Approaches for Probing Allosteric Interactions at 7 Transmembrane Spanning Receptors

Abstract

In recent years, allosteric modulation of 7 transmembrane spanning receptors (7TMRs) has become a highly productive and exciting field of receptor pharmacology and drug discovery efforts. Positive and negative allosteric modulators (PAMs and NAMs, respectively) present a number of pharmacological and therapeutic advantages over conventional orthosteric ligands, including improved receptor-subtype selectivity, a lower propensity to induce receptor desensitization, the preservation of endogenous temporal and spatial activation of receptors, greater chemical flexibility for optimization of drug metabolism and pharmacokinetic parameters, and saturability of effect at target receptors, thus improving safety concerns and risk of overdose. Additionally, the relatively new concept of allosteric modulator-mediated receptor signal bias opens up a number of intriguing possibilities for PAMs, NAMs, and allosteric agonists, including the potential to selectively activate therapeutically beneficial signaling cascades, which could yield a superior tissue selectivity and side effect profile of allosteric modulators. However, there are a number of considerations and caveats that must be addressed when screening for and characterizing the properties of 7TMR allosteric modulators. Mode of pharmacology, methodology used to monitor receptor activity, detection of appropriate downstream analytes, selection of orthosteric probe, and assay time-course must all be considered when implementing any high-throughput screening campaign or when characterizing the properties of active compounds. Yet compared to conventional agonist/antagonist drug discovery programs, these elements of assay design are often a great deal more complicated when working with 7TMRs allosteric modulators. Moreover, for classical pharmacological methodologies and analyses, like radioligand binding and the assessment of compound affinity, the properties of allosteric modulators yield data that are more nuanced than orthosteric ligand–receptor interactions. In this review, we discuss the current methodologies being used to identify and characterize allosteric modulators, lending insight into the approaches that have been most successful in accurately and robustly identifying hit compounds. New label-free technologies capable of detecting phenotypic cellular changes in response to receptor activation are powerful tools well suited for assessing subtle or potentially masked cellular responses to allosteric modulation of 7TMRs. Allosteric modulator-induced receptor signal bias and the assay systems available to probe the various downstream signaling outcomes of receptor activation are also discussed.

Development of allosteric modulators of GPCRs for treatment of CNS disorders

“…Development and optimization of allosteric modulators of GPCRs as drug candidates presents multiple challenges (Conn, 2012; Klein et al, 2013; Melancon et al, 2012). The success of lead compound development for a number of allosteric modulators of GPCRs has established a set of drug optimization strategies for this class.…”

Detection and Quantification of Allosteric Modulation of Endogenous M4 Muscarinic Acetylcholine Receptor Using Impedance-Based Label-Free Technology in a Neuronal Cell Line

“…19 As label-free technologies detect global cellular changes upon receptor activation, they are ideal systems to evaluate and/or screen for allosteric modulation of GPCRs in native systems endogenously expressing the receptor of interest. Indeed, in the past few years, potentiation of orthosteric ligands by allosteric modulators has been measured using both impedance-based 21 and optical-based 22 technologies. However, these experiments were performed in recombinant cells where the receptors of interest have been heterologously expressed.…”

Effect of magnitude and variability of energy of activation in multisite ultrasensitive biochemical processes

“…Our goal in generalizing the MWC model was to explore the qualitative theoretical consequences of allowing the conformational free energies of different sites to vary, and not to make a tool for empirical fitting to data. On this point, however, it should be noted that Stefan et al [ 48 ] have shown how an extended MWC model such as the one developed here can be used in parameter estimation, and experimental methods to measure MWC parameters are constantly improving [ 49 – 51 ]. Another useful recent tool is a method developed by Gruber et al [ 52 ] which facilitates the determination of parameters in the MWC model, by finding a theoretical relationship between the Hill coefficient and model parameters.…”