The current lack of effective treatments for

The current lack of effective treatments for primary or metastatic UM leaves a large therapeutic gap for patients and clinicians underscoring an urgent need for the identification of additional pharmacological targets for therapeutic intervention. As YAP-targeting strategies have remained elusive thus far, the success of FAK inhibition in our in vivo models in the context of previously established success and safety of FAK inhibitors in human clinical trials highlights the translational potential of our findings and establishes FAK as a therapeutic target for the treatment of UM. Toward this end, the application of systems-level and bioinformatics investigation will be a powerful strategy to identify precision treatment options for UM and a myriad of Gαq-driven diseases.
STAR★Methods
Proline-rich tyrosine kinase 2 (Pyk2) is a non-receptor cytoplasmic tyrosine kinase within the focal adhesion kinase (FAK) subfamily. Both Pyk2 and FAK are involved in multiple signaling pathways that regulate cell migration, proliferation and survival., Over the last decade, genetic knockdown of Pyk2 together with inhibition by small molecules have demonstrated the potential role of Pyk2 in the treatment of osteoporosis., Recently, the biological significance of the focal adhesion kinases in promoting the migration and invasion of malignant cells has been of increasing interest., , Glioblastoma multiforme is the most common and most aggressive malignant primary Moxidectin tumor due to the propensity of peripheral glioma cells to migrate and disperse among neighboring normal brain cells. Combinations of surgical resection, radiotherapy and/or chemotherapy with temozolomide are not curative treatments. Over 90% of the estimated new glioblastoma cases in 2015 were found in adults ≥40years old. The 5-year relative survival rate for patients diagnosed with glioblastoma is 5.1%, a statistic which has shown no significant improvement over the past 30years, in spite of recent therapeutic advancements. Given this unmet medical need, we sought to selectively target Pyk2 and evaluate its role in the migration and invasion of malignant glioblastoma cells. Unlike FAK, which is ubiquitously expressed, Pyk2 expression is highly localized in the brain, hematopoietic and nervous systems., Radial migration assays using SF767 glioblastoma cell lines have shown upregulation of Pyk2 in cells at the migratory rim relative to those at the primary core. A direct correlation between the rate of migration and levels of activated Pyk2 was also observed. Conversely, silencing Pyk2 expression with siRNA significantly reduced the migratory potential of glioblastoma cells. A relationship has also been demonstrated between increasing Pyk2 expression/phosphorylation events and advancing tumor WHO grade, thus indicating Pyk2 as a potential mediator of glioblastoma progression. The catalytic domains of Pyk2 and FAK kinases have 73% sequence similarity. The similarity is slightly higher (78%) for residues generally thought to form the ATP binding sites of these two enzymes. Consequently, the discovery of a small molecule inhibitor selective for Pyk2 has been challenging. We initiated efforts toward that goal for proof-of-concept studies and focused our attention on diaminopyrimidine-based inhibitors ,, and compound (). was described as a potent, ATP-competitive and reversible FAK inhibitor (IC=1.5nM) with a ∼9-fold selectivity over Pyk2 (IC=13nM). To better understand the interaction of with Pyk2, we solved a crystal structure of the complex. A comparison in the binding pose of complexed with FAK versus Pyk2 revealed a significant conformational difference in the amino-methyl pyridinyl sulfonamide group (a and b). The rotamer of the benzylic CH-NH bond of observed in the Pyk2-bound structure induced a conformation of the nearby DFG loop in Pyk2 (DFG-in) distinct from that observed in FAK (helical DFG). We hypothesized that a macrocyclic analogue of favoring the Pyk2-bound conformation shown in b would enhance selectivity of the inhibitor over FAK.