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

Abstract: A cyclic CCK8 analogue, cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 (Dpr=L-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 its natural ligand CCK8. The conformational features of cyclo(29,34)[Dpr(29)Lys(34)]-CCK8 hove been determined by NMR spectroscopy in aqueous solution and in water containing DPC-d(38) 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 atoms making a planar ring and the N-terminal tripeptide extending approximately along the plane of this ring. In DPC/ water, the cyclic peptide adopts a "boat-shaped" conformation, which is more compact than that found in aqueous solution. 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 cyclo(29,34)-[Dpr(29),Lys(34)]-CCK8 with the micelles still ploys an 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 underlines that 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 properties of cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 to the N-terminal receptor fragment have been investigated by fluorescence spectroscopy in a micellar environment. Estimates of the apparent dissociation constant, K-d, 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 cyclo(29,34)[Dpr(29),Lys(34)]CCK8 displaces the natural ligand with an IC50 value of 15 muM.