Targeting the Allosteric Pathway That Interconnects the Core-Functional Scaffold and the Distal Phosphorylation Sites for Specific Dephosphorylation of Bcl‑2

Protein
phosphorylation is the most significant post-translational
modification for regulating cellular activities, but site-specific
modulation of phosphorylation is still challenging. Using three-dimensional
NMR spectra, molecular dynamics simulations, and alanine mutations,
we identified that the interaction network between pT69/pS70 and R106/R109
residues prevents the phosphorylation sites from exposure to phosphatase
and subsequent dephosphorylation. A Bcl-2-dephosphorylation probe, S1-6e, was designed by installing a carboxylic acid group
to a Bcl-2 inhibitor. The carboxyl group competitively disrupts the
interaction network between R106/R109 and pT69/pS70 and subsequently
facilitates Bcl-2 dephosphorylation in living cells. As a result, S1-6e manifests a more effective apoptosis induction in pBcl-2-dependent
cancer cells than other inhibitors exhibiting a similar binding affinity
for Bcl-2. We believe that targeting the allosteric pathways interconnecting
the core-functional domain and the phosphorylation site can be a general
strategy for a rational design of site-specific dephosphorylating
probes, since the allosteric pathway has been discovered in a variety
of proteins.