Fragment screening is a popular strategy of generating viable chemical starting points
especially for challenging targets. Although fragments provide a better coverage of
chemical space and they have typically higher chance of binding, their weak affinity
necessitates highly sensitive biophysical assays. Here, we introduce a screening concept
that combines evolutionary optimized fragment pharmacophores with the use of a photoaffinity
handle that enables high hit rates by LC-MS-based detection. The sensitivity of our
screening protocol was further improved by a target-conjugated photocatalyst. We have
designed, synthesized, and screened 100 diazirine-tagged fragments against three benchmark
and three therapeutically relevant protein targets of different tractability. Our
therapeutic targets included a conventional enzyme, the first bromodomain of BRD4,
a protein-protein interaction represented by the oncogenic KRas G12D protein, and
the yet unliganded N -terminal domain of the STAT5B transcription factor. We have
discovered several fragment hits against all three targets and identified their binding
sites via enzymatic digestion, structural studies and modeling. Our results revealed
that this protocol outperforms screening traditional fully functionalized and photoaffinity
fragments in better exploration of the available binding sites and higher hit rates
observed for even difficult targets.