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.

Structure of the complement C5a receptor bound to the extra-helical antagonist NDT9513727.

The complement system is a crucial component of the host response to infection and tissue damage. Activation of the complement cascade generates anaphylatoxins including C5a and C3a. C5a exerts a pro-inflammatory effect via the G-protein-coupled receptor C5a anaphylatoxin chemotactic receptor 1 (C5aR1, also known as CD88) that is expressed on cells of myeloid origin. Inhibitors of the complement system have long been of interest as potential drugs for the treatment of diseases such as sepsis, rheumatoid arthritis, Crohn's disease and ischaemia-reperfusion injuries. More recently, a role of C5a in neurodegenerative conditions such as Alzheimer's disease has been identified. Peptide antagonists based on the C5a ligand have progressed to phase 2 trials in psoriasis and rheumatoid arthritis; however, these compounds exhibited problems with off-target activity, production costs, potential immunogenicity and poor oral bioavailability. Several small-molecule competitive antagonists for C5aR1, such as W-54011 and NDT9513727, have been identified by C5a radioligand-binding assays. NDT9513727 is a non-peptide inverse agonist of C5aR1, and is highly selective for the primate and gerbil receptors over those of other species. Here, to study the mechanism of action of C5a antagonists, we determine the structure of a thermostabilized C5aR1 (known as C5aR1 StaR) in complex with NDT9513727. We found that the small molecule bound between transmembrane helices 3, 4 and 5, outside the helical bundle. One key interaction between the small molecule and residue Trp2135.49 seems to determine the species selectivity of the compound. The structure demonstrates that NDT9513727 exerts its inverse-agonist activity through an extra-helical mode of action.

Corrigendum: Crystal structure of the GLP-1 receptor bound to a peptide agonist.

This corrects the article DOI: 10.1038/nature22800.

Crystal structure of the GLP-1 receptor bound to a peptide agonist.

Glucagon-like peptide 1 (GLP-1) regulates glucose homeostasis through the control of insulin release from the pancreas. GLP-1 peptide agonists are efficacious drugs for the treatment of diabetes. To gain insight into the molecular mechanism of action of GLP-1 peptides, here we report the crystal structure of the full-length GLP-1 receptor bound to a truncated peptide agonist. The peptide agonist retains an α-helical conformation as it sits deep within the receptor-binding pocket. The arrangement of the transmembrane helices reveals hallmarks of an active conformation similar to that observed in class A receptors. Guided by this structural information, we design peptide agonists with potent in vivo activity in a mouse model of diabetes.

Structural insight into allosteric modulation of protease-activated receptor 2.

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.

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.

Extra-helical binding site of a glucagon receptor antagonist.

Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors.

First-in-man study to investigate safety, pharmacokinetics and exploratory pharmacodynamics of HTL0018318, a novel M1 -receptor partial agonist for the treatment of dementias.

AIMS: HTL0018318 is a selective M1 receptor partial agonist currently under development for the symptomatic treatment of cognitive and behavioural symptoms in Alzheimer's disease and other dementias. We investigated safety, tolerability, pharmacokinetics and exploratory pharmacodynamics (PD) of HTL0018318 following single ascending doses. METHODS: This randomized, double-blind, placebo-controlled study in 40 healthy younger adult and 57 healthy elderly subjects, investigated oral doses of 1-35 mg HTL0018318. Pharmacodynamic assessments were performed using a battery of neurocognitive tasks and electrophysiological measurements. Cerebrospinal fluid concentrations of HTL0018318 and food effects on pharmacokinetics of HTL0018318 were investigated in an open label and partial cross-over design in 14 healthy subjects. RESULTS: Pharmacokinetics of HTL0018318 were well-characterized showing dose proportional increases in exposure from 1-35 mg. Single doses of HTL0018318 were associated with mild dose-related adverse events of low incidence in both younger adult and elderly subjects. The most frequently reported cholinergic AEs included hyperhidrosis and increases in blood pressure up to 10.3 mmHg in younger adults (95% CI [4.2-16.3], 35-mg dose) and up to 11.9 mmHg in elderly subjects (95% CI [4.9-18.9], 15-mg dose). There were no statistically significant effects on cognitive function but the study was not powered to detect small to moderate effect sizes of clinical relevance. CONCLUSION: HTL0018318 showed well-characterized pharmacokinetics and following single doses were generally well tolerated in the dose range studied. These provide encouraging data in support of the development for HTL0018318 for Alzheimer's disease and other dementias.

