Prevention of MKK6-Dependent Activation by Binding to p38α 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.

Insights into MAPK p38α DFG flip mechanism by accelerated molecular dynamics

“…3 shows that the local conformation of the DFG motif in the AMD computed pseudo-DFGout conformation is different from the a-DFG-out conformations found in the mutants F169G and F169R described by Bukhtiyarova et al, 18 and from the DFG-'in between' conformation found in some crystal complexes, [19][20][21]10 where the / angle is in the range À60°to À70°and the w is in the range À25°to À55°. The portion of the DFG loop at the N-terminal side of the DFG motive is not completely resolved in these crystal structures, however, a visual inspection of the resolved chain segments reveals that in all the structures the loop is prone to assume a conformation which is very similar among the crystal structures but very different from the AMD pseudo-DFG-out.…”

“…Similar intermediate conformations, also named DFG-'in between', were found depending on the structure of the ligands co-crystallized with the p38a proteins manipulated in positions different from the DFG-motive and under different crystallization conditions. [19][20][21]10 In summary, two mechanisms have been up to now envisaged in the literature for allosteric inhibitors: (1) exclusively binding to proteins in the DFG-out conformation, which, although sampled less frequently, exists in equilibrium with the DFG-in conformation; (2) initial binding to proteins in the DFG-in conformation with subsequent promotion of the DFG-in to -out transition. Besides the recent evidences brought to support this second thesis, no description of the mechanism at the atomic level has yet produced.…”

The design, synthesis, and evaluation of 8 hybrid DFG-out allosteric kinase inhibitors: A structural analysis of the binding interactions of Gleevec®, Nexavar®, and BIRB-796

“…Kinetic data generated during the discovery of BIRB-796 established the slow binding kinetics of DFG-out inhibitors 7 and also highlighted that some allosteric inhibitors can display the ability to induce a conformation in which both the activated enzyme can be potently inhibited and the unphosphorylated enzyme can not be activated by phosphorylation on the activation loop. 30 …”

Affinity-based, biophysical methods to detect and analyze ligand binding to recombinant proteins: Matching high information content with high throughput

“…However, because the concentration response relationship for binding to the non-activating kinase is the same as that for test compound binding to the upstream activating kinase, ITC offers a simpler assay format for measuring affinities. Examples from our work include MEK protein kinase and p38a MAP kinase (VanScyoc et al, 2008;Sullivan et al, 2005).…”