Design, synthesis and evaluation of quinazoline derivatives as Gαq/11 proteins inhibitors against uveal melanoma.

Uveal melanoma (UM) represents the predominant ocular malignancy among adults, exhibiting high malignancy and proclivity for liver metastasis. GNAQ and GNA11 encoding Gαq and Gα11 proteins are key genes to drive UM, making the selective inhibition of Gαq/11 proteins to be a potential therapeutic approach for combating UM. In this study, forty-six quinazoline derivatives were designed, synthesized, and assessed for their ability to inhibit Gαq/11 proteins and UM cells. Compound F33 emerged as the most favorable candidate, and displayed moderate inhibitory activity against Gαq/11 proteins (IC50 = 9.4 µM) and two UM cell lines MP41 (IC50 = 6.7 µM) and 92.1 (IC50 = 3.7 µM). Being a small molecule inhibitor of Gαq/11 proteins, F33 could effectively suppress the activation of downstream signaling pathways in a dose-dependent manner, and significantly inhibits UM in vitro.F33 represents a promising lead compound for developing therapeutics for UM by targeting Gαq/11 proteins.

Discovery of novel indazole derivatives as SOS1 agonists that activate KRAS signaling.

KRAS serves as a vital regulator for cellular signaling and drives tumor pathogenesis after mutation. Despite extensive research efforts spanning several decades, targeting KRAS is still challenging due to the multiple KRAS mutations and the emergence of drug resistance. Interfering the interactions between KRAS and SOS1 is one of the promising approaches for modulating KRAS functions. Herein, we discovered small-molecule SOS1 agonists with novel indazole scaffold. Through structure-based optimization, compound 11 was identified with high SOS1 activation potency (p-ERK EC50 = 1.53 µM). In HeLa cells, compound 11 enhances cellular RAS-GTP levels and exhibits biphasic modulation of ERK1/2 phosphorylation through an on-target mechanism and presents the therapeutic potential to modulate RAS signaling by activating SOS1.

Composite g-C3 N4 @ZnO NP electrostatic self-assembly: enhanced ROS as a key factor for high-efficiency control of tobacco wildfire disease.

BACKGROUND: The utilization of non-metallic inorganic nanomaterials for antimicrobial photocatalytic technology has emerged as a promising approach to combat drug-resistant bacteria. Recently, g-C3 N4 nanosheets have attracted significant attention due to their exceptional stability, degradability, low cost, and remarkable antibacterial properties. In this study, a facile electrostatic self-assembly approach was utilized to functionalize ZnO nanoparticles with g-C3 N4 nanosheets, resulting in the formation of g-C3 N4 @ZnO nanoparticle composites. RESULTS: The Z-shaped heterojunction architecture of these composites facilitates efficient separation of photogenerated electron-hole pairs and enhances visible light catalytic performance. Moreover, the formation of the g-C3 N4 @ZnO heterostructure showed a higher photocatalytic capacity and the generation of reactive oxygen species (ROS) than g-C3 N4 nanosheets. The photocatalytic antibacterial mechanisms of g-C3 N4 @ZnO at the transcriptomic level primarily involve disrupting bacterial membrane synthesis and inhibiting motility and energy metabolism. Therefore, the antibacterial mechanism of g-C3 N4 @ZnO can be attributed to a combination of physical membrane damage, chemical damage (ROS enhancement) and inhibition of chemotaxis, biofilm formation and flagellar motility. CONCLUSION: These findings collectively provide novel high potential and insights into the practical application of photocatalysts in plant disease management. © 2023 Society of Chemical Industry.

Antimicrobial mechanisms of ZnO nanoparticles to phytopathogen Pseudomonas syringae: Damage of cell envelope, suppression of metabolism, biofilm and motility, and stimulation of stomatal immunity on host plant.

Nanoparticles have recently been employed as a new strategy to act as bactericides in agricultural applications. However, the effects and mechanisms of foliar deposition of nanoparticles on bacterial pathogens, plant physiology and particularly plant immunity have not been sufficiently understood. Here, we investigated the effects and mechanisms of ZnO NPs in controlling of tobacco wildfire caused by Pseudomonas syringae pv. tabaci, through the comprehensive analysis of biological changes of both bacteria and plants. The global gene expression changes of Pseudomonas syringae pv. tabaci supported that the functions of "protein secretion", "membrane part", "signal transducer activity", "locomotion", "chemotaxis" and "taxis" in bacteria, as well as the metabolic pathways of "bacterial chemotaxis", "two-component system", "biofilm formation", "ABC transporters" and "valine, leucine and isoleucine degradation" were significantly down-regulated by ZnO NPs. Correspondingly, we reconfirmed that the cell envelope structure, biofilm and motility of Pseudomonas syringae pv. tabaci were directly disrupted or suppressed by ZnO NPs. Different from completely killing Pseudomonas syringae pv. tabaci, ZnO NPs (0.5 mg/mL) potentially improved plant growth and immunity through enzymatic activity and global molecular response analysis. Furthermore, the changes of gene expression in ABA signaling pathway, ABA concentration and stomatal aperture all supported that ZnO NPs can specifically stimulate stomatal immunity, which is important to defend bacterial infection. Taken together, we proposed that both the inhibition or damage of motility, biofilm, metabolisms, virulence and cell envelope on P. syringae pv. tabaci, and the activation of the stomatal immunity formed two-layered antibacterial mechanisms of ZnO NPs on phytopathogenic bacteria.

Fabrication of an alginate-based ZhiNengCong gel showed an enhanced antiviral and plant growth promoting functions.

