Site-Specific Labeling of Endogenous Proteins Using CoLDR Chemistry

Chemical modifications
of native proteins can affect their stability,
activity, interactions, localization, and more. However, there are
few nongenetic methods for the installation of chemical modifications
at a specific protein site in cells. Here we report a covalent ligand
directed release (CoLDR) site-specific labeling strategy, which enables
the installation of a variety of functional tags on a target protein
while releasing the directing ligand. Using this approach, we were
able to label various proteins such as BTK, K-RasG12C,
and SARS-CoV-2 PLpro with different tags. For BTK we have
shown selective labeling in cells of both alkyne and fluorophores
tags. Protein labeling by traditional affinity methods often inhibits
protein activity since the directing ligand permanently occupies the
target binding pocket. We have shown that using CoLDR chemistry, modification
of BTK by these probes in cells preserves its activity. We demonstrated
several applications for this approach including determining the half-life
of BTK in its native environment with minimal perturbation, as well
as quantification of BTK degradation by a noncovalent proteolysis
targeting chimera (PROTAC) by in-gel fluorescence. Using an environment-sensitive
“turn-on” fluorescent probe, we were able to monitor
ligand binding to the active site of BTK. Finally, we have demonstrated
efficient CoLDR-based BTK PROTACs (DC50 < 100 nM), which
installed a CRBN binder onto BTK. This approach joins very few available
labeling strategies that maintain the target protein activity and
thus makes an important addition to the toolbox of chemical biology.