Improved Electrophile Design for Exquisite Covalent Molecule Selectivity

Covalent
inhibitors are viable therapeutics. However, off-target
reactivity challenges the field. Chemists have attempted to solve
this issue by varying the reactivity attributes of electrophilic warheads.
Here, we report the development of an approach to increase the selectivity
of covalent molecules that is independent of warhead reactivity features
and can be used in concert with existing methods. Using the scaffold
of the Bruton’s tyrosine kinase (BTK) inhibitor Ibrutinib for
our proof-of-concept, we reasoned that increasing the steric bulk
of fumarate-based electrophiles on Ibrutinib should improve selectivity
via the steric exclusion of off-targets but retain rates of cysteine
reactivity comparable to that of an acrylamide. Using chemical proteomic
techniques, we demonstrate that elaboration of the electrophile to
a tert-butyl (t-Bu) fumarate ester
decreases time-dependent off-target reactivity and abolishes time-independent
off-target reactivity. While an alkyne-bearing probe analogue of Ibrutinib
has 247 protein targets, our t-Bu fumarate probe
analogue has only 7. Of these 7 targets, BTK is the only time-independent
target. The t-Bu inhibitor itself is also more selective
for BTK, reducing off-targets by 70%. We investigated the consequences
of treatment with Ibrutinib and our t-Bu analogue
and discovered that only 8 proteins are downregulated in response
to treatment with the t-Bu analogue compared to 107
with Ibrutinib. Of these 8 proteins, 7 are also downregulated by Ibrutinib
and a majority of these targets are associated with BTK biology. Taken
together, these findings reveal an opportunity to increase cysteine-reactive
covalent inhibitor selectivity through electrophilic structure optimization.