The Mitogen-Activated Protein Kinase (MAPK) Extracellular-signal Regulated Kinase 5 (ERK5) pathway is involved in almost all the biological features of cancer cells. Indeed, its role in the development and progression of several types of tumours has been demonstrated. Moreover, ERK5 pathway activation is among the resistance mechanisms to RAS/RAF/MEK1/2-ERK1/2 targeted therapy. Hence, several ERK5 kinase activity inhibitors have been developed. Despite their efficacy in reducing cancer cell proliferation, these compounds only work at relatively high concentrations, making them unsuitable for clinical research. In addition, some of these drugs resulted in a paradoxical activation of ERK5, by inducing its nuclear translocation. In sight of this, we aimed at developing new ERK5 kinase inhibitors.

To test the efficacy of the compounds on the kinase activity of ERK5 we conducted in vitro kinase assays. To evaluate the effects of these new potential inhibitors on cancer cell viability we performed cell counting using both wild type and ERK5-knockout (KO) cells.

In silico screening of the commercial database MolPort, that contains 5M compounds, allowed to identify several candidates among which we have tested 12 compounds. With in vitro kinase assay we have individuated one (compound 14) that completely inhibits ERK5 kinase activity up until 1µM and partially inhibits it until 100nM. Interestingly, comparing the effect of compound 14 with the effects of the commercially available ERK5 kinase inhibitor JWG-071, we were able to highlight that the new inhibitor works at lower concentration with respect to JWG-071. Moreover, we studied the effect of these compounds on cell proliferation. In HeLa cells, compound 14 completely inhibits cell proliferation up until 2.5 µM, showing efficacy already at 100 nM. To deepen whether this effect relied, at least partly, on ERK5, we performed the same experiment in control and ERK5-knockout cells. We used two clones of ERK5-KO ovarian cancer cells (OV8) and one ERK5-KO hepatocellular carcinoma cell line (HUH7). Interestingly, in all three models ERK5-KO cells resulted to be less sensitive to compound 14 with respect to the control cells, starting from100 nM up until 10 µM. Comparing these results with the ones obtained for JWG-071, we pointed out a higher specificity of compound 14 in inhibiting cell proliferation

In conclusion, we discovered a new potential ERK5 kinase inhibitor that has an effect at nanomolar concentrations, both in inhibiting ERK5 kinase activity and cell proliferation in an ERK5-dependent manner. Even if further evaluation regarding the capability of this inhibitor not to induce a paradoxical activation of ERK5 will be needed, these results pave the way to a new class of small molecules inhibiting ERK5 activity, that may be able to reach clinical relevance after additional preclinical evaluation.