Carbamate Insecticides Bind to a Novel Allosteric Site on hMT1 Melatonin Receptors
Authors: Jones Anthony J. , Rajnarayanan Rajendram V. , Dubocovich Margarita L. 
Published: June 2021

In silico screening paired with 2‐[125I]‐iodomelatonin competition binding has implicated carbamate insecticides as potential endocrine disruptors due to their affinity for human (h) MT1 and MT2 melatonin receptors (Popovska‐Gorevski et al. 2017). hMT1 and hMT2 receptor modelling predicts that these compounds may be binding to the orthosteric site and/or to previously uncharacterized hydrophobic pockets proximal to the melatonin‐binding pocket. The location of the specific binding site for carbamate insecticides is currently unknown. The goal of this study was to assess the mechanism(s) by which they inhibit orthosteric ligand binding. Melatonin (1 μM), luzindole (a prototypical non‐selective MT1/MT2 competitive melatonin receptor antagonist, 100 μM), and four carbamate insecticides (carbaryl, fenobucarb, bendiocarb, and carbofuran, 1 mM) were screened for binding to allosteric sites by estimating the Maximal Fractional Inhibition (MFI) of a saturating 2‐[125I]‐iodomelatonin concentration (1200 pM) binding to CHO‐hMT1 and CHO‐hMT2 cell membranes. All six compounds at hMT2 and melatonin at hMT1 completely inhibited the saturating concentration of radioligand (MFI≥60%) suggesting an orthosteric binding mechanism, while luzindole and the carbamate insecticides only partially inhibited binding at hMT1 (MFI<60%), alluding to binding through an allosteric mechanism. To confirm these results, full competition binding curves in CHO‐hMT1 and CHO‐hMT2 cells were performed using five different concentrations of 2‐[125I]‐iodomelatonin (50–1200 pM) to assess binding to the receptor orthosteric site (α=0) and to determine the degree of cooperativity (α>0.01) between orthosteric and allosteric ligand binding. For both hMT1 and hMT2 receptors, melatonin (MT1 KB=0.131 nM, MT2 KB=0.154 nM) completely inhibited binding of saturating concentrations of radioligand confirming that both melatonin and 2‐[125I]‐iodomelatonin bind to the same site. Luzindole (MT1 KB=354 nM, MT2 KB=11.3 nM), and the carbamate insecticide with the highest MT1 and MT2 melatonin receptor affinities, carbaryl (MT1 KB=3790 nM, MT2 KB=389 nM), also completely inhibited 1200 pM 2‐[125I]‐iodomelatonin binding at hMT2 confirming that they bind to the orthosteric site. Interestingly, for luzindole and carbaryl at hMT1, the levels of maximal fractional inhibition of binding decreased with increasing concentrations of radioligand. These patterns show that luzindole and carbaryl bind to an allosteric site on hMT1 with strong negative cooperativity (αluzindole=0.037±0.013, n=3; αcarbaryl=0.017±0.001, n=3). Interactions of carbamate insecticides with a melatonin receptor allosteric site may provide novel mechanisms by which environmental melatonin receptor modulators disrupt signaling of the endogenous ligand.Support or Funding InformationSupported by ES 023684 to MLD and RVR.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.