ABSTRACT Derivatives of deoxyribose cyclic dinucleotides (dCDNs) displayed notable advantageous properties in the activation of STING pathway. The chirality of the phosphotriester of dCDN prodrugs may influence the stability, cellular permeability, bioactivity and duration, which remains inadequately explored through systematic evaluations in cellular and animal models. Herein, we isolated and characterized all three diastereoisomers of alkyne‐conjugated esterase‐sensitive dCDN prodrugs, and meticulously examined their difference in stability, cellular uptake and bioactivity. Within THP1 cells, the ( Rp,Rp ) diastereoisomer demonstrated the highest level of activation of STING pathway (EC 50 = 1.7 nM), and induced more robust and prolonged activation pulse than the ( Rp,Sp ) and ( Sp,Sp ) diastereoisomers. In murine models, prodrugs elicited significantly stronger stimulation in the development of an antitumor immune response compared to the parent CDN 3′,3′‐c‐di‐dAMP, as well as the clinically relevant STING agonist ADU‐S100. The ( Rp,Rp ) diastereoisomer exhibited the most pronounced antitumor activity in the context of intravenous administration, significantly suppressing tumor proliferation, extending the survival with a complete response (CR) rate of 90% in a mouse CT26 tumor model, and establishing long‐lasting tumor‐specific immunological memory. These findings underscore the importance of considering the chirality of phosphotriesters in the development of more effective, safer, and sustainable STING agonist in tumor immunotherapy.

The development of dual-target inhibitors represents a cost-effective strategy for integrated pest management. Here, we report the first dual-target inhibitors designed against the evolutionarily conserved domain of phytoene synthase (PSY), a key enzyme in carotenoid biosynthesis. Using comparative genomics, we identified structural conservation between PSY in plants and squalene synthase (erg9) in fungi. Through virtual screening and structure-based optimization of compounds targeting PSY, we identified lead compound 1c, which exhibited potent herbicidal and fungicidal activity. In vitro binding assays confirmed that 1c binds to both PSY and erg9. In plants, 1c treatment reduced chlorophyll content, downregulated photosynthesis-associated genes, and caused substrate accumulation in the carotenoid pathway. In fungi, 1c induced a mycelial morphology identical to erg9 knockout mutants. Molecular dynamics simulations revealed the differential binding conformations of 1c to PSY and erg9, elucidating its mode of action. This work establishes PSY and its homologues as a promising target for the development of novel, broad-spectrum dual-action agrochemicals based on targetome structural similarity.

The development of 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) inhibitors still needs an innovative extension with novel chemotypes to avoid herbicide resistance. Herein, a series of 2,4-diphenylquinoline derivatives were designed, synthesized, and structurally optimized from lead compound 24 via stepwise A/B/C-ring modification. Greenhouse assays identified a-3 (3-CF3 A-ring) as the most potent analogue, exhibiting the higher herbicidal activity (71.4% inhibition on DESSO at 375 g a.i./ha) and dosage tolerance than 24. The mechanism of compound a-3 targeting DXPS proteins has been also identified using surface plasmon resonance binding assay, physiological biomarker measurement, substrate accumulation, and molecular docking. The key active motifs between a-3 and DXPS have been further defined based on the structure-activity relationships. This work will further expand the structural diversity of DXPS inhibitors with higher bioactivity.

ABSTRACT Fast click reactions that operate efficiently in aqueous buffers are highly desirable across many areas of chemistry, yet remain scarce. Herein, we report an u ltrafast 4‐ a zido p yridinium‐based S taudinger (UApS) ligation that proceeds with large bimolecular reaction rate constants (10 3 –10 4 M −1 s −1 ) under catalyst‐free, physiologically relevant conditions. We introduced a new class of substituted 4‐azido‐1‐alkylpyridinium reagents that are both water‐stable and exceptionally reactive toward triarylphosphines, yielding phosphazide adducts that release nitrogen gas and convert into iminophosphoranes. These products were structurally validated by single‐crystal x‐ray diffraction studies and possess good aqueous stability. Mechanistic and computational investigations elucidated the UApS ligation pathway and its fast kinetics arising from the substituted 4‐azidopyridinium scaffolds. We demonstrate that the UApS ligation enables efficient protein labeling and cell‐surface imaging at low reagent concentrations. In combination with the established tetrazine ligation, we demonstrate that the UApS ligation facilitates dual labeling of distinct cellular compartments in living cells. Moreover, the UApS ligation is suitable for live‐cell stimulated emission depletion (STED) super‐resolution imaging of filopodia. Overall, the UApS ligation should extend fast click chemical space, providing a new addition to the bioconjugation toolkit.

