In vitro activity: SL327 is a water soluble, structural homologue of the specific MKK1/2 inhibitor U0126 with IC50 of 0.18 μM and 0.22 μM for MEK1 and MEK2 respectively. SL327 has no effect on a variety of other kinases, including PKA, PKC, or CamKII.

Kinase Assay: Protein kinase assays are performed. All kinase assays are started by adding enzyme to a mixture that included [γ-32P]ATP and substrate. This mixture is then incubated at 30°C or 37°C for 10 min. The reaction is stopped by spotting aliquots of the reaction mixture onto Whatman P-81 phosphocellulose filter paper. The papers are then washed in 150 mM H3PO4, dried, and subjected to scintillation counting. The catalytic subunit of PKA is assayed by measuring [32P]phosphate incorporation into the substrate Kemptide (100 μM). The activity of CaMKII is determined by measuring phosphorylation of the synthetic peptide Autocamtide (100 μM) in the presence of 100 μM Calcium and 10 μg/mL Calmodulin. A synthetic peptide analog of a fragment of neurogranin, NG(28-43) (10 μM) is used as a specific substrate for the catalytic subunit of PKC. In all cases, substrate phosphorylation was linear with respect to time and enzyme concentration.