Molecular mechanisms of inverse agonism via κ-opioid receptor-G protein complexes.

Opioid receptors, a subfamily of G protein-coupled receptors (GPCRs), are key therapeutic targets. In the canonical GPCR activation model, agonist binding is required for receptor-G protein complex formation, while antagonists prevent G protein coupling. However, many GPCRs exhibit basal activity, allowing G protein association without an agonist.

Know your molecule: pharmacological characterization of drug candidates to enhance efficacy and reduce late-stage attrition.

Despite advances in chemical, computational and biological sciences, the rate of attrition of drug candidates in clinical development is still high. A key point in the small-molecule discovery process that could provide opportunities to help address this challenge is the pharmacological characterization of hit and lead compounds, culminating in the selection of a drug candidate. Deeper characterization is increasingly important, because the 'quality' of drug efficacy, at least for G protein-coupled receptors (GPCRs), is now understood to be much more than activation of commonly evaluated pathways such as cAMP signalling, with many more 'efficacies' of ligands that could be harnessed therapeutically.

A Pharmacological perspective on the temporal properties of sweeteners.

Several non-caloric sweeteners exhibit a delay in sweetness onset and a sweetness linger after sampling. These temporal properties are thought to be the result of non-specific interactions with cell membranes and proteins in the oral cavity. Data and analysis presented in this report also support the potential involvement of receptor affinity and binding kinetics to this phenomenon.

Intracellular Allosteric Antagonist of the Olfactory Receptor OR51E2.

Olfactory receptors are members of class A (rhodopsin-like) family of G protein-coupled receptors (GPCRs). Their expression and function have been increasingly studied in nonolfactory tissues, and many have been identified as potential therapeutic targets. In this manuscript, we focus on the discovery of novel ligands for the olfactory receptor family 51 subfamily E2 (OR51E2).

Bias translation: The final frontier?

Biased signalling is a natural result of GPCR allosteric function and should be expected from any and all synthetic and natural agonists. Therefore, it may be encountered in all agonist discovery projects and must be considered as a beneficial (or possible detrimental) feature of new candidate molecules. While bias is detected easily, the synoptic nature of GPCR signalling makes translation of simple in vitro bias to complex in vivo systems problematic.

Allostery: The Good, the Bad, and the Ugly.

With the advent of functional screening, more allosteric molecules are being discovered and developed as possible therapeutic entities. Allosteric proteins are unique because of two specific properties: 1) separate binding sites for allosteric modulators and guests and 2) mandatory alteration of receptor conformation upon binding of allosteric modulators. For G protein-coupled receptors, these properties produce many beneficial effects on pharmacologic systems that are described here.

Neurotensin Receptor Allosterism Revealed in Complex with a Biased Allosteric Modulator.

The NTSR1 neurotensin receptor (NTSR1) is a G protein-coupled receptor (GPCR) found in the brain and peripheral tissues with neurotensin (NTS) being its endogenous peptide ligand. In the brain, NTS modulates dopamine neuronal activity, induces opioid-independent analgesia, and regulates food intake. Recent studies indicate that biasing NTSR1 toward β-arrestin signaling can attenuate the actions of psychostimulants and other drugs of abuse.

Community guidelines for GPCR ligand bias: IUPHAR review 32.

GPCRs modulate a plethora of physiological processes and mediate the effects of one-third of FDA-approved drugs. Depending on which ligand activates a receptor, it can engage different intracellular transducers. This 'biased signalling' paradigm requires that we now characterize physiological signalling not just by receptors but by ligand-receptor pairs.

Development of 3-(4-Chlorophenyl)-1-(phenethyl)urea Analogues as Allosteric Modulators of the Cannabinoid Type-1 Receptor: RTICBM-189 is Brain Penetrant and Attenuates Reinstatement of Cocaine-Seeking Behavior.

