Identification of novel target of quinazolinones active molecules and bioactivity & labeling preference of photocrosslinkers.
This study investigated how different photoaffinity crosslinkers influence the biological activity, labeling efficiency, and target protein preference of quinazolinone-based active molecules—specifically QDAU5, a compound previously shown to promote vascular normalization. The researchers constructed a library of photocrosslinkers and multifunctional photoaffinity probes using structures such as thiophene-substituted α-ketoamide, tetrazole, isoxazole, and 2-nitrobenzyl alcohol, then applied Photoaffinity Labeling–Affinity-Based Protein Profiling (PAL-AfBPP) combined with proteomic analysis to assess probe performance. Prior work had identified EphrinB2 as an intracellular target of QDAU5; this study further validated Thymosin β4 (Tβ4) as an additional potential target and began characterizing how QDAU5 interacts with Tβ4 at a molecular level. The findings suggest that the choice of photocrosslinker meaningfully affects both the retained bioactivity of the parent compound and which proteins are captured, offering practical guidance for PAL-AfBPP probe design. Limitations include that this is primarily a chemical biology and proteomics study conducted at the in vitro/biochemical level, with no human clinical data. The mechanistic link between QDAU5–Tβ4 interaction and angiogenesis regulation requires further validation in vivo.
Why this grade: The study is a chemical biology/proteomics investigation conducted at the biochemical and cell-based in vitro level with no animal models or human subjects reported.
Photoaffinity Labeling-Affinity-Based Protein Profiling (PAL-AfBPP) provides breakthrough solutions in a variety of fields such as target protein discovery. The selection of photoaffinity connectors is the key component of PAL-AfBPP probe design and application. At present, the development of organic chemistry has also made the thiophene-substituted α-ketoamide, tetrazole, isoxazole, 2-nitrobenzyl alcohol and other structures become new photoaffinity ligand candidates for the design of PAL-AfBPP probes. However, how the conjugation of different linkers with active molecules affects the biological activity, labeling efficiency, and target preference of the parent compounds remains unclear. The previous study found that the active quinazolinone molecules represented by QDAU5 had significant vascular normalizing effect, and finally identified that EphrinB2 was an intracellular target protein of QDAU5. At the same time, it was also found that thymosinβ4 (Tβ4) was effectively enriched by QDAU5 probe, which may be another way for QDAU5 to exert biological effects. Herein, we constructed a photocrosslinker library and a multifunctional photoaffinity probe library to verify the effects of different photocrosslinkers on the activity and labeling preference of quinazolinones. Based on proteomic analysis, the potential target Tβ4 was further validated, and the interaction mode between QDAU5 and Tβ4 was preliminarily elucidated. The results of this study provide a practical reference for the precise selection of photocrosslinkers in PAL-AfBPP probe design, and also provide more support for elucidating the mechanism of angiogenesis regulation and vascular normalization by active quinazolinones.
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