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.

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.

Novel Macrocyclic Antagonists of the Calcitonin Gene-Related Peptide Receptor: Design, Realization, and Structural Characterization of Protein-Ligand Complexes.

A series of macrocyclic calcitonin gene-related peptide (CGRP) receptor antagonists identified using structure-based design principles, exemplified by HTL0028016 (1) and HTL0028125 (2), is described. Structural characterization by X-ray crystallography of the interaction of two of the macrocycle antagonists with the CGRP receptor ectodomain is described, along with structure-activity relationships associated with point changes to the macrocyclic antagonists. The identification of non-peptidic/natural product-derived, macrocyclic ligands for a G protein coupled receptor (GPCR) is noteworthy.

3,5-Disubstituted-indole-7-carboxamides: the discovery of a novel series of potent, selective inhibitors of IKK-ß.

The discovery and hit-to-lead exploration of a novel series of selective IKK-ß kinase inhibitors is described. The initial lead fragment 3 was identified by pharmacophore-directed virtual screening. Homology model-driven SAR exploration of the template led to potent inhibitors, such as 12, which demonstrate efficacy in cellular assays and possess encouraging developability profiles.

The discovery and initial optimisation of pyrrole-2-carboxamides as inhibitors of p38alpha MAP kinase.

A novel pyrrole-2-carboxamide series of p38alpha inhibitors, discovered through the application of virtual screening, is presented. Following evaluation of activity, selectivity and developability properties of commercially available analogues, a synthesis program enabled rapid assessment of the series' suitability for further lead optimisation studies.

9,10-Dioxa-1,2-diaza-anthracene derivatives from tetrafluoropyridazine.

Reaction of tetrafluoropyridazine with catechol gives a tricyclic 9,10-dioxa-1,2-diaza-anthracene system by a sequential nucleophilic aromatic substitution ring annelation process, further extending the use of perfluoroheteroaromatic derivatives for the synthesis of unusual polyfunctional heterocyclic architectures. The tricyclic scaffold reacts with amines and sodium ethoxide providing a short series of functional 9,10-dioxa-1,2-diaza-anthracene systems.

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.

Recent Developments in Therapeutic Peptides for the Glucagon-like Peptide 1 and 2 Receptors.

Glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2) are proglucagon derived peptides that are released from gut endocrine cells in response to nutrient intake. These molecules are rapidly inactivated by the action of dipeptidyl peptidase IV (DPP-4) which limits their use as therapeutic agents. The recent emergence of three-dimensional structures of GPCRs such as GLP-1R and glucagon receptor has helped to drive the rational design of innovative peptide molecules that hold promise as novel peptide therapeutics. One emerging area is the discovery of multifunctional molecules that act at two or more pharmacological systems to enhance therapeutic efficacy. In addition, drug discovery efforts are also focusing on strategies to improve patient convenience through alternative routes of peptide delivery. These novel strategies highlight the broad utility of peptide-based therapeutics in human disease settings where unmet needs still exist.

Structure-Based Drug Discovery of N-((R)-3-(7-Methyl-1H-indazol-5-yl)-1-oxo-1-(((S)-1-oxo-3-(piperidin-4-yl)-1-(4-(pyridin-4-yl)piperazin-1-yl)propan-2-yl)amino)propan-2-yl)-2'-oxo-1',2'-dihydrospiro[piperidine-4,4'-pyrido[2,3-d][1,3]oxazine]-1-carboxamide (HTL22562): A Calcitonin Gene-Related Peptide Receptor Antagonist for Acute Treatment of Migraine.

Structure-based drug design enabled the discovery of 8, HTL22562, a calcitonin gene-related peptide (CGRP) receptor antagonist. The structure of 8 complexed with the CGRP receptor was determined at a 1.6 Å resolution. Compound 8 is a highly potent, selective, metabolically stable, and soluble compound suitable for a range of administration routes that have the potential to provide rapid systemic exposures with resultant high levels of receptor coverage (e.g., subcutaneous). The low lipophilicity coupled with a low anticipated clinically efficacious plasma exposure for migraine also suggests a reduced potential for hepatotoxicity. These properties have led to 8 being selected as a clinical candidate for acute treatment of migraine.

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.

Polysubstituted pyridazinones from sequential nucleophilic substitution reactions of tetrafluoropyridazine.

4,5,6-trifluoropyridazin-3(2H)-one can be used as a scaffold for the synthesis of various 4,5- and 4,6-disubstituted and ring-fused pyridazinone systems by sequential nucleophilic aromatic substitution processes. Although the regioselectivity of nucleophilic substitution can be affected by the nature of the nucleophile and the substituent attached to the pyridazinone ring, a variety of polyfunctional systems can be readily accessed by sequential nucleophilic substitution methodology which may have applications in the drug discovery arena. For example, reaction of 4,5,6-trifluoropyridazin-3(2H)-one with nitrogen nucleophiles leads to a mixture of aminated products arising from substitution of fluorine located at the 4- and 5-positions. The ratio of isomers obtained depends on the nucleophile where the 4-isomer is the major product for reaction with primary and secondary amines such as butylamine, morpholine, and aniline derivatives. Subsequent reaction of representative 4-aminated products gave 4,5-disubstituted systems and ring fused derivatives may be formed by reaction of 4,5,6-trifluoropyridazin-3(2H)-one or 4-substituted systems with N,N'-dimethylethylenediamine.

1-Aryl-3,4-dihydroisoquinoline inhibitors of JNK3.

