No abstract

No abstract

Protein kinase inhibitors with enhanced selectivity can be designed by optimizing binding interactions with less conserved inactive conformations because such inhibitors will be less likely to compete with ATP for binding and therefore may be less impacted by high intracellular concentrations of ATP. Analysis of the ATP-binding cleft in a number of inactive protein kinases, particularly in the autoinhibited conformation, led to the identification of a previously undisclosed non-polar region in this cleft. This ATP-incompatible hydrophobic region is distinct from the previously characterized hydrophobic allosteric back pocket, as well as the main pocket. Generalized hypothetical models of inactive kinases were constructed and, for the work described here, we selected the fibroblast growth factor receptor (FGFR) tyrosine kinase family as a case study. Initial optimization of a FGFR2 inhibitor identified from a library of commercial compounds was guided using structural information from the model. We describe the inhibitory characteristics of this compound in biophysical, biochemical, and cell-based assays, and have characterized the binding mode using x-ray crystallographic studies. The results demonstrate, as expected, that these inhibitors prevent activation of the autoinhibited conformation, retain full inhibitory potency in the presence of physiological concentrations of ATP, and have favorable inhibitory activity in cancer cells. Given the widespread regulation of kinases by autoinhibitory mechanisms, the approach described herein provides a new paradigm for the discovery of inhibitors by targeting inactive conformations of protein kinases.It has long been hypothesized that mapping the spatial rearrangements that take place during the cycling between productive (active) and non-productive (inactive) states of kinases should lead to a better understanding of kinase dynamics, structure, function, and regulation (1-3). Furthermore, targeting kinase inhibitors to the inactive state is attractive because that form is more likely to represent a distinct conformation that may in turn lead to the identification of more selective inhibitors (4). However, despite advances in the field, designing inhibitors that target an inactive conformation of a kinase remains challenging (5) and largely empirical. The work described here provides a generally applicable methodology for the design of inhibitors that preferentially bind to the inactive state of a target kinase through developing a better understanding of the interplay between the conformational transitions that take place upon activation (6).Previous approaches have focused on the analysis of socalled "type II inhibitors" (7), which induce a distinct DFG-out conformation and occupy an additional hydrophobic pocket created by this rearrangement. The DFG-out conformation is a prerequisite for designing such type II inhibitors. The success of this strategy relies on the fact that despite the conserved nature of the ATP site, there are sufficient adjacent pockets that can ...