Discovery of a Positive Allosteric Modulator of Cholecystokinin Action at CCK1R in Normal and Elevated Cholesterol

Abstract

Drugs useful in prevention/treatment of obesity could improve health. Cholecystokinin (CCK) is a key regulator of appetite, working through the type 1 CCK receptor (CCK1R); however, full agonists have not stimulated more weight loss than dieting. We proposed an alternate strategy to target this receptor, while reducing likelihood of side effects and/or toxicity. Positive allosteric modulators (PAMs) with minimal intrinsic agonist activity would enhance CCK action, while maintaining spatial and temporal characteristics of physiologic signaling. This could correct abnormal stimulus–activity coupling observed in a high-cholesterol environment observed in obesity. We utilized high-throughput screening to identify a molecule with this pharmacological profile and studied its basis of action. Compound 1 was a weak partial agonist, with PAM activity to enhance CCK action at CCK1R, but not CCK2R, maintained in both normal and high cholesterol. Compound 1 (10 µM) did not exhibit agonist activity or stimulate internalization of CCK1R. It enhanced CCK activity by slowing the off-rate of bound hormone, increasing its binding affinity. Computational docking of Compound 1 to CCK1R yielded plausible poses. A radioiodinatable photolabile analogue retained Compound 1 pharmacology and covalently labeled CCK1R Thr211, consistent with one proposed pose. Our study identifies a novel, selective, CCK1R PAM that binds to the receptor to enhance action of CCK-8 and CCK-58 in both normal and disease-mimicking high-cholesterol environments. This facilitates the development of compounds that target the physiologic spatial and temporal engagement of CCK1R by CCK that underpins its critical role in metabolic regulation.

Screening for positive allosteric modulators of cholecystokinin type 1 receptor potentially useful for management of obesity

“… 32 Pilot screens of a 5,000-compound library in both formats were successful, leading to a CCK1R PAM with minimal intrinsic agonist activity. 12 However, we found the IP-One assay more suitable for a large-scale screening due to its homogeneous 1536-well format. Moreover, a previous study suggested that nonequilibrium formats, such as the calcium assay, are inferior to IP-One equilibrium assays with respect to classification and determination of pharmacological profiles.…”

“…Further, there has been theoretical concern about increasing potency and/or efficacy or duration of action of CCK1R agonists, due to side effects or possible toxicity. Our hypothesis from the start was that PAMs acting at CCK1R that do not possess substantial intrinsic agonist activity would have much more desirable activity and safety profiles 12,26 . Compounds with this type of activity profile have the potential to enhance the signaling profile of endogenous CCK released after a meal, providing temporal control and short duration of action when physiologically appropriate for affecting appetite.…”

“…We found that calcium release and IP-One accumulation represented the two most common functional assays for screening of Gq-coupled receptors 32 . Pilot screens of a 5,000-compound library in both formats were successful, leading to a CCK1R PAM with minimal intrinsic agonist activity 12 . However, we found the IP-One assay more suitable for a large-scale screening due to its homogeneous 1536-well format.…”

“…We have previously made the case for the development of positive allosteric modulators (PAMs) of CCK action at CCK1R, particularly those with minimal endogenous agonist activity, as a strategy to treat obesity without the side effects and toxicities that could be associated with full agonists 12,13 . The only such agent reported to date 12 , came from our effort to establish the high-throughput screening strategy we now describe.…”

“…We have previously made the case for the development of positive allosteric modulators (PAMs) of CCK action at CCK1R, particularly those with minimal endogenous agonist activity, as a strategy to treat obesity without the side effects and toxicities that could be associated with full agonists 12,13 . The only such agent reported to date 12 , came from our effort to establish the high-throughput screening strategy we now describe. That preliminary effort involved the screening of 5,000 compounds of the NATx natural product-inspired compound library, where we evaluated potential screening formats, such Dengler et al,as calcium flux or IP-One assays.…”