1. Introduction
Alzheimer’s disease (AD) is the most common form of dementia, and the symptoms include loss of memory, difficulty dealing with problems, and disorientation regarding time and place [
1]. Although the etiology of AD is unknown, several pathological hallmarks, such as oxidative stress, amyloid-β (Aβ) deposition, tau protein accumulation, and decreased levels of acetylcholine (ACh), show significant roles in the pathophysiology of this disease [
2]. Many hypotheses have been developed to explain the occurrence of these hallmarks. The cholinergic hypothesis was the first theory proposed to explain AD. A decrease in cholinergic activity is usually observed in the brains of AD patients. Previous studies in both humans and primates have proposed a function of ACh in learning and memory [
3,
4]. This hypothesis has led to clinical studies uses a type of cholinergic agonist, acetylcholinesterase inhibitors (AChEIs), which exhibited promise in reversing memory loss in AD patients. Currently, the US Food and Drug Administration (USFDA) has approved treatments to treat the manifestations of AD including four AChEI drugs: rivastigmine, galantamine, donepezil, and a NMDA receptor antagonist called memantine. However, these treatment strategies only delay the progression of symptoms associated with AD [
5]. Turning to the amyloid cascade hypothesis, Aβ is derived from proteolytic processing of the amyloid precursor protein (APP) by the sequential cleavage of beta-site APP cleaving enzyme-1 (BACE1, β-secretase) and γ-secretase [
6]. Consequently, Aβ peptides inherently aggregate into soluble oligomers, form fibrils, and are lastly deposited as senile plaques [
7]. It has been reported that Aβ with 42 amino acid (Aβ42) oligomers induce oxidative damage, promote tau hyperphosphorylation, and result in toxicity in synapses and mitochondria [
2,
8]. Aβ oligomers aggregates are also considered responsible for neuronal and vascular degeneration in AD brains [
9]. The inhibition of BACE1 is an attractive approach for blocking the formation of Aβ, because this enzyme plays a key role in the rate-limiting step of Aβ production [
10].
Melatonin (
N-acetyl-5-methoxytryptamine; MLT) is known as a powerful antioxidant and free radical scavenger [
11]. For neuroprotection and pharmacological activity against AD, MLT showed preventive effects on the death of cerebellar granule cell cultures exposed to kainate, a neuronal toxin [
12]. Neuroblastoma cells incubated with Aβ presented several features of apoptosis—e.g., cellular shrinkage and membrane bleb formation—and exerted anti-amyloid actions through several mechanisms. The addition of MLT to the cultures significantly reduced these features [
13]. Moreover, MLT decreased the level of Aβ in murine neuroblastoma transfected with amyloid precursor protein (N2aAPP) and restored the levels of phosphorylated and non-phosphorylated neurofilaments [
14,
15]. Earlier administrations of MLT reduced the Aβ and abnormal nitration protein levels and increased survival in AD transgenic mice Tg2576 [
16]. Aged mice typically present significant memory and learning impairments and increased expression levels of BACE1 and γ-secretase in their hippocampus. MLT treatment downregulated these proteins levels and restored the impaired memory [
17]. In addition, long-term treatment with 6 mg/day of MLT improved sleep quality and suppressed sundowning; this was an effect seen regardless of the concomitant medication employed to treat the cognitive or behavioral signs of AD in 45 patients with sleep disturbances [
18]. However, MLT presented limitations in pharmacokinetics, such as a low oral bioavailability and short half-life [
19]. Many attempts have been designed and MLT hybrid ligands have been synthesized. The combination of MLT and neurological relevant agents based on a multitarget-directed approach has been designed to enhance the activity against neurodegenerative disorders—for example, tacrine, curcumin, berberine, donepezil, etc. [
20]. However, these interesting strategies have been investigated in pre-clinical studies. In our continuous work on neuroprotective effect of MLT, MLT and 5-methoxytryptamine (5-MT) derivatives with lipophilic substituents (
1–
5) (
Figure 1) were designed and screened for antioxidant activities. Interestingly, all the derivatives showed stronger antioxidant activity than the parent MLT and 5-MT, and one exhibited a neuroprotective effect on P19-derived neurons [
21,
22]. As mentioned earlier, potential therapeutic treatments for AD should act on targeted enzymes, attenuate oxidative stress, and preserve the neuronal integrity or block the progression of the disease in various molecular pathways. Therefore, this study aims to investigate the inhibitory effects against AD-targeted enzymes, such as AChE and BACE1, and the neuroprotective and neuritogenic activities of these new MLT derivatives
1–
5.
4. Discussion
The derivatives of MLT (
1–
5) were evaluated by comparing their activities against AD with those of the parent compound. Similar to their parent compound, none of the derivatives presented an inhibitory effect on AChE activity. Previous studies have reported that the long-term treatment of MLT in APP 695 transgenic mice significantly prevented decreases in choline acetyltransferase (ChAT) activity but did not alter the AChE activity in the frontal cortex or hippocampus [
37]. Therefore, the other targeted enzyme, BACE1, against AD was further investigated in this study.
