The Neutrophil Dynamic Mass Redistribution Assay as a Medium throughput Primary Cell Screening Assay.

In a typical G protein coupled receptor drug discovery campaign, an in vitro primary functional screening assay is often established in a recombinant system overexpressing the target of interest, which offers advantages with respect to overall throughput and robustness of compound testing. Subsequently, compounds are then progressed into more physiologically relevant but lower throughput ex vivo primary cell assays and finally in vivo studies. Here we describe a dynamic mass redistribution (DMR) assay that has been developed in a format suitable to support medium throughput drug screening in primary human neutrophils. Neutrophils are known to express both CXC chemokine receptor (CXCR) 1 and CXCR2 that are thought to play significant roles in various inflammatory disorders and cancer. Using multiple relevant chemokine ligands and a range of selective and nonselective small and large molecule antagonists that block CXCR1 and CXCR2 responses, we demonstrate distinct pharmacological profiles in neutrophil DMR from those observed in recombinant assays but predictive of activity in neutrophil chemotaxis and CD11b upregulation, a validated target engagement marker previously used in clinical studies of CXCR2 antagonists. The primary human neutrophil DMR cell system is highly reproducible, robust, and less prone to donor variability observed in CD11b and chemotaxis assays and thus provides a unique, more physiologically relevant, and higher throughput assay to support drug discovery and translation to early clinical trials. SIGNIFICANCE STATEMENT: Neutrophil dynamic mass redistribution assays provide a higher throughput screening assay to profile compounds in primary cells earlier in the screening cascade enabling a higher level of confidence in progressing the development of compounds toward the clinic. This is particularly important for chemokine receptors where redundancy contributes to a lack of correlation between recombinant screening assays and primary cells, with the coexpression of related receptors confounding results.

A novel somatostatin receptor ligand for human ACTH - and GH -secreting pituitary adenomas.

OBJECTIVE: Somatostatin receptor ligands have come to play a pivotal role in the treatment of both ACTH- and GH-secreting pituitary adenomas. Clinical efficacy averages 30-50%, thus a considerable number of patients with Cushing's disease or acromegaly remain unresponsive to this therapeutic approach. HTL0030310 is a new somatostatin receptor ligand selective for subtype 5 over subtype 2, thus with a different receptor profile compared to clinical somatostatin receptor ligands. DESIGN: Assessment of the effect of HTL0030310 on hormone secretion in human ACTH- and GH-secreting pituitary adenomas in vitro. METHODS: Primary cultures from 3 ACTH-secreting and 5 GH-secreting pituitary adenomas were treated with 1, 10 and 100 nM HTL0030310 alone or with 10 nM CRH or GHRH, respectively. Parallel incubations with 10 nM pasireotide were also carried out. ACTH and GH secretion were assessed after 4 and 24 hour incubation; SSTR2, SSTR3, SSTR5, GH and POMC expression were evaluated after 24 hours. RESULTS: HTL0030310 reduced unchallenged ACTH and POMC levels up to 50% in 2 ACTH-secreting adenomas and blunted CRH-stimulated ACTH/POMC by 20-70% in all 3 specimens. A reduction in spontaneous GH secretion was observed in 4 GH-secreting adenomas and in 2 specimens during GHRH co-incubation. SSTRs expression was detected in all specimens. CONCLUSIONS: This first study on a novel somatostatin receptor 5-preferring ligand indicates that HTL0030310 can inhibit hormonal secretion in human ACTH- and GH-secreting pituitary adenomas. These findings suggest a potential new avenue for somatostatin ligands in the treatment of Cushing's disease and acromegaly.

Targeting the Type 5 Metabotropic Glutamate Receptor: A Potential Therapeutic Strategy for Neurodegenerative Diseases?

The type 5 metabotropic glutamate receptor, mGlu5, has been proposed as a potential therapeutic target for the treatment of several neurodegenerative diseases. In preclinical neurodegenerative disease models, novel allosteric modulators have been shown to improve cognitive performance and reduce disease-related pathology. A common pathological hallmark of neurodegenerative diseases is a chronic neuroinflammatory response, involving glial cells such as astrocytes and microglia. Since mGlu5 is expressed in astrocytes, targeting this receptor could provide a potential mechanism by which neuroinflammatory processes in neurodegenerative disease may be modulated. This review will discuss current evidence that highlights the potential of mGlu5 allosteric modulators to treat neurodegenerative diseases, including Alzheimer's disease, Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Furthermore, this review will explore the role of mGlu5 in neuroinflammatory responses, and the potential for this G protein-coupled receptor to modulate neuroinflammation.

