A high-throughput scintillation proximity assay for sphingosine-1-phosphate lyase.

The emergence of sphingosine-1-phosphate lyase (SPL) as a promising therapeutic target for inflammatory diseases has heightened interest in the identification of small molecules that modulate its activity. The enzymatic activity of SPL is typically measured using radiometric or fluorescence-based assays that require a lipid extraction step, or by direct quantitation of reaction products using mass spectrometry (MS). To facilitate testing large numbers of compounds to identify SPL modulators, we developed a robust scintillation proximity assay (SPA) that is compatible with high-throughput screening (HTS). This assay employs recombinant human full-length SPL in insect cell membrane preparations to catalyze the conversion of biotinylated aminosphingosine-1-[(33)P]phosphate (S1(33)P-biotin) to trans-2-hexadecenal-biotin and ethanolamine [(33)P]phosphate. To validate the SPA and confirm the fidelity of its measurement of SPL enzyme activity, we developed a Rapid-Fire MS method that quantitates nonradiolabeled S1P-biotin. In addition, we developed a simple, scalable method to produce S1(33)P-biotin in quantities sufficient for HTS. The optimized SPA screen in 384-well microplates produced a mean plate-wise Z'-statistic of 0.58 across approximately 3,000 plates and identified several distinct structural classes of SPL inhibitor. Among the inhibitors that the screen identified was one compound with an IC50 of 1.6 µM in the SPA that induced dose-dependent lymphopenia in mice.

Circulating monocytes are reduced by sphingosine-1-phosphate receptor modulators independently of S1P3.

Sphingosine-1-phosphate (S1P) receptors are critical for lymphocyte egress from secondary lymphoid organs, and S1P receptor modulators suppress lymphocyte circulation. However, the role of S1P receptors on monocytes is less clear. To elucidate this, we systematically evaluated monocytes in rats and mice, both in naive and inflammatory conditions, with S1P receptor modulators FTY720 and BAF312. We demonstrate that S1P receptor modulators reduce circulating monocytes in a similar time course as lymphocytes. Furthermore, total monocyte numbers were increased in the spleen and bone marrow, suggesting that S1P receptor modulation restricts egress from hematopoietic organs. Monocytes treated ex vivo with FTY720 had reduced CD40 expression and TNF-α production, suggesting a direct effect on monocyte activation. Similar reductions in protein expression and cytokine production were also found in vivo. Suppression of experimental autoimmune encephalomyelitis in mice and rats by FTY720 correlated with reduced numbers of lymphocytes and monocytes. These effects on monocytes were independent of S1P3, as treatment with BAF312, a S1P1,4,5 modulator, led to similar results. These data reveal a novel role for S1P receptors on monocytes and offer additional insights on the mechanism of action of S1P receptor modulators in disease.

An orally active reversible inhibitor of cathepsin S inhibits human trans vivo delayed-type hypersensitivity.

Cathepsin S is a major histocompatibility complex (MHC) class II associated invariant chain (Ii) degrading enzyme expressed in antigen presenting cells such as B cells and dendritic cells. This enzyme is essential for MHC class II associated antigen processing and presentation to CD4(+) T cells. Compound I, a selective, reversible and orally bioavailable, inhibitor of cathepsin S, with molecular IC(50)=9 nM, has been recently described. We have tested the effects of compound I in a trans vivo model of delayed-type hypersensitivity. Human peripheral blood mononuclear cells (7-10 x 10(6)) from tetanus-sensitized donors were co-injected with tetanus toxoid (0.25 Lf) into C57Bl/6 mouse footpads. At 24 h, significant footpad swelling (+0.024+/-0.001 cm) characterized by an influx of mouse neutrophils and monocytes was observed. Injection of peripheral blood mononuclear cells alone caused negligible swelling (0.002+/-0.0002 cm). Anti-human MHC class II (HLA-DR, DP, DQ) antibody (5 mg/kg, i.p.) inhibited the swelling 91+/-7%, thus demonstrating a role of human antigen presenting cells in this model. Compound I (10, 30, and 100 mg/kg, p.o.) inhibited the response with an ED50 of approximately 18 mg/kg. Compound III, a less active analogue (molecular IC50>20 microM) had no effect. Furthermore, pretreatment of peripheral blood mononuclear cells with 10 nM compound II, an irreversible inhibitor (molecular IC50=11 nM) inhibited swelling 87+/-4%. These findings support the role of cathepsin S in human delayed-type hypersensitivity. Inhibition of cathepsin S with compound I may be useful in the treatment of human autoimmune diseases like rheumatoid arthritis and multiple sclerosis.