INY-06-061

INY-06-061 demonstrates potent and rapid degradation of ERK5 (MAPK7) in cells at sub-micromolar concentrations, but not at supra-micromolar concentrations due to hook effect. Selectivity for ERK5 was shown through cell-free kinome screening and global proteomics profiling analysis in cells. INY-06-089 can serve as an inactive negative control for INY-06-061. Mechanism of action through the ubiquitin-proteasome system was verified using proteasomal and neddylation inhibitors, and indirect evidence of target engagement through cellular competitive binding assays using an ERK5 ligand. Anti-proliferative activity was tested in a panel of 750 cell lines, ERK5-dependent cancer cell lines, and human endothelial cells with little or no effect on proliferation and inflammatory response, indicating a phenotypic discrepancy between genetic knockdown and INY-06-061-induced degradation of ERK5.

INY-06-061 is a potent ERK5 degrader that is well profiled, showing high binding affinity to the target and also dependency on ubiquitin-proteasome system. Complete data on Dmax is not yet established, as well as the time course of the degradation. However, the experiments conducted prove the specific degradation of ERK5 - though it does not stop cell proliferation in cancer models.

This is a potent and selective compound (DC50 = 21 nM, no other proteins significantly downregulated in proteomics after 5h 100 nM treatment). The negative control (inactive VHL isomer) has been shown to be inactive and degradation was prevented by competition with ERK5 inhibitor demonstrating in cell target engagement. Proteasome inhibitor and neddylation inhibitor also prevented degradation. Dmax ~ 93% after 5-hour treatment with 100 nM compound. The compound is mostly non cytotoxic up to 10 uM. The Hook effect has been observed at 5 uM.