and superoxide anion react to yield ONOO−
, which has been implicated in Aβ aggregation observed in the brain of AD patients [28
]. Thus, with the aim to implement both antioxidant and neuroprotective activity to prevent AD, the combined effects of the two compounds were assessed. A study by Booth et al. [29
] reported the inactivity of coumestrol regarding DPPH scavenging, which is contrary to our findings that coumestan-type compounds have definite scavenging potential through hydrogen/electron donation via hydroxyl groups. In addition, recent data indicated that the potent radical scavenging ability of soya beans is attributed to a type of phytoalexin, coumestrol, which has inherent antimicrobial and antioxidant activity, providing additional support for our postulate [30
]. Likewise, a coumestan wedelolactone with a similar ring structure to coumestrol had been reported to exhibit potent DPPH scavenging activity with an IC50
value of 7 µM justifying the potential of coumestrol [31
]. Numerous researchers have speculated that intraneuronal Aβ toxicity (Aβ40 and Aβ42) and secretases (α-secretase and β-secretase) might play important roles in the release of reactive oxygen intermediates and NO•
. Based on the interconnectedness of reactive oxygen intermediates NO•
, and AD, and how an antioxidant mechanism can ultimately alleviate AD, our study evaluated the peroxynitrite scavenging potential through inhibition of ONOO−
-mediated nitrotyrosine formation via western blot analysis.
From a structural point of view, both isoflavonoids are identical with the exception of an aliphatic side chain. Both isoflavonoids potently reduced protein nitration as demonstrated by western blot, thereby inhibiting ChEs and BACE1, suggesting that the benzofuran ring is key to the exhibited anti-oxidant activity. However, the aliphatic side chain in puerarol seemed to decrease activity due to steric hindrance. Coumestrol, which is devoid of the side chain, showed potent antioxidant and anti-AD activities, whereas puerarol had comparatively weaker activity due to the presence of the aliphatic side chain. Interestingly, puerarol showed potent BACE1 activity despite having the side chain; further investigation is needed to explain this observation.
We analyzed the ChE and BACE1 inhibitory properties of coumestrol and puerarol via in vitro inhibitory assays supported by kinetics and computer aided molecular binding analysis. Coumestrol has drawn special attention from researchers, being a potent phytoestrogen. Coumestrol also exhibited potent ChEs inhibitory activity in our current study, which might be correlated with the hypothesis of Castro et al. [16
], that it is a selective estrogen receptor modulator with inherent neuroprotective activity. Coumestrol shares a similar structure with isoflavones like genistein and diadzin that are known to exhibit potent AChE inhibitory activity [32
]. In addition, a similar study by Ahmad et al. [33
] revealed the ability of soybean and Temphe isoflavones including daidzein and genistein along with their glycosides to inhibit BACE1. Coumestrol contains a phenolic group similar to genistein with a similar electrostatic and configurational nature responsible for their activity. The current study identified coumestrol as an active dual inhibitor against ChEs and BACE1. Unlike conventional inhibitors, coumestrol exerts inhibitory activity against both hypothesized mechanisms of AD, providing a greater beneficial advantage. Despite weak BChE inhibitory activity, the BACE1 activity of these compounds is very interesting, which suggests the need for further evaluation.
The cumulative results suggestive of ChE as well as BACE1 inhibitory potential inspired us to further determine the type and mode of interaction via enzyme kinetic study. Coumesterol showed potent ChE activity, in agreement with a low Ki
value determined by kinetic parameters. Puerarol showed potent BACE1 activity, in agreement with a low Ki
value demonstrated by the secondary plot showing mixed type inhibition. Competitive inhibitors bind to catalytic sites of an enzyme and decrease the amount of binding of a substrate or ligand to the enzyme. Coumestrol displayed competitive inhibition against AChE and mixed inhibition against BChE, indicating that it may bind to the enzyme-substrate complex or interact with a specific catalytic or allosteric site of the enzyme. As a mixed inhibitor, coumestrol was able to bind either free BChE or the BChE-substrate complex, while puerarol bound to free BACE1 or the BACE1-substrate complex. In mixed inhibition, at sufficiently high substrate concentrations, the enzyme is exclusively present in the form of enzyme-substrate complex, and an inhibitor acts primarily as an uncompetitive inhibitor, which attempts to bind to the complex. Under this condition, a higher concentration of inhibitor is required to effectively inhibit the enzyme [34
In the present study, molecular docking enabled us to investigate the activity, orientation, and interaction and binding energies. Beyond the goal of using molecular docking to predict binding affinities, modeling also allowed us to confirm the ChE and BACE1 inhibitory activity of both compounds and the inhibition mode through chemical kinetics. The docked ligand molecules were selected based on docking energy and good interaction with active site residues. In our subsequent experiments, 3D docking of the potent ChEs and BACE1 inhibitor, coumestrol, exhibited a minimum docking score for ChEs. A lower docking score indicates a greater binding capacity for the ligand. Hence, the docking scores and binding interactions of coumestrol were significantly associated with its ability to inhibit ChE activity. Regarding the apoptotic potential of coumestrol, a previous report determined the structural stability of coumestrol docked to the estrogen receptor (α and β) by analyzing H-bonds and the interaction energy, suggesting that coumestrol upon interaction with estrogen receptor-α led to a strong substrate binding affinity [35
]. However, in our study, coumestrol was able to bind to the catalytic and allosteric sites of BChE. Coumestrol formed a hydrogen bond with His438, a major catalytic residue of BChE, and with the peripheral anionic site (Asp70 residue) of BChE. On the other hand, coumestrol interacted with His440, an important catalytic residue of AChE, specifically through hydrophobic interactions. Thus, coumestrol exhibited BChE inhibitory activity against the target protein in terms of binding efficiency. In particular, this study is the first to report the ChEs inhibitory activity of coumestrol, derived via enzyme kinetic analysis and molecular docking simulation. Furthermore, the identification of coumestrol as a dual ChE and BACE1 inhibitor could set a benchmark for treating AD. Puerarol exhibited hydrophobic interactions with Asp32 and Asp228, major catalytic residues. On the other hand, it interacted with Thr232, an important allosteric residue, specifically through a hydrogen bonding interaction. Thus, puerarol exhibited inhibitory activity against the target protein in terms of binding efficiency. In particular, this study is the first to report the BACE1 inhibitory activity of puerarol derived via enzyme kinetic analysis and molecular docking simulation.