Synthesis and characterization of new radioligands for the mammalian melanin-concentrating hormone (MCH) receptor

Melanin-concentrating hormone (MCH) is a neuropeptide present in the brain of all vertebrates. For the characterization of MCH receptors, a monoiodinated Phe13, Tyr19]-MCH radioligand analogue was developed. The high susceptibility of 125I]-Phe13, Tyr19]-MCH to oxidative damage and its very lipophilic nature made it necessary to develop new MCH radioligands. To increase the stability, native methionines were replaced by non-sulphur containing amino acid residues. In one analogue, the L-enantiomer of the phenylalanine residue at position 13 was substituted by the D-enantiomer, which increased the relative affinity of the ensuing 125I]-D-Phe13, Tyr19]-MCH about 7-fold. The different analogues were iodinated by an enzymatic reaction and used for binding studies with mouse melanoma cells. 125I]-Met(O)4,8, Phe13, Tyr19]-MCH and 125I]-Hse4,8, Phe13, Tyr19]-MCH showed only about 19% of total binding and 125I]-Ser4,8, Phe13, Tyr19]-MCH displayed about 44% of total binding when compared with 125I]-Phe13, Tyr19]-MCH. Non-specific binding for all tracers was below 11% of total binding of 125I]-Phe13, Tyr19]-MCH binding. 125I]-D-Phe13, Tyr19]-MCH was used for saturation binding studies and revealed a KD of 122.7 +/- 15.3 pmol/l. This radioligand was further characterized by association and dissociation binding studies.