John S. Whoriskey

T-cell co-stimulation through B7RP-1 and ICOS.

T-cell activation requires co-stimulation through receptors such as CD28 (refs 1,2,3) and antigen-specific signalling through the T-cell antigen receptor. Here we describe a new murine co-stimulatory receptor–ligand pair. The receptor, which is related to CD28 and is the homologue of the human protein ICOS, is expressed on activated T cells and resting memory T cells. The ligand, which has homology to B7 molecules and is called B7-related protein-1 (B7RP-1), is expressed on B cells and macrophages. ICOS and B7RP-1 do not interact with proteins in the CD28–B7 pathway, and B7RP-1 co-stimulates T cells in vitro independently of CD28. Transgenic mice expressing a B7RP-1–Fc fusion protein show lymphoid hyperplasia in the spleen, lymph nodes and Peyers patches. Presensitized mice treated with B7RP-1–Fc during antigen challenge show enhanced hypersensitivity. Therefore, B7RP-1 exhibits co-stimulatory activities in vitro and in vivo. ICOS and B7RP-1 define a new and distinct receptor–ligand pair that is structurally related to CD28–B7 and is involved in the adaptive immune response.

G-Protein-Coupled Receptor 35 Is a Target of the Asthma Drugs Cromolyn Disodium and Nedocromil Sodium

We report that the asthma drugs cromolyn disodium and nedocromil sodium are potent G-protein-coupled receptor 35 (GPR35) agonists. We utilized calcium flux and inositol phosphate accumulation assays to examine the pharmacology of these asthma drugs on the human, mouse and rat GPR35. The compounds were more potent on the human GPR35 than on mouse and rat receptors. In contrast, zaprinast, a known GPR35 agonist, was more potent on mouse and rat GPR35 than the human ortholog. We show by quantitative PCR that GPR35 is expressed in human mast cells, human basophils and human eosinophils. We also demonstrate that GPR35 mRNA is upregulated upon challenge with IgE antibodies. We show that, unlike zaprinast, a potent phosphodiesterase 5 (PDE5) inhibitor, cromolyn disodium and nedocromil sodium lack inhibitory activity towards PDE5. These findings suggest that GPR35 may play an important role in mast cell biology and be a potential target for the treatment of asthma.

Potent activity of soluble B7RP-1-Fc in therapy of murine tumors in syngeneic hosts.

We have characterized a receptor:ligand pair, ICOS:B7RP‐1, that is structurally and functionally related to CD28:B7.1/2. We reported previously that B7RP‐1 costimulates T cell proliferation and immune responses (Yoshinaga et al., Nature 1999;402:827–32; Guo et al., J Immunol 2001;166:5578–84; Yoshinaga et al., Int Immunol 2000;12:1439–47). We report that B7RP‐1‐Fc causes rejection or growth inhibition of Meth A, SA‐1 and EMT6 tumors in syngeneic mice. Established Meth A tumors were rejected effectively with a single dose of B7RP‐1‐Fc, however, the treatment was less effective on larger tumors. Mice that rejected Meth A tumors previously by Day 30, also rejected a subsequent Meth A challenge on Day 60, without additional B7RP‐1‐Fc treatment, indicating a long‐lived memory response. Tumor cells believed to be less immunogenic, such as P815 and EL‐4 cells, were less responsive to this treatment. The EL‐4 responsiveness to the B7RP‐1‐Fc treatment was enhanced, however, by pre‐treatment of the mice with cyclophosphamide. As expected, T cells appeared to be targeted by B7RP‐1‐Fc treatment. Thus, the administration of soluble B7RP‐1‐Fc may have therapeutic value in generating or enhancing anti‐tumor activity in a clinical setting.

Discovery and in vivo evaluation of (S)-N-(1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine (AMG319) and related PI3Kδ inhibitors for inflammation and autoimmune disease.

The development and optimization of a series of quinolinylpurines as potent and selective PI3Kδ kinase inhibitors with excellent physicochemical properties are described. This medicinal chemistry effort led to the identification of 1 (AMG319), a compound with an IC50 of 16 nM in a human whole blood assay (HWB), excellent selectivity over a large panel of protein kinases, and a high level of in vivo efficacy as measured by two rodent disease models of inflammation.

