Prevention of MKK6-Dependent Activation by Binding to p38 MAP Kinase
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- 作者：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 R
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：December 20, 2005
- 年：2005
- 卷：44
- 期：50
- 页码：16475 - 16490
- 全文大小：613K
- 年卷期：v.44,no.50(December 20, 2005)
- ISSN：1520-4995
文摘
Inhibition of p38
MAP kinase is a potential approach for the treatment of inflammatorydisorders. MKK6-dependent phosphorylation on the activation loop of p38 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 p38 remaining in a "DFG-in" conformation. We now demonstrate thatselectivity pocket compounds prevent MKK6-dependent activation of p38 in addition to inhibiting catalysisby activated p38. Inhibitors using only the purine site do not prevent MKK6-dependent activation. Wepresent kinetic analyses of seven inhibitors, whose crystal structures as complexes with p38 have beendetermined. This work includes four new crystal structures and a novel assay to measure Kd for nonactivatedp38. Selectivity pocket compounds associate with p38 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, TNF, selectivity pocket compounds decrease levels ofphosphorylated p38 and 
. Stabilization of a DFG-out conformation appears to interfere with recognitionof p38 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 p38.
MAP kinase is a potential approach for the treatment of inflammatorydisorders. MKK6-dependent phosphorylation on the activation loop of p38 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 p38 remaining in a "DFG-in" conformation. We now demonstrate thatselectivity pocket compounds prevent MKK6-dependent activation of p38 in addition to inhibiting catalysisby activated p38. Inhibitors using only the purine site do not prevent MKK6-dependent activation. Wepresent kinetic analyses of seven inhibitors, whose crystal structures as complexes with p38 have beendetermined. This work includes four new crystal structures and a novel assay to measure Kd for nonactivatedp38. Selectivity pocket compounds associate with p38 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, TNF, selectivity pocket compounds decrease levels ofphosphorylated p38 and . Stabilization of a DFG-out conformation appears to interfere with recognitionof p38 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 p38.