1-Deoxy-d-xylulose-5-phosphate synthase (DXPS) is a key-limiting enzyme in the methylerythritol phosphate (MEP) pathway. With only two commercially available herbicides targeting DXPS, there is still a great amount of chemical space to explore new inhibitors targeting DXPS with different skeletons. In this work, we first used CRISPR/Cas9 and virus-induced gene silencing (VIGS) methods to identify the essential function of DXPS. Based on structure-based virtual screening and herbicidal activity assay, we screened two active inhibitors (19 and 24) with novel chemotypes. The analysis of chlorophyll content, surface plasmon resonance (SPR), and molecular docking revealed the mechanism of action of 19 and 24 targeting DXPS. In greenhouse tests, the postemergence and pre-emergence herbicidal activities of 24 (100 and 65%) and Bixlozone (100 and 61%) on BRACL (Brassica chinensis L.) at 750 g a.i./ha were comparable. This work provides a novel chemical framework and optimization basis for the development of herbicides based on DXPS.

Phytoene desaturase (PDS) is an important target for the development of bleaching herbicides. The lack of the simple and efficient method to directly measure PDS enzyme activity has been the major hurdle to in vitro screening of potent PDS inhibitors. Here, we propose to substitute the traditionally used two‐phase reaction including water and organic solvents with the one‐phase enzymatic reaction as a simple and efficient method to determine PDS enzyme activity. Through combining the fast substrate extraction and high‐performance liquid chromatography, the K m and V max of the Syn PDS enzyme from Synechococcus elongatus was calculated as 24.6 µM and 0.06 µM/min, respectively. Furthermore, the inhibitory activity of seven commercial PDS herbicides was also determined, which was consistent with the molecular docking results. This one‐phase‐based in vitro PDS enzyme activity assay method effectively improved the substrate‐enzyme interaction and reduce the adverse effect of organic solvent on enzyme activity. It will provide a fast and direct evaluation method for the high‐throughput screen of potent PDS inhibitors rather than using the protein‐binding assay.

The development of dual-target herbicides provides an effective approach to emerging weed resistance. Given the high structural similarity of phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS), it is possible to explore the dual-inhibitors targeting PDS and ZDS with high potency. In this work, two series of compounds (I-1 to I-20; II-1 to II-20) were designed and synthesized from our previously reported lead compound 8e. The bioassay results indicated that II-13 exhibited the highest postemergence herbicidal activity (95%) against three dicotyledonous weeds and 85% against the monocotyledonous weed POLFU at 750 g a.i./ha. Meanwhile, compound I-20 displayed the best pre-emergence herbicidal activity at 187.5 g a.i./ha. Subsequently, surface plasmon resonance and molecular dynamics simulation assays further validated the dual binding capacity of II-13 to PDS and ZDS. These results demonstrate that it is feasible to target two key enzymes in the carotenoid biosynthesis pathway to enhance herbicidal activity.

Plant viral diseases cause great harm to crops in terms of yield and quality. Natural products have been providing an excellent source of novel chemistry, inspiring the development of novel synthetic pesticides. The Amaryllidaceae alkaloids crinasiadine (3a), trisphaeridine (4a), and bicolorine (5a) were selected as parent structures, and a series of their derivatives were designed, synthesized, and investigated for their anti-plant virus effects for the first time. Compounds 13b and 18 exhibited comparable inhibitory activities to ningnanmycin against tobacco mosaic virus (TMV). Preliminary research into the mechanism, involving transmission electron microscopy and molecular docking studies, suggests that compound 18 may interfere with the elongation phase of the TMV assembly process. This study provides some important information for the research and development of agrochemicals with phenanthridine structures.

