Strategies for the NMR‐Based Identification and Optimization of Allosteric Protein Kinase Inhibitors

Abstract

Protein kinases are important drug targets, but kinase inhibitors ought to be selective and specific in order to avoid side effects in the clinic. Kinase inhibitors that do not target the highly conserved ATP-binding site, but that target an allosteric site, are generally expected to be more selective for the target kinase and thus have a better clinical profile. Here we propose an NMR-based strategy to discover and optimize allosteric kinase inhibitors. The approach uses a spin-labeled adenine analogue to detect allosteric kinase ligands by paramagnetic relaxation enhancement. Protein kinases comprise a large family of enzymes that catalyze the transfer of the terminal phosphate from ATP (adenosine triphosphate) to protein substrates, specifically to the hydroxyl group of serine or threonine (Ser/Thr kinases) or tyrosine (Tyr kinases). Protein kinases play a crucial role in signal transduction and thereby regulate central cellular processes such as cell-cycle control, growth control, apoptosis, and transcriptional activation. Kinase activity is generally tightly regulated, but can get out of control with overactive or constitutionally activated kinases. Several pathological states or diseases, such as cancer, can be a consequence of kinase overactivation. Small molecules that can modulate kinase activity in vivo are therefore of high therapeutic interest, and those kinases with a central and specific role in a particular disease are pharmaceutically highly relevant drug targets. 3] A recent example of successful target selection and inhibitor design is the clinical success of Gleevec;, a low-molecular-weight inhibitor of the constitutionally activated tyrosine kinase, Bcr-Abl. Protein kinases generally consist of a catalytic (SH1) domain and one or several regulatory (e.g. SH2 or SH3) domains. The catalytic domains have a conserved three-dimensional fold with a bilobed structure: an N-terminal lobe consisting mainly of b sheets and a C-terminal helical lobe (Figure 1). The catalytic site is located near a hinge region that connects these two domains. Kinases can adopt multiple conformational states that are associated with the degree of catalytic activity : fully active kinases are generally phosphorylated in their activation loop, which adopts a conformation that allows for optimal binding of ATP/Mg and substrate protein, and for efficient transfer of the phosphate group of ATP. There are several regulatory mechanisms by which a kinase becomes down-regulated or “inactive”. The conformational consequence of kinase downregulation can be movement of the activation loop or other components so that the substrate cannot be efficiently bound to the kinase catalytic domain. Besides the ATP-binding site and the substrate binding site, allosteric binding sites occur in kinases, often at sites with regulatory control function. More than 500 kinases are estimated to be encoded in the human genome. All of them bind ATP/Mg , and the ATP-binding site is highly conserved both in amino acid sequence and in three-dimensional structure. Kinase inhibitors that target the ATP site in an active kinase conformation (type I inhibitors) might therefore have a higher risk of clinical liabilities due to lack of selectivity against other kinases. The ATP-binding site changes shape and becomes structurally less conserved when it is in a down-regulated conformation. Kinase inhibitors that target the ATP-binding site in a down-regulated conformation (type II inhibitors) might therefore have better selectivity and specificity, and hopefully a better clinical profile. Glivec/Gleevec is such a type II kinase inhibitor. While not being perfectly selective, it targets the ATP site of Bcr-Abl in its down-regulated conformation. The best selectivity profile might be possible for inhibitors that bind outside the ATP site, at the substrate site or an allosteric binding site. These sites are not generally conserved, and high selectivity against other kinases can hopefully be achieved. Most known kinase inhibitors are type I or II inhibitors. 14] This is probably due to the fact that most kinase inhibitor screens are performed by using biochemical functional assays with purified and activated recombinant kinase. Allosteric kinase inhibitors are not identified by these assay types if they do not inhibit kinase catalysis per se, although they might inhibit kinase activation or signal transduction. Allosteric kinase Figure 1. Principle of the experiment. Spin-labeled adenine analogue 3 is bound to the ATP-binding site. Any ligand that binds simultaneously but at a different binding site feels the paramagnetic relaxation enhancement mediated by 3. The structure of MEK2 in complex with PD334581 and ATP is shown here solely to illustrate the technique.