Nicholas Paul Barton

Distinct Conformations of the Chemokine Receptor CCR4 with Implications for Its Targeting in Allergy

CC chemokine receptor 4 (CCR4) is expressed by Th2 and regulatory T cells and directs their migration along gradients of the chemokines CCL17 and CCL22. Both chemokines and receptor are upregulated in allergic disease, making CCR4 a therapeutic target for the treatment of allergy. We set out to assess the mechanisms underlying a previous report that CCL22 is a dominant ligand of CCR4, which may have implications for its therapeutic targeting. Human T cells expressing endogenous CCR4 and transfectants engineered to express CCR4 were assessed for receptor function, using assays of calcium release, chemotaxis, receptor endocytosis, and ligand binding. Despite the two ligands having equal potency in calcium flux and chemotaxis assays, CCL22 showed dominance in both receptor endocytosis assays and heterologous competitive binding assays. Using two different CCR4-specific Abs, we showed that CCR4 exists in at least two distinct conformations, which are differentially activated by ligand. A major population is activated by both CCL17 and CCL22, whereas a minor population is activated only by CCL22. Mutation of a single C-terminal residue K310 within a putative CCR4 antagonist binding site ablated activation of CCR4 by CCL17, but not by CCL22, despite having no effect on the binding of either ligand. We conclude that CCL17 and CCL22 are conformationally selective ligands of CCR4 and interact with the receptor by substantially different mechanisms. This finding suggests that the selective blockade of CCR4 in allergy may be feasible when one CCR4 ligand dominates, allowing the inhibition of Th2 signaling via one ligand while sparing regulatory T cell recruitment via another.

Synthesis and Structure–Activity Relationships of Indazole Arylsulfonamides as Allosteric CC-Chemokine Receptor 4 (CCR4) Antagonists

A series of indazole arylsulfonamides were synthesized and examined as human CCR4 antagonists. Methoxy- or hydroxyl-containing groups were the more potent indazole C4 substituents. Only small groups were tolerated at C5, C6, or C7, with the C6 analogues being preferred. The most potent N3-substituent was 5-chlorothiophene-2-sulfonamide. N1 meta-substituted benzyl groups possessing an α-amino-3-[(methylamino)acyl]-group were the most potent N1-substituents. Strongly basic amino groups had low oral absorption in vivo. Less basic analogues, such as morpholines, had good oral absorption; however, they also had high clearance. The most potent compound with high absorption in two species was analogue 6 (GSK2239633A), which was selected for further development. Aryl sulfonamide antagonists bind to CCR4 at an intracellular allosteric site denoted site II. X-ray diffraction studies on two indazole sulfonamide fragments suggested the presence of an important intramolecular interaction in the active conformation.

Discovery of Novel Irreversible Inhibitors of Interleukin (IL)-2-inducible Tyrosine Kinase (Itk) by Targeting Cysteine 442 in the ATP Pocket

Background: Itk is considered an important target for anti-inflammatory drug discovery, particularly asthma. Results: We have designed an irreversible covalent inhibitor of the kinase by targeting Cys-442 in the ATP pocket. Conclusion: We have produced a potent, selective inhibitor with a long duration of action. Significance: We describe a novel drug discovery strategy with specific design features for inhaled delivery. IL-2-inducible tyrosine kinase (Itk) plays a key role in antigen receptor signaling in T cells and is considered an important target for anti-inflammatory drug discovery. In order to generate inhibitors with the necessary potency and selectivity, a compound that targeted cysteine 442 in the ATP binding pocket and with an envisaged irreversible mode of action was designed. We incorporated a high degree of molecular recognition and specific design features making the compound suitable for inhaled delivery. This study confirms the irreversible covalent binding of the inhibitor to the kinase by x-ray crystallography and enzymology while demonstrating potency, selectivity, and prolonged duration of action in in vitro biological assays. The biosynthetic turnover of the kinase was also examined as a critical factor when designing irreversible inhibitors for extended duration of action. The exemplified Itk inhibitor demonstrated inhibition of both TH1 and TH2 cytokines, was additive with fluticasone propionate, and inhibited cytokine release from human lung fragments. Finally, we describe an in vivo pharmacodynamic assay that allows rapid preclinical development without animal efficacy models.

The identification and optimisation of novel and selective diamide neuropeptide Y Y2 receptor antagonists

A novel small molecule NPY Y2 antagonist (3) identified from high throughput screening is described. A subsequent SAR study and optimisation programme based around this molecule is also described, leading to the identification of potent and soluble pyridyl analogue 36.

Antagonism of human CC-chemokine receptor 4 can be achieved through three distinct binding sites on the receptor

Chemokine receptor antagonists appear to access two distinct binding sites on different members of this receptor family. One class of CCR4 antagonists has been suggested to bind to a site accessible from the cytoplasm while a second class did not bind to this site. In this report, we demonstrate that antagonists representing a variety of structural classes bind to two distinct allosteric sites on CCR4. The effects of pairs of low‐molecular weight and/or chemokine CCR4 antagonists were evaluated on CCL17‐ and CCL22‐induced responses of human CCR4+ T cells. This provided an initial grouping of the antagonists into sets which appeared to bind to distinct binding sites. Binding studies were then performed with radioligands from each set to confirm these groupings. Some novel receptor theory was developed to allow the interpretation of the effects of the antagonist combinations. The theory indicates that, generally, the concentration‐ratio of a pair of competing allosteric modulators is maximally the sum of their individual effects while that of two modulators acting at different sites is likely to be greater than their sum. The low‐molecular weight antagonists could be grouped into two sets on the basis of the functional and binding experiments. The antagonistic chemokines formed a third set whose behaviour was consistent with that of simple competitive antagonists. These studies indicate that there are two allosteric regulatory sites on CCR4.

The identification of a series of novel, soluble non-peptidic neuropeptide Y Y2 receptor antagonists

The identification and subsequent optimisation of a selective non-peptidic NPY Y2 antagonist series is described. This led to the development of amine 2, a selective, soluble NPY Y2 receptor antagonist with enhanced CNS exposure.

Evolution of a Novel, Orally Bioavailable Series of PI3Kδ Inhibitors from an Inhaled Lead for the Treatment of Respiratory Disease

A four-step process of high-quality modeling of existing data, deconstruction, identification of replacement cores, and an innovative synthetic regrowth strategy led to the rapid discovery of a novel oral series of PI3Kδ inhibitors with promising selectivity and excellent in vivo characteristics.

A practical drug discovery project at the undergraduate level.

In this article, we describe a practical drug discovery project for third-year undergraduates. No previous knowledge of medicinal chemistry is assumed. Initial lecture workshops cover the basic principles; then students, in teams, seek to improve the profile of a weakly potent, insoluble phosphatidylinositide 3-kinase delta (PI3Kδ) inhibitor (1) through compound array design, molecular modelling, screening data analysis and the synthesis of target compounds in the laboratory. The project benefits from significant industrial support, including lectures, student mentoring and consumables. The aim is to make the learning experience as close as possible to real-life industrial situations. In total, 48 target compounds were prepared, the best of which (5b, 5j, 6b and 6ap) improved the potency and aqueous solubility of the lead compound (1) by 100-1000 fold and ≥tenfold, respectively.

Discovery and optimisation of a potent and selective tertiary sulfonamide oxytocin antagonist

The optimisation of a tertiary sulfonamide high-throughput screening hit is described. A combination of high-throughput chemistry, pharmacophore analysis and in silico PK profiling resulted in the discovery of potent sulfonamide oxytocin receptor antagonists with oral exposure and good selectivity over vasopressin receptors.