Mapping protein-protein interaction surfaces by photoactivable molecular fragments

Protein-protein interactions (PPIs) represent viable therapeutic targets against diseases under transcriptional, translational and signal transduction control. PPIs, however, are still considered as challenging targets for small molecules due to their extended and dynamic interacting surfaces providing only shallow trenches and grooves with distant binding hot spots. We introduced a new 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. We have designed, synthesized, and screened 100 diazirine-tagged small molecule fragments against protein-protein interactions represented by the oncogenic KRasG12D protein, and the yet unliganded N-terminal domain of the STAT5B transcription factor. We have discovered several new fragment hits against all these targets and identified their binding sites via enzymatic digestion, structural studies and modelling. These results revealed that the protocol outperforms screening traditional PPI targeted libraries in better exploration of the available binding sites and higher hit rates observed for even difficult targets.
Our approach was recently extended to the development of proteomimetics screening a 100-membered photoreactive foldamer peptide fragment library. The members of this library act as local surface mimetics and identified functionally relevant protein interfaces, allosteric and previously unexplored targetable regions on the surface of both PPI targets. Target-templated dynamic fragment linking of foldamer hits resulted in two orders of magnitude affinity improvement in a single step. The dimeric K-Ras ligand mimicked protein-like catalytic functions. The photo-foldamer approach thus enables the highly efficient mapping of protein-protein interaction sites and provides a viable starting point for proteomimetic ligand development without a priori structural hypotheses.