Identification of Novel Compounds Inhibiting the Kinase Activity of the CDK5/p25 Complex via Direct Binding to p25

Tamoxifen, the gold standard drug for endocrine therapy for breast cancer, modulates the phosphorylation status of the TAU protein in Alzheimer's disease by inhibiting CDK5 kinase activity. Its binding to p25 prevents CDK5/p25 complexation and hence a decrease of CDK5 activity. In breast tumors, this complex is involved in the proliferation and survival of cancer cells, as well as in the disease's prognosis. Still, the molecular stability of the CDK5/p25 complex following tamoxifen exposure in this cancer type has not yet been clearly deciphered. Here, we report the functional characterization of CDK5 and its p25 regulatory subunit in the absence and presence of tamoxifen. In addition, two novel inhibitors of the kinase activity of the CDK5/p25 complex are identified, both of which would reduce the risk of recurrence of estrogen receptor-positive (ER+) breast cancers and prevent drawbacks induced by tamoxifen exposure. Accordingly, 6His-CDK5 and 6His-p25 have been expressed and purified. Fluorescence anisotropy measurements have been used to assess that the two proteins do form an active complex, and thermodynamic parameters of their interaction were measured. It was also confirmed that tamoxifen directly binds to p25 and inhibits CDK5 kinase activity. Similar observations were obtained using 4-hydroxytamoxifen, an active metabolized form of tamoxifen. Two novel compounds have been identified here that harbor a benzofuran moiety and were shown to target directly p25, and their bindings resulted in decreased CDK5 kinase activity. This encouraging alternative opens the way to the ensuing chemical optimization of this scaffold. It also promises a more specific therapeutic approach that may both tackle the pathological signaling in breast cancer and provide a potential new drug for Alzheimer's disease.