Diagrammatic outline of p38 mitogen-activated protein kinase (MAPK) pathways in glia and neurons. Disease-relevant stressors or stimuli can activate a variety of cross-talking and interacting signal transduction pathways, some of which can converge on activation of the p38 MAPK signaling cascades. For example, a typical p38 MAPK cascade consists of a three-tiered series of protein kinases: a MAPK (p38) and two upstream components (a MAPK kinase (MEK) and a MAPKK kinase (MEKK)) that activate the p38 MAPK by a series of activating phosphorylations. Activation of p38 MAPK and phosphorylation of its downstream substrates in activated glia (primarily microglia) can lead to up-regulation of proinflammatory cytokine production. Proinflammatory cytokines can act back on glia to stimulate multiple intracellular signaling pathways. Neurons can also respond to proinflammatory cytokine or other stressors/stimuli and activate neuronal p38 MAPK, culminating in neuron damage. The consequences of p38 MAPK activation in glia and neurons depend on the set of upstream signals, the isoform of p38 MAPK, the cell type, and the set of substrates that are stimulated.

Strategy for development of a p38α mitogen-activated protein kinase (MAPK) inhibitor. (A) The p38α MAPK inhibitor MW01-2-069A-SRM was developed using a structure-assisted and computational modeling design strategy, along with consideration of compound molecular properties. The inhibitor is based on a 3-amino-6-phenyl pyridazine scaffold (MW01-3-183WH) found in other central nervous system (CNS)-active compounds [46,68]. A pyridinyl pharmacophore characteristic of many p38 MAPK inhibitors was added to the scaffold. Modeling forecasted that MW01-2-069A-SRM could be accommodated by the p38α MAPK structure. The smaller Thr106 gatekeeper residue allowed the phenyl ring of the compound to occupy a hydrophobic pocket while the nitrogen of the pyridine ring could make a critical H-bond interaction with the amide bond between Met109 and Gly110. These interactions are important for selectivity and affinity for the p38α MAPK isoform. MW01-2-069A-SRM was a p38α MAPK inhibitor, with an IC₅₀ of approximately 0.8 μM, and was relatively selective for p38α MAPK; at 20 μM, the compound showed complete inhibition of p38α MAPK, partial inhibition of p38β MAPK, and no inhibition of p38δ MAPK, p38γ MAPK, or 40 other protein kinases [41]. (B) Validation of the design approach and modeling of predicted interactions was done by testing analogs that did not show the predicted interactions with the kinase. For example, the scaffold compound MW01-3-183WH, which lacks the pyridine ring, is inactive. The inactive analog MW01-4-199SRM has a pyridine nitrogen in a different structural orientation, which should compromise activity due to distance constraints and altered electronegativity. Another inactive analog, MW01-6-189WH, has an identical composition to MW01-2-069A-SRM, but has its pyridine ring in a different position on the pyridazine scaffold. These data show that the pyridine pharmacophore must be introduced adjacent to the phenyl group in the molecular context of the scaffold, as found in MW01-2-069A-SRM, to produce a p38α MAPK inhibitor with good affinity and selectivity.