Laura H. Heitman

Structural basis for allosteric regulation of GPCRs by sodium ions.

GPCR Close-Up Structures of G protein–coupled receptors (GPCRs) determined in the past few years, have provided insight into the function of this important family of membrane proteins. Liu et al. (p. 232) used a protein-engineering strategy to produce a stabilized version of the human A2Aadenosine receptor (A2AAR). The high-resolution structure reveals the position of about 60 internal waters, which suggests an almost continuous channel in the GPCR and can explain the allosteric effects of Na+ on ligand binding and how cholesterol may contribute to GPCR stabilization. A protein-engineering strategy yields a closer look at the receptor-bound water, sodium, and lipid molecules. Pharmacological responses of G protein–coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR structures. We reengineered the human A2A adenosine receptor by replacing its third intracellular loop with apocytochrome b562RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp2.50. Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function.

Drug-target residence time--a case for G protein-coupled receptors.

A vast number of marketed drugs act on G protein‐coupled receptors (GPCRs), the most successful category of drug targets to date. These drugs usually possess high target affinity and selectivity, and such combined features have been the driving force in the early phases of drug discovery. However, attrition has also been high. Many investigational new drugs eventually fail in clinical trials due to a demonstrated lack of efficacy. A retrospective assessment of successfully launched drugs revealed that their beneficial effects in patients may be attributed to their long drug‐target residence times (RTs). Likewise, for some other GPCR drugs short RT could be beneficial to reduce the potential for on‐target side effects. Hence, the compounds’ kinetics behavior might in fact be the guiding principle to obtain a desired and durable effect in vivo. We therefore propose that drug‐target RT should be taken into account as an additional parameter in the lead selection and optimization process. This should ultimately lead to an increased number of candidate drugs moving to the preclinical development phase and on to the market. This review contains examples of the kinetics behavior of GPCR ligands with improved in vivo efficacy and summarizes methods for assessing drug‐target RT.

Functional efficacy of adenosine A2A receptor agonists is positively correlated to their receptor residence time

BACKGROUND AND PURPOSE The adenosine A2A receptor belongs to the superfamily of GPCRs and is a promising therapeutic target. Traditionally, the discovery of novel agents for the A2A receptor has been guided by their affinity for the receptor. This parameter is determined under equilibrium conditions, largely ignoring the kinetic aspects of the ligand‐receptor interaction. The aim of this study was to assess the binding kinetics of A2A receptor agonists and explore a possible relationship with their functional efficacy.

The Role of a Sodium Ion Binding Site in the Allosteric Modulation of the A2A Adenosine G Protein-Coupled Receptor

The function of G protein-coupled receptors (GPCRs) can be modulated by a number of endogenous allosteric molecules. In this study, we used molecular dynamics, radioligand binding, and thermostability experiments to elucidate the role of the recently discovered sodium ion binding site in the allosteric modulation of the human A(2A) adenosine receptor, conserved among class A GPCRs. While the binding of antagonists and sodium ions to the receptor was noncompetitive in nature, the binding of agonists and sodium ions appears to require mutually exclusive conformational states of the receptor. Amiloride analogs can also bind to the sodium binding pocket, showing distinct patterns of agonist and antagonist modulation. These findings suggest that physiological concentrations of sodium ions affect functionally relevant conformational states of GPCRs and can help to design novel synthetic allosteric modulators or bitopic ligands exploiting the sodium ion binding pocket.

Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists.

CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer. These disease associations have motivated numerous preclinical studies and clinical trials (see http://www.clinicaltrials.gov) in search of therapies that target the CCR2–chemokine axis. To aid drug discovery efforts, here we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein–protein interactions, receptor–chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.

Cannabinoid CB2 receptor ligand profiling reveals biased signalling and off-target activity

The cannabinoid CB2 receptor (CB2R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB2R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB2R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB2R agonists to study the role of CB2R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research.

