Carbamate Insecticides Target Human Melatonin Receptors

Abstract

Carbaryl (1-naphthyl methylcarbamate) and carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) are among the most toxic insecticides, implicated in a variety of diseases including diabetes and cancer among others. Using an integrated pharmacoinformatics based screening approach, we have identified these insecticides to be structural mimics of the neurohormone melatonin and were able to bind to the putative melatonin binding sites in MT₁ and MT₂ melatonin receptors in silico. Carbaryl and carbofuran then were tested for competition with 2-[¹²⁵I]-iodomelatonin (300 pM) binding to hMT₁ or hMT₂ receptors stably expressed in CHO cells. Carbaryl and carbofuran showed higher affinity for competition with 2-[¹²⁵I]-iodomelatonin binding to the hMT₂ compared to the hMT₁ melatonin receptor (33 and 35-fold difference, respectively) as predicted by the molecular modeling. In the presence of GTP (100 μM), which decouples the G-protein linked receptors to modulate signaling, the apparent efficacy of carbaryl and carbofuran for 2-[¹²⁵I]-iodomelatonin binding for the hMT₁ melatonin receptor was not affected but significantly decreased for the hMT₂ melatonin receptor compatible with receptor antagonist/inverse agonist and agonist efficacy, respectively. Altogether, our data points to a potentially new mechanism through which carbamate insecticides carbaryl and carbofuran could impact human health by altering the homeostatic balance of key regulatory processes by directly binding to melatonin receptors.

Figure 1

Chemical clustering of environmental chemicals from Chem2Risk Knowledgebase. Structural distances from the seed ligand melatonin (Dx, Dy) were computed using ChemMine tools. Melatonin, carbaryl, and carbofuran are marked A, B, and C, respectively.

Figure 2

Chemical structures of (A) melatonin, (B) carbaryl, and (C) carbofuran. Three dimensional pharmacophoric similarities of carbamate insecticides (E) melatonin, (D) carbaryl, and (E) carbofuran. HA = hydrogen bond acceptor; HD = hydrogen bond donor; AR = aromatic moiety; and HYD = hydrophobic moiety.

Figure 3

Carbaryl and carbofuran docking to and competition for 2-[¹²⁵I]-iodomelatonin binding to the hMT₁ and hMT₂ melatonin receptors. Binding poses of carbaryl-hMT₁ (A) hMT₂ (B) or carbofuran-hMT₁ (D) and hMT₂ complexes (E). The ordinate represents 2-[¹²⁵I]-iodomelatonin binding to either the hMT₁ or hMT₂ receptors expressed as percent of total binding (C). Membranes from CHO cells stably expressing hMT₁ and hMT₂ receptors were incubated with 2-[¹²⁵I]-iodomelatonin (300 pM) in the absence (open circle) and presence (closed diamond for MT₁; closed circle for MT₂) of different concentrations of carbaryl (0.1 nM to 1 mM). The ordinate represents 2-[¹²⁵I]-iodomelatonin binding to either the hMT₁ or hMT₂ melatonin receptors expressed as percent of total binding (F). Membranes from CHO cells stably expressing hMT₁ and hMT₂ melatonin receptors were incubated with 2-[¹²⁵I]-iodomelatonin (300 pM) in the absence (open circle) and presence (closed square for MT₁; closed triangle for MT₂) of different concentrations of carbofuran (0.1 nM to 1 mM). The effect of vehicle (ethanol or DMSO) used to dissolve the carbamate compounds in competition for 2-[¹²⁵I]-iodomelatonin binding to the hMT₁ and hMT₂ was negligible and hence was not plotted. Values are the mean ± SEM of 8 independent experiments.

Figure 4

Carbaryl and carbofuran competition for 2-[¹²⁵I]-iodomelatonin binding to hMT₁ and hMT₂ melatonin without (control) and with (GTP) G protein inactivation. The ordinate represents 2-[¹²⁵I]-iodomelatonin binding to either the hMT₁ (A,C) or the hMT₂ (B,D) melatonin receptors expressed as a percent of total binding. Membranes from CHO cells stably expressing hMT₁ and hMT₂ melatonin receptors were incubated with 2-[¹²⁵I]-iodomelatonin (300 pM) in the absence (closed diamond for MT₁; closed circle for MT₂) and presence (open diamond for MT₁; open circle for MT₂) of GTP (100 μM) and different concentrations (0.1 nM to 1 mM) of carbaryl (A,B) or carbofuran (C,D). Binding was performed using Tris buffer (Tris-HCl 50 mM and 10 mM MgCl₂ at pH 7.4), with the absence or presence of 100 μM GTP, 1 mM EDTA, and 150 mM NaCl, at 25 °C for 1 h. The effect of vehicle (ethanol or DMSO) used to dissolve the carbamate compounds in competition for 2-[¹²⁵I]-iodomelatonin binding to the melatonin receptors was negligible and hence was not plotted. Data shown are the mean ± SEM of 5–8 independent experiments, expressed as percentage of total binding. Ratio (K_iGTP/K_iControl) refers to the relationship between the K_i values obtained in the presence (K_iGTP) and absence (K_iControl) of GTP.