Safety, pharmacokinetics and pharmacodynamics of HTL0009936, a selective muscarinic M1 -acetylcholine receptor agonist: A randomized cross-over trial.

AIMS: HTL0009936 is a selective M1 muscarinic receptor agonist in development for cognitive dysfunction in Alzheimer's disease. Safety, tolerability and pharmacokinetics and exploratory pharmacodynamic effects of HTL0009936 administered by continuous IV infusion at steady state were investigated in elderly subjects with below average cognitive functioning (BACF). METHODS: Part A was a four-treatment open label sequential study in healthy elderly investigating 10-83 mg HTL0009936 (IV) and a 24 mg HTL0009936 single oral dose. Part B was a five-treatment randomized, double-blind, placebo and physostigmine controlled cross-over study with IV HTL0009936 in elderly subjects with BACF. Pharmacodynamic assessments were performed using neurocognitive and electrophysiological tests. RESULTS: Pharmacokinetics of HTL0009936 showed dose-proportional increases in exposure with a mean half-life of 2.4 hours. HTL0009936 was well-tolerated with transient dose-related adverse events (AEs). Small increases in mean systolic blood pressure of 7.12 mmHg (95% CI [3.99-10.24]) and in diastolic of 5.32 mmHg (95% CI [3.18-7.47]) were noted at the highest dose in part B. Overall, there was suggestive, but no definitive, positive or negative pharmacodynamic effects. Statistically significant effects were observed on P300 with HTL0009936 and adaptive tracking with physostigmine. CONCLUSIONS: HTL0009936 showed well-characterized pharmacokinetics and single doses were safe and generally well-tolerated in healthy elderly subjects. Due to physostigmine tolerability issues and subject burden, the study design was changed and some pharmacodynamic assessments (neurocognitive) were performed at suboptimal drug exposures. Therefore no clear conclusions can be made on pharmacodynamic effects of HTL0009936, although an effect on P300 is suggestive of central target engagement.

Visualizing GPCR 'Megaplexes' Which Enable Sustained Intracellular Signaling.

A range of cutting-edge techniques have been employed to visualize 'megaplexes' consisting of a G protein-coupled receptor (GPCR) bound to ß-arrestin in intracellular endosomes following agonist-induced internalization. Surprisingly, the complex includes simultaneous binding of the heterotrimeric G protein, which retains full functional activity and supports sustained signaling from within the cell.

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.

Identification of a novel allosteric GLP-1R antagonist HTL26119 using structure- based drug design.

A series of novel allosteric antagonists of the GLP-1 receptor (GLP-1R), exemplified by HTL26119, are described. SBDD approaches were employed to identify HTL26119, exploiting structural understanding of the allosteric binding site of the closely related Glucagon receptor (GCGR) (Jazayeri et al., 2016) and the homology relationships between GCGR and GLP-1R. The region around residue C3476.36b of the GLP-1R receptor represents a key difference from GCGR and was targeted for selectivity for GLP-1R.

Structurally Enabled Discovery of Adenosine A2A Receptor Antagonists.

Over the past decade there has been a revolution in the field of G protein-coupled receptor (GPCR) structural biology. Many years of innovative research from different areas have come together to fuel this significant change in the fortunes of this field, which for many years was characterized by the paucity of high-resolution structures. The determination to succeed has been in part due to the recognized importance of these proteins as drug targets, and although the pharmaceutical industry has been focusing on these receptors, it can be justifiably argued and demonstrated that many of the approved and commercially successful GPCR drugs can be significantly improved to increase efficacy and/or reduce undesired side effects. In addition, many validated targets in this class remain to be drugged. It is widely recognized that application of structure-based drug design approaches can help medicinal chemists a long way toward discovering better drugs. The achievement of structural biologists in providing high-resolution insight is beginning to transform drug discovery efforts, and there are a number of GPCR drugs that have been discovered by use of structural information that are in clinical development. This review aims to highlight the key developments that have brought success to GPCR structure resolution efforts and exemplify the practical application of structural information for the discovery of adenosine A2A receptor antagonists that have potential to treat multiple conditions.

