A Cyclic CCK8 Analogue Selective for the Cholecystokinin Type A Receptor: Design, Synthesis, NMR Structure and Binding Measurements

A cyclic CCK8 analogue, cyclo29,34[Dpr29,Lys34]-CCK8 (DprL-2,3-diaminopropionic acid), has been designed on the basis of the NMR structure of the bimolecular complex between the N-terminal fragment of the CCKA receptor and itsnatural ligand CCK8. The conformational featuresof cyclo29,34[Dpr29,Lys34]-CCK8 have been determined by NMR spectroscopy in aqueous solution and in water containing DPC-d38 micelles(DPC dodecylphosphocholine). The structure of the cyclic peptide in aqueous solution is found to be in a relaxed conformation, with the backbone and Dpr29 side chain atomsmakin g a planar ring and the N-terminal tripeptide extending approximately along the plane of thisring. In DPC/water, the cyclic peptide adoptsa ™boat-shaped∫ conformation, which ismore compact than that found in aqueouss olution. The cyclic constraint between the Dpr29 side chain and the CCK8 carboxyl terminus (Lys34) introduces a restriction in the backbone conformational freedom. However, the interaction of cyclo29,34-[Dpr29,Lys34]-CCK8 with the micelless till playsan important role in the stabilisation of the bioactive conformation. A careful comparison of the NMR structure of the cyclic peptide in a DPC micelle aqueous solution with the structure of the rationally designed model underlinesthat the turn-like conformation in the Trp30 - Met31 region is preserved, such that the Trp30 and Met31 side chains can adopt the proper spatial orientation to interact with the CCKA receptor. The binding propertiesof cyclo29,34[Dpr29,Lys34]-CCK8 to the N-terminal receptor fragment have been investigated by fluorescence spectroscopy in a micellar environment. Estimates of the apparent dissociation constant, Kd, were in the range of 70-150 nM, with a mean value of 120±27 nM. Preliminary nuclear medicine studies on cell lines transfected with the CCKA receptor indicate that the sulfated-Tyr derivative of cyclo29,34[Dpr29,Lys34]-CCK8 displaces the natural ligand with an IC50 value of 15 microM.