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Summary Agroinfiltrated Nicotiana benthamiana is a flexible and scalable platform for recombinant protein (RP) production, but its great potential is hampered by plant proteases that degrade RPs. Here, we tested 29 candidate protease inhibitors (PIs) in agroinfiltrated N. benthamiana leaves for enhancing accumulation of three unrelated RPs: glycoenzyme α‐Galactosidase; glycohormone erythropoietin (EPO); and IgG antibody VRC01. Of the previously described PIs enhancing RP accumulation, we found only cystatin SlCYS8 to be effective. We identified three additional new, unrelated PIs that enhance RP accumulation: N. benthamiana NbPR4, NbPot1 and human HsTIMP, which have been reported to inhibit cysteine, serine and metalloproteases, respectively. Remarkably, accumulation of all three RPs is enhanced by each PI similarly, suggesting that the mechanism of degradation of unrelated RPs follows a common pathway. Inhibitory functions HsTIMP and SlCYS8 are required to enhance RP accumulation, suggesting that their target proteases may degrade RPs. Different PIs additively enhance RP accumulation, but the effect of each PI is dose‐dependent. Activity‐based protein profiling (ABPP) revealed that the activities of papain‐like Cys proteases (PLCPs), Ser hydrolases (SHs) or vacuolar processing enzymes (VPEs) in leaves are unaffected upon expression of the new PIs, whereas SlCYS8 expression specifically suppresses PLCP activity only. Quantitative proteomics indicates that the three new PIs affect agroinfiltrated tissues similarly and that they all increase immune responses. NbPR4, NbPot1 and HsTIMP can be used to study plant proteases and improve RP accumulation in molecular farming.

Summary Infiltration of disarmed Agrobacterium tumefaciens into leaves of Nicotiana benthamiana (agroinfiltration) facilitates quick and safe production of antibodies, vaccines, enzymes and metabolites for industrial use (molecular farming). However, yield and purity of proteins produced by agroinfiltration are hampered by unintended proteolysis, restricting industrial viability of the agroinfiltration platform. Proteolysis may be linked to an immune response to agroinfiltration, but understanding of the response to agroinfiltration is limited. To identify the proteases, we studied the transcriptome, extracellular proteome and active secretome of agroinfiltrated leaves over a time course, with and without the P19 silencing inhibitor. Remarkably, the P19 expression had little effect on the leaf transcriptome and no effect on the extracellular proteome. 25% of the detected transcripts changed in abundance upon agroinfiltration, associated with a gradual up‐regulation of immunity at the expense of photosynthesis. By contrast, 70% of the extracellular proteins increased in abundance, in many cases associated with increased efficiency of extracellular delivery. We detect a dynamic reprogramming of the proteolytic machinery upon agroinfiltration by detecting transcripts encoding for 975 different proteases and protease homologs. The extracellular proteome contains peptides derived from 196 proteases and protease homologs, and activity‐based proteomics displayed 17 active extracellular Ser and Cys proteases in agroinfiltrated leaves. We discuss unique features of the N. benthamiana protease repertoire and highlight abundant extracellular proteases in agroinfiltrated leaves, being targets for reverse genetics. This data set increases our understanding of the plant response to agroinfiltration and indicates ways to improve a key expression platform for both plant science and molecular farming.

