Computational Design of Lysine Targeting Covalent Binders Using Rosetta

Chemical probes that form a covalent bond with their
target protein
have been established as a powerful tool for investigating proteins
and modulating their activity, but until recently were mostly targeting
cysteine residues. Covalent binders that target lysine residues are
increasingly reported. Covalent binding to lysine involves challenges
such as the increased pKa of the side
chain and its considerable flexibility. Here, we describe two computational
methods to derivatize lysine-binding covalent small-molecules based
on known noncovalent binders, approaching the design problem from
two opposite directions. In a “ligand-side” approach,
we scan different ligand positions to install an electrophile and
dock these derivatized ligands into the target protein. In a “protein-side”
approach, we install an electrophile on the target lysine and model
its conformational space to find suitable installation vectors on
the ligand. We applied both of these protocols retrospectively to
a data set of electrophilic ligands and to a data set of vitamin B6
covalently bound to a receptor lysine residue. Our ligand-side protocol
successfully identified the known covalent binder in 80% and 86% of
cases, while the protein-side protocol achieved identification rates
of 56% and 82%, respectively. We prospectively validated these protocols
by designing and testing a new lysine-targeting MKK7 inhibitor. Mass-spectrometry
and crystallography validated the covalent binding to the target lysine.
Applying these protocols to a data set of known kinase inhibitors
identified high-confidence covalent candidates for more than 200 human
kinases, demonstrating the potential impact of our protocols.