Targeted protein degradation, which is a strategy to destroy cancer-causing proteins, has emerged in recent years as one of the hottest fields in drug discovery. Small molecules known as PROteolysis-TArgeting Chimeras (PROTACs) utilize our cells’ own mechanism for recycling proteins, the ubiquitin-proteasome system (UPS), to eliminate unwanted proteins. Normally, the UPS modifies a protein with several copies of a small regulatory protein known as ubiquitin. The ubiquitylated protein is recognized by the UPS and degraded. PROTACs are a powerful strategy to redirect the UPS to degrade proteins at will. Typically, a PROTAC is composed of two ligands joined by a linker: one ligand recruits and binds the target protein, while the other recruits and binds the ubiquitin ligase complex, which leads to marking of the target protein with ubiquitin. As soon as the target protein has been tagged with ubiquitin, the UPS degrades it as it would any other ubiquitylated protein.
This approach is especially attractive in drug development to target protein kinases that drive tumorigenesis through their catalytic and scaffolding mechanisms. As an example, focal adhesion kinase (FAK) is a non-receptor, cytoplasmic tyrosine kinase with nuclear and scaffolding functions that plays an important role in many cellular processes. In many cancer types including pancreatic and breast cancer, FAK is overexpressed, resulting in increased cell proliferation, migration, and adhesion through downstream mechanisms. To date, the field has largely focused on developing small molecule inhibitors of FAK kinase activity as cancer therapeutics. In a recent publication in ChemBioChem, Dr. Behnam Nabet, an Assistant Professor in the Human Biology Division, and his team took a different approach and developed a selective FAK PROTAC, called BSJ-04-146. “Several FAK inhibitors have been developed, which unfortunately have shown limited efficacy in clinical trials as cancer therapeutics,” Dr. Nabet said. “These inhibitors target the enzymatic activities of FAK and do not disrupt its non-enzymatic functions.” Unlike traditional kinase inhibitors, PROTACs can eliminate both catalytic and scaffolding functions, offering a new strategy for targeting oncoproteins. Dr. Nabet aims to use PROTACs to “disrupt all FAK functions required for improved anti-cancer responses.”
Initially, Dr. Nabet and his team conjugated the FAK inhibitor VS-4718 with a ubiquitin ligase recruiting ligand and tested its ability to degrade FAK in pancreatic ductal adenocarcinoma (PDAC) cell lines. Although VS-4718-conjugated PROTACs degraded FAK, they also exhibited off-target effects on several important kinases. For instance, BSJ-03-136 caused significant FAK degradation and decreased phosphorylation of AKT, a downstream target of FAK, and also depleted kinases such as WEE1 and AURKA. To further enhance the selectivity of these FAK PROTACs, the authors performed structure-guided design to improve the kinase-targeting profile of VS-4718 yielding BSJ-04-175. This inhibitor demonstrated profound specificity towards FAK in comparison to VS-4718 and BSJ-03-136.
To compare biological responses to FAK inhibition with complete FAK loss, the authors converted BSJ-04-175 into a FAK PROTAC, called BSJ-04-146. FAK degradation was induced by BSJ-04-146 at doses as low as 10 nM and showed a significant functional impact on downstream FAK signaling. The effects were observed as soon as one hour following the treatment. Unlike the VS-4718-conjugated PROTACs, BSJ-04-146 did not induce off-target kinase degradation and was remarkably selective. Moreover, the authors evaluated changes in the phosphoproteome following FAK degradation versus FAK inhibition. The degradation of FAK resulted in significant changes in the phosphoproteome, which were not observed when FAK was inhibited. These observations are consistent with the notion that FAK functions both as an enzyme and as a protein scaffold. Using BSJ-04-146, the authors demonstrated that FAK degradation showed a markedly improved decrease in the proliferation of pancreatic and breast cancer cells grown as 3D-spheroids as well as the migration of breast cancer cells, compared to FAK inhibitors. Lastly, BSJ-04-146 was tested for its ability to degrade FAK in murine models. Excitingly, Dr. Nabet and his team confirmed that BSJ-04-146 exhibits significant degradation of FAK in vivo. Dr. Nabet’s work showed that “targeting FAK for degradation leads to enhanced biological responses, compared to inhibiting FAK activity.” Targeted FAK degradation is "an advantageous strategy that warrants further development as a novel therapeutic approach in pancreatic cancer” he added.
Dr. Nabet is excited about bringing “the first PROTACs forward as treatments for difficult-to-treat cancers including pancreatic cancer.” Now, Dr. Nabet is interested in identifying "the degradation-specific biomarkers of response to FAK PROTACs” as well as identifying “the non-enzymatic functions of FAK that are important for the enhanced responses to targeted degradation.” Additionally, he is interested in investigating how to “improve the delivery of FAK PROTACs in vivo.” Stay tuned for more PROTAC updates
The spotlighted research was supported by grants from the National Cancer Institute, the Claudia Adams Barr Program in Innovative Basic Cancer Research, and the Robert L. Fine Cancer Research Foundation, and Fred Hutchinson Cancer Center start-up funds. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Robert L. Fine Cancer Research Foundation.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Dr. Behnam Nabet contributed to this work.
Koide E, Mohardt ML, Doctor ZM, Yang A, Hao M, Donovan KA, Kuismi CC, Nelson AJ, Abell K, Aguiar M, Che J, Stokes MP, Zhang T, Aguirre AJ, Fischer ES, Gray NS, Jiang B, Nabet B. Development and characterization of selective FAK inhibitors and PROTACs with in vivo activity. Chembiochem. 2023 Apr 23:e202300141.