BACE1 is an attractive target for therapeutic agents against AD. The earlier generation of BACE1 inhibitors which are peptidomimetic molecules encountered pharmacokinetic problems such as absorption, half-life, and blood-brain barrier (BBB) penetration [
38]. Although the small molecules inhibitors had improved pharmacokinetic properties, most of them were the substrates of P-glycoprotein, resulting in the drugs not reaching therapeutic concentration at the target site [
39]. Having the appropriate partition coefficient (logP), not being the efflux pump substrate and distributing widely to BBB, the pineal hormone, MLT, is an interesting molecule for the development of BACE1 inhibitor. [
19]. Moreover, the derivatives
1–
5 were predicted their physicochemical properties by the SwissADME program [
40]. Their logP values were in the range of 1.99 to 4.17 and were not substrates of P-glycoprotein, indicating these derivatives would permeate the membrane and cross the BBB. Therefore, we focused on the inhibitory effects of MLT and its derivatives on the BACE1 activity. The results from the BACE1 fluorescence assay in
Table 1 presented that all the compounds had a significantly higher BACE1 inhibitory activity (67–88%) than that of quercetin (48%) at the same concentration (5 µM). The results also showed that the modifications
1–
5, including their parents MLT and 5-MT, can be considered to have a potent inhibitory effect against BACE1 [
23,
41]. Our in silico molecular docking study revealed the interactions and binding energy of quercetin. Quercetin, a well-known BACE1 inhibitor, was predicted to have no interaction with the catalytic residues. It established hydrogen bonds with flap region residue (Gln73) and allosteric residues resulting in higher binding energy than all the tested compounds. According to the in vitro experiment, quercetin presented a lower inhibitory effect on BACE1 than MLT and its derivatives, which is in accordance with the in silico prediction. The un-substituted at
N1-indole core structures including MLT, 5-MT, and
5 could interact with the catalytic residue Asp32 and the flap region residue by hydrogen bonds. The addition of
N1-substituted groups (
1–
4) changed the orientation of the structure and affected the binding mode with the enzyme. These compounds interacted with the allosteric residue (Thr232) instead of the catalytic site, and the additional aromatic rings of
2,
3, and
5 played important roles for π-π interactions. Recently, the investigation of the BACE1 inhibitory modes found that the inhibition at the catalytic site would relate to mechanism-based side effects in mice. Allosteric inhibition would be alternative target site for the design of BACE1 inhibitors [
38,
42,
43]
MLT and the derivatives were also investigated for their neuroprotective and neuritogenic effects on the cell viability of P19-derived neurons. The P19 cell line is a suitable cell line for AD testing because it can differentiate into neurons after treatment with RA for 4–5 days. These neurons are irreversibly postmitotic and present the antigen in the nucleus of neurons of the central nervous system [
30]. At a cell density of 10
4 cells/cm
2, these cells differentiate into cholinergic neurons, where the neurotransmitter ACh was found [
44]. From phase-contrast microscope, the morphology of RA-treated cells changed from a polygonal shape to round cell bodies with a long branching process (
Figure 4). From our previous study, MLT was evaluated the cytotoxicity on P19-derived neurons at a concentration 1–10,000 nM and it was found that the trend of toxicity was concentration-dependent [
21]. Therefore, the lowest non-cytotoxic concentration at 1 nM was selected for neuroprotective and neuritogenic studies. FBS was withdrawn from neuron cells, then the oxidative stress was generated [
31,
45]. This condition caused a cell death of almost 50% in the control group. MLT,
1,
3, and
5 significantly increased the neuronal viability in the oxidative stress condition compared with the control group (
Figure 3). From a previous study, MLT,
3, and
5 exhibited good antioxidant capacities, as shown in the ORAC assay [
22]. Our results indicated the neuroprotective effect of MLT and its derivative effect by the antioxidant. For a neuritogenic study, all the tested compounds significantly increased in neurite number by 113–206% compared with the control (
Table 3). Moreover, the bromobenzoly-modified compounds
3 and
5 has a significantly greater number of neuron cells than the parent compounds, MLT and 5-MT, and standard quercetin. Interestingly, derivatives
1,
3, and
5 had significantly longer neurite lengths than quercetin. The neuron cells incubated with
1,
3, and
5 increased the neurite length up to 34–46% compared with the control. This corresponded to the study of Lui et al. [
46], which showed that MLT was able to enhance the proliferation and neurite outgrowth of PC12 cells. From previous results, the increasing of the number and length of neuron cells was related to the BACE1 inhibitory activity. This result might be the effect of ratio of soluble amyloid precursor protein alpha (sAPPα). The increasing sAPPα could bind to p75 neurotrophin receptor (p75
NTR) and stimulate neurite outgrowth with a lower stress on neuron cells [
47,
48]. Therefore, other proposed neurite outgrowth mechanisms of derivatives
1,
3, and
5 will be performed in a further study.
Our MLT derivatives were developed to overcome the pharmacokinetic limitation of MLT by the substitution of more lipophilic groups. The derivatives were proposed to have a longer half-life than MLT, but still present pharmacological activities, especially antioxidant and neuroprotective activities, similar to their parent compound. From all experiments, the derivatives 1, 3, and 5 showed outstanding abilities comparable to those of MLT and had potential as lead compounds for neuroprotective agents. In addition, other mechanisms against AD, such as ChAT activity, Aβ aggregation, and tau protein accumulation, should be investigated and the toxicities of these molecules should be tested both in vivo and in vitro.
5. Conclusions
MLT, 5-MT, and all derivatives 1–5 showed potent inhibitory effects on BACE1 at 5 µM compared with the positive inhibitor, quercetin. In silico molecular modelling predicted that MLT, 5-MT, and 5 interact with BACE1 at the catalytic site and the flap region, whereas 1–4 interacted with allosteric residue Thr232 and the flap region. The additional π-π interactions between the benzene ring of 2, 3, and 5 with the Tyr71 residue promoted ligand-enzyme binding. MLT, 1, 3, and 5 significantly protected cell death in oxidative stress conditions. Furthermore, these derivatives exhibited neuritogenic effects on the neurite number and length. From our study, the derivatives 1, 3, and 5 presented the prominent BACE1 inhibitory activity, exhibited neuroprotective ability against oxidative stress, and promoted neurite outgrowth, which brought new insights into the future of new MLT derivatives as candidates for therapeutics against AD and neuroprotective agents.