We have shown that CB receptor negative allosteric modulators (NAMs) attenuated the reinstatement of cocaine-seeking behaviors in rats. In an effort to further define the structure-activity relationships and assess the druglike properties of the 3-(4-chlorophenyl)-1-(phenethyl)urea-based CB NAMs that we recently reported, we introduced substituents of different electronic properties and sizes to the phenethyl group and evaluated their potency in CB calcium mobilization, cAMP, and GTPγS assays. We found that 3-position substitutions such as Cl, F, and Me afforded enhanced CB potency, whereas 4-position analogues were generally less potent.

Biased signaling as allosteric probe dependence.

Signaling 'bias' is a phenomenon whereby the natural allosteric probe dependence of seven transmembrane receptors allows different receptor conformations (stabilized by different agonists) to activate some signaling pathways (coupled to pleiotropically coupled receptors) more than others at the expense of those other pathways. There are a number of relevant scenarios where such an activity could be therapeutically beneficial therefore there are practical reasons why this property of receptors should be exploited. This paper discusses recent ideas around attempts to harness this potentially useful idea and also the limitations around the current methods available to do so.

Differential Roles of Extracellular Histidine Residues of GPR68 for Proton-Sensing and Allosteric Modulation by Divalent Metal Ions.

GPR68, an orphan G-protein coupled receptor, senses protons, couples to multiple G-proteins, and is also activated or inhibited by divalent metal ions. It has seven extracellular histidine residues, although it is not clear how these histidine residues play a role in both proton-sensing and metal ion modulation. Here we demonstrate that divalent metal ions are allosteric modulators that can activate or inhibit proton activity in a concentration- and pH-dependent manner.

The function and allosteric control of the human sweet taste receptor.

Humans perceive sweet taste via activation of a specific taste receptor expressed at the surface of taste receptor cells located on the tongue and soft palate papillae. The sweet taste receptor functions as an obligate heterodimer, comprising two different class C GPCR subunits. This receptor is unique in that it is activated or modulated by a plethora of ligands from highly diverse chemical classes, from small molecules to peptides and proteins and interacting with topologically distinct sites on each of its subunits.

Interdependent allosteric free fatty acid receptor 2 modulators synergistically induce functional selective activation and desensitization in neutrophils.

The non-activating allosteric modulator AZ1729, specific for free fatty acid receptor 2 (FFAR2), transfers the orthosteric FFAR2 agonists propionate and the P2YR specific agonist ATP into activating ligands that trigger an assembly of the neutrophil superoxide generating NADPH-oxidase. The homologous priming effect on the propionate response and the heterologous receptor cross-talk sensitized ATP response mediated by AZ1729 are functional characteristics shared with Cmp58, another non-activating allosteric FFAR2 modulator. In addition, AZ1729 also turned Cmp58 into a potent activator of the superoxide generating neutrophil NADPH-oxidase, and in agreement with the allosteric modulation concept, the effect was reciprocal in that Cmp58 turned AZ1729 into a potent activating allosteric agonist.

Virtual discovery of melatonin receptor ligands to modulate circadian rhythms.

The neuromodulator melatonin synchronizes circadian rhythms and related physiological functions through the actions of two G-protein-coupled receptors: MT and MT. Circadian release of melatonin at night from the pineal gland activates melatonin receptors in the suprachiasmatic nucleus of the hypothalamus, synchronizing the physiology and behaviour of animals to the light-dark cycle. The two receptors are established drug targets for aligning circadian phase to this cycle in disorders of sleep and depression.

Characterization of the various functional pathways elicited by synthetic agonists or antagonists at the melatonin MT and MT receptors.

Melatonin is a neurohormone that translates the circadian rhythm to the peripheral organs through a series of binding sites identified as G protein-coupled receptors MT and MT. Due to minute amounts of receptor proteins in target organs, the main tool of studies of the melatoninergic system is recombinant expression of the receptors in cellular hosts. Although a number of studies exist on these receptors, studies of several signaling pathways using a large number of melatoninergic compounds are rather limited.

Emergent Concepts of Receptor Pharmacology.

Pharmacology, the chemical control of physiology, emerged as an offshoot of physiology when the physiologists using chemicals to probe physiological systems became more interested in the probes than the systems. Pharmacologists were always, and in many ways still are, bound to study drugs in systems they do not fully understand. Under these circumstances, null methods were the main ways in which conclusions about biologically active molecules were made.

