Discovery and Pharmacological Characterization of Environmental Melatonin Receptor Modulators
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Melatonin targets MT1 and MT2 melatonin receptors that are expressed in brain and peripheral tissues in order to signal “time-of-day” messages for the synchronization of circadian rhythms. In 2017, Popovska-Gorevski et al. demonstrated via in-silico screening paired with competition binding that environmental carbamate insecticides with similar pharmacophores to melatonin, carbaryl and carbofuran, may be human (h) MT1 and MT2 melatonin receptor disruptors based on their affinity for melatonin recombinant receptors. Carbaryl (Ki hMT1 = 3.3 µM, hMT2 = 0.16 µM) and carbofuran (Ki hMT1 = 94 µM, hMT2 = 3.6 µM) are only two of many environmental pollutants identified by in-silico chemical clustering to have a similar structure to melatonin. It is most likely that melatonin receptor modulators with different pharmacological activities than melatonin will be the most disruptive for rhythmic homeostatic processes, such as timekeeping in the suprachiasmatic nucleus or pancreatic insulin release. Therefore, we hypothesized that further screening in-vitro would identify melatonin receptor-specific environmental toxins that exhibit signaling profiles distinct from melatonin. The objectives of this study were to identify environmental melatonin receptor modulators using integrated pharmacoinformatics and competition binding, to determine pharmacological properties of melatonin receptor ligands at hMT1 and hMT2 receptors in-vitro, and to assess the ability of unique environmental melatonin receptor modulators to alter melatonin receptor-mediated processes in target tissues. Based on the ability to inhibit 2-[125I]-iodomelatonin binding to MT1 and MT2 melatonin receptors in-vitro these studies implicate carbamate insecticides, fenobucarb (Ki hMT1 = 10 µM, hMT2 = 0.55 µM) and bendiocarb (Ki hMT1 = 42 µM, hMT2 = 3.0 µM), and eight low molecular weight phthalate diesters, including ubiquitous diethyl phthalate (DEP; Ki hMT1 = 48 µM, hMT2 = 3.3 µM), butylbenzyl phthalate (BBzP; Ki hMT1 = 3.3 µM, hMT2 = 1.1 µM), and dibutyl phthalate (DBP; Ki hMT1 = 22 µM, hMT2 = 6.6 µM) as environmental melatonin receptor modulators. Saturation and kinetic binding studies verified that carbamate insecticides were orthosteric ligands for both receptor types. Further, all compounds displayed different profiles from hMT1 and hMT2 agonist melatonin as intrinsic efficacy at recombinant hMT1 and hMT2 melatonin receptors were determined for higher affinity compounds by GTP-shift membrane assays. Specifically, carbaryl (Emax hMT1 = -8.7% MLT, hMT2 > 100% MLT), fenobucarb (hMT1 = 17%, hMT2 > 100%), bendiocarb (hMT1 = 11%, hMT2 > 100%), carbofuran (hMT1 = -6.7%, hMT2 > 100%), and DEP (hMT1 = -41%, hMT2 > 100%) classified as hMT1 antagonists and hMT2 agonists while BBzP (hMT1 = -43%, hMT2 = -14%) and DBP (hMT1 = -62%, hMT2 = -66%) were determined to be antagonists at both receptors. Measures of efficacy in the forskolin-stimulated cAMP live cell assays, once again determined the four carbamate insecticides to be weak agonists or antagonists for hMT1 (inhibition at 10 µM = 3.7-41% MLT) and hMT2 agonists (inhibition at 10 µM = 40-61%). Notably, carbaryl displayed picomolar potency for activation of hMT2 (hMT1 IC50 = 1.6 µM, Imax = 45% MLT; hMT2 IC50 = 95 pM, Imax = 67% MLT), equal to the effect of melatonin. Lastly, carbaryl was able to inhibit forskolin-stimulated insulin release in the pancreatic β-cell line, INS-1E (inhibition at 10 µM = 96% MLT). Considering the wide range of influences melatonin receptors have in regulating metabolism, circadian rhythms, mood, immune system, and other critical biological functions, disruption of these receptor pathways could contribute to associated disease pathologies. Based on these results, we propose that environmental compound screening involving high-throughput in-vitro bioassays for critical melatonin receptor-mediated processes should be implemented in chemical risk assessment platforms.