Structure-activity relationships of thiadiazole agonists of the human secretin receptor.

Agonists of the secretin receptor have potential applications for diseases of the cardiovascular, gastrointestinal, and metabolic systems, yet no clinically-active non-peptidyl agonists of this receptor have yet been developed. In the current work, we have identified a new small molecule lead compound with this pharmacological profile. We have prepared and characterized a systematic structure-activity series around this thiadiazole scaffold to better understand the molecular determinants of its activity.

Cholesterol-dependent dynamic changes in the conformation of the type 1 cholecystokinin receptor affect ligand binding and G protein coupling.

Development of optimal therapeutics for disease states that can be associated with increased membrane cholesterol requires better molecular understanding of lipid modulation of the drug target. Type 1 cholecystokinin receptor (CCK1R) agonist actions are affected by increased membrane cholesterol, enhancing ligand binding and reducing calcium signaling, while agonist actions of the closely related CCK2R are not. In this work, we identified a set of chimeric human CCK1R/CCK2R mutations that exchange the cholesterol sensitivity of these 2 receptors, providing powerful tools when expressed in CHO and HEK-293 model cell lines to explore mechanisms.

Mechanistic insights from the atomic-level quaternary structure of short-lived GPCR oligomers in live cells.

The functional significance of the interactions between proteins in living cells to form short-lived quaternary structures cannot be overemphasized. Yet, quaternary structure information is not captured by current methods, neither can those methods determine structure within living cells. The dynamic versatility, abundance, and functional diversity of G protein-coupled receptors (GPCRs) pose myriad challenges to existing technologies but also present these proteins as the ideal testbed for new technologies to investigate the complex inter-regulation of receptor-ligand, receptor-receptor, and receptor-downstream effector interfaces in living cells.

Impact of secretin receptor homo-dimerization on natural ligand binding.

Class B G protein-coupled receptors can form dimeric complexes important for high potency biological effects. Here, we apply pharmacological, biochemical, and biophysical techniques to cells and membranes expressing the prototypic secretin receptor (SecR) to gain insights into secretin binding to homo-dimeric and monomeric SecR. Spatial proximity between peptide and receptor residues, probed by disulfide bond formation, demonstrates that the secretin N-terminus moves from adjacent to extracellular loop 3 (ECL3) at wild type SecR toward ECL2 in non-dimerizing mutants.

Mechanism of Action and Structure-Activity Relationships of Tetracyclic Small Molecules Acting as Universal Positive Allosteric Modulators of the Cholecystokinin Receptor.

As part of an ongoing effort to develop a drug targeting the type 1 cholecystokinin receptor (CCK1R) to help prevent and/or treat obesity, we recently performed a high throughput screening effort of small molecules seeking candidates that enhanced the action of the natural agonist, CCK, thus acting as positive allosteric modulators without exhibiting intrinsic agonist action. Such probes would be expected to act in a temporally finite way to enhance CCK action to induce satiety during and after a meal and potentially even modulate activity at the CCK1R in a high cholesterol environment present in some obese patients. The current work focuses on the best scaffold, representing tetracyclic molecules identified through high throughput screening we previously reported.

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

Obesity has become a prevailing health burden globally and particularly in the US. It is associated with many health problems, including cardiovascular disease, diabetes and poorer mental health. Hence, there is a high demand to find safe and effective therapeutics for sustainable weight loss.

Secretin Amino-Terminal Structure-Activity Relationships and Complementary Mutagenesis at the Site of Docking to the Secretin Receptor.

Class B1 G protein-coupled receptors are activated by peptides, with amino-terminal regions critical for biologic activity. Although high resolution structures exist, understanding of key features of the peptide activation domain that drive signaling is limited. In the secretin receptor (SecR) structure, interactions are observed between peptide residues His and Ser and seventh transmembrane segment (TM7) receptor residue E373.

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

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.

Structure of an AMPK complex in an inactive, ATP-bound state.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility.

Environment-Sensitive Fluorescence of 7-Nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-Labeled Ligands for Serotonin Receptors.

Serotonin is a neurotransmitter that plays a crucial role in the regulation of several behavioral and cognitive functions by binding to a number of different serotonin receptors present on the cell surface. We report here the synthesis and characterization of several novel fluorescent analogs of serotonin in which the fluorescent NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group is covalently attached to serotonin. The fluorescent ligands compete with the serotonin receptor specific radiolabeled agonist for binding to the receptor.

Roles of Cholecystokinin in the Nutritional Continuum. Physiology and Potential Therapeutics.

Cholecystokinin is a gastrointestinal peptide hormone with important roles in metabolic physiology and the maintenance of normal nutritional status, as well as potential roles in the prevention and management of obesity, currently one of the dominant causes of direct or indirect morbidity and mortality. In this review, we discuss the roles of this hormone and its receptors in maintaining nutritional homeostasis, with a particular focus on appetite control. Targeting this action led to the development of full agonists of the type 1 cholecystokinin receptor that have so far failed in clinical trials for obesity.

Structures of the human cholecystokinin 1 (CCK1) receptor bound to Gs and Gq mimetic proteins provide insight into mechanisms of G protein selectivity.

G protein-coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here, we used cryo-electron microscopy (cryo-EM) to determine structures of the cholecystokinin (CCK) type 1 receptor (CCK1R) bound to the CCK peptide agonist, CCK-8 and 2 distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs.

Single-molecule FRET imaging of GPCR dimers in living cells.

Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells.

Discovery of small molecule positive allosteric modulators of the secretin receptor.

