Human Remyelination Promoting Antibody Stimulates Astrocytes Proliferation Through Modulation of the Sphingolipid Rheostat in Primary Rat Mixed Glial Cultures.

Remyelination promoting human IgMs effectively increase the number of myelinated axons in animal models of multiple sclerosis. Hence, they ultimately stimulate myelin production by oligodendrocytes (OLs); however, their exact mechanism of action remains to be elucidated, and in particular, it remains unclear whether they are directly targeting OLs, or their action is mediated by effects on other cell types. We assessed the effect of remyelination promoting antibody rHIgM22 on the proliferative response and on the ceramide/sphingosine 1-phosphate rheostat in mixed glial cell cultures (MGCs). rHIgM22 treatment caused a time-dependent increase in PDGFαR protein in MGCs. Forty-eight hours of treatment with rHIgM22 induced a dose-dependent proliferative response (evaluated as total cell number and as EdU(+) cell number) in MGCs. When the proliferation response of MGCs to rHIgM22 was analyzed as a function of the cell types, the most significant proliferative response was associated with GLAST(+) cells, i.e., astrocytes. In many cell types, the balance between different sphingolipid mediators (the "sphingolipid rheostat"), in particular ceramide and sphingosine 1-phosphate, is critical in determining the cell fate. rHIgM22 treatment in MGCs induced a moderate but significant inhibition of total acidic sphingomyelinase activity (measured in vitro on cell lysates), the main enzyme responsible for the stimulus-mediated production of ceramide, when treatment was performed in serum containing medium, but no significant differences were observed when antibody treatment was performed in the absence of serum. Moreover, rHIgM22 treatment, either in the presence or in absence of serum, had no effects on ceramide levels. On the other hand, rHIgM22 treatment for 24 h induced increased production and release of sphingosine 1-phosphate in the extracellular milieu of MGC. Release of sphingosine 1-phosphate upon rHIgM22 treatment was strongly reduced by a selective inhibitor of PDGFαR. Increased sphingosine 1-phosphate production does not seem to be mediated by regulation of the biosynthetic enzymes, sphingosine kinase 1 and 2, since protein levels of these enzymes and phosphorylation of sphingosine kinase 1 were unchanged upon rHIgM22 treatment. Instead, we observed a significant reduction in the levels of sphingosine 1-phosphate lyase 1, one of the key catabolic enzymes. Remarkably, rHIgM22 treatment under the same experimental conditions did not induce changes in the production and/or release of sphingosine 1-phosphate in pure astrocyte cultures. Taken together, these data suggest that rHIgM22 indirectly influences the proliferation of astrocytes in MGCs, by affecting the ceramide/sphingosine 1-phosphate balance. The specific cell population directly targeted by rHIgM22 remains to be identified, however our study unveils another aspect of the complexity of rHIgM22-induced remyelinating effect.

Potent MCH-1 receptor antagonists from cis-1,4-diaminocyclohexane-derived indane analogs.

Benzimidazole and indane are the two key fragments in our potent and selective MCH-1 receptor (MCHR1) antagonists. To identify novel linkers connecting the two fragments, we investigated diamino-cycloalkane-derived analogs and discovered highly potent antagonists with cis-1,4-diaminocyclohexane as a unique spacer in this chemical class. Structural overlay suggested that cis-1-substituted-4-aminocyclohexane functions as a bioisostere of 4-substituted-piperidine and that the active conformation adopts a U-shaped orientation.

Potent, selective MCH-1 receptor antagonists.

This paper describes the lead optimization of a new series of potent, selective, orally bioavailable, brain-penetrant MCH-1 receptor antagonists. A major focus of the work was to achieve a selectivity profile appropriate for in vivo efficacy studies and safety.

Inhibitors of Ras/Raf-1 interaction identified by two-hybrid screening revert Ras-dependent transformation phenotypes in human cancer cells.

The interaction of activated Ras with Raf initiates signaling cascades that contribute to a significant percentage of human tumors, suggesting that agents that specifically disrupt this interaction might have desirable chemotherapeutic properties. We used a subtractive forward two-hybrid approach to identify small molecule compounds that block the interaction of Ras with Raf. These compounds (MCP1 and its derivatives, 53 and 110) reduced serum-induced transcriptional activation of serum response element as well as Ras-induced transcription by way of the AP-1 site. They also inhibited Ras-induced Raf-1 activation in human embryonic kidney 293 cells, Raf-1 and mitogen-activated protein kinase kinase 1 activities in HT1080 fibrosarcoma cells, and epidermal growth factor-induced Raf-1 activation in A549 lung carcinoma cells. The MCP compounds caused reversion of ras-transformed phenotypes including morphology, in vitro invasiveness, and anchorage-independent growth of HT1080 cells. Decreased level of matrix metalloproteinases was also observed. Further characterization showed that MCP compounds restore actin stress fibers and cause flat reversion in NIH 3T3 cells transformed with H-Ras (V12) but not in NIH 3T3 cells transformed with constitutively active Raf-1 (RafDeltaN). Finally, we show that MCP compounds inhibit anchorage-independent growth of A549 and PANC-1 cells harboring K-ras mutation. Furthermore, MCP110 caused G(1) enrichment of A549 cells with the decrease of cyclin D level. These results highlight potent and specific effects of MCP compounds on cancer cells with intrinsic Ras activation.