Prevention of MKK6-Dependent Activation by Binding to p38alpha MAP Kinase.

Inhibition of p38α MAP kinase is a potential approach for the treatment of inflammatory disorders. MKK6-dependent phosphorylation on the activation loop of p38α increases its catalytic activity and affinity for ATP. An inhibitor, BIRB796, binds at a site used by the purine moiety of ATP and extends into a “selectivity pocket”, which is not used by ATP. It displaces the Asp168-Phe169-Gly170 motif at the start of the activation loop, promoting a “DFG-out” conformation. Some other inhibitors bind only in the purine site, with p38α remaining in a “DFG-in” conformation. We now demonstrate that selectivity pocket compounds prevent MKK6-dependent activation of p38α in addition to inhibiting catalysis by activated p38α. Inhibitors using only the purine site do not prevent MKK6-dependent activation. We present kinetic analyses of seven inhibitors, whose crystal structures as complexes with p38α have been determined. This work includes four new crystal structures and a novel assay to measure Kd for nonactivated p38...