The secretin receptor (SCTR) is a prototypic Class B1 G protein-coupled receptor (GPCR) that represents a key target for the development of therapeutics for the treatment of cardiovascular, gastrointestinal, and metabolic disorders. However, no non-peptidic molecules targeting this receptor have yet been disclosed. Using a high-throughput screening campaign directed at SCTR to identify small molecule modulators, we have identified three structurally related scaffolds positively modulating SCTRs.

Evidence that specific interactions play a role in the cholesterol sensitivity of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol's general effects on the membrane is not well-understood. We performed coarse-grained molecular dynamics (CGMD) simulations coupled with structural bioinformatics approaches on the β-adrenergic receptor (βAR) and the cholecystokinin (CCK) receptor subfamily. The βAR has been shown to be sensitive to membrane cholesterol and cholesterol molecules have been clearly resolved in numerous βAR crystal structures.

Development of a Testing Funnel for Identification of Small-Molecule Modulators Targeting Secretin Receptors.

The secretin receptor (SCTR), a prototypical class B G protein-coupled receptor (GPCR), exerts its effects mainly by activating Gαs proteins upon binding of its endogenous peptide ligand secretin. SCTRs can be found in a variety of tissues and organs across species, including the pancreas, stomach, liver, heart, lung, colon, kidney, and brain. Beyond that, modulation of SCTR-mediated signaling has therapeutic potential for the treatment of multiple diseases, such as heart failure, obesity, and diabetes.

Rational development of a high-affinity secretin receptor antagonist.

The secretin receptor is a prototypic class B GPCR with substantial and broad pharmacologic importance. The aim of this project was to develop a high affinity selective antagonist as a new and important pharmacologic tool and to aid stabilization of this receptor in an inactive conformation for ultimate structural characterization. Amino-terminal truncation of the natural 27-residue ligand reduced biological activity, but also markedly reduced binding affinity.

Kinetics of Gallbladder Emptying During Cholecystokinin Cholescintigraphy as an Indicator of In Vivo Hormonal Sensitivity.

Cholecystokinin cholescintigraphy is used clinically to quantify gallbladder ejection fraction as an indicator of functional gallbladder disorder. It can also provide the opportunity to quantify an individual's responsiveness to the physiologic stimulant of gallbladder contraction, cholecystokinin, which is a major regulator of appetite and postprandial satiety. In the current work, we use cholecystokinin cholescintigraphy to quantify the kinetics of gallbladder emptying, including average and peak rates, in response to a standard cholecystokinin infusion.

Crystal structure of the Frizzled 4 receptor in a ligand-free state.

Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms. As central mediators in this complex signalling pathway, FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research. Here we present an atomic-resolution structure of the human Frizzled 4 receptor (FZD4) transmembrane domain in the absence of a bound ligand.

Bioluminescence Resonance Energy Transfer (BRET) Assay for Determination of Molecular Interactions in Living Cells.

The bioluminescence resonance energy transfer (BRET) assay can be used as an indicator of molecular approximation and/or interaction. A significant resonance energy transfer signal is generated when the acceptor, having the appropriate spectral overlap with the donor emission, is approximated with the donor. In the example provided, proteins tagged with bioluminescent luciferase (Rlu) as donor and yellow fluorescent protein (YFP) as acceptor were co-expressed in cells.

Streptavidin Bead Pulldown Assay to Determine Protein Homooligomerization.

Pulldown assay is a conventional method to determine protein-protein interactions . Expressing a protein of interest with two different tags allows testing whether both versions can be captured via one of the two tags as homooligomeric complex. This protocol is based on streptavidin bead capture of a biotinylated protein and co-associated Flag-tagged protein using Streptavidin MagBeads.

Detection of Membrane Protein Interactions by Cell-based Tango Assays.

The Tango assay is a protein-protein interaction assay, in which a transcription factor (rTA) is fused to a membrane-bound protein via a linker that contains a cleavage site for TEV protease, whereas a soluble interaction partner is fused to TEV protease (Barnea , 2008). Association between the two interaction partners leads to an efficient cleavage of the transcription factor, allowing it to translocate to the nucleus and activate a luciferase reporter gene as measurement of the interactions. In this modified assay, we fused one copy of the membrane-spanning amyloid precursor protein (APP) C99 region to TEV site-rTA (C99-TEV site-rTA) and a second copy to TEV protease (C99-TEV) to analyze intramembrane C99-C99 interaction in live cells.

γ-Secretase Epsilon-cleavage Assay.

γ-Secretase epsilon-cleavage assay is derived from the cell-based Tango assay (Kang ., 2015), and is a fast and sensitive method to determine the initial cleavage of C99 by γ-secretase. In this protocol, we use HTL cells, which are HEK293 cells with a stably integrated luciferase reporter under the control of the bacterial tetO operator element, in which C99 C terminally fused to a reversed tetracyclin-inducible activator (rTA) transcriptional activator is expressed.

Dimerization of the transmembrane domain of amyloid precursor protein is determined by residues around the γ-secretase cleavage sites.

One of the hallmarks of Alzheimer's disease is the formation of extracellular amyloid plaques that consist mainly of abnormally aggregated forms of amyloid β (Aβ) peptides. These peptides are generated by γ-secretase-catalyzed cleavage of a dimeric membrane-bound C-terminal fragment (C99) of the amyloid precursor protein. Although C99 homodimerization has been linked to Aβ production and changes in the aggregation-determining Aβ42/Aβ40 ratio, the motif through which C99 dimerizes has remained controversial.