Identification and characterization of an atypical Gαs-biased ß2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis.

G protein-coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For ß2-adrenergic receptors (ß2AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and ß-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable ß-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling-a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: ß2AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from ß-arrestin, in contrast to albuterol and C5-S C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from ß-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of ß2AR actions favorable for treating obstructive lung disease.

Predicted structure and cell signaling of TAS2R14 reveal receptor hyper-flexibility for detecting diverse bitter tastes.

The 25 human bitter taste receptors (TAS2Rs) are expressed on taste and extra-oral cells representing an integrated chemosensory system. The archetypal TAS2R14 is activated by > 150 topographically diverse agonists, raising the question of how this uncharacteristic accommodation is achieved for these GPCRs. We report the computationally derived structure of TAS2R14 with binding sites and energies for five highly diverse agonists. Remarkably, the binding pocket is the same for all five agonists. The energies derived from molecular dynamics are consistent with experiments determining signal transduction coefficients in live cells. TAS2R14 accommodates agonists through the breaking of a TMD3 H-bond instead of the prototypic strong salt bridge, a TMD1,2,7 interaction different from Class A GPCRs, and agonist-promoted TMD3 salt bridges for high affinity (which we confirmed by receptor mutagenesis). Thus, the broadly tuned TAS2Rs accommodate diverse agonists via a single (vs multiple) binding pocket through unique TM interactions for sensing disparate micro-environments.

Biased ß-Agonists Favoring Gs over ß-Arrestin for Individualized Treatment of Obstructive Lung Disease.

Signals from G-protein-coupled receptors (GPCRs) are the most frequently targeted pathways of currently prescribed therapeutics. Rather than being a simple switch, it is now evident that a given receptor can directly initiate multiple signals, and biasing to achieve signal selectivity based on agonist structure is possible. Biased agonists could direct therapeutically favorable pathways while avoiding counterproductive or adverse reaction pathways. For obstructive lung diseases, ß2-adrenergic receptor (ß2AR) agonists act at these receptors on airway smooth muscle (ASM) cells to open the airways by relaxing ASM, improving airflow and morbidity. However, these receptors signal to the G protein Gs (increasing cAMP and promoting relaxation), but also to ß-arrestin (promoting desensitization and a loss of effectiveness). Indeed, ß-agonist use is associated with adverse events in asthma pathogenesis and clinical outcomes which are related to desensitization. ß-agonists favoring Gs coupling over ß-arrestin binding would provide a means of tailoring bronchodilator therapy. In this review, we show how combinatorial methods with a 40 million compound agnostic library led to a new class of biased ß-agonists that do not desensitize, providing an opportunity to personalize therapy in patients who experience poor efficacy or adverse effects from traditional balanced agonists.

Selective Signal Capture from Multidimensional GPCR Outputs with Biased Agonists: Progress Towards Novel Drug Development.

G protein coupled receptors (GPCRs) are a superfamily of transmembrane-spanning receptors that are activated by multiple endogenous ligands and are the most common target for agonist or antagonist therapeutics across a broad spectrum of diseases. Initial characterization within the superfamily suggested that a receptor activated a single intracellular pathway, depending on the G protein to which it coupled. However, it has become apparent that a given receptor can activate multiple different pathways, some being therapeutically desirable, while others are neutral or promote deleterious signaling. The activation of pathways that limit effectiveness of a primary pathway or promote unwanted signals has led to abandonment of some GPCRs as drug targets. However, it is now recognized that the conformation of the receptor in its ligand-bound state can be altered by the structure of the agonist or antagonist to achieve pathway selectivity, a property termed biased signaling. Biased ligands could dramatically expand the number of novel drugs acting at GPCRs for new indications. However, the field struggles with the complexity and uncertainty of these structure-functions relationships. In this review we define the theoretical underpinnings of the biased effect, discuss the methods for measuring bias, and the pitfalls that can lead to incorrect assignments of bias. Using the recent elucidation of a ß2-adrenergic receptor agonist that is biased in favor of Gs coupling over ß-arrestin binding, we provide an example of how large libraries of compounds that are impartial to preconceived notions of agonist binding can be utilized to discover pathway-specific agonists. In this case, an agonist that lacks tachyphylaxis for the treatment of obstructive lung diseases was uncovered, with a structure that was distinctly different from other agonists. We show how biased characteristics were ascertained analytically, and how molecular modeling and simulations provide a structural basis for a restricted signaling repertoire.