Small Molecules as Potential Inhibitors of the 14-3-3/c-Abl Interaction for the Treatment of CML ^†

c-Abl is a tyrosine kinase implicated in the regulation of proliferation, adhesion, motility, and cell survival. After phosphorylation on the Thr735 residue, it binds to 14-3-3 proteins that sequester c-Abl into the cytoplasm where it induces proliferation and survival. DNA damage or oxidative stress lead to disruption of the c-Abl/14-3-3 complex, nuclear translocation of the c-Abl protein, and activation of apoptosis. The oncogenic form of c-Abl, Bcr-Abl, is the cause of the development and progression of chronic myeloid leukemia (CML) and is localized exclusively in the cytoplasm, inducing proliferation and inhibiting apoptotic cell death.

The aim of the project is to find small molecules able to disrupt the 14-3-3/c-Abl interaction and thus promote c-Abl nuclear translocation and apoptosis of CML cells.

After an X-ray crystallography screening of an in-house library of small molecules containing a phosphate group, the coenzyme pyridoxal phosphate (PLP) and the nucleotide inosine monophosphate (IMP) were crystallized and identified as potential modulators of the 14-3-3σ. Analysis of the crystal structures revealed that the phosphate group of both molecules could mimic the phosphorylated motif of the protein partners, hence acting as potential inhibitors.

Biophysical analyses were performed in order to validate the former approach, more specifically with the NMR, FP, and SPR techniques.

After validation of the 14-3-3/c-Abl interaction, the role of PLP and IMP as weak inhibitors was confirmed. Using IMP and PLP evaluation procedures as reference and positives controls, an FP-based screening of a small library of 48 compounds was carried out. Four compounds (compound 3, 8, 29 and 31) were found to interact, showing similar binding affinities as the reference molecules and evidencing their behavior as soft inhibitors of the 14-3-3/c-Abl complex. The 6 potential inhibitors were assessed against K562 CML cell lines, and the biological effects on Bcr-Abl expressing cells were promising.