Estimation of Agonist Activity at G Protein-Coupled Receptors: Analysis of M₂ Muscarinic Receptor Signaling through G_i/o,G_s, and G₁₅

Abstract

We developed novel methods for analyzing the concentrationresponse curve of an agonist to estimate the product of observed affinity and intrinsic efficacy, expressed relative to that of a standard agonist. This parameter, termed intrinsic relative activity (RA i ), is most applicable for the analysis of responses at G protein-coupled receptors. RA i is equivalent to the potency ratios that agonists would exhibit in a hypothetical, highly sensitive assay in which all agonists behave as full agonists, even those with little intrinsic efficacy. We investigated muscarinic responses at the M 2 receptor, including stimulation of phosphoinositide hydrolysis through G Drug discovery often involves testing compounds in isolated tissues and high throughput assays to determine activity at target receptors. In the case of agonists, the measured parameters are usually EC 50 and E max , whereas the parameters of greater relevance to drug design are observed affinity and intrinsic efficacy.1 If the receptor is a ligand-gated ion channel, then receptor activation can be measured directly as whole cell current, and the EC 50 and E max are reasonable estimates of observed affinity and intrinsic efficacy provided that desensitization is not excessive. It would seem that, in most instances, the observed affinity and intrinsic efficacy of an agonist for a ligand-gated ion channel would be constant, regardless of the tissue or cell in which the receptor is expressed.The situation is more complex for a G protein-coupled receptor (GPCR). First, GPCRs are inactive in isolation and must interact with a G protein to elicit a response. It is possible that the G protein with which the receptor interacts selects for a receptor conformation having a unique agonist profile and that the observed affinity and intrinsic efficacy of the agonist-receptor complex may be G protein-specific or influenced by other proteins interacting with the receptor (Leff et al., 1997;Berg et al., 1998). Second, because it is difficult to measure receptor activation directly, most assays involve measuring a downstream response, and the corresponding EC 50 and E max values may vary, depending on the point in the signaling cascade at which the experimenter measures the response. Therefore, the magnitude of the response elicited by a GPCR is usually not proportional to receptor activation, and EC 50 and E max are not equivalent to observed affinity and intrinsic efficacy. Rather, EC 50 and 1 Here and throughout the article, we use the term "observed intrinsic efficacy" to refer to Furchgott's definition of "intrinsic efficacy" (Furchgott, 1966), which denotes the amount of activated receptors. As described previously (Ehlert, 2000), it is useful to discriminate between the latter definition of observed intrinsic efficacy and the ratio of agonist affinity constants for ground and active conformations of the receptor (intrinsic efficacy).

Biased Type 1 Cannabinoid Receptor Signaling Influences Neuronal Viability in a Cell Culture Model of Huntington Disease

“…and STHdh Q111/Q111 datasets and constrained to be greater than -15 (Griffin et al, 2007;Ehlert, 2015). Relative activity (DlogR) was calculated in Prism as the difference between transduction coefficients [logR (t/K A )] values for two ligands, a "test" ligand, and a reference ligand (here WIN) as measured between sample-matched replicates (Kenakin et al, 2012) (eq.…”

“…Although it is often considered ideal to choose the endogenous receptor agonist as a reference ligand (Kenakin and Christopoulos, 2013), WIN was chosen as a reference ligand for these studies because: 1) it is a widely used reference compound to study CB 1 -dependent signaling (Lauckner et al, 2005); 2) it acted as an agonist in all assays with nonsignificant differences in EC 50 observed between assays; and 3) we wanted to determine whether the two endogenous cannabinoids, AEA and 2-AG, were inherently biased either in wild-type (STHdh Q7/Q7 ) or mHtt-expressing (STHdh Q111/Q111 ) cells. Concentration-response curves for ERK, BRET 2 (CB 1 /b-arrestin1), CREB, phospholipase C (PLC)b3, and Akt are presented as % of WIN E max in STHdh Q7/Q7 cells (Griffin et al, 2007). Concentration-response curves were fit to nonlinear regression with variable slope (four-parameter) model to determine pEC 50 and E max (Table 1), or global nonlinear regression using the operational model (Black and Leff, 1983;Ehlert et al, 2011;Kenakin et al, 2012) (eq.…”

The Effective Application of Biased Signaling to New Drug Discovery

“…Such a scale can be derived from the Black/Leff operational model of agonism (Black and Leff, 1983), which furnishes estimates of affinity in the form of K A values (equilibrium dissociation constants of agonist-receptor complexes) and agonist efficacy (in the form of t values for a given signaling pathway). A null method to derive such a factor (termed RA i denoted as receptor activity) has been published (Ehlert et al, 1999) and subsequently applied to agonist bias (Griffin et al, 2007;Figueroa et al, 2009;Tran et al, 2009;Ehlert et al, 2011).…”

Biased Ligands for Better Cardiovascular Drugs

“…Several attempts have been made to standardize nomenclature and criteria for determining ligand bias, 71,134 and recently several groups have introduced quantitative methods to assess ligand bias. [135][136][137] Further effort to develop these approaches is warranted, as is diligence in recognizing their assumptions and applying their criteria, to most efficiently move the field of ligand bias and functional selectivity forward to clinical relevance.…”