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

Stefania De Luca, Raffaele Ragone, Chiara Bracco, Giuseppe Digilio, Luigi Aloj, Diego Tesauro, Michele Saviano, Carlo Pedone, Giancarlo Morelli

Research output: Contribution to journalArticle

Abstract

A cyclic CCK8 analogue, cyclo29,34[Dpr29,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 cyclo29,34[Dpr29,Lys 34]-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 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[Dpr29,Lys34]-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 CCKA receptor. The binding properties of 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 μM.

Original languageEnglish
Pages (from-to)1176-1187
Number of pages12
JournalChemBioChem
Volume4
Issue number11
DOIs
Publication statusPublished - Nov 7 2003

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Keywords

  • Binding studies
  • CCK analogues
  • Conformation analysis
  • Cyclic peptides
  • Ligand design

ASJC Scopus subject areas

  • Drug Discovery
  • Organic Chemistry
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

De Luca, S., Ragone, R., Bracco, C., Digilio, G., Aloj, L., Tesauro, D., Saviano, M., Pedone, C., & Morelli, G. (2003). A Cyclic CCK8 Analogue Selective for the Cholecystokinin Type A Receptor: Design, Synthesis, NMR Structure and Binding Measurements. ChemBioChem, 4(11), 1176-1187. https://doi.org/10.1002/cbic.200300635