Structural studies on radiopharmaceutical DOTA-minigastrin analogue (CP04) complexes and their interaction with CCK2 receptor

Piotr F J Lipiński,Nils Metzler-Nolte,Raphael Stoll,Renata Mikołajczak,Monika Orzełowska,Piotr Garnuszek,Artur Wodyński,Marta K Dudek,Jan Cz Dobrowolski,Michał Maurin

doi:10.1186/s13550-018-0387-3

Abstract

BackgroundThe cholecystokinin receptor subtype 2 (CCK-2R) is an important target for diagnostic imaging and targeted radionuclide therapy (TRNT) due to its overexpression in certain cancers (e.g., medullary thyroid carcinoma (MTC)), thus matching with a theranostic principle. Several peptide conjugates suitable for the TRNT of MTC have been synthesized, including a very promising minigastrin analogue DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (CP04). In this contribution, we wanted to see whether CP04 binding affinity for CCK-2R is sensitive to the type of the complexed radiometal, as well as to get insights into the structure of CP04-CCK2R complex by molecular modeling.ResultsIn vitro studies demonstrated that there is no significant difference in CCK-2R binding affinity and specific cellular uptake between the CP04 conjugates complexed with [68Ga]Ga3+ or [177Lu]Lu3+. In order to investigate the background of this observation, we proposed a binding model of CP04 with CCK-2R based on homology modeling and molecular docking. In this model, the C-terminal part of the molecule enters the cavity formed between the receptor helices, while the N-terminus (including DOTA and the metal) is located at the binding site outlet, exposed in large extent to the solvent. The radiometals do not influence the conformation of the molecule except for the direct neighborhood of the chelating moiety.ConclusionsThe model seems to be in agreement with much of structure-activity relationship (SAR) studies reported for cholecystokinin and for CCK-2R-targeting radiopharmaceuticals. It also explains relative insensitivity of CCK-2R affinity for the change of the metal. The proposed model partially fits the reported site-directed mutagenesis data.

Highlights

The cholecystokinin receptor subtype 2 (CCK-2R) is an important target for diagnostic imaging and targeted radionuclide therapy (TRNT) due to its overexpression in certain cancers (e.g., medullary thyroid carcinoma (MTC)), matching with a theranostic principle
In vitro binding properties With the purpose of checking the sensitivity of CCK-2R/ CP04 interaction to the change of metal coupled to the peptide, [177Lu]Lu- and [68Ga]Ga-CP04 complexes were prepared and assayed for affinity to human CCK-2R by competition binding experiments in A431-CCK2R(+) cells
Both complexes efficiently internalized in A431-CCK2R(+) cells after 1-h incubation at 37 °C, showing a minor, 4% portion of radioactivity bound on the cell membrane, as expected for receptor agonists, with high internalization (Table 1)

Summary

Introduction

The cholecystokinin receptor subtype 2 (CCK-2R) is an important target for diagnostic imaging and targeted radionuclide therapy (TRNT) due to its overexpression in certain cancers (e.g., medullary thyroid carcinoma (MTC)), matching with a theranostic principle. Several peptide conjugates suitable for the TRNT of MTC have been synthesized, including a very promising minigastrin analogue DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (CP04). In this contribution, we wanted to see whether CP04 binding affinity for CCK-2R is sensitive to the type of the complexed radiometal, as well as to get insights into the structure of CP04-CCK2R complex by molecular modeling. Several suitable peptide conjugates have been reported with some of them showing high receptor affinity and ability for internalization [4] One of these is a minigastrin (MG; H-Leu-(Glu)5-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2) analogue CP04: DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-MetAsp-Phe-NH2 (Fig. 1). These encouraging results prompted further clinical evaluation of [111In]In-CP04 within the ERA-NET project GRAN-T-MTC [8, 9]

Methods

Results

Conclusion