Elucidating the Structure–Activity Relationship of the Pentaglutamic Acid Sequence of Minigastrin with Cholecystokinin Receptor Subtype 2

Abstract

Derivatized minigastrin analogues make up a promising class of candidates for targeting cholecystokinin receptor subtype 2 (CCK2R), which is overexpressed on cancer cells of various neuroendocrine tumors. The pentaglutamic acid sequence of minigastrin influences its biological properties. In particular, it plays a crucial role in the kidney reuptake mechanism. However, the importance of the binding affinity and interaction of this region with the receptor on a molecular level remains unclear. To elucidate its structure–activity relationship with CCK2R, we replaced this sequence with various linkers differing in their amount of anionic charge, structural characteristics, and flexibility. Specifically, a flexible aliphatic linker, a linker with only three d-Glu residues, and a structured linker with four adjacent β3-glutamic acid residues were evaluated and compared to the lead compound PP-F11N (DOTA-[d-Glu1–6,Nle11]­gastrin-13). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the minigastrin derivatives, which allowed radiolabeling with Lutetium-177. The levels of In vitro internalization into MZ-CRC1 cells and in vivo tumor uptake as well as human blood plasma stability increased in the following order: aliphatic linker < three d-Glu < (β3-Glu)4 < (d-Glu)6. The in vitro and in vivo behavior was therefore significantly improved with anionic charges. Computational modeling of a CCK2 receptor–ligand complex revealed ionic interactions between cationic residues (Arg and His) of the receptor and anionic residues of the ligand in the linker.

Evaluation of Actinium-225 Labeled Minigastrin Analogue [225Ac]Ac-DOTA-PP-F11N for Targeted Alpha Particle Therapy

“…Both [ 225 Ac]Ac-PP-F11N and [ 177 Lu]Lu-PP-F11N showed high internalization efficiency in vitro. Ritler et al reported [ 177 Lu]Lu-PP-F11N internalization rate of 50.2% ± 8.9 after 2 h incubation in CCKBR positive cells recently [ 16 ], which is similar to the internalization rate of [ 225 Ac]Ac-PP-F11N (45.9% ± 1.6) in our study.…”

Cholecystokinin 2 Receptor Agonist 177Lu-PP-F11N for Radionuclide Therapy of Medullary Thyroid Carcinoma: Results of the Lumed Phase 0a Study

“…The evaluation of the different radiolabeled minigastrin derivatives differing in both, the Cterminal peptide binding moiety and the structural characteristics of the linker, revealed that the compounds with six D-glutamic acid residues within the amino acid chain belong to the most promising substances with an reasonable balance of tumor uptake and decreased kidney reabsorption (11,12,16). The pronounced hydrophilicity of such compounds is most likely the reason for the fast renal excretion and shorter blood half-life of 177 Lu-PP-F11N in comparison to…”

“…jnm.snmjournals.org Downloaded from effect. Another approach to reduce nephrotoxicity is to modify the compound itself: Amino acid chains with more than 5 glutamic acids in their sequence play an important role in the kidney reuptake mechanism and may reduce kidney uptake (10,11). Such an approach was implemented by several groups and resulted in the development of a library of improved radiolabeled minigastrin analogues (12)(13)(14)(15)(16)(17).…”

Permanent Photodynamic Activation of the Cholecystokinin 2 Receptor

“…Out of the 447 residues in the full CCK2R sequence, the following are susceptible to 1 O 2 oxidation: 10 Met residues-Met1, Met67, Met73, Met108, Met117, Met134, Met186, Met234, Met334, and Met393; 15 Cys residues-Cys22, Cys39, Cys107, Cys127, Cys157, Cys205, Cys279, Cys293, Cys345, Cys384, Cys391, Cys401, Cys405, Cys408, and Cys409; 6 His residues-His170, His207, His274, His364, His376, and His394; 6 Trp residues-Trp165, Trp179, Trp209, Trp218, Trp346, and Trp355; and 12 Tyr residues-Tyr61, Tyr132, Tyr153, Tyr189, Tyr192, Tyr238, Tyr246, Tyr294, Tyr350, Tyr380, Tyr390, Tyr438, Tyr61, Tyr189, Tyr192, and Tyr350 ( Figure A1 in Appendix A). The CCK2R residues that have been found to be essential for CCK binding (as determined by site-directed mutagenesis and photoaffinity labeling) are as follows: Arg57, Tyr61, Thr111, Thr119, Phe120, Phe122, Tyr189, Tyr192, Thr193, His207, Phe227, Phe342, Trp346, Val349, Tyr350, Asn353, Arg356, and His376 [65][66][67][68][69][70][71]. The overlapping residues in CCK2R are Tyr61, Tyr189, Tyr192, His207, Trp346, Tyr350, and His376.…”