Prevention of MKK6-Dependent Activation by Binding to p38α 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 p38α. Selectivity pocket compounds associate with p38α over 30-fold more slowly than purine site compounds, apparently due to low abundance of the DFG-out conformation. At concentrations that inhibit cellular production of an inflammatory cytokine, TNFα, selectivity pocket compounds decrease levels of phosphorylated p38α and ?. Stabilization of a DFG-out conformation appears to interfere with recognition of p38α as a substrate by MKK6. ATP competes less effectively for prevention of activation than for inhibition of catalysis. By binding to a different conformation of the enzyme, compounds that prevent activation offer an alternative approach to modulation of p38α.