Cysteine
-­
reactive small molecules are used as chemical
probes of biological systems and as medicines. Identifying
high
-­
quality covalent ligands requires comprehensive kinetic analysis to
distinguish selective binders from pan
-­
reactive compounds. Here we
describe quantitative irreversible tethering
(qIT)
, a general method
for screening cysteine
-­
reactive small molecules
based upon the
maximization of kinetic selectivity. We apply this method
prospectively to discover covalent fragments that
target
the clinically
important cell cycle regulator Cdk2.
Crystal structures of the inhibitor
complexes validate the approach and g
uide further optimization.
The
power of this technique is highlighted by the identification of a Cdk2
-­
selective allosteric (type IV) kinase inhibitor whose novel mode
-­
of
-­
action could be exploited therapeutically.