Harikumar, Kaleeckal G

G protein-coupled receptors (GPCRs) are among the most common drug targets, with numerous agonists and antagonists approved for clinical use. More recently, it has been appreciated that drugs can also modulate the action of natural agonists of these receptors, thus providing unique clinical advantages. Here, we describe the identification and characterization of a small molecule positive allosteric modulator (PAM) of secretin action at the class B G protein-coupled secretin receptor. This phenyl(trifluoromethyl)-pyrimidine can occupy the secretin receptor without stimulating its internalization, yet priming it to enhance both the potency and efficacy of the action of natural secretin. This is also shown to exhibit its effects on cells expressing low numbers of these receptors, without enhancing the effects of other structurally related hormones acting at other class B GPCRs. The mechanism responsible for this PAM effect is the slowing of the off-rate of receptor-bound secretin. This compound can serve as a lead to the development of other drugs that enhance the action of natural endogenous secretin and can be utilized to explore the potential therapeutic utility of such compounds.

Abstract 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. Here, we present development and use of a novel method capable of overcoming existing challenges by combining distributions (or spectrograms) of FRET efficiencies from populations of fluorescently tagged proteins associating into oligomeric complexes in live cells with diffusion-like trajectories of FRET donors and acceptors obtained from molecular dynamics (MD) simulations. Our approach provides an atom-level picture of the binding interfaces within oligomers of the human secretin receptor (hSecR) in live cells and allows for extraction of mechanistic insights into the function of GPCRs oligomerization. This FRET-MD spectrometry approach is a robust platform for investigating protein-protein binding mechanisms and opens a new avenue for investigating stable as well as fleeting quaternary structures of any membrane proteins in living cells.

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. Extensive characterization of the two top “hits” from the previous effort demonstrated them to fulfill the desired pharmacologic profile. We undertook analog-by-catalog expansion of this scaffold using 65 commercially available analogs. In this effort, we were able to eliminate an off-target effect observed for this scaffold while retaining its activity as a positive allosteric modulator of CCK1R in both normal and high cholesterol membrane environments. These insights should be useful in the rational medicinal chemical enhancement of this scaffold and in the future development of candidates to advance to pre-clinical proof-of-concept and to clinical trials.

How to catch a dynamic state AMP-activated protein kinase (AMPK) is a key sensor of energy status in eukaryotes. Its dynamic structure is regulated by allosteric factors including phosphorylation and binding of nucleotides and metabolites. Yan et al. developed conformation-specific antibodies that trap AMPK in a fully inactive state that has experienced a large, domain-level rotation. Biophysical experiments in cells and in vitro are consistent with the structural work and support a model in which the activation loop is fully exposed in the completely inactive, dephosphorylated state. These structures inform our understanding of the complex allosteric behavior in this crucial metabolic regulator. Science , abe7565, this issue p. 413

Previous studies have indicated that the G-protein-coupled secretin receptor is present as a homodimer, organized through symmetrical contacts in transmembrane domain IV, and that receptor dimerization is critical for high-potency signalling by secretin. However, whether all of the receptor exists in the dimeric form or if this is regulated is unclear. We used measures of quantal brightness of the secretin receptor tagged with monomeric enhanced green fluorescent protein (mEGFP) and spatial intensity distribution analysis to assess this. Calibration using cells expressing plasma membrane-anchored forms of mEGFP initially allowed us to demonstrate that the epidermal growth factor receptor is predominantly monomeric in the absence of ligand and while wild-type receptor was rapidly converted into a dimeric form by ligand, a mutated form of this receptor remained monomeric. Equivalent studies showed that, at moderate expression levels, the secretin receptor exists as a mixture of monomeric and dimeric forms, with little evidence of higher-order complexity. However, sodium butyrate-induced up-regulation of the receptor resulted in a shift from monomeric towards oligomeric organization. In contrast, a form of the secretin receptor containing a pair of mutations on the lipid-facing side of transmembrane domain IV was almost entirely monomeric. Down-regulation of the secretin receptor-interacting G-protein Gαs did not alter receptor organization, indicating that dimerization is defined specifically by direct protein–protein interactions between copies of the receptor polypeptide, while short-term treatment with secretin had no effect on organization of the wild-type receptor but increased the dimeric proportion of the mutated receptor variant.

ABSTRACT Angiotensin (ANGII) and secretin (SCT) share overlapping, interdependent osmoregulatory functions in brain, where SCT peptide/receptor function is required for ANGII action, yet the molecular basis is unknown. Since receptors for these peptides (AT1aR, SCTR) are coexpressed in osmoregulatory centers, a possible mechanism is formation of a cross‐class receptor heterocomplex. Here, we demonstrate such a complex and its functional importance to modulate signaling. Association of AT1aR with SCTR reduced ability of SCT to stimulate cyclic adenosine monophosphate (cAMP), with signaling augmented in presence of ANGII or constitutively active AT1aR. Several transmembrane (TM) peptides of these receptors were able to affect their conformation within complexes, reducing receptor BRET signals. AT1aR TM1 affected only formation and activity of the heterocomplex, without effect on homomers of either receptor, and reduced SCT‐stimulated cAMP responses in cells expressing both receptors. This peptide was active in vivo by injection into mouse lateral ventricle, thereby suppressing water‐drinking behavior after hyperosmotic shock, similar to SCTR knockouts. This supports the interpretation that active conformation of AT1aR is a key modulator of cAMP responses induced by SCT stimulation of SCTR. The SCTR/AT1aR complex is physiologically important, providing differential signaling to SCT in settings of hyperosmolality or food intake, modulated by differences in levels of ANGII.—Lee, L. T. O., Ng, S. Y. L., Chu, J. Y. S., Sekar, R., Harikumar, K. G., Miller, L. J., Chow, B. K. C. Transmembrane peptides as unique tools to demonstrate the in vivo action of a cross‐class GPCR heterocomplex. FASEB J . 28, 2632–2644 (2014). www.fasebj.org

The glucagon-like peptide-1 receptor (GLP-1R) is a family B G protein-coupled receptor and an important drug target for the treatment of type II diabetes, with activation of pancreatic GLP-1Rs eliciting glucose-dependent insulin secretion. Currently, approved therapeutics acting at this receptor are peptide based, and there is substantial interest in small molecule modulators for the GLP-1R. Using a variety of resonance energy transfer techniques, we demonstrate that the GLP-1R forms homodimers and that transmembrane helix 4 (TM4) provides the primary dimerization interface. We show that disruption of dimerization using a TM4 peptide, a minigene construct encoding TM4, or by mutation of TM4, eliminates G protein-dependent high-affinity binding to GLP-1(7-36)NH 2 but has selective effects on receptor signaling. There was <10-fold decrease in potency in cAMP accumulation or ERK1/2 phosphorylation assays but marked loss of intracellular calcium mobilization by peptide agonists. In contrast, there was near-complete abrogation of the cAMP response to an allosteric agonist, compound 2, but preservation of ERK phosphorylation. Collectively, this indicates that GLP-1R dimerization is important for control of signal bias. Furthermore, we reveal that two small molecule ligands are unaltered in their ability to allosterically modulate signaling from peptide ligands, demonstrating that these modulators act in cis within a single receptor protomer, and this has important implications for small molecule drug design.