TAK1, a transforming growth factor-β-activated kinase, is involved in numerous signalling pathways, thus regulating inflammatory response and cell survival. It is activated after stimulus of a variety of signalling molecules, which leads to pro-inflammatory cytokines and antiapoptotic factors gene induction through different post-translational mechanisms. Consequently, TAK1 is involved in the development of inflammatory and malignant diseases. However, its effects are cell-type dependent, rendering TAK1 a very complex therapeutic target. Out of a few known TAK1 inhibitors, takinib has shown the highest kinome selectivity and has demonstrated promising results in in vitro biological assays. Unfortunately, its low bioavailability represents an obstacle for further research of its therapeutic significance. The quest of finding new ways to modulate TAK1 activity is gaining an importance, which is why we focused on exploring targeted protein degradation of TAK1 using the novel PROTAC technology in our work. PROTAC molecules are heterobifunctional molecules, which induce proteasomal degradation of a protein of interest by drawing a chosen E3 ligase in its close proximity. These molecules are assembled of an E3 ligase ligand and a protein of interest ligand, which are connected by a linker. The latter enables regulating the physicochemical properties of the final molecules. Compared to classic enzyme inhibitors, this technology offers several advantages. Namely, PROTACs are able to target the so-called undruggable proteins with no active site. Additionally, due to the event-driven model of their pharmacology, they can be used in substoichiometric concentrations, which consequently lowers the occurrence of off-site side effects.
During the course of our research, we synthesized four PROTAC molecules with a ligand for protein of interest TAK1 and ligands for CRBN, VHL and IAP E3 ligases. We also focused on the importance of the linker's length and binding site on the overall functionality of the final compounds. We evaluated the final PROTAC molecules first by in vitro enzyme inhibition assay, followed by MTS test of cell viability and western blot, which were both performed using the THP-1 cell line. Out of four final compounds, all exhibited high binding affinity towards TAK1, and one of them had a great impact on lowering cell viability and TAK1 concentration in the cells.
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