Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

Abstract

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38α (involved in the formation of TNFα and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional (1)H/(13)C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38α both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.

Kinases as Novel Therapeutic Targets in Asthma and Chronic Obstructive Pulmonary Disease

“…Allosteric kinase inhibitors, such as trametinib, are now in clinical development, at least for the treatment of cancer. For example, allosteric inhibitors that bind to a distal site have now been discovered for p38 MAPK (Comess et al, 2011). Inhaled delivery may also reduce the risk of adverse effects by targeting the drug to the airways.…”

25 Years of Small Molecular Weight Kinase Inhibitors: Potentials and Limitations

“…Type 3 inhibitors that are further away from the ATP site are the myristate (myr) pocket binders located in the bottom of the C-lobe of ABL (Adrian et al, 2006;Zhang et al, 2009;Fabbro et al, 2010), CHK1 inhibitors that occupy, in part, the substrate-binding site (Converso et al, 2009), and JNK1 inhibitors that occupy the mitogen-activated protein kinase insert region and A-loop (Comess et al, 2011), to only cite a few. A more comprehensive review on type 3 inhibitors has at ASPET Journals on May 9, 2018 molpharm.aspetjournals.org…”

Computational Insights for the Discovery of Non-ATP Competitive Inhibitors of MAP Kinases

“…A unique binding site was confirmed by NMR, utilizing 13 C-methyl-labeled JNK1α1 (residues 1-364, T183E and Y185E) and by crystallization of the complex of JNK1 and 15 [71]. The binding site for 15 was shown to be located in the region of the MAP insert region ( Figure 6A).…”

“…This work highlights the difficulty of identifying highly specific ATP-competitive protein kinase inhibitors. Another limitation of such inhibitors is that they must compete with ~1 mM endogenous ATP, which has quite high affinity for MAP kinases [71]. Finally, more than 10,000 patents or patent applications for protein kinase inhibitors have been filed since 2001.…”

“…A lipid-binding site on p38 MAPK-Recently, a novel lipid-binding site was noted in the C-terminal lobe of p38 MAPK formed by the MAP kinase insert [71,100,101]. In 2009, Perry et al applied AutoLigand to examine both the optimal ligand size and the affinity of compounds that may bind this site [101], which represents a promising target for the design of inhibitors since it is a hydrophobic pocket furnishing hydrogen-bond acceptors.…”