Structure of class B GPCR corticotropin-releasing factor receptor 1.

Structural analysis of class B G-protein-coupled receptors (GPCRs), cell-surface proteins that respond to peptide hormones, has been restricted to the amino-terminal extracellular domain, thus providing little understanding of the membrane-spanning signal transduction domain. The corticotropin-releasing factor receptor type 1 is a class B receptor which mediates the response to stress and has been considered a drug target for depression and anxiety. Here we report the crystal structure of the transmembrane domain of the human corticotropin-releasing factor receptor type 1 in complex with the small-molecule antagonist CP-376395. The structure provides detailed insight into the architecture of class B receptors. Atomic details of the interactions of the receptor with the non-peptide ligand that binds deep within the receptor are described. This structure provides a model for all class B GPCRs and may aid in the design of new small-molecule drugs for diseases of brain and metabolism.

From G Protein-coupled Receptor Structure Resolution to Rational Drug Design.

A number of recent technical solutions have led to significant advances in G protein-coupled receptor (GPCR) structural biology. Apart from a detailed mechanistic view of receptor activation, the new structures have revealed novel ligand binding sites. Together, these insights provide avenues for rational drug design to modulate the activities of these important drug targets. The application of structural data to GPCR drug discovery ushers in an exciting era with the potential to improve existing drugs and discover new ones. In this review, we focus on technical solutions that have accelerated GPCR crystallography as well as some of the salient findings from structures that are relevant to drug discovery. Finally, we outline some of the approaches used in GPCR structure based drug design.

Structure-based drug design for G protein-coupled receptors.

Our understanding of the structural biology of G protein-coupled receptors has undergone a transformation over the past 5 years. New protein-ligand complexes are described almost monthly in high profile journals. Appreciation of how small molecules and natural ligands bind to their receptors has the potential to impact enormously how medicinal chemists approach this major class of receptor targets. An outline of the key topics in this field and some recent examples of structure- and fragment-based drug design are described. A table is presented with example views of each G protein-coupled receptor for which there is a published X-ray structure, including interactions with small molecule antagonists, partial and full agonists. The possible implications of these new data for drug design are discussed.

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.

Preparation of purified GPCRs for structural studies.

Since the publication of the first X-ray structure of a GPCR (G-protein couple receptor) in 2000, the rate at which subsequent ones have appeared has steadily increased. This has required the development of new methodology to overcome the challenges presented by instability of isolated GPCRs, combined with a systematic optimization of existing approaches for protein expression, purification and crystallization. In addition, quality control measures that are predictive of successful outcomes have been identified. Repeated attempts at solving the structures of GPCRs have highlighted experimental approaches that are most likely to lead to success, and have allowed definition of a first-pass protocol for new receptors.

Targeting impaired adult hippocampal neurogenesis in ageing by leveraging intrinsic mechanisms regulating Neural Stem Cell activity.

Deficits in adult neurogenesis may contribute to the aetiology of many neurodevelopmental, psychiatric and neurodegenerative diseases. Genetic ablation of neurogenesis provides proof of concept that adult neurogenesis is required to sustain complex and dynamic cognitive functions, such as learning and memory, mostly by providing a high degree of plasticity to neuronal circuits. In addition, adult neurogenesis is reactive to external stimuli and the environment making it particularly susceptible to impairment and consequently contributing to comorbidity. In the human brain, the dentate gyrus of the hippocampus is the main active source of neural stem cells that generate granule neurons throughout life. The regulation and preservation of the pool of neural stem cells is central to ensure continuous and healthy adult hippocampal neurogenesis (AHN). Recent advances in genetic and metabolic profiling alongside development of more predictive animal models have contributed to the development of new concepts and the emergence of molecular mechanisms that could pave the way to the implementation of new therapeutic strategies to treat neurological diseases. In this review, we discuss emerging molecular mechanisms underlying AHN that could be embraced in drug discovery to generate novel concepts and targets to treat diseases of ageing including neurodegeneration. To support this, we review cellular and molecular mechanisms that have recently been identified to assess how AHN is sustained throughout life and how AHN is associated with diseases. We also provide an outlook on strategies for developing correlated biomarkers that may accelerate the translation of pre-clinical and clinical data and review clinical trials for which modulation of AHN is part of the therapeutic strategy.