Discovery of drug target for vascular normalization through multifunctional photoaffinity probes.

The development of drug resistance in tumors attributed to vascular abnormality hinders anti-angiogenic treatments. While vascular normalization strategy offer promise, the absence of clearly defined targets and drugs remains a notable challenge. In a prior study, our research team pinpointed QDAU5, a quinazolinone active compound with vascular-normalizing properties, which was found to selectively bind to and inhibit the phosphorylation of EphrinB2, thereby EphrinB2 emerges as a promising therapeutic target for drug development. Herein, we constructed two types of multifunctional photoaffinity probes based on QDAU5 by in situ assembly of diazirine photosensitizing motifs and bio-orthogonal functional motif, demonstrating concurrent antitumor and vascular normalization activities. Through the incorporation of bioorthogonal labeling/purification motifs, we have achieved the labeling, purification and identification of the target protein EphrinB2, and analyzed its binding site, thus elucidating EphrinB2 as a drug target for tumor vascular normalization therapeutic strategy.