From structure to clinic: Design of a muscarinic M1 receptor agonist with potential to treatment of Alzheimer's disease.

Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.

Discovery and Characterization of Novel Antagonists of the Proinflammatory Orphan Receptor GPR84.

GPR84 is a poorly characterized, nominally orphan, proinflammatory G protein-coupled receptor that can be activated by medium chain length fatty acids. It is attracting considerable interest as a potential therapeutic target for antagonist ligands in both inflammatory bowel diseases and idiopathic pulmonary fibrosis. Successful screening of more than 300 000 compounds from a small molecule library followed by detailed analysis of some 50 drug-like hits identified 3-((5,6-bis(4-methoxyphenyl)-1,2,4-triazin-3-yl)methyl)-1H-indole as a high affinity and highly selective competitive antagonist of human GPR84. Tritiation of a di-iodinated form of the core structure produced [3H]3-((5,6-diphenyl-1,2,4-triazin-3-yl)methyl)-1H-indole, which allowed effective measurement of receptor levels in both transfected cell lines and lipopolysaccharide-treated THP-1 monocyte/macrophage cells. Although this compound series lacks significant affinity at mouse GPR84, homology modeling and molecular dynamics simulations provided a potential rationale for this difference, and alteration of two residues in mouse GPR84 to the equivalent amino acids in the human orthologue, predicted to open the antagonist binding pocket, validated this model. Sequence alignment of other species orthologues further predicted binding of the compounds as high affinity antagonists at macaque, pig, and dog GPR84 but not at the rat orthologue, and pharmacological experiments confirmed these predictions. These studies provide a new class of GPR84 antagonists that display species selectivity defined via receptor modeling and mutagenesis.

Characterisation of inverse agonism of the orphan-G protein-coupled receptor GPR52 by cannabinoid ligands Cannabidiol and O-1918.

The identification of cannabinoid ligands Cannabidiol and O-1918 as inverse agonists of the orphan receptor GPR52 is reported. Detailed characterisation of GPR52 pharmacology and modelling of the proposed receptor interaction is described. The identification of a novel and further CNS pharmacology for the polypharmacological agent and marketed drug Cannabidiol is noteworthy.

Understanding Exposure-Receptor Occupancy Relationships for Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators across a Range of Preclinical and Clinical Studies.

The metabotropic glutamate receptor 5 (mGlu5) is a recognized central nervous system therapeutic target for which several negative allosteric modulator (NAM) drug candidates have or are continuing to be investigated for various disease indications in clinical development. Direct measurement of target receptor occupancy (RO) is extremely useful to help design and interpret efficacy and safety in nonclinical and clinical studies. In the mGlu5 field, this has been successfully achieved by monitoring displacement of radiolabeled ligands, specifically binding to the mGlu5 receptor, in the presence of an mGlu5 NAM using in vivo and ex vivo binding in rodents and positron emission tomography imaging in cynomolgus monkeys and humans. The aim of this study was to measure the RO of the mGlu5 NAM HTL0014242 in rodents and cynomolgus monkeys and to compare its plasma and brain exposure-RO relationships with those of clinically tested mGlu5 NAMs dipraglurant, mavoglurant, and basimglurant. Potential sources of variability that may contribute to these relationships were explored. Distinct plasma exposure-response relationships were found for each mGlu5 NAM, with >100-fold difference in plasma exposure for a given level of RO. However, a unified exposure-response relationship was observed when both unbound brain concentration and mGlu5 affinity were considered. This relationship showed <10-fold overall difference, was fitted with a Hill slope that was not significantly different from 1, and appeared consistent with a simple Emax model. This is the first time this type of comparison has been conducted, demonstrating a unified brain exposure-RO relationship across several species and mGlu5 NAMs with diverse properties. SIGNIFICANCE STATEMENT: Despite the long history of mGlu5 as a therapeutic target and progression of multiple compounds to the clinic, no formal comparison of exposure-receptor occupancy relationships has been conducted. The data from this study indicate for the first time that a consistent, unified relationship can be observed between exposure and mGlu5 receptor occupancy when unbound brain concentration and receptor affinity are taken into account across a range of species for a diverse set of mGlu5 negative allosteric modulators, including a new drug candidate, HTL0014242.

Structure-based discovery and development of metabotropic glutamate receptor 5 negative allosteric modulators.

