Theodore C. Jessop

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.

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.

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.

Lead optimization and structure-based design of potent and bioavailable deoxycytidine kinase inhibitors

A series of deoxycytidine kinase inhibitors was simultaneously optimized for potency and PK properties. A co-crystal structure then allowed merging this series with a high throughput screening hit to afford a highly potent, selective and orally bioavailable inhibitor, compound 10. This compound showed dose dependent inhibition of deoxycytidine kinase in vivo.

Reaction profiling by ultra high-pressure liquid chromatography/time-of-flight mass spectrometry in support of the synthesis of DNA-encoded libraries

An ultra high-pressure liquid chromatography/mass spectrometry (UHPLC/MS) separation and analysis method has been devised for open access analysis of synthetic reactions used in the production of DNA-encoded chemical libraries. The aqueous mobile phase is 100mM hexafluoroisopropanol and 8.6mM triethylamine; the organic mobile phase is methanol. The UHPLC separation uses a C18 OST column (50mm×2.1mm×1.7μm) at 60°C, with a flow rate of 0.6mL/min. Gradient concentration is from 10 to 40% B in 1.0min, increasing to 95% B at 1.2min. Cycle time was about 5min. This method provides a detection limit of a 20-mer oligonucleotide by mass spectrometry of better than 1pmol on-column. Linear UV response for 20-mer extends from 2 to 200pmol/μL in concentration, same-day relative average deviations are less than 5% and bias (observed minus expected) is less than 10%. Deconvoluted mass spectra are generated for components in the predicted mass range using a maximum entropy algorithm. Mass accuracy of deconvoluted spectra is typically 20ppm or better for isotopomers of oligonucleotides up to 7000Da.

5-Fluorocytosine derivatives as inhibitors of deoxycytidine kinase

A series of potent piperidine-linked cytosine derivatives were prepared as inhibitors of deoxycytidine kinase (dCK). Compound 9h was discovered to be a potent inhibitor of dCK and shows a good combination of cellular potency and pharmacokinetic parameters. Compound 9h blocks the incorporation of radiolabeled cytosine into mouse T-cells in vitro, as well as in vivo in mice following a T-cell challenge.

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.