S100B sellckchem and synaptophysin levels were measured by ELISA as previ ously described. Statistical analyses of in vivo results Experimental and control groups were compared using one way ANOVA with Newman Keuls post hoc analysis using GraphPad Prism, version 4. 00 statistical software. Inhibitors,Modulators,Libraries Significance was assumed when p 0. 05. Results and Discussion Development and characterization of a novel p38 MAPK inhibitor with potential use for CNS studies The synthetic scheme and design strategy for the p38 MAPK inhibitor 069A were based on a chem ical diversification of the inactive 3 phenyl 6 piperazin 1 ylpyridazine scaffold, used in previous development of CNS penetrant, Inhibitors,Modulators,Libraries orally bioavailable, non toxic, experimental therapeutics.

Computational modeling predicted that the scaffold should fit into the p38 MAPK structure, with the phenyl ring occupying a hydrophobic pocket in the kinase. To create the potential for further interaction with the p38 MAPK active site, we introduced into the scaffold a pyridinyl pharmacophore found in a variety of p38 MAPK inhibitors. The nitrogen of the pyridine ring is potentially Inhibitors,Modulators,Libraries able to make a critical H bond interaction with the amide bond formed between Met109 and Gly110 of p38 MAPK. Therefore, we Inhibitors,Modulators,Libraries incor porated this feature into the design of 069A. The activity of 069A as a p38 MAPK inhibitor was tested in an in vitro protein kinase assay over a wide concentra tion range. As shown in Fig. 3A, 069A inhibits p38 MAPK activity in a concentration depend ent manner with an estimated IC50 of 0. 8M. As shown in Fig.

3B, the starting scaffold lacks inhibitory activity, consistent with the model in Fig. 2. Therefore, introduction of the pyridinyl pharmacophore adjacent to the phenyl ring of the inactive scaffold to generate 069A results in a greater than 100 fold increase in inhibitory activity. Inhibitors,Modulators,Libraries To further validate the design approach and specificity of the inhibitor kinase interaction, we examined in more detail the finding that introduction of the pyridinyl phar macophore into the inactive scaffold generated kinase inhibitory activity. The attainment of p38 MAPK inhibi tory activity is dependent on the molecular context of the pyridinyl pharmacophore placement. For example, we synthesized an analog, called MW01 4 119SRM, to test the activity importance of the potential H bond interac tion with the Met109 peptide backbone of the kinase.

We used the same synthetic scheme as used for 069A, but a different pyridinylboronic acid, allowing a different structural orientation of the nitrogen in the pyri dine selleck chemicals llc ring. If the proposed interaction involves such an H bond, one would anticipate that activity would be compromised due to distance constraints and altered electronegativity. As shown in Fig. 3B, there was a major loss of activity back toward that seen with the scaffold alone.