Background/Objectives: Astragalus root is a classical qi-tonifying traditional Chinese medicine that has demonstrated potential therapeutic efficacy in type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD). However, the precise mechanisms underlying its effects on the comorbidity of these two disorders remain unclear. This study investigated the molecular mechanisms by which Astragalus root ameliorated T2DM-NAFLD comorbidity. Methods: Network pharmacology, molecular docking, molecular dynamics simulation, and in vitro experiments were employed to elucidate the potential roles and mechanisms of Astragalus root in the management of T2DM-NAFLD comorbidity. Results: A total of 25 bioactive constituents and 152 corresponding targets associated with Astragalus root were identified. PPI network analysis revealed the top ten core candidate targets, among which six possessed suitable crystal structures for molecular docking, including interleukin-6 (IL-6), threonine-protein kinase 1(AKT1), transcription factor AP-1(JUN), tumor necrosis factor (TNF), cysteine-dependent aspartate-specific protease 3 (CASP3), and estrogen Receptor 1(ESR1). Kyoto encyclopedia of genes and genomes (KEGG) analysis further identified the phosphatidylinositol 3-kinase (PI3K)-AKT as the most significantly enriched pathway. Molecular docking validated the potential binding modes of formononetin to the six core targets, a finding that was further confirmed by molecular dynamics simulations, which proved the stability of the resulting complexes. In vitro experiments demonstrated that formononetin obviously decreased lipid droplet accumulation, downregulated total cholesterol (TC) and triglyceride (TG) levels, suppressed the expression of TNF-α, IL-6, and interleukin-1β (IL-1β), decreased reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and enhanced glutathione (GSH) content and superoxide dismutase (SOD) activity. These therapeutic effects were achieved through inhibition of protein expression within the PI3K/AKT/mechanistic target of rapamycin (mTOR) signaling pathway. Conclusions: This study determined the potential therapeutic targets and underlying mechanisms of formononetin derived from Astragalus root in the T2DM-NAFLD management, thereby providing a scientific basis for its clinical application.

The optimization of membrane permeability is a pivotal approach for mitigating late-stage failures in peptide drug development. By leveraging linker chemical diversity, stapled peptides utilize linker engineering to precisely modulate key physicochemical parameters—such as lipophilicity and conformational constraints—to overcome the desolvation energy penalty. This review systematically evaluates linker-based strategies for enhancing the permeability of stapled peptides, categorized into two primary dimensions: (1) high-throughput screening (HTS) compatibility, focusing on the integration of functionalized linkers into mRNA display, phage display, and DNA-encoded libraries (DELs) to identify lead scaffolds with inherent permeability potential during early discovery; and (2) post-screening structural refinement, covering rational design strategies including intramolecular hydrogen-bond (IMHB) shielding, “chameleonic” adaptations, and stimuli-responsive reversible stapling. Furthermore, we analyze the paradigm shift in assessment methodologies from qualitative imaging to quantitative cytosolic delivery assays, which have deepened our understanding of mechanisms such as the charge/lipophilicity threshold balance and metabolism-driven trapping. Overall, linker engineering provides a robust technical roadmap for developing the next generation of cell-permeable stapled peptide therapeutics.

