Alan Main

Inhibition of Sphingosine 1-Phosphate Lyase for the Treatment of Rheumatoid Arthritis: Discovery of (E)-1-(4-((1R,2S,3R)-1,2,3,4-Tetrahydroxybutyl)-1H-imidazol-2-yl)ethanone Oxime (LX2931) and (1R,2S,3R)-1-(2-(Isoxazol-3-yl)-1H-imidazol-4-yl)butane-1,2,3,4-tetraol (LX2932)

Sphingosine 1-phosphate lyase (S1PL) has been characterized as a novel target for the treatment of autoimmune disorders using genetic and pharmacological methods. Medicinal chemistry efforts targeting S1PL by direct in vivo evaluation of synthetic analogues of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI, 1) led to the discovery of 2 (LX2931) and 4 (LX2932). The immunological phenotypes observed in S1PL deficient mice were recapitulated by oral administration of 2 or 4. Oral dosing of 2 or 4 yielded a dose-dependent decrease in circulating lymphocyte numbers in multiple species and showed a therapeutic effect in rodent models of rheumatoid arthritis (RA). Phase I clinical trials indicated that 2, the first clinically studied inhibitor of S1PL, produced a dose-dependent and reversible reduction of circulating lymphocytes and was well tolerated at dose levels of up to 180 mg daily. Phase II evaluation of 2 in patients with active rheumatoid arthritis is currently underway.

Modulation of Peripheral Serotonin Levels by Novel Tryptophan Hydroxylase Inhibitors for the Potential Treatment of Functional Gastrointestinal Disorders

The discovery of a novel class of peripheral tryptophan hydroxylase (TPH) inhibitors is described. This class of TPH inhibitors exhibits excellent potency in in vitro biochemical and cell-based assays, and it selectively reduces serotonin levels in the murine intestine after oral administration without affecting levels in the brain. These TPH1 inhibitors may provide novel treatments for gastrointestinal disorders associated with dysregulation of the serotonergic system, such as chemotherapy-induced emesis and irritable bowel syndrome.

Inhibition of Sphingosine-1-Phosphate Lyase for the Treatment of Autoimmune Disorders

During nearly a decade of research dedicated to the study of sphingosine signaling pathways, we identified sphingosine-1-phosphate lyase (S1PL) as a drug target for the treatment of autoimmune disorders. S1PL catalyzes the irreversible decomposition of sphingosine-1-phosphate (S1P) by a retro-aldol fragmentation that yields hexadecanaldehyde and phosphoethanolamine. Genetic models demonstrated that mice expressing reduced S1PL activity had decreased numbers of circulating lymphocytes due to altered lymphocyte trafficking, which prevented disease development in multiple models of autoimmune disease. Mechanistic studies of lymphoid tissue following oral administration of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI) 3 showed a clear relationship between reduced lyase activity, elevated S1P levels, and lower levels of circulating lymphocytes. Our internal medicinal chemistry efforts discovered potent analogues of 3 bearing heterocycles as chemical equivalents of the pendant carbonyl present in the parent structure. Reduction of S1PL activity by oral administration of these analogues recapitulated the phenotype of mice with genetically reduced S1PL expression.

Substituted 3-(4-(1,3,5-triazin-2-yl)-phenyl)-2-aminopropanoic acids as novel tryptophan hydroxylase inhibitors.

Tryptophan hydroxylase (TPH) is a key enzyme in the synthesis of serotonin. As a neurotransmitter, serotonin plays important physiological roles both peripherally and centrally. Here we describe the discovery of substituted triazines as a novel class of tryptophan hydroxylase inhibitors. This class of TPH inhibitors can selectively reduce serotonin levels in murine intestine after oral administration without affecting levels in the brain. These TPH inhibitors may provide novel treatments for gastrointestinal disorders associated with dysregulation of the serotonergic system, such as chemotherapy-induced emesis and irritable bowel syndrome.

Genetic Deletion of Mst1 Alters T Cell Function and Protects against Autoimmunity

Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1−/− B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1−/− CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.