Fragment Screening of GPCRs Using Biophysical Methods: Identification of Ligands of the Adenosine A2A Receptor with Novel Biological Activity

Fragment-based drug discovery (FBDD) has proven a powerful method to develop novel drugs with excellent oral bioavailability against challenging pharmaceutical targets such as protein-protein interaction targets. Very recently the underlying biophysical techniques have begun to be successfully applied to membrane proteins. Here we show that novel, ligand efficient small molecules with a variety of biological activities can be found by screening a small fragment library using thermostabilized (StaR) G protein-coupled receptors (GPCRs) and target immobilized NMR screening (TINS). Detergent-solubilized StaR adenosine A(2A) receptor was immobilized with retention of functionality, and a screen of 531 fragments was performed. Hits from the screen were thoroughly characterized for biochemical activity using the wild-type receptor. Both orthosteric and allosteric modulatory activity has been demonstrated in biochemical validation assays. Allosteric activity was confirmed in cell-based functional assays. The validated fragment hits make excellent starting points for a subsequent hit-to-lead elaboration program.

Dual-Point Competition Association Assay: A Fast and High-Throughput Kinetic Screening Method for Assessing Ligand-Receptor Binding Kinetics

The concept of ligand-receptor binding kinetics is emerging as an important parameter in the early phase of drug discovery. Since the currently used kinetic assays are laborious and low throughput, we developed a method that enables fast and large format screening. It is a so-called dual-point competition association assay, which measures radioligand binding at two different time points in the absence or presence of unlabeled competitors. Specifically, this assay yields the kinetic rate index (KRI), which is a measure for the binding kinetics of the unlabeled ligands screened. As a prototypical drug target, the adenosine A1 receptor (A1R) was chosen for assay validation and optimization. A screen with 35 high-affinity A1R antagonists yielded seven compounds with a KRI value above 1.0, which indicated a relatively slow dissociation from the target. All other compounds had a KRI value below or equal to 1.0, predicting a relatively fast dissociation rate. Several compounds were selected for follow-up kinetic quantifications in classical kinetic assays and were shown to have kinetic rates that corresponded to their KRI values. The dual-point assay and KRI value may have general applicability at other G-protein-coupled receptors, as well as at drug targets from other protein families.

Controlling the Dissociation of Ligands from the Adenosine A2A Receptor through Modulation of Salt Bridge Strength.

The association and dissociation kinetics of ligands binding to proteins vary considerably, but the mechanisms behind this variability are poorly understood, limiting their utilization for drug discovery. This is particularly so for G protein-coupled receptors (GPCRs) where high resolution structural information is only beginning to emerge. Engineering the human A2A adenosine receptor has allowed structures to be solved in complex with the reference compound ZM241385 and four related ligands at high resolution. Differences between the structures are limited, with the most pronounced being the interaction of each ligand with a salt bridge on the extracellular side of the receptor. Mutagenesis experiments confirm the role of this salt bridge in controlling the dissociation kinetics of the ligands from the receptor, while molecular dynamics simulations demonstrate the ability of ligands to modulate salt bridge stability. These results shed light on a structural determinant of ligand dissociation kinetics and identify a means by which this property may be optimized.

A Series of 2,4-Disubstituted Quinolines as a New Class of Allosteric Enhancers of the Adenosine A3 Receptor

The adenosine receptor subfamily consists of the adenosine A(1), A(2A), A(2B), and A(3) receptors, which are localized in a variety of tissues throughout the human body. It is, therefore, a challenge to develop receptor specific ligands with improved tissue selectivity. Allosteric modulators could have these therapeutic advantages over orthosteric ligands. In the present study, a series of 2,4-disubstituted quinolines were synthesized on the basis of the structure of LUF6000 (34). Compound 27 (LUF6096) was able to allosterically enhance agonist binding to a similar extent as 34. In addition, this new compound showed low, if any, orthosteric affinity for any of the adenosine receptors. In a functional assay, compound 27 showed improved activity in comparison to 34, as it increased both the intrinsic efficacy and the potency of the reference agonist Cl-IB-MECA at the human adenosine A(3) receptor.