Jonathan Swaffield

Incomplete Inhibition of Sphingosine 1-Phosphate Lyase Modulates Immune System Function yet Prevents Early Lethality and Non-Lymphoid Lesions

Background S1PL is an aldehyde-lyase that irreversibly cleaves sphingosine 1-phosphate (S1P) in the terminal step of sphingolipid catabolism. Because S1P modulates a wide range of physiological processes, its concentration must be tightly regulated within both intracellular and extracellular environments. Methodology In order to better understand the function of S1PL in this regulatory pathway, we assessed the in vivo effects of different levels of S1PL activity using knockout (KO) and humanized mouse models. Principal Findings Our analysis showed that all S1PL-deficient genetic models in this study displayed lymphopenia, with sequestration of mature T cells in the thymus and lymph nodes. In addition to the lymphoid phenotypes, S1PL KO mice (S1PL−/−) also developed myeloid cell hyperplasia and significant lesions in the lung, heart, urinary tract, and bone, and had a markedly reduced life span. The humanized knock-in mice harboring one allele (S1PLH/−) or two alleles (S1PLH/H) of human S1PL expressed less than 10 and 20% of normal S1PL activity, respectively. This partial restoration of S1PL activity was sufficient to fully protect both humanized mouse lines from the lethal non-lymphoid lesions that developed in S1PL−/− mice, but failed to restore normal T-cell development and trafficking. Detailed analysis of T-cell compartments indicated that complete absence of S1PL affected both maturation/development and egress of mature T cells from the thymus, whereas low level S1PL activity affected T-cell egress more than differentiation. Significance These findings demonstrate that lymphocyte trafficking is particularly sensitive to variations in S1PL activity and suggest that there is a window in which partial inhibition of S1PL could produce therapeutic levels of immunosuppression without causing clinically significant S1P-related lesions in non-lymphoid target organs.

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.

Development of a highthroughput yeast-based assay for detection of metabolically activated genotoxins

The potential genotoxicity of drug candidates is a serious concern during drug development. Therefore, it is important to assess the potential genotoxicity and mutagenicity of a compound early in the discovery phase of drug development. AMES Salmonella assay is the most widely used assay for the assessment of mutagenicity and genotoxicity. However, the AMES assay is not readily adaptable to highthroughput screening and several strains of Salmonella must be employed to ensure that different types of DNA damage can be studied. Therefore, an additional robust highthroughput genotoxicity screen would be of significant value in the early detection and elimination of genotoxicity. The complexity of DNA damage requires numerous cellular pathways, thus using single model organism to predict genotoxicity in early stage is challenging. Another critical component of such screens is that they incorporate the capability of metabolic activation to ensure that no genotoxic metabolites are generated. We have developed a novel highthroughput reporter assay for DNA repair that detects genotoxicity, and which incorporates metabolic activation. The assay has a low compound requirement as compared to Ames, and relies upon two different reporter genes cotransfected into a yeast strain. The gene encoding Renilla luciferase is fused to the constitutive 3-phosphoglycerate kinase (PGK1) promoter and integrated into the yeast genome to provide a control for cell numbers. The firefly luciferase gene is fused to the RAD51 (bacterial RecA homolog) promoter and used to report an increase in DNA repair activity. A dual luciferase assay is performed by measuring the firefly and Renilla luciferase activities in the same sample. The result is expressed as the ratio of the two luciferase activities; changes from the base level (control) are interpreted as induction of the RAD51 promoter and evidence of DNA repair activity in eukaryote cells due to DNA damage. The yeast dual luciferase reporter has been characterized with and without S-9 activation using positive and negative control agents. This assay is efficient, requires little time and low amounts of compound. The assay is compatible with metabolic activation, adaptable to a highthroughput platform, and yields data that accurately and reproducibly detects DNA damage. Whereas the normal yeast cell wall, plasma membrane composition and the presence of active transporters can prevent the entry or persistence of some compounds internally in yeast cells, our assay did show concordance with regulatory mutagenicity assays, many of which require metabolic activation and are poorly detected by bacterial mutagenicity assays. Although there were false negative results, in our hands this assay performs as well as or better than other commercially available genetox assays. Furthermore, the RAD51 gene is strongly inducible by homologous intrachromosomal recombination; thus this assay may provide a means to detect clastogens. The RAD51 promoter fused dual luciferase assay represents a valuable addition to the armamentarium for the early detection of genotoxic compounds.

Novel Potent Inhibitors of Deoxycytidine Kinase Identified and Compared by Multiple Assays

Deoxycytidine kinase (dCK) phosphorylates deoxycytidine, deoxyguanosine, and deoxyadenosine and plays an important role in the salvage pathway of nucleoside metabolism. dCK is also required for the phosphorylation of several antiviral and anticancer nucleoside drugs, with resistance to these agents often being associated with a loss or decrease in dCK activity. Data also indicate a role for dCK in immune function, and dCK inhibitors may provide treatment for immune disorders. To identify novel dCK inhibitors, the authors evaluated 2 existing biochemical assays, adapted both to high-throughput screening, and identified several series of hits. They also compared the potency of the hits between purified recombinant and endogenous enzyme. Meanwhile, they also developed a novel cell-based assay that rests on the rescue of cells from dCK-dependent cytotoxic agents such as AraC. A large number of compounds were tested using the 3 assays, and a strong correlation in potency was observed between the biochemical assay using endogenous enzyme and the cell-based assay. The hits identified in these screens have proved to be good starting points for the synthesis of much more potent tool compounds to further investigate the physiological functions of dCK and potentially lead to the development of therapeutic agents.

Identification of Small Molecules That Selectively Inhibit Diacylglycerol Lipase-α Activity

Recent genetic evidence suggests that the diacylglycerol lipase (DAGL-α) isoform is the major biosynthetic enzyme for the most abundant endocannabinoid, 2-arachidonoyl-glycerol (2-AG), in the central nervous system. Revelation of its essential role in regulating retrograde synaptic plasticity and adult neurogenesis has made it an attractive therapeutic target. Therefore, it has become apparent that selective inhibition of DAGL-α enzyme activity with a small molecule could be a strategy for the development of novel therapies for the treatment of disease indications such as depression, anxiety, pain, and cognition. In this report, the authors present the identification of small-molecule inhibitor chemotypes of DAGL-α, which were selective (≥10-fold) against two other lipases, pancreatic lipase and monoacylglycerol lipase, via high-throughput screening of a diverse compound collection. Seven chemotypes of interest from a list of 185 structural clusters, which included 132 singletons, were initially selected for evaluation and characterization. Selection was based on potency, selectivity, and chemical tractability. One of the chemotypes, the glycine sulfonamide series, was prioritized as an initial lead for further medicinal chemistry optimization.

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.

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.