Obesity has reached epidemic proportions, contributing to similar increases in type 2 diabetes mellitus and its
multiple co-morbidities, accounting for immense expense, suffering, and early mortality. Modification of diet
and lifestyle, while effective for acute weight loss, is not durable, and bariatric surgery, while effective for
morbid obesity, is not scalable for this need. Therefore, new safe and effective pharmacotherapies for obesity
are needed. This proposal is responsive to PAR-16-374 soliciting assays for discovery of such therapeutics.
Our OBJECTIVE is to identify molecules with activity as positive allosteric modulators (PAMs) of the type 1
cholecystokinin receptor (CCK1R) that do not exhibit intrinsic agonist activity and that can be safe and effective
across the full spectrum of potential patients, from those tending to gain weight to the morbidly obese with
metabolic syndrome. This type of drug does not now exist. It would increase signaling responses to
endogenous hormone released after a meal, when a greater biological response can enhance satiety, while
overcoming side effects and potential toxicity caused by full agonists. It would also have the unique property of
correcting a defect in stimulus-activity coupling now recognized to be present in a subset of these patients.
Sequential dual screening strategies will be directed toward the natural wild type (WT) CCK1R, as well as this
receptor in an abnormal conformation induced by elevated cholesterol in the membrane, with the latter
mimicked by a CCK1R mutant. Enhancing and/or recalibrating CCK1R to be normally responsive to CCK in
these settings could accentuate the hormonal effect on satiety in a gentle and safe manner. This represents a
partnership between Dr. Miller at Mayo Clinic, with expertise in molecular pharmacology of CCK1R, and Dr.
Sergienko at Sanford Burnham Prebys, with expertise in high throughput screening (HTS) and chemical
genomics. There are 3 specific AIMS: (i) Primary HTS of a small molecule library using intracellular calcium
response assays in CHO cells expressing WT CCK1R, performed in two modes to examine (a) intrinsic agonist
activity and (b) PAM activity that enhances the responsiveness to CCK. Hits will be confirmed in orthogonal IP-
1 assays and counter-screened using parental CHO cells; (ii) Hit validation and functional profiling will be
performed using analogous assays with CHO cells expressing a mutant CCK1R that mimics the abnormal
conformation of this receptor in a high cholesterol environment. Also, assays will be performed to ensure that
compounds do not stimulate CCK1R internalization, interfere with natural hormone binding and biological
activity, or cause cytotoxicity; and (iii) Lead identification and functional characterization will be performed
using a neuronal cell line that naturally expresses a physiologic density of CCK1R, CHP212 cells, to ensure
absence of intrinsic agonist and trophic activity, and to reconfirm ability to enhance the responses to CCK.
Other characterization will include studying effects on CCK-58, and those at CCK1R from various species and
on CCK2R, as well as a panel of GPCRs, and a broad survey of signaling responses and ADME/Tox assays.