Inter- and intraspecies polymorphisms in the cholecystokinin-B/gastrin receptor alter drug efficacy

Abstract

The brain cholecystokinin-B͞gastrin receptor (CCK-BR) is a major target for drug development because of its postulated role in modulating anxiety, memory, and the perception of pain. Drug discovery efforts have resulted in the identification of small synthetic molecules that can selectively activate this receptor subtype. These drugs include the peptide-derived compound PD135,158 as well as the nonpeptide benzodiazepine-based ligand, L-740,093 (S enantiomer). We now report that the maximal level of receptor-mediated second messenger signaling that can be achieved by these compounds (drug efficacy) markedly differs among species homologs of the CCK-BR. Further analysis reveals that the observed differences in drug efficacy are in large part explained by single or double aliphatic amino acid substitutions between respective species homologs. This interspecies variability in ligand efficacy introduces the possibility of species differences in receptor-mediated function, an important consideration when selecting animal models for preclinical drug testing. The finding that even single amino acid substitutions can significantly affect drug efficacy prompted us to examine ligandinduced signaling by a known naturally occurring human CCK-BR variant (glutamic acid replaced by lysine in position 288; 288 E 3 K). When examined using the 288 E 3 K receptor, the efficacies of both PD135,158 and L-740,093 (S) were markedly increased compared with values obtained with the wild-type human protein. These observations suggest that functional variability resulting from human receptor polymorphisms may contribute to interindividual differences in drug effects.

Identification of a series of CCK-2 receptor nonpeptide agonists: Sensitivity to stereochemistry and a receptor point mutation

“…It is well documented that interspecies differences in GPCRs may alter ligand activity (12). Ligand-induced IP production was assessed in transiently transfected COS-7 cells expressing the mouse CCK-2R (18). The efficacies of 1S and 2S were 88 Ϯ 6.2% and 92 Ϯ 7.5% (mean Ϯ SEM of three independent experiments), respectively, of the CCK-8-induced maximum.…”

Partial Agonism, Neutral Antagonism, and Inverse Agonism at the Human Wild-Type and Constitutively Active Cholecystokinin-2 Receptors

“…Several of them were reported as true antagonists and even, for one of them (L740093R), as an inverse agonist on a constitutively active human CCK2R mutant . Concerning the peptoid PD135,158, despite initial studies demonstrating its anxiolytic activity, this compound was further showed to stimulate second-messenger formation in cells transiently expressing the human, mouse, or dog CCK2R (Hughes et al, 1990;Kopin et al, 1997;Kopin et al, 2000). Moreover, PD135,158 was found to stimulate rat pancreatic acinar cell secretion through the CCK1R (Hocker et al, 1993).…”

“…Indeed, aside from multiple peripheral and central effects linked to its shortterm activation, CCK2R is endowed with oncogenic potential and/or growth-promoting activity (Dufresne et al, 2006). Further molecular investigations pointed out an interspecies genetic polymorphism regarding the CCK2R that markedly affect both affinity and activity of synthetic ligands without altering endogenous ligand-induced activity (Kopin et al, 1997Blaker et al, 2000). These interspecies differences toward synthetic ligands were shown to be caused by sequence variations within the binding site of the CCK2R, thus pointing out the high importance of in vivo and in vitro tests with human protein targets before clinical evaluation of the molecules in humans .…”

Cholecystokinin and Gastrin Receptors

“…Mutation of nonconserved amino acids from transmembrane helices led to the identification of a single amino acid in the canine sequence (Leu355, TM VI) that is responsible for this reversal of specificity (38). In general, studies with so-called nonpeptide antagonists of CCK2R revealed that although the amino acid sequence homology of CCK2R in the different species is near 90%, the efficacy of the nonpeptide molecules to stimulate phospholipase C varied from 0 to 60% of the CCK-induced maximal response according to the species and the compound tested (L365,260, L740,093, YM022, PD135158, PD136,450, PD134,308) (39, 47, 48, 227,232,255,256). Incorporation of nonconservative amino acids in the human CCK2R sequence enabled identification of amino acids in transmembrane helices that account for these variations (47, 48).…”