Environmental Phthalate Diesters as hMT1 and hMT2 Melatonin Receptor Ligands

Abstract

Phthalates esters are commonly used plasticizers that have been linked to endocrine disruption and developmental alterations. Humans are exposed to these ubiquitous synthetic compounds via inhalation of ambient air, dermal contact with consumer products, and ingestion through food and drinking water. It is estimated from urinary metabolite levels that in the US, median adult daily exposure values for phthalate levels are about 8.5–16.8 μg/kg bw/day (Wittassek et al., Mol Nutr Food Res. 55:7–31, 2011). Using integrated pharmacoinformatics‐molecular docking approaches we discovered that several phthalate diesters show structural similarities to melatonin receptor ligands and bind with relatively high docking scores to melatonin binding pockets on in silico hMT1 and hMT2 melatonin receptor models. An assortment of phthalates including three monoesters and ten diesters with alkyl and/or aromatic substituents were tested for competition with 2‐[125I]‐iodomelatonin (100 nM) binding to hMT1 and hMT2 receptors stably expressed in CHO cells to assess binding affinity (Ki) as well as apparent intrinsic efficacy in the presence of GTP (100 μM). In GPCR binding assays, GTP decouples G proteins from receptors causing decreased affinity (rightward shift) for agonists but not for antagonists. Phthalate diesters with aromatic substituents (BBzP: Benzyl Butyl Phthalate; DPP: Diphenyl Phthalate) exhibited the highest affinities toward both hMT1 (BBzP Ki: 2.6 ± 0.3 μM, n=8; DPP Ki: 2.9 ± 0.7 μM, n=8) and hMT2 (BBzP Ki: 1.3 ± 0.2 μM, n=9; DPP Ki: 0.66 ± 0.10 μM, n=9) receptors. Among phthalates with alkyl substituents, four carbon (C4; DBP: Dibutyl Phthalate) and six carbon (C6; DHP: Dihexyl Phthalate) chain length diesters best competed for binding to hMT1 receptors with affinities ranging from 5 to 8 μM. hMT2 receptors did not show any preference to chain lengths of C6 or shorter (DEP: Diethyl Phthalate; DMP: Dimethyl Phthalate; DPrP: Dipropyl Phthalate; DBP; DHP), as all Ki values were between 2 and 7 μM. DBP and BBzP bound to melatonin receptors with affinities in the low micromolar range, however their respective monoester metabolites, MBP (Monobutyl Phthalate) and MBzP (Monobenzyl Phthalate), did not compete for binding to either receptor. The presence of GTP caused a leftward shift of the binding competition curves for both BBzP and DPP at hMT1 melatonin receptors (BBzP KiGTP/KiControl: 3.8; DPP KiGTP/KiControl: 2.6) suggesting inverse agonist efficacy. Little or no shift (BBzP KiGTP/KiControl: 1.3; DPP KiGTP/KiControl: 1.5) on competition curves for hMT2 melatonin receptors suggests antagonist efficacy. Together these data suggest that phthalate diesters could interact with hMT1 and hMT2 receptors to disrupt melatonin receptor‐mediated signaling processes in target tissues.Support or Funding InformationSupported by ES 023684 to MLD and RVR.