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.

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.

The optimization of membrane permeability is a decisive strategy for mitigating late-stage failures in peptide drug development. By leveraging linker chemical diver-sity, stapled peptides utilize linker engineering to precisely modulate key physico-chemical parameters—such as lipophilicity and conformational constraints—to over-come the desolvation energy penalty. This review systematically evaluates link-er-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, cover-ing rational design strategies including intramolecular hydrogen bond (IMHB) shield-ing, "chameleonic" adaptations, and stimuli-responsive reversible stapling . Further-more, we analyze the paradigm shift in assessment methodologies from qualitative imaging to quantitative cytosolic delivery assays, which have deepened our under-standing of mechanisms such as the charge/lipophilicity threshold balance and meta-bolic-driven trapping. Overall, linker engineering provides a robust technical roadmap for developing the next generation of cell-permeable stapled peptide therapeutics.

The c-Jun N-terminal kinase (JNK) signaling cascade is a central regulator of cellular stress responses and a validated therapeutic target for diverse pathologies, including neurodegeneration, fibrosis, and cancer. However, the development of direct JNK inhibitors has been impeded by challenges regarding isoform selectivity and on-target toxicity. Consequently, medicinal chemistry efforts have shifted upstream to the "gatekeepers" of the pathway: the dual-specificity kinases MKK4 and MKK7. This review provides a comprehensive analysis of the structural biology, physiological functions, and pharmacological inhibition of these critical signaling nodes. We highlight the non-redundant therapeutic potential of these kinases, contrasting the role of MKK4 inhibition in unlocking liver regeneration with the utility of MKK7 inhibition in suppressing fibrosis and chronic inflammation. A systematic overview of small-molecule inhibitors is presented. For MKK4, we discuss the evolution from early promiscuous chemotypes to the first-in-class clinical candidate HRX215, which has demonstrated safety and efficacy in promoting hepatocyte proliferation. For MKK7, we examine the design of covalent inhibitors exploiting the unique hinge-region cysteine (Cys218), as well as emerging modalities such as lysine-targeting binders and peptide inhibitors disrupting protein-protein interactions. Finally, we discuss remaining challenges and future opportunities, including the development of dual inhibitors and proteolysis-targeting chimeras (PROTACs), to fully exploit the therapeutic value of the MKK4/7-JNK axis.

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.