Kinetic Optimization of Lysine-Targeting Covalent Inhibitors of HSP72

The covalent inhibition mechanism of action, which overcomes
competition
with high-affinity, high-abundance substrates of challenging protein
targets, can deliver effective chemical probes and drugs. The success
of this strategy has centered on exposed cysteine residues as nucleophiles
but the low abundance of cysteine in the proteome has limited its
application. We have recently reported our discovery that lysine-56
in the difficult-to-drug target HSP72 could form a covalent bond with
a small-molecule inhibitor. We now disclose the optimization of these
targeted covalent inhibitors using rational design. Essential to our
optimization was the development of a new covalent fluorescence polarization
assay, which allows for the direct measurement of the key kinetic
parameter in covalent inhibitor design, kinact/KI, extrapolation of
the underlying parameters, kinact and Ki, and direct comparison to
reversible analogues. Using our approach, we demonstrate a >100-fold
enhancement in covalent efficiency and key learnings in lysine-selective
electrophile optimization.