Effect of Melatonin Receptor Ligands on Acetylcholinesterase Activity
Klatt, Emily S.
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The hormone melatonin is primarily synthesized in the pineal gland and retina, and released according to a circadian rhythm with high levels at night. It shows antioxidant properties, binds to the MT 1 and MT 2 G protein-coupled receptors, and possibly to a third membrane-bound melatonin binding site. While melatonin plays a protective role in neurodegenerative disease, several studies have indicated that decreased acetylcholine levels play a role in neurodegeneration. Acetylcholinesterase (AChE) is an enzyme that hydrolyzes the neurotransmitter acetylcholine into acetate and choline, subsequently inactivating acetylcholine. Recently, melatonin-tacrine analogues and synthetic melatonin- N,N -dibenzyl( N -methyl)amine hybrids have been shown to inhibit human AChE (hAChE), in addition to stimulating several other neuroprotective properties. Furthermore, the natural decline in melatonin levels with aging has been associated with the development of neurodegenerative diseases including Alzheimer’s Disease (AD). Currently, four therapeutic options for the treatment of AD are available: three are AChE inhibitors and one is an NMDA receptor antagonist. Since AChE is an essential enzyme involved in nerve transmission, and AD is characterized by cholinergic deficiency, inhibition of AChE could effectively increase the level of acetylcholine in the brain. Therefore, melatonin analogues that simultaneously target both MT1 and MT2 and the binding site on AChE may provide additional treatment options for neurodegenerative disease. To test this concept, we first modeled binding of melatonin receptor ligands and tacrine analogues into the AChE enzyme using computational docking to assess interactions between the ligands and their respective binding sites in the enzyme. We then tested these candidate ligands in vitro using a modified Ellman’s AChE activity assay to elucidate their effects on AChE activity in homogenates from whole C3H/HeN mouse brain. The peripheral anionic site (PAS) in hAChE, which is required for binding of acetylcholine, is composed of several aromatic amino acid residues and a negatively charged residue. Melatonin ligands and tacrine analogues with aromatic moieties and a protonated nitrogen could interact with the PAS via π-π stacking and ion-ion interactions, thus providing a novel pocket with favorable binding interactions and elucidating a previously uncharacterized binding site. Several melatonin receptor ligands and tacrine analogues were evaluated for affinity at human melatonin receptors (hMT 1 and hMT 2 ) and inhibitory activity on AChE. In addition to carbaryl, which directly binds to hMT 1 and hMT 2 and competes for 2-[ 125 I]-iodomelatonin binding at hMT1 with a K i of 2.4 μM and hMT2 with a K i of 0.16 μM, and inhibits AChE activity with an IC 50 of 1.5 μM, we discovered ESK-1, a tacrine analogue, which competes for binding at hMT 1 with a K i of 7.2 μM and inhibits AChE activity with an IC 50 of 21 μM. Currently, we are the only group to have identified such compounds that can target both melatonin receptors and AChE. A single compound that could target melatonin receptors and inhibit AChE would be an important, novel advancement towards the ongoing drug discovery efforts in identifying potential treatments of pathologically complex neurodegenerative diseases.