Effect of Melatonin Receptor Ligands on Acetylcholinesterase Activity in C3H/HeN Mouse Brain
Authors: Klatt Emily S. , Rajnarayanan Rajendram V. , Dubocovich Margarita L. 
Published: November 2023

The hormone melatonin (N‐acetyl‐5‐methoxytryptamine) is primarily synthesized in the pineal gland and retina, and released following a circadian rhythm with high levels at night. It shows antioxidant properties (Reiter et al., Adv Med Sci. 52: 11, 2007), binds to the MT1 and MT2 G protein‐coupled receptors, and possibly to a third membrane melatonin binding site (Dubocovich et al., Pharmacol Rev. 62: A, 2010). Hybrid compounds containing melatonin or its indole structure along with an acetylcholinesterase (AChE) inhibitor chemical moiety within the molecule show both anticholinergic properties and antioxidant effects (Fernandez‐Bachiller et al., Chem Med Chem. 4: 828, 2009; Lopez‐Iglesias et al., J Med Chem. 57: 3773, 2014). Our hypothesis is that melatonin receptor ligands modulate the activity of AChE by binding to a novel site also modulated by AChE inhibitors. We first modeled melatonin receptor ligands into the AChE enzyme using computational docking to assess binding interactions between the ligand and the potential melatonin binding site. We then tested these melatonin ligands in vitro using a modified Ellman's AChE activity assay to elucidate the effect of ligands on AChE activity in homogenates from whole C3H/HeN mouse brain. Briefly, homogenates were incubated with ligand, substrate (acetylthiocholine iodide) (500 μM), and DTNB (500 μM) for an appropriate length of time at 37°C as described (Ellman et al., Biochem Pharmacol. 7:88, 1961). There are two binding sites on human AChE required for binding of acetylcholine: the catalytic active site (CAS) and the peripheral anionic site (PAS). While the active site is composed of the catalytic triad (Ser203, Glu334, and His447), the peripheral site is composed of several aromatic amino acid residues (Tyr72, Tyr124, Trp286, and Tyr341) and a negatively charged residue (Asp74). Melatonin ligands with aromatic moieties and a protonated nitrogen could interact with the PAS via π‐π stacking and ion‐ion interactions. These molecular features provide a pocket for favorable binding interactions between melatonin ligands and AChE inhibitors, creating a potentially new and novel binding site. Melatonin ligands that could be accommodated in the PAS, could modify the enzyme and allosterically modulate the binding of an AChE inhibitor in the CAS. Characterization and validation of the AChE assays in the C3H/HeN brain using a Lineweaver‐Burk double‐reciprocal plot demonstrated Km (101.97 μM) and Vmax (0.2534 units/min) values. The carbamates (0.1 to 100 μM), carbaryl and fenobucarb inhibit AChE activity in a concentration dependent manner with IC50 of 1.7 μM and 1.1 μM, respectively. Melatonin (0.1 – 100 μM) and 2‐iodomelatonin (0.1 – 100 μM) alone did not significantly modulate AChE activity (n=3). Results on potentiation of AChE activity resulting from interactions between AChE inhibitors (piperidines) and melatonin receptor ligands (bivalent indoles) will be reported. Increased inhibition of AChE by combining melatonin and AChE inhibitors would be an important advancement towards the treatment of pathologically complex neurodegenerative diseases such as Alzheimer's Disease.

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Supported by ES 023684 to MLD and RVR.