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Prevention of MKK6-Dependent Activation by Binding to p38α MAP Kinase

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journal contribution
posted on 20.12.2005, 00:00 by Jane E. Sullivan, Geoffrey A. Holdgate, Douglas Campbell, David Timms, Stefan Gerhardt, Jason Breed, Alexander L. Breeze, Alun Bermingham, Richard A. Pauptit, Richard A. Norman, Kevin J. Embrey, Jon Read, Wendy S. VanScyoc, Walter H. J. Ward
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 β. 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.