Discovery of Highly Specific Catalytic-Site-Targeting Fluorescent Probes for Detecting Lysosomal PDE10A in Living Cells

A challenge
for sensors targeting specific enzymes of interest
in their native environment for direct imaging is that they rationally
exploit a highly selective fluorescent probe with a high binding affinity
to provide real-time detection. Immunohistochemical staining, proteomic
analysis, or recent enzymatic fluorescent probes are not optimal for
tracking specific enzymes directly in living cells. Herein, we introduce
the concept of designing a highly effective fluorescent probe (BVQ1814) targeting phosphodiesterase 10A with a highly potent
affinity and a >1000-fold subfamily selectivity by gaining insights
into the three-dimensional structural information of the active site
of the catalytic pocket. BVQ1814 showed an outstanding
binding affinity for PDE10A in vitro and specifically
detected PDE10A in living cells, indicating that most PDE10A was probably
distributed in the lysosomes. We validated the PDE10A distribution
in stable mCherry-PDE10A-overexpressing HepG2 cells.
This probe delineated the profile of PDE10A in tissue sections and
exhibited a remarkable therapeutic effect as a PDE10A inhibitor for
treating pulmonary arterial hypertension. This concept will open up
a new avenue for designing a highly effective fluorescent probe for
tracking receptor proteins by taking full advantage of the structural
information in the ligand-binding pocket of the target of interest.