Identification of Protein Targets of Bioactive Small Molecules Using Randomly Photomodified Probes

Identifying protein
targets of bioactive small molecules often
requires complex, lengthy development of affinity probes. We present
a method for stochastic modification of small molecules of interest
with a photoactivatable phenyldiazirine linker. The resulting isomeric
mixture is conjugated to a hydrophilic copolymer decorated with biotin
and a fluorophore. We validated this approach using known inhibitors
of several medicinally relevant enzymes. At least a portion of the
stochastic derivatives retained their binding to the target, enabling
target visualization, isolation, and identification. Moreover, the
mix of stochastic probes could be separated into fractions and tested
for binding affinity. The structure of the active probe could be determined
and the probe resynthesized to improve binding efficiency. Our approach
can thus enable rapid target isolation, identification, and visualization,
while providing information required for subsequent synthesis of an
optimized probe.