OP0195 Genetic and Pharmacologic Inhibition of MST1 Blocks Lymphocyte Function and Protects Against Inflammation and Autoimmunity

Background The mammalian sterile 20-like kinase 1 (MST1) is a MAP4 kinase which acts upstream of a wide range of signal transduction pathways. It can modify signaling events mediated by Jnk, p38, histone 2B, hSav, FOXO3, and LFA-1; therefore it is involved in diverse cellular responses, including apoptosis, oxidative stress, integrin LFA-1 clustering, and lymphocyte adhesion and trafficking (1-3). Objectives We have explored the contribution of MST1 to immune function and disease development by performing a comprehensive phenotypic analysis of MST1-deficient (-/-) mice and challenging them in various disease models. We have also discovered potent inhibitors of MST1 and assessed their potential therapeutic utility in mouse models of arthritis. Methods Details of the phenotypic analysis applied to knockout lines generated in our facilities is described in Reference 4. It includes complete blood cell counts and phenotyping, in vitro cell activation assays, and in vivo challenges to assess immune system function during disease development. Results MST1-/- mice exhibited marked lymphopenia in the blood and peripheral lymphoid tissues compared to wild type littermates. Absence of MST1 reduced CD3/CD28-mediated in vitro T-cell proliferation and cytokine production, blocked cell cycle progression, elevated apoptosis of T cells, and altered expression of T-cell activation markers. In addition, MST1-/- B cells showed decreased responses to B-cell mitogens in vitro and deficient anti-ovalbumin Ig production in vivo. Consistent with altered T- and B-cell function, MST1 knockout mice were resistant to disease induction in a number of autoimmune and inflammatory disease models, including collagen-induced and anti-collagen antibody-induced arthritis. These findings have prompted the discovery of novel compounds that are potent inhibitors of MST1 and exhibit oral bioavailability. Administration of these compounds to mice recapitulated the disease-resistant phenotype of MST1-deficient mice. Conclusions MST1 controls multiple aspects of lymphocyte physiology and is essential for disease induction in a number of autoimmune and inflammatory disease models. References Nehme NT, et al. Blood. 119:777-85, 2012. Mou F, et al. J. Exp. Med. 209:741-59, 2012. Avruch J, et al. Semin. Cell. Dev. Biol. 23:770-84, 2012. Beltrandelrio H et al. in Model Organisms in Drug Discovery. P. M. Carroll, and K. Fitzgerald, eds. John Wiley & Sons, Chichester, United Kingdom, pp. 251–278, 2003. Disclosure of Interest T. Oravecz Shareholder of: Lexicon Pharmaceuticals, Inc., Employee of: Lexicon Pharmaceuticals, Inc., W. Chang Shareholder of: Lexicon Pharmaceuticals, Inc., Employee of: Lexicon Pharmaceuticals, Inc., K. Jhaver Shareholder of: Lexicon Pharmaceuticals, Inc., Employee of: Lexicon Pharmaceuticals, Inc., A. Al-Shami Shareholder of: Lexicon Pharmaceuticals, Inc., Employee of: Lexicon Pharmaceuticals, Inc., T. Jessop Shareholder of: Lexicon Pharmaceuticals, Inc., Employee of: Lexicon Pharmaceuticals, Inc., B. Hamman Shareholder of: Lexicon Pharmaceuticals, Inc., Employee of: Lexicon Pharmaceuticals, Inc., J. Bagdanoff Shareholder of: Lexicon Pharmaceuticals, Inc., Employee of: Lexicon Pharmaceuticals, Inc., D. Augeri Shareholder of: Lexicon Pharmaceuticals, Inc., Employee of: Lexicon Pharmaceuticals, Inc., P. Vogel Shareholder of: Lexicon Pharmaceuticals, Inc., Employee of: Lexicon Pharmaceuticals, Inc., J. Swaffield Shareholder of: Lexicon Pharmaceuticals, Inc., Employee of: Lexicon Pharmaceuticals, Inc., A. Wilson Shareholder of: Lexicon Pharmaceuticals, Inc., Employee of: Lexicon Pharmaceuticals, Inc., K. Carson Shareholder of: Lexicon Pharmaceuticals, Inc., Employee of: Lexicon Pharmaceuticals, Inc., A. Main Shareholder of: Lexicon Pharmaceuticals, Inc., Employee of: Lexicon Pharmaceuticals, Inc., B. Zambrowicz Shareholder of: Lexicon Pharmaceuticals, Inc., Employee of: Lexicon Pharmaceuticals, Inc.