Glycogen synthase kinase‐3 (GSK3), consisting of GSK3α and GSK3β subtypes, is a complex protein kinase that regulates numerous substrates. Research has observed increased GSK3 expression in the brains of Alzheimer's disease (AD) patients and models. AD is a neurodegenerative disorder with diverse pathogenesis and notable cognitive impairments, characterized by Aβ aggregation and excessive tau phosphorylation. This article provides an overview of GSK3's structure and regulation, extensively analyzing its relationship with AD factors. GSK3 overactivation disrupts neural growth, development, and function. It directly promotes tau phosphorylation, regulates amyloid precursor protein (APP) cleavage, leading to Aβ formation, and directly or indirectly triggers neuroinflammation and oxidative damage. We also summarize preclinical research highlighting the inhibition of GSK3 activity as a primary therapeutic approach for AD. Finally, pending issues like the lack of highly specific and affinity‐driven GSK3 inhibitors, are raised and expected to be addressed in future research. In conclusion, GSK3 represents a target in AD treatment, filled with hope, challenges, opportunities, and obstacles.

Identification of therapeutic targets can directly elucidate the mechanism and effect of drug therapy, which is a central step in drug development. The disconnect between protein targets and phenotypes under complex mechanisms hampers comprehensive target understanding. Metabolomics, as a systems biology tool that captures phenotypic changes induced by exogenous compounds, has emerged as a valuable approach for target identification. A comprehensive overview was provided in this review to illustrate the principles and advantages of metabolomics, delving into the application of metabolomics in target identification. This review outlines various metabolomics‐based methods, such as dose–response metabolomics, stable isotope‐resolved metabolomics, and multiomics, which identify key enzymes and metabolic pathways affected by exogenous substances through dose‐dependent metabolite–drug interactions. Emerging techniques, including single‐cell metabolomics, artificial intelligence, and mass spectrometry imaging, are also explored for their potential to enhance target discovery. The review emphasizes metabolomics' critical role in advancing our understanding of disease mechanisms and accelerating targeted drug development, while acknowledging current challenges in the field.

Benzo[α]pyrene (Bap) is recognized as a ubiquitous environmental pollutant among the polycyclic aromatic hydrocarbons (PAHs) class. Previous studies have shown that the hepatotoxicity of Bap is mainly caused by its metabolites, although it remains unclear whether Bap itself induces such damage. This study integrated metabolomics and chemical proteomics approaches to comprehensively identify the potential target proteins affected by Bap in liver cells. The results from the metabolomics showed that the significant changed metabolites were related with cellular redox homeostasis. CEllular Thermal Shift Assay (CETSA) showed that Bap induced protein thermal displacement of superoxide dismutase 3 (SOD3) and glutathione peroxidase 4 (GPX4), which are closely related to oxidative homeostasis. Further validation through in vitro CETSA and drug affinity response target stability (DARTS) revealed that Bap directly affected the stability of SOD3 and GPX4 proteins. The binding affinities of Bap to the potential target proteins were further evaluated using molecular docking, while the isothermal titration calorimetry (ITC) interaction measurements indicated nanomolar-level Kd values. Importantly, we found that Bap weakened the antioxidant capacity by destroying the activities of SOD3 and GPX4, which provided a new understanding of the mechanism of hepatotoxicity induced by Bap. Moreover, our provided workflow integrating metabolomics and label-free chemical proteomics, can be regarded as a practical way to identify the targets and inter-mechanisms for the various environmental compounds.