Opportunities and Challenges of Screening for Allosteric Modulators of G-Protein Coupled Receptors

Abstract

G-protein coupled receptors (GPCRs) represent the predominant class of targets of drug discovery, but current therapeutics are almost exclusively orthosteric ligands of Family A GPCRs. Recent advances in assay techniques have opened up areas of opportunity at previously poorly tractable GPCR families, whilst a deeper understanding of ligand binding sites on the receptor distinct from those of the native ligand has enabled the progression of a new class of druggable molecules, the allosteric ligand. I describe some of the experimental challenges in progressing allosteric ligands through drug discovery and highlight some of the potential advantages of this approach over classical orthosteric ligands.

Introduction

G-protein coupled receptors (GPCRs) represent the predominant class of targets in drug discovery [1]. Approximately one-third of marketed drugs are active at GPCRs; GPCR specific drugs are marketed in almost every therapeutic area and generate significant sales for the pharmaceutical industry [2, 3]. However, current drugs target only 10% of identified GPCRs.

GPCRs can be classified in three main families based on sequence homology [4] and downstream signalling pathways. To date, only Family A (“rhodopsin like”), which includes receptors for small molecules (such as histamine or chemokines), neurotransmitters (such as dopamine or serotonin), and small peptides (such as motilin) have proved highly tractable as drug targets.

GPCRs exert their physiological effects by transmitting an extracellular stimulus or signal across the cell membrane [1, 4]. When a ligand binds to the receptor it causes a conformational change in the receptor resulting in the activation of downstream signalling pathways. In this respect, GPCRs are prototypic allosteric proteins [5]. They possess multiple binding sites – for ligands and G-protein – and each binding event results in a conformation change to the receptor. Allosteric ligands are those that bind to any site on the receptor which is topographically distinct from that of the endogenous “orthosteric” ligand. They exert their effects by modulating the binding and/or signalling properties of the receptor [6].

There are several advantages of targeting allosteric ligands clinically demonstrated to some extent by cinacalcet (a calcium receptor modulator) [7] and galantamine (a nicotinic acetyl choline receptor modulator) [8]. Orthosteric sites may be poorly chemically tractable due to their conservation across families. Allosteric ligands may generate a lower side effect profile clinically as they only exert their effect in the presence of endogenous ligand, they may show improved selectivity as they can target less conserved regions on receptors, and they can act to modulate physiological responses positively or negatively [9-12].

Tools and Methods to Facilitate Studies of Allosteric Modulators of GPCRs

Functional Assay Formats

The development of functional screening methodologies where information can be derived about the result of the interaction of orthosteric ligand, compound of interest, and target has been the single biggest contributing factor to the emergence of allosteric modulators as tools for exploiting previously intractable drug targets. This development came about as a result of dramatic improvements in sensitivity and throughput of plate based readers capable of measuring surrogate end-points of GPCR activation and enabled allosteric molecules to be uncovered and characterized [12, 13]. Assay systems, which detect positive allosteric modulators which act to enhance activity or negative allosteric modulators whose action attenuates the activity of the orthosteric ligand (see figure 1), reveal the potential range of activities of compounds acting at sites distinct from that occupied by the orthosteric ligand.

Figure 1: Typical timecourse of an orthosteric agonist induced cellular response potentiated in the presence of a positive allosteric modulator or attenuated in the presence of a negative allosteric modulator

Figure 2 illustrates a particularly important attribute of allosteric ligands. It shows a rightward shift of the agonist curve with negative allosteric modulators and a leftward shift with positive allosteric modulators. The example in figure 2 allows us to differentiate negative allosteric modulation from orthosteric antagonism as the effect is saturable, such that a point is reached where adding additional modulator has no further observable effect. This example shows modulators which affect the potency but not efficacy of the orthosteric ligand, although examples have been found of compounds affecting potency and efficacy or efficacy alone [10].

Figure 2: Orthosteric agonist concentration effect curve shifted to the left in the presence of a positive allosteric modulator or the right in the presence of a negative allosteric modulator

Assays can be designed following a “dual addition” methodology where the target of choice is challenged with modulator then orthosteric agonist. The resulting output is shown in figure 3 where response to orthosteric agonist challenge can be monitored for potentiation.