Cellular senescence plays a critical role in repeated ultraviolet (UV) exposure‐induced skin photoaging. Currently, from the perspective of regulating senescent cells, potent compounds or reliable protein targets that could effectively prevent skin photoaging have not yet been reported. Herein, we demonstrated that chlorogenic acid (CGA) significantly inhibited UVA‐induced senescence of human dermis skin fibroblasts (HDF) cells by screening the natural product library. The activity‐based protein profiling (ABPP) result revealed that Enolase 1 (ENO1) is one of the direct targets of CGA in HDF cells. Further mechanism research indicated that CGA covalently binds to ENO1, and prevented UVA‐induced cellular senescence by suppressing the activity of ENO1 protein to block the glycolytic pathway. Importantly, we found that CGA dose‐dependently reduced the skin wrinkle score, alleviated skin pathological features and inhibited senescent characteristics in a photoaging mouse model. The proteomic analysis revealed that CGA treatment effectively inhibited senescence‐associated secretory phenotype (SASP) secretion and glycolysis in skin samples of mice. Collectively, our study not only demonstrated that inhibiting cell senescence is an effective anti‐skin photoaging strategy, but also revealed that ENO1 is a promising protein target to prevent photoaging. A schematic depiction of chlorogenic acid inhibited UVA‐induced cellular senescence and mouse skin photoaging by suppressing the activity of Enolase 1 (ENO1) protein to block glycolytic pathway.

Current challenges in controlling phytopathogenic bacteria lie in widespread chemical resistance, biosafety concerns, and the scarcity of novel biomacromolecule targets. While transposable elements have emerged as critical drivers of genetic variability and virulence in plant pathogens, their potential as druggable targets remains unexplored. Here, we report the first discovery of ISXoo15 transposase in Xanthomonas oryzae pv. oryzae (Xoo) as the bactericidal receptor for J9, a pyrimidine‐substituted pleuromutilin derivative. In vitro assays demonstrate J9's superior anti‐Xoo activity, with an EC50 of 0.12 mg/L—significantly lower than commercial agents thiodiazole copper (86.39 mg/L) and zinc thiazole (26.15 mg/L). In vivo pot trials reveal enhanced curative and protective efficacy of J9 against rice bacterial leaf blight compared to these metal‐based controls. A photoaffinity probe, P‐J9, is synthesised and coupled with activity‐based protein profiling to unequivocally identify ISXoo15 transposase (encoded by PXO_03433) as J9's specific target. Reverse transcription‐quantitative PCR confirmed significant downregulation of PXO_03433 expression in J9‐treated Xoo. Physiological and virulence‐related functional analyses of a homologous recombination‐mediated PXO_03433‐knockout strain (ΔPXO_03433) showed markedly attenuated virulence and impaired pathogenicity. Conversely, PXO_03433‐complemented strain CΔPXO_03433 possessed substantial restoration of pathogenicity‐related traits. Proteomic profiling revealed significant downregulation of pathways associated with DNA repair, recombination and binding proteins in both J9‐treated and mutant strains. ISXoo15 transposase may serve as a key regulator in enabling the homeostasis of the DNA metabolic network in the bacteria. This study provides pioneering evidence for targeting bacterial transposases as a novel antibacterial strategy, establishing a foundation for effective management of phytopathogenic bacteria. The study identifies the previously unknown transposase ISXoo15 as an antiphytobacterial target. By constructing mutant strains, this enzyme's essential role in regulating bacterial pathogenicity and virulence is revealed.

In recent years, cannabidiol (CBD), a non‐psychotropic cannabinoid, has garnered substantial interest in drug development due to its broad pharmacological activity and multi‐target effects. Diabetes is a chronic metabolic disease that can damage multiple organs in the body, leading to the development of complications such as abnormal kidney function, vision loss, neuropathy, and cardiovascular disease. CBD has demonstrated significant therapeutic potential in treating diabetes mellitus and its complications owing to its various pharmacological effects. This work summarizes the role of CBD in diabetes and its impact on complications such as cardiovascular dysfunction, nephropathy, retinopathy, and neuropathy. Strategies for discovering molecular targets for CBD in the treatment of diabetes and its complications are also proposed. Moreover, ways to optimize the structure of CBD based on known targets to generate new CBD analogues are explored.