The metabotropic glutamate (mGlu) receptors are a family of eight class C G protein-coupled receptors (GPCRs) which modulate cell signaling and synaptic transmission to the major excitatory neurotransmitter l-glutamate (l-glutamic acid). Due to their role in modulating glutamate response, their widespread distribution in the central nervous system (CNS) and some evidence of dysregulation in disease, the mGlu receptors have become attractive pharmacological targets. As the orthosteric (glutamate) binding site is highly conserved across the eight mGlu receptors, it is difficult not only to generate ligands with subtype selectivity but, due to the nature of the binding site, with suitable drug-like properties to allow oral bioavailability and CNS penetration. Selective pharmacological targeting of a single receptor subtype can be achieved by targeting alternative (allosteric) binding sites. The nature of the allosteric binding pockets allows ligands to be developed that have good physical chemical properties as evidenced by several allosteric modulators of mGlu receptors entering clinical trials. The first negative allosteric modulators of the metabotropic glutamate 5 (mGlu5) receptor were discovered from high throughput screening activities. An alternative approach to drug discovery is to use structural knowledge to enable structure-based drug design (SBDD), which allows the design of molecules in a more rational, rather than empirical, fashion. Here we will describe the process of SBDD in the discovery of the mGlu5 negative allosteric modulator HTL0014242 and describe how knowledge of receptor structure can also be used to gain insights into the receptor activation mechanisms.

Comparison of Orexin 1 and Orexin 2 Ligand Binding Modes Using X-ray Crystallography and Computational Analysis.

The orexin system, which consists of the two G protein-coupled receptors OX1 and OX2, activated by the neuropeptides OX-A and OX-B, is firmly established as a key regulator of behavioral arousal, sleep, and wakefulness and has been an area of intense research effort over the past two decades. X-ray structures of the receptors in complex with 10 new antagonist ligands from diverse chemotypes are presented, which complement the existing structural information for the system and highlight the critical importance of lipophilic hotspots and water molecules for these peptidergic GPCR targets. Learnings from the structural information regarding the utility of pharmacophore models and how selectivity between OX1 and OX2 can be achieved are discussed.

Intracellular allosteric antagonism of the CCR9 receptor.

Chemokines and their G-protein-coupled receptors play a diverse role in immune defence by controlling the migration, activation and survival of immune cells. They are also involved in viral entry, tumour growth and metastasis and hence are important drug targets in a wide range of diseases. Despite very significant efforts by the pharmaceutical industry to develop drugs, with over 50 small-molecule drugs directed at the family entering clinical development, only two compounds have reached the market: maraviroc (CCR5) for HIV infection and plerixafor (CXCR4) for stem-cell mobilization. The high failure rate may in part be due to limited understanding of the mechanism of action of chemokine antagonists and an inability to optimize compounds in the absence of structural information. CC chemokine receptor type 9 (CCR9) activation by CCL25 plays a key role in leukocyte recruitment to the gut and represents a therapeutic target in inflammatory bowel disease. The selective CCR9 antagonist vercirnon progressed to phase 3 clinical trials in Crohn's disease but efficacy was limited, with the need for very high doses to block receptor activation. Here we report the crystal structure of the CCR9 receptor in complex with vercirnon at 2.8 Å resolution. Remarkably, vercirnon binds to the intracellular side of the receptor, exerting allosteric antagonism and preventing G-protein coupling. This binding site explains the need for relatively lipophilic ligands and describes another example of an allosteric site on G-protein-coupled receptors that can be targeted for drug design, not only at CCR9, but potentially extending to other chemokine receptors.

Fragment and Structure-Based Drug Discovery for a Class C GPCR: Discovery of the mGlu5 Negative Allosteric Modulator HTL14242 (3-Chloro-5-[6-(5-fluoropyridin-2-yl)pyrimidin-4-yl]benzonitrile).

Fragment screening of a thermostabilized mGlu5 receptor using a high-concentration radioligand binding assay enabled the identification of moderate affinity, high ligand efficiency (LE) pyrimidine hit 5. Subsequent optimization using structure-based drug discovery methods led to the selection of 25, HTL14242, as an advanced lead compound for further development. Structures of the stabilized mGlu5 receptor complexed with 25 and another molecule in the series, 14, were determined at resolutions of 2.6 and 3.1 Å, respectively.

Structures of mGluRs shed light on the challenges of drug development of allosteric modulators.

The metabotropic glutamate receptor family includes many potential therapeutic targets for a wide range of neurological disorders however to date no approved drugs have progressed to market. For some receptor subtypes it has been difficult to separate therapeutic benefit from undesirable side effects. For others finding suitable drug like molecules has been challenging. Chemotypes identified from screening have been limited and difficult to optimise away from undesirable groups. Frequently within related series, compounds have switched from agonist to antagonists. Recently the structures of the transmembrane domain of mGlu1 and mGlu5 have been solved revealing the binding site of allosteric modulators which provides an understanding of the difficulties to date and an opportunity for future structure based approaches to drug design.

Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain.

Metabotropic glutamate receptors are class C G-protein-coupled receptors which respond to the neurotransmitter glutamate. Structural studies have been restricted to the amino-terminal extracellular domain, providing little understanding of the membrane-spanning signal transduction domain. Metabotropic glutamate receptor 5 is of considerable interest as a drug target in the treatment of fragile X syndrome, autism, depression, anxiety, addiction and movement disorders. Here we report the crystal structure of the transmembrane domain of the human receptor in complex with the negative allosteric modulator, mavoglurant. The structure provides detailed insight into the architecture of the transmembrane domain of class C receptors including the precise location of the allosteric binding site within the transmembrane domain and key micro-switches which regulate receptor signalling. This structure also provides a model for all class C G-protein-coupled receptors and may aid in the design of new small-molecule drugs for the treatment of brain disorders.

Pharmacology and structure of isolated conformations of the adenosine A2A receptor define ligand efficacy.

Using isolated receptor conformations crystal structures of the adenosine A2A receptor have been solved in active and inactive states. Studying the change in affinity of ligands at these conformations allowed qualitative prediction of compound efficacy in vitro in a system-independent manner. Agonist 5'-N-ethylcarboxamidoadenosine displayed a clear preference to bind to the active state receptor; inverse agonists (xanthine amine congener, ZM241385, SCH58261, and preladenant) bound preferentially to the inactive state, whereas neutral antagonists (theophylline, caffeine, and istradefylline) demonstrated equal affinity for active and inactive states. Ligand docking into the known crystal structures of the A2A receptor rationalized the pharmacology observed; inverse agonists, unlike neutral antagonists, cannot be accommodated within the agonist-binding site of the receptor. The availability of isolated receptor conformations opens the door to the concept of "reverse pharmacology" whereby the functional pharmacology of ligands can be characterized in a system-independent manner by their affinity for a pair (or set) of G protein-coupled receptor conformations.

Structure of the adenosine A(2A) receptor in complex with ZM241385 and the xanthines XAC and caffeine.

Methylxanthines, including caffeine and theophylline, are among the most widely consumed stimulant drugs in the world. These effects are mediated primarily via blockade of adenosine receptors. Xanthine analogs with improved properties have been developed as potential treatments for diseases such as Parkinson's disease. Here we report the structures of a thermostabilized adenosine A(2A) receptor in complex with the xanthines xanthine amine congener and caffeine, as well as the A(2A) selective inverse agonist ZM241385. The receptor is crystallized in the inactive state conformation as defined by the presence of a salt bridge known as the ionic lock. The complete third intracellular loop, responsible for G protein coupling, is visible consisting of extended helices 5 and 6. The structures provide new insight into the features that define the ligand binding pocket of the adenosine receptor for ligands of diverse chemotypes as well as the cytoplasmic regions that interact with signal transduction proteins.

Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation.

Adenosine receptors and ß-adrenoceptors are G-protein-coupled receptors (GPCRs) that activate intracellular G proteins on binding the agonists adenosine or noradrenaline, respectively. GPCRs have similar structures consisting of seven transmembrane helices that contain well-conserved sequence motifs, indicating that they are probably activated by a common mechanism. Recent structures of ß-adrenoceptors highlight residues in transmembrane region 5 that initially bind specifically to agonists rather than to antagonists, indicating that these residues have an important role in agonist-induced activation of receptors. Here we present two crystal structures of the thermostabilized human adenosine A(2A) receptor (A(2A)R-GL31) bound to its endogenous agonist adenosine and the synthetic agonist NECA. The structures represent an intermediate conformation between the inactive and active states, because they share all the features of GPCRs that are thought to be in a fully activated state, except that the cytoplasmic end of transmembrane helix 6 partially occludes the G-protein-binding site. The adenine substituent of the agonists binds in a similar fashion to the chemically related region of the inverse agonist ZM241385 (ref. 8). Both agonists contain a ribose group, not found in ZM241385, which extends deep into the ligand-binding pocket where it makes polar interactions with conserved residues in H7 (Ser 277(7.42) and His 278(7.43); superscripts refer to Ballesteros-Weinstein numbering) and non-polar interactions with residues in H3. In contrast, the inverse agonist ZM241385 does not interact with any of these residues and comparison with the agonist-bound structures indicates that ZM241385 sterically prevents the conformational change in H5 and therefore it acts as an inverse agonist. Comparison of the agonist-bound structures of A(2A)R with the agonist-bound structures of ß-adrenoceptors indicates that the contraction of the ligand-binding pocket caused by the inward motion of helices 3, 5 and 7 may be a common feature in the activation of all GPCRs.