Figure 3: Typical timecourse observed in a “dual addition” protocol measuring increases in intracellular calcium in response to G-protein coupled receptor stimulation. First addition of compound results in no change in signal, but subsequent addition of orthosteric agonist results in a potentiation of signal

In this type of experimental design, the concentration of orthosteric ligand is critical. If the selected agonist concentration is too low or too high then the potentiation signal window is compromised and it becomes impossible to robustly characterize compound activity over the kinds of prolonged timeframes required for drug development. Since the degree of potentiation will vary from modulator to modulator depending on its mechanism and whether it alters efficacy, potency, or both for the orthosteric agonist, there remains a trade off between signal and the variability of both agonist and modulator responses in the test system.

Probe Dependency

The effect of an allosteric ligand is intrinsically linked to the orthosteric agonist it is acting with. The impact of probe dependency was illustrated using the M2 muscarinic acetyl choline receptor as a model system [15]. In this example, the modulator eburnamonine has profoundly different effects depending on the orthosteric agonist used. The potency of arecoline is increased fifteenfold, the potency of pilocarpine drops thirtyfold, whilst carbachol and APE are barely affected. Thus, assays intended to predict the activity of allosteric ligands in vivo must use the endogenous ligand agonist whose activity is to be targeted in vivo.

Allosteric Ligands with Intrinsic Efficacy

Early small molecule Glucagon-like Peptide 1 (GLP-1) agonists highlighted another potentially useful facet of allosteric compounds. They acted as activators of the GLP-1 receptor in their own right but did not interfere with the binding of the endogenous agonist [16, 17]. They are behaving as allosteric agonists. However, they are also capable of potentiating the effect of the orthosteric agonist GLP-1.

Controllable Receptor Expression

Study of a number of poorly tractable targets, such as the metabotropic glutamate receptors (mGluRs), has been severely hampered by the fact that prolonged surface expression of the target receptor leads to cellular toxicity. In the case of mGluRs, this is exacerbated by the production of glutamate by cells in culture leading to receptor desensitization and downregulation of expression. The development of several systems where expression of receptor is kept under tight control and induced on demand at the time of assay has allowed these previously troublesome targets to be characterised [18-21]. The use of these systems highlighted that receptor expression level is critical to reported activity. As the level of receptor expression increases, so the possibility that a positive modulator will show the type of allosteric agonism described above increases. In functional assay systems such allosteric agonism can cause receptor desensitization and resulting potentiation can be missed. It is critical that receptor expression at the time of assay be tailored to closely match that seen in native systems. In such situations it is advantageous to utilize an expression system where expression level can be fine-tuned by altering the concentration of induction agent or altering the transfection or transduction conditions until the desired level is achieved.

Conclusions

G-protein coupled receptors (GPCRs) continue to be a major focus of interest for drug discovery, and attention has turned from small molecule orthosteric antagonists of highly tractable family A GPCRs to historically less tractable but equally physiologically important targets.

It has long been established that GPCRs contain allosteric binding sites topographically distinct from the orthosteric ligand binding pocket, where a binding event causes a conformational change that affects the magnitude and duration of a downstream signalling event. Allosteric ligands identified using the approaches and tools described are simply ligands which take advantage of this promiscuous property of GPCRs.

What has become clear is that ligands that target these allosteric sites are attractive due to the fact that they can enable selectivity, are probe dependent, act cooperatively with orthosteric ligands, and can affect affinity with or without effects on efficacy, affording the possibility of exquisite fine-tuning of the native biological response being targeted.

The opportunities afforded by this class of compound are clear, and the approaches described have enabled the pharmaceutical industry to begin to probe the attributes of this previously poorly understood class of compounds and apply it to the development of drugs. The challenge which remains is one which faces all such efforts: the reliable detection of activity; the validation of the mechanism as a druggable target; and the discovery of compounds which clear the hurdles of efficacy and safety required to make a medicine.

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