title: Prevention of MKK6-dependent activation by binding to p38alpha MAP kinase.
abstract: Inhibition of p38alpha MAP kinase is a potential approach for the treatment of inflammatory disorders. MKK6-dependent phosphorylation on the activation loop of p38alpha 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 p38alpha remaining in a "DFG-in" conformation. We now demonstrate that selectivity pocket compounds prevent MKK6-dependent activation of p38alpha in addition to inhibiting catalysis by activated p38alpha. 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 p38alpha have been determined. This work includes four new crystal structures and a novel assay to measure K(d) for nonactivated p38alpha. Selectivity pocket compounds associate with p38alpha 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, TNFalpha, selectivity pocket compounds decrease levels of phosphorylated p38alpha and beta. Stabilization of a DFG-out conformation appears to interfere with recognition of p38alpha 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 p38alpha.
authors.affiliation: Molecular Enzymology, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK.
authors.forename: Jane E
authors.initials: JE
authors.lastname: Sullivan
authors.forename: Geoffrey A
authors.initials: GA
authors.lastname: Holdgate
authors.forename: Douglas
authors.initials: D
authors.lastname: Campbell
authors.forename: David
authors.initials: D
authors.lastname: Timms
authors.forename: Stefan
authors.initials: S
authors.lastname: Gerhardt
authors.forename: Jason
authors.initials: J
authors.lastname: Breed
authors.forename: Alexander L
authors.initials: AL
authors.lastname: Breeze
authors.forename: Alun
authors.initials: A
authors.lastname: Bermingham
authors.forename: Richard A
authors.initials: RA
authors.lastname: Pauptit
authors.forename: Richard A
authors.initials: RA
authors.lastname: Norman
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Embrey
authors.forename: Jon
authors.initials: J
authors.lastname: Read
authors.forename: Wendy S
authors.initials: WS
authors.lastname: VanScyoc
authors.forename: Walter H J
authors.initials: WH
authors.lastname: Ward
mesh_terms: D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D048669:MAP Kinase Kinase 6 / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D048051:p38 Mitogen-Activated Protein Kinases; D048669:MAP Kinase Kinase 6; C482566:MAP2K6 protein, human