Summary Co‐expression of protease inhibitors like the tomato cystatin SlCYS8 is useful to increase recombinant protein production in plants, but key proteases involved in protein proteolysis are still unknown. Here, we performed activity‐based protein profiling to identify proteases that are inhibited by SlCYS8 in agroinfiltrated Nicotiana benthamiana. We discovered that SlCYS8 selectively suppresses papain‐like cysteine protease (PLCP) activity in both apoplastic fluids and total leaf extracts, while not affecting vacuolar‐processing enzyme and serine hydrolase activity. A robust concentration‐dependent inhibition of PLCPs occurred in vitro when purified SlCYS8 was added to leaf extracts, indicating direct cystatin–PLCP interactions. Activity‐based proteomics revealed that nine different Cathepsin‐L/‐F‐like PLCPs are strongly inhibited by SlCYS8 in leaves. By contrast, the activity of five other Cathepsin‐B/‐H‐like PLCPs, as well as 87 Ser hydrolases, was unaffected by SlCYS8. SlCYS8 expression prevented protein degradation by inhibiting intermediate and mature isoforms of granulin‐containing proteases from the Resistant‐to‐Desiccation‐21 (RD21) PLCP subfamily. Our data underline the key role of endogenous PLCPs on recombinant protein degradation and reveal candidate proteases for depletion strategies.

The salinilactones, volatile marine natural products secreted from Salinispora arenicola, feature a unique [3.1.0]‐lactone ring system and cytotoxic activities through a hitherto unknown mechanism. To find their molecular target, an activity‐based protein profiling with a salinilactone‐derived probe is applied that disclosed the protein disulfide‐isomerases (PDIs) as the dominant mammalian targets of salinilactones, and thioredoxin (TRX1) as secondary target. The inhibition of protein disulfide‐isomerase A1 (PDIA1) and TRX1 is confirmed by biochemical assays with recombinant proteins, showing that (1S,5R)‐salinilactone B is more potent than its (1R,5S)‐configured enantiomer. The salinilactones bound covalently to C53 and C397, the catalytically active cysteines of the isoform PDIA1 according to tandem mass spectrometry. Reactions with a model substrate demonstrated that the cyclopropyl group is opened by an attack of the thiol at C6. Fluorophore labeling experiments showed the cell permeability of a salinilactone‐BODIPY (dipyrrometheneboron difluoride) conjugate and its co‐localization with PDIs in the endoplasmic reticulum. The study is one of the first to pinpoint a molecular target for a volatile microbial natural product, and it demonstrates that salinilactones can achieve high selectivity despite their small size and intrinsic reactivity. Molecular targets of volatile natural products are largely unknown. The identification of protein disulfide‐isomerases (PDIs) are presented as a target protein family of the salinilactones, volatile metabolites produced by marine Salinispora bacteria. The unnatural (1S,5R)‐salinilactone B enantiomer exhibits high target selectivity by binding covalently to protein disulfide‐isomerase A1 (PDIA1) at C53 and C397 residues and localizes at the endoplasmic reticulum of A549 cells.

Rheumatoid arthritis (RA), a prevalent and incurable autoimmune disease globally, is characterized by the immune system attacking the body's own tissues, leading to joint inflammation and damage. Capsaicin (CAP), from Capsicum annuum L., is known for its burning sensation‐inducing property and has shown various pharmacological effects, yet its specific mechanisms and targets in RA treatment remain largely unclear. This study aimed to investigate the role of CAP in RA by synthesizing CAP probes and using activity‐based protein profiling. We found that CAP reduced joint swelling in arthritic mice and exerted anti‐inflammatory and antiproliferative effects on fibroblast‐like synoviocytes. We identified that CAP binds to PRDX2, inhibiting its antioxidant function and inducing oxidative stress and apoptosis, contributing to the antiarthritic effects. These results suggest that PRDX2 is a potential target for CAP in RA treatment, providing new insights into the molecular mechanisms and potential therapeutic strategies for RA. Capsaicin attenuating arthritis by inhibiting AKT phosphorylation and activating the NRF2–HO‐1 axis via targeting PRDX2

