Multiple strategies targeting c-Jun N-terminal kinases: synthesis of novel inhibitors and development of a new binding assay methodology

Abstract

In the realm of drug discovery protein kinases have emerged as extremely promising therapeutic targets as witnessed by the increasing amount of FDA-approved kinase inhibitors. In particular, the presented thesis work is focused in the design and synthesis of novel inhibitors of the c-Jun N-terminal kinases (JNKs). Such enzymes, belonging to the family of mitogen-activated protein kinases (MAPKs), regulate the cell response to a variety of extracellular stress stimuli. The three existing isoforms (JNK1, 2, and 3) present different tissue distribution and, most likely, distinct physiological functions. The abnormal activity of these protein kinases has been connected to diverse diseases ranging from neurodegenerative disorders to cancer and inflammatory or metabolic disturbs. However, despite the intense endeavor devoted in the research of novel inhibitors, to date no clinical candidate has reached approval in therapy. The first part of this thesis work concerns the development of a competition binding assay as a tool to efficiently evaluate the affinity of novel inhibitors for the three JNK isoforms, as well as for the closely related p38α MAPK. Such assay is based on the principle of fluorescence polarization (FP) and required the synthesis and characterization of a fluorescently labeled probe based on a pyridinylimidazole scaffold. After obtaining a potent probe displaying Kd values in the low nM range for all the target enzymes, the assay conditions were optimized in order to improve the assay performance. The method was then validated by employing known inhibitors and comparing the measured affinities with results from different assays. Finally, the suitability for the high throughput screening format was confirmed, making the developed assay emerge as a fast and relatively inexpensive method for a rapid screening of novel inhibitors. The second part of this thesis work consists instead in the optimization of a dual JNK3/p38α MAPK pyridinylimidazole-based lead compound. In details, the substitution at different positions of the scaffold, as well as the nature of the five-membered core were alternatively modified in order to shift the selectivity towards the JNK3. Among the different attempts, the presence of a simple methyl group at the imidazole-C4 position, together with a 2-methylsulfanyl moiety permitted to abolish the activity on the p38α MAPK while maintaining the inhibition on the JNK3. The best inhibitor of the series inhibits the JNK3 in the triple digit nanomolar range, with a > 27-fold selectivity over the p38α MAPK. The achieved information concerning the structure-activity relationship (SAR) together with the crystal structure determination of the best inhibitor in complex with the JNK3 could aid the design of following optimization strategies.