Search

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in the United States. PqsE, a thioesterase enzyme, is vital for virulence of P. aeruginosa, making PqsE an attractive target for inhibition. Neither the substrate nor the product of PqsE catalysis has been identified. A library of 550 million DNA-encoded drug-like small molecules was screened for those that bind to the purified PqsE protein. The structures of the bound molecules were identified by high throughput sequencing of the attached DNA barcodes. Putative PqsE binders with the strongest affinity features were examined for inhibition of PqsE thioesterase activity in vitro. The most potent inhibitors were resynthesized off DNA and examined for the ability to alter PqsE thermal melting and for PqsE thioesterase inhibition. Here, we report the synthesis, biological activity, mechanism of action, and early structure-activity relationships of a series of 2-(phenylcarbamoyl)benzoic acids that noncompetitively inhibit PqsE. A small set of analogs designed to probe initial structure-activity relationships showed increases in potency relative to the original hits, the best of which has an IC50 = 5 μM. Compound refinement is required to assess their in vivo activities as the current compounds do not accumulate in the P. aeruginosa cytosol. Our strategy validates DNA-encoded compound library screening as a rapid and effective method to identify catalytic inhibitors of the PqsE protein, and more generally, for discovering binders to bacterial proteins revealed by genetic screening to have crucial in vivo activities but whose biological functions have not been well-defined.

BackgroundImmunotherapies aiming to boost anti-tumor cell responses in cancer patients has been proven successful by checkpoint inhibitors targeting PD1 or CTLA-4, but the majority of cancer patients do not garner durable benefit. Co-stimulation through the CD28 pathway is one potential approach to maximize the benefits of immunotherapies. The E3 ubiquitin ligase Cbl-b (casitas b-lineage lymphoma proto-oncogene b) has been established as a master negative regulator of T-cells and NK cells and plays an important role in immune suppression. Genetic ablation of Cbl-b or functional inactivation of its E3 ligase activity in mice resulted in CD8 T-cell-mediated rejection of primary tumors in several mouse models. Based on the overwhelming evidence supporting the role of Cbl-b in immune suppression, targeting Cbl-b with small molecule inhibitors is attractive for cancer immunotherapy.MethodsCbl-b is activated by tyrosine kinases and undergoes a large conformational change from closed inactive form to open active form. Historically, it had been difficult to identify inhibitors of Cbl-b. Through the utilization of our proprietary SpotFinder platform, a druggable phosphoregulatory pocket was identified in the inactive form of Cbl-b. Learnings from the platform allowed for the development of screening assays utilizing specifically designed protein constructs. Assays were developed to identify inhibitors that bind to the hotspot and lock Cbl-b in its inactive form.ResultsHere we report on a member of our lead series of inhibitors, a low nanomolar potent inhibitor identified via application of our SpotFinder platform. This inhibitor binds to the inactive form of Cbl-b, its binding mode in the identified hotspot confirmed by co-crystal structures. It inhibits the phosphorylation of Cbl-b by kinases, inhibits the E3 ligase activity of Cbl-b, promotes cytokine release and enhances T cell proliferation well as NK cell activation and killing. In vivo, our CBL-B inhibitors efficaciously augmented the T cell response in anti-CD3 treated mice.ConclusionsWe herein demonstrated the validation of our proprietary SpotFinder platform via the prediction and drugging of a regulatory hotspot on an important immune oncology target that has to date been very difficult to drug.