Endogenously generated reactive sulfur species (RSS) play critical roles in various physiological processes. RSS donors can enhance our understanding of RSS chemical biology and open new avenues for treating RSS-associated diseases. Nevertheless, general strategies for the controllable release of distinct RSS remain lacking. Herein, we present the first general platform for controllable release of RSS with sulfur oxidation states ranging from -2 to +4, based on the intramolecular thiol-promoted decomposition of cysteine ester (ITPDC). We first rationally designed ITPDC-based hydrogen sulfide (H2S) donors that avoid electrophilic byproducts and exhibit high H2S release efficiencies (>50%). Mechanistic investigations and density functional theory calculations elucidated the detailed pathways of pH-controllable H2S release from ITPDC, and computational studies also predicted other H2S-related RSS release from the ITPDC-based motifs. Importantly, we developed a series of ITPDC-based donors capable of releasing various RSS, including persulfide, hydrogen persulfide, sulfenic acid, sulfinic acid, and sulfur dioxide (SO2). Moreover, fluorescent imaging demonstrated the successful cellular delivery of H2S, persulfide, and SO2 from these donors, and the ITPDC-based motif was employed to create a light-triggered donor. We anticipate that these innovative chemistries will provide valuable tools for studying sulfur biology and for developing new RSS donors and bio-orthogonal cleavage techniques.

Cyclic dinucleotides (CDNs) play important physiological roles in bacteria, mammals and insects as a novel class of signaling molecules. However, the application of CDNs in agricultural pest control has not been reported yet. To explore the potential bioactivity of CDNs on agricultural pests, we synthesized ten kinds of CDNs containing adenine and guanine bases with different internucleotide linkages (3',3'; 2',3'; 2',2'). The target CDNs were used to determine the antifeedant and insecticidal activity against common Lepidoptera pests including S. frugiperda, M. separata, and H. armigera. The bioassay tests indicated that 3',3'-c-di-AMP showed the highest antifeedant activity (EC50 = 0.59 mg/L) against M. separata larvae among all the tested CDNs. Regarding insecticidal activity, 2',3'-c-di-AMP showed higher insecticidal activity against M. separata larvae with LC50 of 55.4 mg/L. RNA-seq further revealed that 3',3'-c-di-AMP and 2',3'-c-di-AMP exhibited a significant effect on the growth and development process of insects. More importantly, the bioactivity of 3',3'-c-di-AMP was also closely correlated with the stimulation of insect immune inflammation. These results indicated that cyclic dinucleotides can affect the normal physiological process of insects, providing a new direction for managing pests.

Prodrug 14a exhibited the highest activity and could activate all five most common STING variants.

For non-small-cell lung cancer (NSCLC), the ubiquitous occurrence of concurrent multiple genomic alterations poses challenges to single-gene therapy. To increase therapeutic efficacy, we used the branch-PCR method to develop a multigene nanovector, NP-TP53-BIM-PTEN, that carried three therapeutic gene expression cassettes for coexpression. NP-TP53-BIM-PTEN exhibited a uniform size of 104.8 ± 24.2 nm and high serum stability. In cell transfection tests, NP-TP53-BIM-PTEN could coexpress TP53, BIM, and PTEN in NCI-H1299 cells and induce cell apoptosis with a ratio of up to 94.9%. Furthermore, NP-TP53-BIM-PTEN also inhibited cell proliferation with a ratio of up to 42%. In a mouse model bearing an NCI-H1299 xenograft tumor, NP-TP53-BIM-PTEN exhibited a stronger inhibitory effect on the NCI-H1299 xenograft tumor than the other test vectors without any detectable side effects. These results exhibited the potential of NP-TP53-BIM-PTEN as an effective and safe multigene nanovector to enhance NSCLC therapy efficacy, which will provide a framework for genome therapy with multigene combinations.