Gram‐negative bacteria are particularly prone to developing antimicrobial resistance (AMR), as evidenced by the WHO's ESKAPEE list of high‐priority pathogens. One strategy that has increased is the use of antibiotic enhancers, which can re‐empower abandoned or poorly active antibiotics against the resistant strain of interest. In this study, the polyamino‐isoprenyl antibiotic enhancer, NV716, was tested in combination with two families of multi‐target Ser/Cys‐based enzyme inhibitors, the oxadiazolone derivatives (OX) and the Cyclipostins and Cyclophostin analogs (CyC), which are inactive against Gram‐negative ESKAPEE bacteria, to potentiate their antibacterial activity and thus make them active against these bacteria. We demonstrated that NV716 potentiates some OX and CyC compounds by permeabilizing the outer membrane and thus by increasing the inhibitor accumulation, as shown by fluorescence microscopy. By using the click‐chemistry activity‐based protein profiling (ABPP) approach coupled with proteomic analysis, we also confirmed the multi‐target nature of the best OX and CyC inhibitors by identifying their target proteins on a bacterial culture of Enterobacter cloacae. Remarkably, a large set of these identified proteins had already been captured in previous ABPP experiments conducted on Mycobacterium tuberculosis and/or Mycobacterium abscessus culture. Furthermore, we showed that five of the identified target proteins were present in a total lysate of Pseudomonas aeruginosa. Importantly, these latter enzymes are highly conserved among Gram‐negative bacteria, with two of them annotated as essential for bacterial survival. These results provide proof of concept that both OX and CyC, if successfully potentiated, could be used against ESKAPEE Gram‐negative bacteria. Impact statement The increasing incidence of resistant Gram‐negative bacteria and the lack of new drugs underscore the urgent need for new antimicrobial agents. A promising strategy to overcome this problem is the use of antibiotic adjuvants, which can revive poorly active antibiotics against resistant strains of interest. Here, we report the potentiating effect of the polyamino‐isoprenyl adjuvant NV716 in activating the antimicrobial activities of two families of multi‐target inhibitors, initially ineffective on Gram‐negative bacteria, against the ESKAPEE bacterium Enterobacter cloacae. The mechanism of action of NV716 and the potential target enzymes of our inhibitors in En. cloacae were also elucidated, highlighting the antibacterial potential of our inhibitors against bacterial pathogens.

Meisoindigo (Mei) has been clinically utilized for the treatment of chronic myeloid leukemia (CML), yet the precise molecular targets by which it exerts effects remain unclear. Through activity‐based protein profiling (ABPP), the protein kinase, membrane‐associated tyrosine/threonine 1 (PKMYT1) is identified as a direct target of Mei. Specifically, Mei forms a selective and reversible covalent bond with the Cys301 residue of PKMYT1, triggering its K48‐linked polyubiquitination and accelerating proteasomal degradation, which is mediated by the E3 ligase TRIM25. The study reveals that Mei acts as a molecular glue, enhancing the interaction between PKMYT1 and TRIM25 by approximately 30‐fold, thereby facilitating efficient PKMYT1 degradation. Further investigations reveal the pivotal role of PKMYT1 in cell growth. Knockdown of PKMYT1 in K562 cells induces G2/M phase arrest, enhances early apoptosis, and inhibits cell proliferation. In an orthotopic xenograft model, PKMYT1 knockdown delays leukemia progression and reduces lymph node metastasis, reinforcing its role in CML progression and metastasis. These findings provide a molecular rationale for the clinical efficacy of Mei and highlight PKMYT1 as a promising therapeutic target for CML. Additionally, it offers a valuable scaffold and inspiration for the development of novel molecular glue‐based protein degraders. Meisoindigo targets PKMYT1 for degradation by acting as a molecular glue that enhances PKMYT1‐TRIM25 interaction, leading to K48‐linked ubiquitination and subsequent proteasomal degradation, thereby exerting therapeutic effects in chronic myeloid leukemia.