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in the United States. PqsE, a thioesterase enzyme, is vital for virulence of P. aeruginosa, making PqsE an attractive target for inhibition. Neither the substrate nor the product of PqsE catalysis has been identified. A library of 550 million DNA-encoded drug-like small molecules was screened for those that bind to the purified PqsE protein. The structures of the bound molecules were identified by high throughput sequencing of the attached DNA barcodes. Putative PqsE binders with the strongest affinity features were examined for inhibition of PqsE thioesterase activity in vitro. The most potent inhibitors were re-synthesized off DNA and examined for the ability to alter PqsE thermal melting and for PqsE thioesterase inhibition. Here, we report the synthesis, biological activity, mechanism of action, and early structure-activity relationships of a series of 2-(phenylcarbamoyl)benzoic acids that non-competitively inhibit PqsE. A small set of analogs designed to probe initial structure-activity relationships showed increases in potency relative to the original hits, the best of which has an IC(50) = 5 μM. Compound refinement is required to assess their in vivo activities as the current compounds do not accumulate in the P. aeruginosa cytosol. Our strategy validates DNA-encoded compound library screening as a rapid and effective method to identify catalytic inhibitors of the PqsE protein, and more generally, for discovering binders to bacterial proteins revealed by genetic screening to have crucial in vivo activities but whose biological functions have not been well defined.

The increasing number of people afflicted with diabetes throughout the world is a major health issue. Inhibitors of the sodium-dependent glucose cotransporters (SGLT) have appeared as viable therapeutics to control blood glucose levels in diabetic patents. Herein we report the discovery of LX2761, a locally acting SGLT1 inhibitor that is highly potent in vitro and delays intestinal glucose absorption in vivo to improve glycemic control.

To identify novel targets for neuropathic pain, 3097 mouse knockout lines were tested in acute and persistent pain behavior assays. One of the lines from this screen, which contained a null allele of the adapter protein-2 associated kinase 1 (AAK1) gene, had a normal response in acute pain assays (hot plate, phase I formalin), but a markedly reduced response to persistent pain in phase II formalin. AAK1 knockout mice also failed to develop tactile allodynia following the Chung procedure of spinal nerve ligation (SNL). Based on these findings, potent, small-molecule inhibitors of AAK1 were identified. Studies in mice showed that one such inhibitor, LP-935509, caused a reduced pain response in phase II formalin and reversed fully established pain behavior following the SNL procedure. Further studies showed that the inhibitor also reduced evoked pain responses in the rat chronic constriction injury (CCI) model and the rat streptozotocin model of diabetic peripheral neuropathy. Using a nonbrain-penetrant AAK1 inhibitor and local administration of an AAK1 inhibitor, the relevant pool of AAK1 for antineuropathic action was found to be in the spinal cord. Consistent with these results, AAK1 inhibitors dose-dependently reduced the increased spontaneous neural activity in the spinal cord caused by CCI and blocked the development of windup induced by repeated electrical stimulation of the paw. The mechanism of AAK1 antinociception was further investigated with inhibitors of α2 adrenergic and opioid receptors. These studies showed that α2 adrenergic receptor inhibitors, but not opioid receptor inhibitors, not only prevented AAK1 inhibitor antineuropathic action in behavioral assays, but also blocked the AAK1 inhibitor-induced reduction in spinal neural activity in the rat CCI model. Hence, AAK1 inhibitors are a novel therapeutic approach to neuropathic pain with activity in animal models that is mechanistically linked (behaviorally and electrophysiologically) to α2 adrenergic signaling, a pathway known to be antinociceptive in humans.

A group of small molecule thienochromenes inhibitors of Notum Pectinacetylesterase are described. We developed SAR on three series based on carbon, oxygen and sulfur replacement of the 5-position. In each series, highly potent Notum Pectinacetylesterase inhibitors were identified.

Small molecule modulators of GPR88 activity (agonists, antagonists, or modulators) are of interest as potential agents for the treatment of a variety of psychiatric disorders including schizophrenia. A series of phenylglycinol and phenylamine analogs have been prepared and evaluated for their GPR88 agonist activity and pharmacokinetic (PK) properties.

