| Abstract
| - Inhibition of p38α MAP kinase is a potential approach for the treatment of inflammatorydisorders. MKK6-dependent phosphorylation on the activation loop of p38α increases its catalytic activityand affinity for ATP. An inhibitor, BIRB796, binds at a site used by the purine moiety of ATP andextends into a “selectivity pocket”, which is not used by ATP. It displaces the Asp168-Phe169-Gly170motif at the start of the activation loop, promoting a “DFG-out” conformation. Some other inhibitorsbind only in the purine site, with p38α remaining in a “DFG-in” conformation. We now demonstrate thatselectivity pocket compounds prevent MKK6-dependent activation of p38α in addition to inhibiting catalysisby activated p38α. Inhibitors using only the purine site do not prevent MKK6-dependent activation. Wepresent kinetic analyses of seven inhibitors, whose crystal structures as complexes with p38α have beendetermined. This work includes four new crystal structures and a novel assay to measure Kd for nonactivatedp38α. Selectivity pocket compounds associate with p38α over 30-fold more slowly than purine sitecompounds, apparently due to low abundance of the DFG-out conformation. At concentrations that inhibitcellular production of an inflammatory cytokine, TNFα, selectivity pocket compounds decrease levels ofphosphorylated p38α and β. Stabilization of a DFG-out conformation appears to interfere with recognitionof p38α as a substrate by MKK6. ATP competes less effectively for prevention of activation than forinhibition of catalysis. By binding to a different conformation of the enzyme, compounds that preventactivation offer an alternative approach to modulation of p38α.
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