2-[¹²⁵I]iodo-5-methoxycarbonylamino-N-acetyltryptamine: A selective radioligand for the characterization of melatonin ML₂ binding sites

We now describe the preparation and characterization of a novel radioligand, 2-[¹²⁵I]iodo-5-methoxy-carbonylamino-N-acetyltryptamine (2-[¹²⁵I]MCA-NAT), with high affinity and pharmacological selectivity for melatonin ML₂ receptor sites. 2-[¹²⁵I]MCA-NAT was prepared by introducing an [¹²⁵I]iodine molecule on carbon 2 of 5-MCA-NAT (5-methoxycarbonylamino N-acetyltryptamine), a selective melatonin ML₂ receptor ligand. The specific binding (88%) of 2-[¹²⁵I]MCA-NAT (50 pM) to whole washed hamster brain membranes showed rapid kinetics of association/dissociation, and was of high affinity and saturable (K_d value = 116 ± 14 pM; B_max value = 15.5 ± 1.8 fmol/mg protein, n = 3). 2-[¹²⁵I]MCA-NAT showed no affinity for melatonin ML₁ receptors of chicken retina. Competition curves of various melatonin analogues for 2-[¹²⁵I]MCA-NAT binding to hamster brain, testes and kidney were monophasic and showed a pharmacological order of affinities (K_i values for brain, nM) identical to that of the ML₂ sites [2-iodomelatonin (0.77) > 6-chloro-2-methyl-melatonin (2.56) > 6-chloromelatonin (6.8) > prazosin (21.7) ≥ N-acetylserotonin (23.3 nM) ≥ 5-MCA-NAT (29.5) ≥ melatonin (83.9) > luzindole (1687) > serotonin (2120)]. Affinity constants for competition of melatonin analogues on [¹²⁵I]MCA-NAT binding to hamster brain, testes, and kidney correlated significantly [r = 0.962, P < 0.001, n = 9; r = 0.982, P < 0.0001, n = 13; r = 0.975, P < 0.0001, n = 9, respectively) with the affinities determined on 2-[¹²⁵I]iodomelatonin binding to ML₂ sites (hamster brain) but not to ML₁ sites (chicken retina, r = 0.33, P > 0.05, n = 16). In conclusion, 2-[¹²⁵I]MCA-NAT is a specific radioligand for the identification and characterization of ML₂ binding sites in brain and peripheral tissues.