LX2761 is a potent sodium/glucose cotransporter 1 inhibitor restricted to the intestinal lumen after oral administration. Studies presented here evaluated the effect of orally administered LX2761 on glycemic control in preclinical models. In healthy mice and rats treated with LX2761, blood glucose excursions were lower and plasma total glucagon-like peptide-1 (GLP-1) levels higher after an oral glucose challenge; these decreased glucose excursions persisted even when the glucose challenge occurred 15 hours after LX2761 dosing in ad lib-fed mice. Further, treating mice with LX2761 and the dipeptidyl-peptidase 4 inhibitor sitagliptin synergistically increased active GLP-1 levels, suggesting increased LX2761-mediated release of GLP-1 into the portal circulation. LX2761 also lowered postprandial glucose, fasting glucose, and hemoglobin A1C, and increased plasma total GLP-1, during long-term treatment of mice with either early- or late-onset streptozotocin-diabetes; in the late-onset cohort, LX2761 treatment improved survival. Mice and rats treated with LX2761 occasionally had diarrhea; this dose-dependent side effect decreased in severity and frequency over time, and LX2761 doses were identified that decreased postprandial glucose excursions without causing diarrhea. Further, the frequency of LX2761-associated diarrhea was greatly decreased in mice either by gradual dose escalation or by pretreatment with resistant starch 4, which is slowly digested to glucose in the colon, a process that primes the colon for glucose metabolism by selecting for glucose-fermenting bacterial species. These data suggest that clinical trials are warranted to determine if LX2761 doses and dosing strategies exist that provide improved glycemic control combined with adequate gastrointestinal tolerability in people living with diabetes.

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.

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 dCKdependent 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. (Journal of Biomolecular Screening 2010:72-79)

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.

A group of small molecule thienopyrimidine inhibitors of Notum Pectinacetylesterase are described. We explored both 2-((5,6-thieno[2,3-d]pyrimidin-4-yl)thio)acetic acids and 2-((6,7-thieno[3,2-d]pyrimidin-4-yl)thio)acetic acids. In both series, highly potent, orally active Notum Pectinacetylesterase inhibitors were identified.

Interleukin-8 modulation is implicated in many inflammatory and cancer diseases. Starting from a mass-screening hit, the synthesis and structure-activity relationship of 2-amino-3-heteroarylquinoxalines as non-peptide, small molecule interleukine-8 receptor antagonists have been developed. The optimized derivatives, PD 0210293 (13y) and PD 0220245 (13r), show inhibition of both IL-8 receptor binding and IL-8-mediated neutrophil chemotaxis.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of oral anti-diabetic agents that improve glycemic control by inhibiting SGLT2-mediated renal glucose reabsorption. Currently available agents increase urinary glucose excretion (UGE) to 50% of filtered glucose when SGLT2 is completely inhibited. This led us to test whether LP-925219, a small molecule dual SGLT1/SGLT2 inhibitor, increases UGE to maximal values in wild-type (WT) mice. We first tested LP-925219 inhibition of glucose transport by HEK293 cells expressing SGLT1 or SGLT2, and then characterized LP-925219 pharmacokinetics. We found that LP-925219 was a potent inhibitor of mouse SGLT1 (IC50 = 22.6 nmol/L) and SGLT2 (IC50 = 0.5 nmol/L), and that a 10 mg/kg oral dose was bioavailable (87%) with a long half-life (7 h). We next delivered LP-925219 by oral gavage to WT, SGLT1 knockout (KO), SGLT2 KO, and SGLT1/SGLT2 double KO (DKO) mice and measured their 24-h UGE. We found that, in vehicle-treated mice, DKO UGE was maximal and SGLT2 KO, SGLT1 KO, and WT UGEs were 30%, 2%, and 0.2% of maximal, respectively; we also found that LP-925219 dosed at 60 mg/kg twice daily increased UGE of SGLT1 KO, SGLT2 KO, and WT mice to DKO UGE levels. These findings show that orally available dual SGLT1/SGLT2 inhibitors can maximize 24-h UGE in mammals, and suggest that such agents merit further evaluation for their potential, in diabetic patients, to achieve better glycemic control than is achieved using selective SGLT2 inhibitors.

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.

The amino acid sequence L-D-T-S-L has been shown to be a significant recognition motif involved in the interaction of integrin α4β7 with its endothelial ligand, MAdCAM-1. Based on the known active peptide, a series of sulfonamide peptidomimetics was synthesized. Sulfonopeptide 2, Ac-L-ψD-V-NH2, was shown to inhibit leukocyte adhesion.