A series of 1-aryl-3,4-dihydroisoquinoline inhibitors of JNK3 are described. Compounds 20 and 24 are the most potent inhibitors (pIC50 7.3 and 6.9, respectively in a radiometric filter binding assay), with 10- and 1000-fold selectivity over JNK2 and JNK1, respectively, and selectivity within the wider mitogen-activated protein kinase (MAPK) family against p38alpha and ERK2. X-ray crystallography of 16 reveals a highly unusual binding mode where an H-bond acceptor interaction with the hinge region is made by a chloro substituent.

Structure-Based Optimization Strategies for G Protein-Coupled Receptor (GPCR) Allosteric Modulators: A Case Study from Analyses of New Metabotropic Glutamate Receptor 5 (mGlu5) X-ray Structures.

Two interesting new X-ray structures of negative allosteric modulator (NAM) ligands for the mGlu5 receptor, M-MPEP (3) and fenobam (4), are reported. The new structures show how the binding of the ligands induces different receptor water channel conformations to previously published structures. The structure of fenobam, where a urea replaces the acetylenic linker in M-MPEP and mavoglurant, reveals a binding mode where the ligand is rotated by 180° compared to a previously proposed docking model. The need for multiple ligand structures for accurate GPCR structure-based drug design is demonstrated by the different growing vectors identified for the head groups of M-MPEP and mavoglurant and by the unexpected water-mediated receptor interactions of a new chemotype represented by fenobam. The implications of the new structures for ligand design are discussed, with extensive analysis of the energetics of the water networks of both pseudoapo and bound structures providing a new design strategy for allosteric modulators.

Perhalogenated pyrimidine scaffolds. Reactions of 5-chloro-2,4,6-trifluoropyrimidine with nitrogen centred nucleophiles.

BACKGROUND: Highly functionalised pyrimidine derivatives are of great importance to the life-science industries and there exists a need for efficient synthetic methodology that allows the synthesis of polysubstituted pyrimidine derivatives that are regioselective in all stages to meet the demands of RAS techniques for applications in parallel synthesis. 5-Chloro-2,4,6-trifluoropyrimidine may be used as a scaffold for the synthesis of polyfunctional pyrimidine systems if sequential nucleophilic aromatic substitution processes are regioselective. RESULTS: Use of 5-chloro-2,4,6-trifluoropyrimidine as a core scaffold for the synthesis of functionalised pyrimidine systems is assessed in reactions with a small range of nitrogen centred nucleophiles. Mixtures of products arising from nucleophilic aromatic substitution processes are formed, reflecting the activating effect of ring nitrogen and the steric influences of the chlorine atom. CONCLUSIONS: 5-Chloro-2,4,6-trifluoropyrimidine is not an ideal scaffold for analogue synthesis or for multiple substitution processes because purification must be performed to remove the 2-substituted regioisomer from the mixture before further reactions can be carried out. However, 4-amino derivatives can be isolated in acceptable yields using this methodology.

3,5-Disubstituted-indole-7-carboxamides as IKKß Inhibitors: Optimization of Oral Activity via the C3 Substituent.

IκB kinase ß (IKKß or IKK2) is a key regulator of nuclear factor kappa B (NF-κB) and has received attention as a therapeutic target. Herein we report on the optimization of a series of 3,5-disubstituted-indole-7-carboxamides for oral activity. In doing so, we focused attention on potency, ligand efficiency (LE), and physicochemical properties and have identified compounds 24 and (R)-28 as having robust in vivo activity.

Potential for the Rational Design of Allosteric Modulators of Class C GPCRs.

Class C G protein-coupled receptors encompass a range of promising therapeutic targets for a variety of diseases, yet to date only two members of this sub-family of GPCRs have been drugged. Recent advances in structural biology have revealed the X-ray crystallographic structures of allosteric ligands bound to two Class C metabotropic glutamate (mGlu) receptors, mGlu1 and mGlu5. Herein, we review how this information can be leveraged to help understand some of the historical challenges of mGlu receptor allosteric modulator drug discovery, and discuss how the structural enablement can be prospectively used for structurebased drug discovery approaches across Class C GPCR targets in general.

The use of conformationally thermostabilised GPCRs in drug discovery: application to fragment, structure and biophysical techniques.

Recent developments in receptor stabilisation have facilitated major advances in G protein-coupled receptor (GPCR) research, notably structural biology, over the past eight years. Here we review the application of fragment, structure and biophysical techniques using stabilised GPCRs (StaR proteins), and their impact in the drug discovery process. These techniques have, most recently, been utilised in the discovery of the non-alkyne mGlu5 negative allosteric modulator HTL14242, in addition to the dual orexin receptor antagonist HTL6641, with differentiated residence time kinetics.

Orexin Receptor Antagonists: Historical Perspectives and Future Opportunities.

The orexin receptors OX1 and OX2 play important roles in the regulation of sleep-wake cycles, feeding, reward and energy homeostasis. Since these G protein-coupled receptors were deorphanised in 1998, more than 200 patents containing orexin receptor antagonists have been filed and, in 2014, suvorexant (Belsomra®) became the first of these compounds to receive approval from the FDA. Suvorexant is a dual orexin receptor antagonist (DORA) which is available for the treatment of insomnia. This review provides a historical perspective on the discovery and development of DORAs as well as selective OX1 receptor antagonists (1-SORAs) and selective OX2 receptor antagonists (2-SORAs). 2-SORAs are under clinical evaluation for their ability to modulate sleep, and 1-SORAs have shown promise for the treatment of addiction in pre-clinical animal models. Detailed medicinal chemistry case studies are presented and future opportunities for orexin receptor antagonists are considered.

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.

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.