Background/Objectives: Astragalus root is a classical qi-tonifying traditional Chinese medicine that has demonstrated potential therapeutic efficacy in T2DM and NAFLD. However, the precise mechanisms underlying its effects on the comorbidity of these two disorders remain unclear. This study investigated the molecular mechanisms by which astragalus root ameliorated T2DM-NAFLD comorbidity. Methods: Network pharmacology, molecular docking, molecular dynamics simulation, and in vitro experiments were employed to elucidate the potential roles and mechanisms of astragalus root in the management of T2DM-NAFLD comorbidity. Results: A total of 25 bioactive constituents and 152 corresponding targets associated with astragalus root were identified. PPI network analysis revealed the top ten core candidate targets, among which six possessed suitable crystal structures for molecular docking, including IL-6, AKT1, JUN, TNF, CASP3, and ESR1. KEGG analysis further identified the PI3K-AKT as the most significantly en-riched pathway. Molecular docking of the principal bioactive constituent formononetin from astragalus root with the six core targets was conducted using AutoDock4 software. Molecular dynamics simulations verified the stability of the interactions between for-mononetin and each of the six core target proteins. In vitro experiments demonstrated that formononetin obviously decreased lipid droplet accumulation, downregulated TC and TG levels, suppressed the expression of TNF-α, IL-6, and IL-1β, decreased ROS and MDA levels, and enhanced GSH content and SOD activity. These therapeutical effects were achieved through inhibition of protein expression within the PI3K/AKT/mTOR signaling pathway. Conclusions: This study determined the potential therapeutic targets and underlying mechanisms of formononetin derived from astragalus root in the T2DM-NAFLD management, thereby providing a scientific basis for its clinical application.

The c-Jun N-terminal kinase (JNK) pathway is a central driver of fibrosis, inflammation, and neurodegeneration. While direct JNK inhibitors have shown therapeutic promise, achieving high isoform selectivity remains a significant medicinal chemistry challenge. Furthermore, targeting the upstream ‘gatekeepers’ MKK4 and MKK7 offers a distinct mechanism to modulate pathway output with greater precision. Consequently, medicinal chemistry efforts have shifted upstream to the dual-specificity kinases MKK4 and MKK7. This review critically evaluates the structural biology and pharmacological evolution of small-molecule inhibitors targeting these nodes. We contrast the distinct therapeutic landscapes of the two kinases: while MKK4 inhibition has emerged as a breakthrough strategy for unlocking liver regeneration (exemplified by the first-in-class clinical candidate HRX215), MKK7 inhibition is primarily pursued for its anti-fibrotic and anti-inflammatory potential. Special attention is given to structure-based design strategies, including the exploitation of the unique hinge-region cysteine (Cys218) for MKK7-specific covalent targeting and the optimization of scaffold selectivity against off-targets like BRAF. Finally, we discuss emerging modalities, such as PROTACs and dual inhibitors, outlining a roadmap for the next generation of precision therapeutics targeting the MKK–JNK axis.

As the key enzyme catalyzing the final step in heme and chlorophyll biosynthesis, protoporphyrinogen oxidase (PPO) is a crucial target for herbicide development. To date, over 40 PPO inhibitors have been commercialized. They offer high efficacy, environmental safety, low application rates, and broad-spectrum weed control. Recently, significant progress has been made in PPO structural biology, with several crystal structures resolved. Despite decades of use, the emergence of resistant weeds necessitates the continuous innovation of novel PPO inhibitors. This review systematically summarizes PPO three-dimensional structures, enzyme-inhibitor interaction mechanisms, and quantitative structure–activity relationships (QSARs). Finally, we outline rational molecular design strategies for the next generation of PPO inhibitors.

As the key enzyme catalyzing the final step in the biosynthesis of heme and chlorophyll, protoporphyrinogen oxidase (PPO) has become a crucial target for herbicide development. To date, more than 40 PPO-inhibiting herbicides have been developed, exhibiting multiple advantageous characteristics: they combine high efficacy with environmental friendliness, feature low effective concentrations, rapid action, long-lasting effects, and excellent control of both monocotyledonous and dicotyledonous weeds. In recent years, significant progress has been made in the structural biology of PPO—five crystal structures from tobacco, humans, and various bacteria have been resolved, most of which are presented as enzyme-inhibitor complexes. Although the development of such herbicides spans over five decades, novel PPO inhibitors still hold broad potential for innovation due to the resistance of early applied PPOs. This review systematically summarizes the three-dimensional structures of PPO from different sources, the interaction mechanisms between the enzyme and inhibitors, studies on quantitative structure-activity relationships of inhibitors, and outlines molecular design directions for the next generation of PPO inhibitors.