Sodium triacetoxyborohydride is presented as a general reducing agent for the reductive amination of aldehydes and ketones. Procedures for using this mild and selective reagent have been developed for a wide variety of substrates. The scope of the reaction includes aliphatic acyclic and cyclic ketones, aliphatic and aromatic aldehydes, and primary and secondary amines including a variety of weakly basic and nonbasic amines. Limitations include reactions with aromatic and unsaturated ketones and some sterically hindered ketones and amines. 1,2-Dichloroethane (DCE) is the preferred reaction solvent, but reactions can also be carried out in tetrahydrofuran (THF) and occasionally in acetonitrile. Acetic acid may be used as catalyst with ketone reactions, but it is generally not needed with aldehydes. The procedure is carried out effectively in the presence of acid sensitive functional groups such as acetals and ketals; it can also be carried out in the presence of reducible functional groups such as C-C multiple bonds and cyano and nitro groups. Reactions are generally faster in DCE than in THF, and in both solvents, reactions are faster in the presence of AcOH. In comparison with other reductive amination procedures such as NaBH(3)CN/MeOH, borane-pyridine, and catalytic hydrogenation, NaBH(OAc)(3) gave consistently higher yields and fewer side products. In the reductive amination of some aldehydes with primary amines where dialkylation is a problem we adopted a stepwise procedure involving imine formation in MeOH followed by reduction with NaBH(4).

Synthetic 1-morpholino-1-deoxyceramides were designed to inhibit glucosylceramide synthase. The most potent inhibitor 2a possesses the unnatural R,R-configuration of D-threo-sphingosine.

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.

Sodium triacetoxyborohydride is a reagent of choice in the reductive amination of aldehydes and saturated aliphatic ketones with primary and secondary amines. Sodium triacetoxyborohydride was successfully used in the reductive amination of variety of aldehydes and ketones with primary and secondary amines.

Synthetic 1-morpholino-1-deoxyceramides were designed to inhibit glucosylceramide synthase. The most potent inhibitor 2a possesses the unnatural R,R-configuration of D-threo-sphingosine.

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.

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.

Le traitement de l'o-quinone monoimide N-(2,4-dichloro-6-oxo-2,4-cyclohexadien-1-ylidene)-4-nitrobenzamide (I) avec les 2,6-dimethyl- et 2,6-dichlorophenols donne des composes d'addition formes par attaque nucleotide du phenol sur l'azote imido de I. Certains de ces composes d'addition peuvent etre ensuite oxydes par I ou par la DDQ pour conduire a la N-aryl-p-iminoquinone selon une nouvelle migration acyle N→O

The previously unknown 3-aminomethyl-1,3-dihydro-5-(2′-fluorophenyl)-2H,4-benzodiazepin-2-one, 3a, was synthesized in two steps as a racemate. In the chiral series, 3(S)-azidocarbonylmethyl-1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one, 12b, was prepared from Nα-Cbz-β-methylaspartate in five synthetic operations and subjected to Curtius rearrangement. The intermediate isocyanate was trapped intramolecularly by the 5-imine nitrogen of the benzodiazepine ring in 12b. This unanticipated result runs counter to the generally held dictum that the isocyanate group has a strictly linear shape.

Sodium triacetoxyborohydride is a reagent of choice in the reductive amination of aldehydes and saturated aliphatic ketones with primary and secondary amines. Sodium triacetoxyborohydride was successfully used in the reductive amination of variety of aldehydes and ketones with primary and secondary amines.

The identification, optimization, and structure-activity relationship (SAR) of small-molecule CCR4 antagonists is described. An initial screening hit with micromolar potency was identified that was optimized to sub-micromolar binding potency by enantiomer resolution, halogenation of the naphthalene ring, and extension of the alkyl chain linker between the central piperidine ring and the terminal aryl group. An antagonist was identified that showed good cross-reactivity against the mouse receptor and inhibited CCR4-based cell recruitment in dose-dependent fashion.

