Improved Electrophile Design for Exquisite Covalent Molecule Selectivity

16 November 2021, Version 2


Covalent inhibitors continue to show therapeutic promise. However, off-target reactivity challenges the field. Extensive efforts have been exerted to solve this issue by varying the reactivity attributes of electrophilic warheads, with features such as reversibility or metabolic vulnerability. Here we report the development of a new approach to increase the selectivity of covalent probes and small molecule inhibitors that is independent of warhead reactivity features and can be used in concert with already-existing methods. Using the Bruton’s Tyrosine Kinase (BTK) inhibitor Ibrutinib scaffold 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 ideally 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 significantly decreases time-dependent off-target reactivity and abolishes time-independent off-target reactivity but retains BTK target engagement. While an alkyne-bearing probe analog of Ibrutinib has 247 protein targets, our t-Bu fumarate Ibrutinib probe analog has only 7 protein targets. Of these 7 targets, BTK is the only time-independent target. This 2-order-of-magnitude increase in selectivity is also conferred to the t-Bu inhibitor itself. By shotgun proteomics, we investigated the consequences of treatment with Ibrutinib and our t-Bu analog and discovered that only 8 proteins are downregulated in response to treatment with the t-Bu analog 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 a previously-unappreciated opportunity to increase cysteine-reactive covalent inhibitor selectivity through electrophilic structure optimization.


Supplementary materials

Supplemental Tables
Mass spectrometry data
Supporting Information
Supplemental figures and methods


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