Inhibition mechanism of Focal Adhesion Kinase is a modulator of in vivo enzyme function

The non-receptor tyrosine kinase, focal adhesion kinase (FAK) controls signalling pathways that manage cell proliferation, apoptosis and migration.1 The FAK is a player in signalling processes through phosphorylation of tyrosine residues and binding events. Auto-phosphorylation of tyrosine-397 has been demonstrated to modulate FAK function. This behaviour makes FAK an interesting target for anti-tumour agents and components for tissue culture systems. The FAK enzyme is inhibited by small molecules binding at two interaction sites; interaction at the ATP binding site and interaction at an allosteric binding site.2

Kinase inhibitors often have cross reactivity between kinases, limiting their utility and making the development of specific inhibitors a difficult but rewarding task.3 Targeting inhibitors to the allosteric inhibition site is a potentially productive way to develop new FAK inhibitors.

Allosteric Inhibition of FAK. There has been a lot of high quality medicinal chemistry work directed at the synthesis of FAK inhibitors and their evaluation using crystallography and solution studies. Several different designs of FAK inhibitors have been synthesised and crystallised in co-crystals with FAK, this has produced structures where inhibitors bind in either the ATP or allosteric binding sites, providing an indication of inhibitor mechanisms. Using crystallography as a guide, studies of FAK inhibitors have demonstrated both the ATP site binding inhibition and allosteric site inhibition in solution, using carefully designed experiments with recombinant FAK.4 The structural data in these studies revealed the importance of hydrophobic interactions in the inhibition of FAK through binding to the allosteric site. The binding of an inhibitor to the allosteric site causes the transfer of information in a negative allosteric cooperativity through conformational changes, which reduces the ability of ATP to bind to the ATP binding site.5 The magnitude of this negative cooperative effect can be quantified through analysis of inhibitor (K1) and ATP association constants (K2), the ratio of these constants, α = K1/K2, provides a measure of this cooperative effect.

Novel strategies to modulate kinase activity will be developed using libraries of allosteric binding site inhibitors, whose allosteric inhibition is well studied in vitro. These inhibitors would provide useful tools for those working in the bio-medicinal sciences and would be applied to important questions. Hydrophobic interactions are controlled by surface area and steric effects that create hydrophobic contacts and modulate binding.6 The binding studies would improve understanding of inhibition mechanisms involving negative allosteric cooperativity.