Preclinical Testing of Nalfurafine as an Opioid-sparing Adjuvant that Potentiates Analgesia by the Mu Opioid Receptor-targeting Agonist Morphine.

Mu opioid receptor (MOR)-targeting analgesics are efficacious pain treatments, but notorious for their abuse potential. In preclinical animal models, coadministration of traditional kappa opioid receptor (KOR)-targeting agonists with MOR-targeting analgesics can decrease reward and potentiate analgesia. However, traditional KOR-targeting agonists are well known for inducing antitherapeutic side effects (psychotomimesis, depression, anxiety, dysphoria).

Discovery and Characterization of a Cellular Potent Positive Allosteric Modulator of the Polycomb Repressive Complex 1 Chromodomain, CBX7.

Polycomb-directed repression of gene expression is frequently misregulated in human diseases. A quantitative and target-specific cellular assay was utilized to discover the first potent positive allosteric modulator (PAM) peptidomimetic, UNC4976, of nucleic acid binding by CBX7, a chromodomain methyl-lysine reader of Polycomb repressive complex 1. The PAM activity of UNC4976 resulted in enhanced efficacy across three orthogonal cellular assays by simultaneously antagonizing H3K27me3-specific recruitment of CBX7 to target genes while increasing non-specific binding to DNA and RNA.

G-Protein/Receptor inhibitors as blockers of receptor signaling.

This paper describes the behavior of binding and functional receptor systems where an antagonist of the receptor/G protein binding reaction is added as a blocker of agonist-induced receptor function. For agonist radioligands, the reduction of G protein receptor interaction leads to a possible change in the binding affinity of the agonist radioligand to the receptor. Reciprocally, the allosteric cooperativity between the agonist and the G protein binding site antagonist (quantified by the factor γ) affects the potency of the G protein antagonist modulator; this model presents the various profiles that would be expected for modulators that reduce (γ = 0.

The pharmacologic characterization of allosteric molecules: Gq protein activation.

Drugs such as positive allosteric modulators (PAMs) produce complex behaviors when acting on tissues in different physiological contexts . This study describes the use of functional assays of varying receptor sensitivity to unveil the various behaviors of PAMs and thus quantify allosteric effect through system independent scales. Muscarinic receptor activation with acetylcholine (ACh) was used to the demonstrate activity of the PAM agonist 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, Benzyl quinolone carboxylic acid (BQCA) in terms of direct agonism, potentiation of ACh affinity, and ACh efficacy.

Design, Synthesis, and Characterization of Ogerin-Based Positive Allosteric Modulators for G Protein-Coupled Receptor 68 (GPR68).

G protein-coupled receptor 68 (GPR68) is an understudied orphan G protein-coupled receptor (GPCR). It is expressed most abundantly in the brain, potentially playing important roles in learning and memory. Pharmacological studies with GPR68 have been hindered by lack of chemical tools that can selectively modulate its activity.

Prescient Indices of Activity: The Application of Functional System Sensitivity to Measurement of Drug Effect.

Through pharmacological procedures, indices of drug activity can be obtained that transcend the systems in which they are measured. If (i) affinity, (ii) efficacies, (iii) orthosteric versus allosteric interaction, and (iv) rate of receptor offset can be determined, activity can be predicted in all systems. This can yield more detailed profiles (fingerprints) of efficacy to better define the required activities of follow-up molecules should the original candidates fail in the clinic.

Biased Receptor Signaling in Drug Discovery.

A great deal of experimental evidence suggests that ligands can stabilize different receptor active states that go on to interact with cellular signaling proteins to form a range of different complexes in varying quantities. In pleiotropically linked receptor systems, this leads to selective activation of some signaling pathways at the expense of others (biased signaling). This article summarizes the current knowledge about the complex components of receptor systems, the evidence that biased signaling is used in natural physiology to fine-tune signaling, and the current thoughts on how this mechanism may be applied to the design of better drugs.