Determination of the Ki values proved that compounds with proline in position P1 are highly potent inhibitors of PREP. The remaining compounds with a sub stituted moiety in that region showed very poor PREP inhibition. While PREP inhibitor compounds HAK 1, HAK 2, HAK 5, HAK 6 and HAK 7 significantly reduced OSM KPT-330 FDA induced IL 6 secretion, there was no intimate correla tion between the extent of PREP inhibition and the potency to suppress the IL 6 expression for different HAK compounds. For example, compound HAK 8 is a potent PREP inhibitor but does not reduce OSM stimulated IL 6 secretion. On the other hand, compounds HAK 3 and HAK 4 are poor PREP inhibitors but significantly reduced OSM stimulated IL 6 secretion. This indicates that HAKs reduce IL 6 secretion independent from their PREP inhibiting activity.

Inhibitors,Modulators,Libraries In contrast to PREP inhibition, the proline residue at position P1 can be replaced by other amino acid residues like alanine or leucine without loss the bioactivity to reduce IL 6 expression. To clarify the role of PREP in the regulation of IL 6 expression, PREP was knocked down by siRNA technique in U343 cells. The remaining mRNA expression level of PREP was lower than 15% in comparison to mock and to non target con trol siRNA sample. Interestingly, 6 h after onset of OSM stimulation, a 2 fold higher PREP mRNA level was obtained in non OSM treated cells compared to OSM stimulated NTC and mock samples. The biological Inhibitors,Modulators,Libraries basis of PREP up regulation under these experimental conditions is not known, but could involve similar mechanisms contributing to Inhibitors,Modulators,Libraries induction of PREP in glial cells in experimental animals.

Compound HAK 2 did not have an effect on this phenomenon, allowing to investigate the effect of siRNA mediated PREP knock down on OSM stimulated IL 6 expression. Contrary to compound HAK Inhibitors,Modulators,Libraries 2, neither IL 6 mRNA level nor IL 6 protein level in the conditioned medium was significantly reduced after specific knock down of PREP. Inhibitors,Modulators,Libraries This result strongly indicates that PREP is not involved in regulation of IL 6 expres sion by HAKs. Therefore, we conclude that HAKs exert their effects on IL 6 expression independent from PREP inhibition by modulating at least a second molecular target. Effect of HAK compounds on OSM induced IL 6 mRNA expression To reveal whether bioactivity of HAK compounds is based on suppression of IL 6 protein biosynthesis or on interference with IL 6 mRNA expression OSM treated U343 cells were incubated with 20 uM of compound HAK 2 for different periods of time.

Time course ana lyses revealed a strong inhibition of the OSM induced IL 6 mRNA expression by compound HAK 2. Notably, http://www.selleckchem.com/products/pacritinib-sb1518.html only the second peak in IL 6 mRNA synthesis at 6 h post stimulation was affected, whereas the first peak 1 h post stimulation was insensitive to HAK 2 treatment.