Discovery, Optimization, and in Vivo Evaluation of Benzimidazole Derivatives AM-8508 and AM-9635 as Potent and Selective PI3Kδ Inhibitors

Lead optimization efforts resulted in the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 1 (AM-8508) and 2 (AM-9635), with good pharmacokinetic properties. The compounds inhibit B cell receptor (BCR)-mediated AKT phosphorylation (pAKT) in PI3Kδ-dependent in vitro cell based assays. These compounds which share a benzimidazole bicycle are effective when administered in vivo at unbound concentrations consistent with their in vitro cell potency as a consequence of improved unbound drug concentration with lower unbound clearance. Furthermore, the compounds demonstrated efficacy in a Keyhole Limpet Hemocyanin (KLH) study in rats, where the blockade of PI3Kδ activity by inhibitors 1 and 2 led to effective inhibition of antigen-specific IgG and IgM formation after immunization with KLH.

Spontaneous Colitis Occurrence in Transgenic Mice with Altered B7-Mediated Costimulation

The B7 costimulatory molecules govern many aspects of T cell immune responses by interacting with CD28 for costimulation, but also with CTLA-4 for immune suppression. Although blockade of CTLA-4 with Ab in humans undergoing cancer immune therapy has led to some cases of inflammatory bowel disease, spontaneous animal models of colitis that depend upon modulation of B7 interactions have not been previously described. In this study, we demonstrate that mice expressing a soluble B7-2 Ig Fc chimeric protein spontaneously develop colitis that is dependent on CD28-mediated costimulation of CD4+ T cells. We show that the chimeric protein has mixed agonistic/antagonist properties, and that it acts in part by blocking the cell intrinsic effects on T cell activation of engagement of CTLA-4. Disease occurred in transgenic mice that lack expression of the endogenous B7 molecules (B7 double knock-out mice), because of the relatively weak costimulatory delivered by the chimeric protein. Surprisingly, colitis was more severe in this context, which was associated with the decreased number of Foxp3+ regulatory T cells in transgenic B7 double knock-out mice. This model provides an important tool for examining how B7 molecules and their effects on CTLA-4 modulate T cell function and the development of inflammatory diseases.

Discovery and in Vivo Evaluation of the Potent and Selective PI3Kδ Inhibitors 2-((1S)-1-((6-Amino-5-cyano-4-pyrimidinyl)amino)ethyl)-6-fluoro-N-methyl-3-(2-pyridinyl)-4-quinolinecarboxamide (AM-0687) and 2-((1S)-1-((6-Amino-5-cyano-4-pyrimidinyl)amino)ethyl)-5-fluoro-N-methyl-3-(2-pyridinyl)-4-quinolinecarboxamide (AM-1430)

Optimization of the potency and pharmacokinetic profile of 2,3,4-trisubstituted quinoline, 4, led to the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 6a (AM-0687) and 7 (AM-1430). On the basis of their improved profile, these analogs were selected for in vivo pharmacodynamic (PD) and efficacy experiments in animal models of inflammation. The in vivo PD studies, which were carried out in a mouse pAKT inhibition animal model, confirmed the observed potency of 6a and 7 in biochemical and cellular assays. Efficacy experiments in a keyhole limpet hemocyanin model in rats demonstrated that administration of either 6a or 7 resulted in a strong dose-dependent reduction of IgG and IgM specific antibodies. The excellent in vitro and in vivo profiles of these analogs make them suitable for further development.

The anthelmintic niclosamide is a potent TMEM16A antagonist that fully bronchodilates airways

There is an unmet need in severe asthma where approximately 40% of patients exhibit poor β-agonist responsiveness, suffer daily symptoms and show frequent exacerbations. Antagonists of the Ca2+-activated-Cl− channel, TMEM16A, offers a new mechanism to bronchodilate airways and block the multiple contractiles operating in severe disease. To identify TMEM16A antagonists we screened a library of ~580,000 compounds. The anthelmintics niclosamide, nitazoxanide and related compounds were identified as potent TMEM16A antagonists that blocked airway smooth muscle depolarization and contraction. To evaluate whether TMEM16A antagonists resist use- and inflammatory-desensitization pathways limiting β-agonist action, we tested their efficacy under harsh conditions using maximally contracted airways or airways pretreated with a cytokine cocktail. Stunningly, TMEM16A antagonists fully bronchodilated airways, while the β-agonist isoproterenol showed only partial effects. Thus, antagonists of TMEM16A and repositioning of niclosamide and nitazoxanide represent an important additional treatment for patients with severe asthma and COPD that is poorly controlled with existing therapies. It is of note that drug repurposing has also attracted wide interest in niclosamide and nitazoxanide as a new treatment for cancer and infectious disease. For the first time we identify TMEM16A as a molecular target for these drugs and thus provide fresh insights into their mechanism for the treatment of these disorders in addition to respiratory disease.