Thermostabilisation of an agonist-bound conformation of the human adenosine A(2A) receptor.

The adenosine A(2A) receptor (A(2A)R) is a G-protein-coupled receptor that plays a key role in transmembrane signalling mediated by the agonist adenosine. The structure of A(2A)R was determined recently in an antagonist-bound conformation, which was facilitated by the T4 lysozyme fusion in cytoplasmic loop 3 and the considerable stabilisation conferred on the receptor by the bound inverse agonist ZM241385. Unfortunately, the natural agonist adenosine does not sufficiently stabilise the receptor for the formation of diffraction-quality crystals. As a first step towards determining the structure of A(2A)R bound to an agonist, the receptor was thermostabilised by systematic mutagenesis in the presence of the bound agonist [(3)H]5'-N-ethylcarboxamidoadenosine (NECA). Four thermostabilising mutations were identified that when combined to give mutant A(2A)R-GL26, conferred a greater than 200-fold decrease in its rate of unfolding compared to the wild-type receptor. Pharmacological analysis suggested that A(2A)R-GL26 is stabilised in an agonist-bound conformation because antagonists bind with up to 320-fold decreased affinity. None of the thermostabilising mutations are in the ZM241385 binding pocket, suggesting that the mutations affect ligand binding by altering the conformation of the receptor rather than through direct interactions with ligands. A(2A)R-GL26 shows considerable stability in short-chain detergents, which has allowed its purification and crystallisation.

When simple agonism is not enough: emerging modalities of GPCR ligands.

Recent advances in G protein-coupled receptors have challenged traditional definitions of agonism, antagonism, affinity and efficacy. The discovery of agonist functional selectivity and receptor allosterism has meant researchers have an expanded canvas for designing and discovering novel drugs. Here we describe modes of agonism emerging from the discovery of functional selectivity and allosterism. We discuss the concept of ago-allosterism, where ligands can initiate signaling by themselves and influence the actions of another ligand at the same receptor. We introduce the concept of dualsteric ligands that consist of distinct elements which bind to each of the orthosteric and an allosteric domain on a single receptor to enhance subtype selectivity. Finally, the concept that efficacy should be defined by the activity of an endogenous ligand will be challenged by the discovery that some ligands act as 'super-agonists' in specific pathways or at certain receptor mutations.

Growth hormone secretagogues and growth hormone releasing peptides act as orthosteric super-agonists but not allosteric regulators for activation of the G protein Galpha(o1) by the Ghrelin receptor.

Some growth hormone secretagogues act as agonists at the ghrelin receptor and have been described as "ago-allosteric" ligands because of an ability to also modulate the maximum efficacy and potency of ghrelin (Holst et al., 2005). In membranes prepared from cells coexpressing the human ghrelin receptor and the G protein Galpha(o1), N-[1(R)-1, 2-dihydro-1-ethanesulfonylspiro-3H-indole-3,4'-piperidin)-1'-yl]carbonyl-2-(phenylmethoxy)-ethyl-2-amino-2-methylpropanamide (MK-677), growth hormone-releasing peptide 6 (GHRP-6), and the 2(R)-hydroxypropyl derivative of 3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-([2'-(1H-tetrazol-5-yl) (1,1'-biphenyl)-4-yl]methyl)-1H-1-benzazepin-3(R)-yl)-butanamide (L-692,585) each functioned as direct agonists, and each displayed higher efficacy than ghrelin. The effect of multiple, fixed concentrations of each of these ligands on the function and concentration-dependence of ghrelin and the effect of multiple, fixed concentrations of ghrelin on the action of MK-677, GHRP-6, and L-692,585 was analyzed globally according to a modified version of an operational model of allosterism that accounts for allosteric modulation of affinity, efficacy, and allosteric agonism. Each of the data sets was best fit by a model of simple competition between a partial and a full agonist. Both positive and negative allosteric modulators are anticipated to alter the kinetics of binding of an orthosteric agonist. However, none of the proposed ago-allosteric regulators tested had any effect on the dissociation kinetics of (125)I-[His]-ghrelin, and GHRP-6 and MK-677 were able to fully displace (125)I-[His]-ghrelin from the receptor. At least in the system tested, each of the ligands acted in a simple competitive fashion with ghrelin as demonstrated by analysis according to a model whereby ghrelin is a partial agonist with respect to each of the synthetic agonists tested.