Prevention of MKK6-dependent activation by binding to p38α MAP kinase

Inhibition of p38 alpha MAP kinase is a potential approach for the treatment of inflammatory disorders. MKK6-dependent phosphorylation on the activation loop of p38 alpha 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 p38 alpha remaining in a "DFG-in" conformation. We now demonstrate that selectivity pocket compounds prevent MKK6-dependent activation of p38 alpha in addition to inhibiting catalysis by activated p38 alpha. 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 p38 alpha have been determined. This work includes four new crystal structures and a novel assay to measure K-d for nonactivated p38 alpha. Selectivity pocket compounds associate with p38 alpha 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, TNF alpha, selectivity pocket compounds decrease levels of phosphorylated p38 alpha and beta. Stabilization of a DFG-out conformation appears to interfere with recognition of p38 alpha 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 p38 alpha.