A cyclic CCK8 analogue selective for the cholecystokinin type A receptor: design, synthesis, NMR structure and binding measurements.
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 CCK(A) receptor and its natural ligand CCK8. The conformational features of cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 have 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 plays 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 CCK(A) 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 CCK(A) receptor indicate that the sulfated-Tyr derivative of cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 displaces the natural ligand with an IC(50) value of 15 microM.
De Luca
Stefania
S
Centro Interuniversitario per la Ricerca sui Peptidi Bioattivi (CIRPeB), & Istituto di Biostrutture e Bioimmagini del CNR, Via Mezzocannone 6/8, 80134 Naples, Italy.
Ragone
Raffaele
R
Bracco
Chiara
C
Digilio
Giuseppe
G
Aloj
Luigi
L
Tesauro
Diego
D
Saviano
Michele
M
Pedone
Carlo
C
Morelli
Giancarlo
G
D002478:Cells, Cultured; D015195:Drug Design; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D043886:Receptor, Cholecystokinin A / Q000737:chemistry* / Q000378:metabolism*
D010446:Peptide Fragments; D010456:Peptides, Cyclic; D043886:Receptor, Cholecystokinin A