A cyclic CCK8 analogue selective for the cholecystokinin type A receptor: design, synthesis, NMR structure and binding measurements(472 views) De Luca S, Ragone R, Bracco C, Digilio G, Aloj L, Tesauro D, Saviano M, Pedone C, Morelli G
Chembiochem (ISSN: 1439-7633, 1439-4227, 1439-7633electronic), 2003 Nov 7; 4(11): 1176-1187.
Keywords: Binding Studies, Cck Analogues, Conformation Analysis, Cyclic Peptides, Ligand Design, Cholecystokinin A Receptor, Cholecystokinin Derivative, Cyclopeptide, Dodecylphosphorylcholine, Methionine, Tryptophan, Water, Amino Terminal Sequence, Aqueous Solution, Article, Carboxy Terminal Sequence, Dissociation Constant, Fluorescence Spectroscopy, Human, Human Cell, Micelle, Nuclear Magnetic Resonance Spectroscopy, Priority Journal, Receptor Binding, Spatial Orientation, Cultured, Drug Design, Models, Molecular, Molecular Structure, Peptide Fragments, Protein Binding, Protein Conformation, Chemical Synthesis, Chemistry, Pharmacology, Metabolism,
Affiliations: *** IBB - CNR ***
CIRPeB, Ist. Biostrutture/Bioimmagini CNR, Via Mezzocannone, 6/8, 80134 Napoli, Italy
Dipto. di Biochimica e Biofisica, Seconda Università di Napoli, Via Costantinopoli, 16, 80138 Napoli, Italy
Bioindustry Park del Canavese, Via Ribes, 5, 10010 Colleretto Giacosa (TO), Italy
Centro Interuniversitario per la Ricerca sui Peptidi Bioattivi (CIRPeB), & Istituto di Biostrutture e Bioimmagini del CNR, Via Mezzocannone 6/8, 80134 Naples, Italy.,
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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.
A cyclic CCK8 analogue selective for the cholecystokinin type A receptor: design, synthesis, NMR structure and binding measurements