Activation of CCR8 by its ligand CCL1 may play an important role in diseases such as asthma, multiple sclerosis, and cancer. The study of small molecule CCR8 antagonists will help establish the validation of these hypotheses. We report the design, synthesis, and progress toward optimization of potent small molecule CCR8 antagonists identified from a high-throughput screen. These analogues exhibit good potency in binding and chemotaxis assays, show good selectivity versus the hERG channel, and have good eADME (early absorption, distribution, metabolism, and excretion) profiles.

Publisher Summary This chapter analyzes the role of chemokines in therapeutics. The study of the chemokine superfamily has provided a clearer understanding of some of the mechanisms by which the migration of leukocytes is controlled, during normal immune function as well as in pathology. The human repertoire of chemokines comprises a family of approximately 50 small proteins that share a high degree of homology in sequence, tertiary structure, and function. The family is comprised of four sub-classes based on the pattern of conserved cysteine residues. This classification also tends to parallel the shared cell type activities. The activity of chemokines is mediated by cell surface receptors that comprise a subfamily of the G protein-coupled receptor (GPCR) superfamily. Chemokines have generally been characterized as being proinflammatory factors. Some chemokines have been found to influence normal leukocyte trafficking and immune function, promote or inhibit angiogenesis, and may play a role in the growth and metastasis of cancer cells. The chapter focuses on the progress made towards identifying low molecular weight antagonists for CCR1, one of the receptors for MIP-lα and RANTES, CXCR2, one of the IL-8 receptors, and CCR2, the MCP-1 receptor (CCR2). An overview of CCR1 receptor antagonists is presented and the CXCR2 receptor antagonists are also analyzed.

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.

The ceramide metabolite sphingosine 1-phosphate (S1P) is an important modulator of a wide variety of physiologic functions via its action as a second messenger in intracellular signaling pathways and its extracellular interaction with specific G protein-coupled receptors on the cell surface. In the immune system, interaction of S1P with one of its receptors, S1P1, leads to inhibition of lymphocyte egress from primary and secondary lymphoid tissues resulting in peripheral lymphopenia, a mechanism with demonstrated effects in recent human clinical studies using the S1P1 receptor agonist, FTY720. S1P lyase (S1PL), which catalyzes the only irreversible step in sphingosine metabolism, the degradation of S1P to hexadecenal and ethanolamine phosphate, has recently been suggested to be a target of 2-acetyl-4-tetrahydroxybutylimidazole (THI), a compound known to increase tissue S1P levels and alter lymphocyte trafficking. In the current study, genetic inactivation of S1PL in knockout (KO) mice resulted in severe depletion of lymphocytes in the peripheral blood accompanied by accumulation of mature T cells in the thymus. This abnormal pattern of lymphocyte distribution suggests that results previously seen in THI treated mice were indeed due to THI’s inhibition of S1PL rather than another unknown activity. S1PL KO mice also demonstrated significantly reduced viability, perhaps due to increased numbers of myeloid cells which infiltrated the lungs. In a S1PL humanized allele, in which the human S1PL has been introduced into S1PL KO mice and restores ∼17% of wild type S1PL activity, the reduced viability trait is rescued while peripheral lymphopenia is maintained. Since the phenotypes are genetically separable by an allele that reduces S1PL activity, the lack of observed toxicity at doses of THI resulting in lymphopenia is likely due to its partial inhibition of S1PL activity at those doses, perhaps due to differences in S1P or S1P receptor expression or metabolism in different cell types. The potential for separation of lymphopenia and myeloid cell lung pathology effects by chemical inhibition was confirmed by chronic treatment of mice with the oral, small molecule S1PL inhibitor, LX2931, for 47 days during which no effects on viability were observed while the altered profile of lymphocyte migration seen in the partially inhibited humanized S1PL allele was maintained. Rats and monkeys treated with LX2931 also exhibited peripheral lymphopenia suggesting that the S1PL signaling pathway is conserved across mammalian species. Both S1PL KO and LX2931 treated mice were protected in the oxazolone challenge assay of delayed type hypersensitivity and the collagen induced model of rheumatoid arthritis, demonstrating that the reduction of peripheral lymphocytes previously observed with S1PL inhibition can beneficially affect the development of autoimmune disease.