Phytoene desaturase (PDS) is a key rate-limiting enzyme in the carotenoid biosynthesis pathway. Although commercial PDS inhibitors have been developed for decades, it remains necessary to develop novel PDS inhibitors with higher bioactivity. In this work, we used the scaffold hopping and linker modification approaches to design and synthesize a series of compounds (7a-7o, 8a-8l, and 14a-14d). The postemergence application assay demonstrated that 8e and 7e separately showed the best herbicidal activity at 750 g a.i./ha and lower doses (187.5 g, 375g a.i./ha) without no significant toxicity to maize and wheat. The surface plasmon resonance revealed strong binding affinity between 7e and Synechococcus PDS (SynPDS). The HPLC analysis confirmed that 8e at 750 g a.i./ha caused significant phytoene accumulation in Arabidopsis seedlings. This work demonstrates the efficacy of structure-guided optimization through scaffold hopping and linker modification to design potent PDS inhibitors with enhanced bioactivity and crop safety.

To improve the chemical regulation on the activity of cyclic dinucleotides (CDNs), we here designed a reduction-responsive dithioethanol (DTE)-based dCDN prodrug 9 (DTE-dCDN). Prodrug 9 improved the cell permeability with the intracellular levels peaking in 2 h in THP-1 cells. Under the reductive substance such as GSH or DTT, prodrug 9 could be quickly decomposed in 30 min to release the parent dCDN. In THP1-Lucia cells, prodrug 9 also retained a high bioactivity with the EC50 of 0.96 μM, which was 51-, 43-, and 3-fold more than the 2′,3′-cGAMP (EC50 = 48.6 μM), the parent compound 3′,3′-c-di-dAMP (EC50 = 41.3 μM), and ADU-S100 (EC50 = 2.9 μM). The high bioactivity of prodrug 9 was validated to be highly correlated with the activation of the STING signaling pathway. Furthermore, prodrug 9 could also improve the transcriptional expression levels of IFN-β, CXCL10, IL-6, and TNF-α in THP-1 cells. These results will be helpful to the development of chemically controllable CDN prodrugs with a high cellular permeability and potency.

Improving nitrogen (N) assimilation efficiency without yield penalties is important to sustainable food security. The chemical regulation approach of N assimilation efficiency is still less explored. We previously found that the co-application of brassinolide (BL) and pyraclostrobin (Pyr) synergistically boosted biomass and yield via regulating photosynthesis in Arabidopsis thaliana. However, the synergistic effect of BL and Pyr on N metabolism remains unclear. In this work, we examined the N and protein contents, key N assimilatory enzyme activities, and transcriptomic and metabolomic changes in the four treatments (untreated, BL, Pyr, and BL + Pyr). Our results showed that BL + Pyr treatment synergistically improved N and protein contents by 56.2% and 58.0%, exceeding the effects of individual BL (no increase) or Pyr treatment (36.4% and 36.1%). Besides synergistically increasing the activity of NR (354%), NiR (42%), GS (62%), and GOGAT (62%), the BL + Pyr treatment uniquely coordinated N metabolism, carbon utilization, and photosynthesis at the transcriptional and metabolic levels, outperforming the effects of individual BL or Pyr treatments. These results revealed that BL + Pyr treatments could synergistically improve N assimilation efficiency through improving N assimilatory enzyme activities and coordinated regulation of N and carbon metabolism. The identified genes and metabolites also informed potential targets and agrochemical combinations to enhance N assimilation efficiency.

Applying brassinolide (BL, a phytohormone) in combination with pyraclostrobin (Pyr, a fungicide) has shown effective disease control in field trials. However, the mechanism by which BL + Pyr control disease remains uncertain. This work compared the disease control and defense responses of three pretreatments (BL, Pyr, and BL + Pyr) in Arabidopsis thaliana. We found that BL + Pyr improved control against Pyr-sensitive Hyaloperonospora arabidopsidis and Botrytis cinerea by 19 and 17% over Pyr, respectively, and achieved 29% control against Pyr-resistant B. cinerea. Furthermore, BL + Pyr outperformed BL or Pyr in boosting transient H2O2 accumulation, and the activities of POD, APX, GST, and GPX. RNA-seq analysis revealed a more potent activation of defense genes elicited by BL + Pyr than by BL or Pyr. Overall, BL + Pyr controlled disease by integrating the elicitation of plant innate disease resistance with the fungicidal activity of Pyr.

