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Using chemoproteomic techniques, we first identified EIF2AK2, eEF1A1, PRDX3 and VPS4B as direct targets of berberine (BBR) for its synergistically anti-inflammatory effects. Of them, BBR has the strongest affinity with EIF2AK2 via two ionic bonds, and regulates several key inflammatory pathways through EIF2AK2, indicating the dominant role of EIF2AK2. Also, BBR could subtly inhibit the dimerization of EIF2AK2, rather than its enzyme activity, to selectively modulate its downstream pathways including JNK, NF- κ B, AKT and NLRP3, with an advantage of good safety profile. In EIF2AK2 gene knockdown mice, the inhibitory IL-1 β , IL-6, IL-18 and TNF- α secretion of BBR was obviously attenuated, confirming an EIF2AK2-dependent anti-inflammatory efficacy. The results highlight the BBR's network mechanism on anti-inflammatory effects in which EIF2AK2 is a key target, and inhibition of EIF2AK2 dimerization has a potential to be a therapeutic strategy against inflammation-related disorders. EIF2AK2 is proved to be a key target for BBR's network mechanism, and inhibition of EIF2AK2 dimerization has a potential to be a therapeutic strategy against inflammation-related disorders. [Display omitted] [ABSTRACT FROM AUTHOR]

Target identification of bioactive compounds is important for understanding their mechanisms of action and provides critical insights into their therapeutic utility. While it remains a challenge, unbiased chemoproteomics strategy using clickable photoaffinity probes is a useful and validated approach for target identification. One major limitation of this approach is the efficient synthesis of appropriately substituted clickable photoaffinity probes. Herein, we describe an efficient and consistent method to prepare such probes. We further employed this method to prepare a highly stereo-congested probe based on naturally occurring triterpenoid betulinic acid. With this photoaffinity probe, we identified tropomyosin as a novel target for betulinic acid that can account for the unique biological phenotype on cellular cytoskeleton induced by betulinic acid. A new method was developed to reliably prepare stereo-hindered clickable diazirines for target identification. Tropomyosin was identified as a new target for betulinic acid. [Display omitted] [ABSTRACT FROM AUTHOR]

Rice exposed to organic pollutants such as polybrominated diphenyl ethers (PBDEs) usually experiences reduced biomass and increased soluble sugar content. This study showed that 2, 2′, 4, 4′-tetrabromodiphenyl ether (BDE-47) led to increased glucose, fructose, and sucrose in rice leaves, accompanied by decreased photosynthetic rate and biomass. In order to identify the key enzyme that BDE-47 interacted with, a diazirine-alkynyl photoaffinity probe was designed, and photoaffinity labeling based chemoproteomics was conducted. Among all differentially expressed proteins, fructose-1, 6-bisphosphate aldolase (FBA) involved in carbohydrate metabolism was most likely the target protein of BDE-47. Spectral techniques and molecular docking analysis further revealed that the pollutant-protein interaction was driven by hydrophobic force. BDE-47 inhibited FBA catalytic efficiency by competing with its substrate, fructose-1, 6-diphosphate (F-1, 6-P), leading to soluble sugar accumulation, photosynthetic rate decline and biomass reduction. This study unraveled the influencing mechanism of PBDEs on rice by combining the novel photoaffinity labeling-based chemoproteomics with conventional proteomics. The improved knowledge on direct interaction between organic pollutants and proteins will help alleviate the harmful effects of soil pollution on plants. [Display omitted] • Biomass and total sugar content of rice changed upon BDE-47 exposure. • Soluble sugar accumulation led to photosynthesis inhibition and total sugar change. • Carbohydrate metabolism was the main metabolic process affected by BDE-47. • Photoaffinity labeling based chemoproteomics identified BDE-47 target proteins. • Binding of BDE-47 with FBA in glycolysis caused soluble sugar accumulation. [ABSTRACT FROM AUTHOR]