Photoaffinity labeling has gained widespread popularity in new interdisciplines including chemical biology, medicinal chemistry, biological physics and biomaterial science. In particular, as a powerful tool, photoaffinity labeling utilizes photoreactive groups, also known as photo-cross-linkers, to capture biological interactions inside native cellular environments upon photo triggering. Despite the widely recognition and application of the favored diazirine-based photo-cross-linkers and other newely-developed photo-cross-linkers, drawbacks such as interference on bioactivity of ligands, ambiguous cross-linking mechanism, low cross-linking yield and poor efficiency remain inevitable, thus limiting their practicability. Herein, we integrated and summarized the photoreaction mechanisms of a frequently-used pharmacophore, isoxazole, and managed to apply it to photoaffinity labeling. Specifically, a small-molecular library containing nine fully functionalized isoxazole probes have been designed and synthesized. In vivo and in situ labeling profiles of these probes demonstrated superior cross-linking potency and high labeling efficiency of the isoxazole group as photo-cross-linker. Hence, two isoxazole-containing drugs, Danazol and Luminespib, were privileged to uncover their cellular protein targets via isoxazole-based photoaffinity labeling and affinity-based protein profiling. Briefly, 48 proteins hits including TMF1, P53 and PDIAs were identified as the potential cellular targets of Danazol. While Hsp90, EFTS, CD63, CD147 as well as other 16 proteins were revealed as the primary targets of Luminespib. Besides, various potential interactomes and pathways, for example, interactions among chaperones, co-chaperones and clients, have been proximally labeled and characterized to investigate their cellular functions and clinical side effects. Surprisingly, among those primary cellular targets of Luminespib, we found a membrane glycoprotein, CD147, concerned to cancer proliferation and served as a receptor protein that facilitates invasion of plasmodium, human immunodeficiency virus, coronavirus etc. This promising druggable target might provide opportunities for developing novel therapeutics against cancer or those severe pathogens such as SARS-CoV-2. Additionally, the Luminespib-based probe exhibited high selectivity and sensitivity in detecting and imaging endogenous Hsp90 in live cells. Considering that Hsp90 is widely regarded as a cancer biomarker, this probe could be further applied to diverse cancer diagnoses and therapies.

To further provide robust solutions for combating cancer, we have developed a novel small molecules-based nanoplatform for combinational cancer diagnosis and therapy, namely phototheranostic. A versatile probe has been designed and synthesized based on methylene blue, the first FDA-approved near-infrared fluorophore. Owing to the rational design, this probe owns several favorable characteristics, including GSH activatable in cancer cells, biotin-guided cancer targeting ability, self-assembled to form nanoparticles in aqueous solution. As a consequence, this probe exhibited remarkable reduced cytotoxicity over normal cells and improved therapeutic efficiency towards cancer cells. Hence, we have applied this probe to fluorescence-guided cancer phototheranostic. In vivo near-infrared fluorescence imaging offered significant information for guiding phototherapy. Satisfyingly, in vivo photodynamic therapy demonstrated superior capacity and efficiency in ablation of tumor. By contrast, extremely low toxicity and neglectable side effects were observed for normal tissues and organs.

Overall, we explored the possibilities of utilizing light-mediated chemistry approaches to solve biological problems. We hope these powerful probes and interesting results could benefit drug discovery and cancer treatment in the future.