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A Cyclic CCK8 Analogue Selective for the Cholecystokinin Type A Receptor: Design, Synthesis, NMR Structure and Binding Measurements
Author(s) -
De Luca Stefania,
Ragone Raffaele,
Bracco Chiara,
Digilio Giuseppe,
Aloj Luigi,
Tesauro Diego,
Saviano Michele,
Pedone Carlo,
Morelli Giancarlo
Publication year - 2003
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200300635
Subject(s) - cholecystokinin , chemistry , cyclic peptide , stereochemistry , cholecystokinin receptor , receptor , combinatorial chemistry , biochemistry , peptide
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 n M , with a mean value of 120±27 n M . 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 μ M .