Fast and Site-Specific Covalent Targeting of Proteins by Arylfluorosulfate-Modified Aptamers

Sulfur fluoride exchange (SuFEx) is a versatile click
chemistry
platform with emerging applications in covalent inhibitor development,
among which arylfluorosulfate (AFS) derivatives have shown great promise
because of their exclusive selectivity to target proteins. Nevertheless,
due to the inherently mild electrophilicity of AFS, the high target
specificity often comes at the expense of target reactivity. Here,
we challenge this conventional view and show that in vitro selected
AFS-modified covalent aptamers (AFS-Aps) can be both highly target
selective and reactive. One such AFS-Ap is able to site-specifically
modify the extracellular domain (ECD) of human epidermal growth factor
receptor 3 (HER3) with a “click” reaction half-life
(t1/2) as short as 1 min and covalently
inhibit the protein–protein interaction (PPI) of HER3/EGFRs.
Another AFS-Ap targeting human vascular endothelial growth factor
(VEGF) can also rapidly cross-link VEGF165 and block the PPI of VEGF/VEGFR.
The inactivation rate constants (kinact) of these AFS-Aps are one to two orders of magnitude higher than
many reported AFS-based covalent molecules. This unexpectedly fast
and site-specific covalent targeting requires a precisely organized
aptamer–protein binding interface, where the AFS structure,
rather than electrophilicity, is the decisive factor. We believe the
target reactivity potential of AFS derivatives may have long been
underestimated in covalent inhibitor development. AFS-adapted combinatorial
techniques are therefore invaluable for discovering covalent inhibitors
with both high target reactivity and selectivity.