Benzodiazepine ligands can act as allosteric modulators of the Type 1 cholecystokinin receptor

Abstract

The cholecystokinin (CCK(1)) receptor is a G protein-coupled receptor important for nutrient homeostasis. The molecular basis of CCK-receptor binding has been debated, with one prominent model suggesting occupation of the same region of the intramembranous helical bundle as benzodiazepines. Here, we used a specific assay of allosteric ligand interaction to probe the mode of binding of devazepide, a prototypic benzodiazepine ligand. Devazepide elicited marked slowing of dissociation of pre-bound CCK, only possible through binding to a topographically distinct allosteric site. This effect was disrupted by chemical modification of a cysteine in the benzodiazepine-binding pocket. Application of an allosteric model to the equilibrium interaction between a series of benzodiazepine ligands and CCK yielded quantitative estimates of each modulator's affinity for the allosteric site, as well as the degree of negative cooperativity for the interaction between occupied orthosteric and allosteric sites. The allosteric nature of benzodiazepine binding to the CCK(1) receptor provides new opportunities for small molecule drug development.

Figure 1

Dissociation of CCK₁ receptor-bound [¹²⁵I]CCK in the absence (■) or presence (●) of devazepide (10 nM) from CHO-CCKR cell membranes.

Figure 2

Dissociation of CCK₁ receptor-bound [¹²⁵I]CCK in the absence (■) or presence (●) of devazepide (10 nM) from Alexa-MTSEA-derivatized CHO-CCKR cell membranes.

Figure 3

Effect of CCK (●), devazepide (○), compound 6 (□) or compound 8 (△) on the equilibrium binding of [¹²⁵I]CCK. Data for compounds 6 and 8 have been re-plotted from that presented in reference . Curves through the points represent the best fit of a competitive model (CCK) or an allosteric ternary complex model (devazepide, compounds 6 and 8).

Figure 4

Panel A. CCK₁ receptor residues proposed to interact with the carboxyl terminus of CCK in two distinct models. Trp³⁹ (cyan) was identified by photoaffinity labeling and places the CCK carboxyl terminus at the extracellular face of the receptor; Phe²¹⁸, Trp³²⁷, Ile³³⁰ and Phe³³¹ (brown) are residues identified via mutagenesis data that have been used to alternatively place the CCK carboxyl terminus deep within the transmembrane helical bundle domain. Panel B. Computational docking of CCK (green) and devazepide (yellow) to the CCK₁ receptor.