In the context of global food crisis, applying the phytohormone-brassinosteroids (BRs) in combination with the fungicide-pyraclostrobin (Pyr) was beneficial for plant quality and productivity in several field trials. However, in addition to the benefits of disease control due to the innate fungicidal activity of Pyr, it remains to be understood whether the coapplication of BL+ Pyr exerts additional growth-promoting effects. For this purpose, the effects of BL treatment, Pyr treatment, and BL+ Pyr treatment in Arabidopsis thaliana were compared. The results showed that the yield increased at a rate of 25.6% in the BL+Pyr group and 9.7% in the BL group, but no significant change was observed in the Pyr group. Furthermore, the BL+Pyr treatment increased the fresh weight of both the leaves and the inflorescences. In contrast, the Pyr and BL treatments only increased the fresh weight of leaves and inflorescences, respectively. Additionally, the BL + Pyr treatment increased the Pn, Gs, Tr, Vc, max, Jmax, VTPU, ETR, Fv'/Fm', ΦPSII, Rd, AYE and Rubisco enzyme activity by 26%, 38%, 40%, 16%, 19%, 15%, 9%, 10%, 17%, 179%, 18% and 32%, respectively. While, these paraments did not change significantly by the BL or Pyr treatments. Treatment with BL + Pyr and Pyr, rather than BL, improved the chlorophyll a and chlorophyll b contents by upregulating genes related to chlorophyll biosynthesis and downregulating genes related to chlorophyll degradation. Additionally, according to transcriptomic and metabolomic analysis, the BL+ Pyr treatment outperformed the individual BL or Pyr treatments in activating the transcription of genes involved in photosynthesis and increasing sugar accumulation. Our results first validated that the combined usage of BL and Pyr exerted striking synergistic effects on enhancing plant biomass and yield by increasing photosynthetic efficiency. These results might provide new understanding for the agricultural effects by the co-application of BL and Pyr, and it might stimulate the efforts to develop new environment-friendly replacement for Pyr to minimize the ecotoxicology of Pyr.

Gene therapy is a potential approach to deal with complicated diseases that are now untreatable. A critical step in the effective gene therapy is the successful delivery of therapeutic genes into target cells. The fast development of gene regulation toolboxes and diverse gene vectors greatly increase the likelihood of delivering functional gene modules to multiple organs and expand the therapeutic scope of complex diseases. In the design of the optimal gene vectors for therapeutic applications, the gene loading capacity, shape, size, and safety are critical factors to be considered. In this chapter, the design, construction, function, applications, and future prospects of a new type of nonviral gene nanovector with Archimedes solid-like truncated octahedron nanostructures (ASN-TO) constructed from polymerase chain reaction based on branched primers (branch-PCR) were described. ASN-TO gene nanovector exhibits its unique feature in the improvement of high DNA loading capacity, serum stability, particle size controllability, and long-lasting bioactivity, especially multiplex gene regulation. ASN-TO gene nanovector offers us new opportunity to develop the strategy of genome therapy through integrating various kinds of gene regulation toolboxes (gene overexpression, gene silencing, gene editing, etc.) as an all-in-one vector, fine-tuning gene expression in multiple levels and synergistically adjusting network targets in multiple signaling pathways, which is different from single gene therapy and would potentially expand therapeutic applications. For complex diseases involving multiple genes, ASN-TO gene nanovector would hopefully act as a chromosome-like payload to carry diverse artificially designed gene regulation toolboxes to synergistically regulate the network targets for genome therapy.

Dual labeling of an RNAcan provide Forster resonance energy transfer (FRET) sensors for studying RNA folding, miRNA maturation, and RNA-protein interactions. Here, we report the development of a highly efficient strategy for direct dual-terminal labeling of any RNA of interest. We explored newMichael cycloaddition for facile labeling of 5'-terminal RNA with improved efficiency. Direct chemical tetrazinylation of RNA at the 3'-terminus was achieved with the highly efficient and catalysis-free tetrazine-cycloalkyne ligation. Both single-terminal labeling methods were combined for dual-terminal labeling of an RNA including short hairpin RNA, pre-miRNA, riboswitch, and noncoding RNA. Notably, these dual-labeled RNA-based FRET sensors were used to monitor RNA-ligand interactions in vitro and in live cells. It is anticipated that these universal RNA labeling strategies will be useful to study RNA structures and functions.