Tobacco mosaic disease is a worldwide viral disease that can cause huge economic losses. Plant immune inducers have become the main force in the prevention and treatment of viral disease own to their high efficiency and rapid effect. However, since tobacco mosaic disease can occur at any point in the plant growth cycle, a single application period cannot guarantee the completely management. In this study, an extract from Paecilomyces variotii named ZhiNengCong (ZNC), which can fight against tobacco mosaic disease with 65% control effect, and improve the promotion of tobacco stem girth, was selected from five commercial antiviral medicines, and a sustained release sodium alginate (Alg)-based ZNC (ZNC@Alg) was prepared by physical absorption. ZNC@Alg, who contains only 5 mg/mL ZNC, can release ZNC for 7 consecutive days, and displayed an enhanced effect in inducing the PAL-mediated salicylic acid signaling pathway activation to participate in the inhibition of green fluorescent protein (GFP)-tagged tobacco mosaic virus (TMV-GFP) infection, even after 7 days of the application. Notably, field experiments showed that the control effect of ZNC@Alg was up to 88%, which was significantly better than that of ZNC with the same concentration (10 µg per plant). In addition, ZNC@Alg exhibited a stronger growth-promoting effect than ZNC, which significantly increased the wet weight of tobacco. Taken together, we screened out a plant immune inducer ZNC that can effectively inhibit tobacco virus disease, and created ZNC@Alg with higher control effect and growth promotion effect, laying a foundation for effective field management of tobacco mosaic disease.

Fabrication of polydopamine reduced CuO nanoparticle-alginate composite nanogels for management of Pseudomonas synringae pv. tabaci in tobacco.

BACKGROUND: The wildfire disease on tobacco can seriously hinder plants. Meanwhile, its pathogen, Pseudomonas syringae, can also infect over 200 plants and threat agriculture production. However, the disease usually occurs after summer rains which washes away most copper (Cu)-based bactericides, allowing the disease to invade. Therefore, we fabricate a new nanogel with high disease control and anti-erosion ability and study the effects of the reductant on the performance of the copper oxide nanoparticle (CuONP) composite nanogel. RESULTS: Polydopamine (PDA) is a polycation for both in situ reduction of CuONP in alginate nanogels and for adjusting the copper ion (Cu2+ ) releasing rate in this work. The composite nanogel fabricated by PDA (PDA-CuONP@ALGNP@CTAC) had a higher Cu2+ releasing rate, damaging the pathogen membrane more efficiently, allowing for better disease control and plant growth promotion when compared to sodium borohydride (SBH)-fabricated nanogel (SBH-CuONP@ALGNP@CTAC) or the commercial bactericide, thiodiazole copper. The PDA-CuONP@ALGNP@CTAC had a high anti-erosion ability and could remain adhered to the leaf surface even after five rain event simulations. CONCLUSION: The addition of polycations (like PDA) into CuONP composite nanogel could increase the Cu2+ releasing rate, resulting in improved disease management when compared to SBH-CuONP@ALGNP@CTAC or thiodiazole copper. The PDA containing gel had an improved anti-erosion ability and water resistance. This new composite nanogel has a high potential for wildfire disease control, improving agricultural production. © 2022 Society of Chemical Industry.

Identification of novel Pyrrolo[2,3-d]Pyrimidine-based KRAS G12C inhibitors with anticancer effects.

Oncogene KRAS plays predominant roles in human cancers by regulating cell proliferation, differentiation, and migration. Recent progress revealed that directly target KRAS G12C with allosteric inhibitors that covalently bind to the switch Ⅱ pocket is feasible. Herein, series of pyrrolo[2,3-d]pyrimidine derivatives were designed and synthesized through systematic structural optimization, leading to the discovery of compound 2-((S)-1-acryloyl-4-(2-(((2R,7aS)-2-fluorohexahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-7-methyl-6-(8-methylnaphthalen-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperazin-2-yl)acetonitrile (50) with high KRAS/SOS1 inhibitory potency (IC50 = 0.21 µM) and strong anti-proliferation activities on cancer cells harboring KRAS p.G12C. Compound 50 also exhibited satisfactory selectivity, moderate pharmacokinetic characters, and good anticancer effects in vivo. Meaningfully, the identification of these compounds highlights the necessity of an appropriate conformational constraint for acquiring the applicable binding pose in the cryptic pocket of KRAS, and the results support efforts toward design of KRAS inhibitors with novel skeleton and binding mechanism could be beneficial for targeting the acquired drug resistance.

Tobacco mosaic virus hijacks its coat protein-interacting protein IP-L to inhibit NbCML30, a calmodulin-like protein, to enhance its infection.

Calcium is an important plant immune signal that is essential for activating host resistance, but how RNA viruses manipulate calcium signals to promote their infections remains largely unknown. Here, we demonstrated that tobacco mosaic virus (TMV) coat protein (CP)-interacting protein L (IP-L) associates with calmodulin-like protein 30 (NbCML30) in the cytoplasm and nucleus, and can suppress its expression at the nucleic acid and protein levels. NbCML30, which lacks the EF-hand conserved domain and cannot bind to Ca2+ , was located in the cytoplasm and nucleus and was downregulated by TMV infection. NbCML30 silencing promoted TMV infection, while its overexpression inhibited TMV infection by activating Ca2+ -dependent oxidative stress in plants. NbCML30-mediated resistance to TMV mainly depends on IP-L regulation as the facilitation of TMV infection by silencing NbCML30 was canceled by co-silencing NbCML30 and IP-L. Overall, these findings indicate that in the absence of any reported silencing suppressor activity, TMV CP manipulates IP-L to inhibit NbCML30, influencing its Ca2+ -dependent role in the oxidative stress response. These results lay a theoretical foundation that will enable us to engineer tobacco (Nicotiana spp.) with improved TMV resistance in the future.

Synthesis and evaluation of imidazo[1,2-a]pyrazine derivatives as small molecule Gαq/11 inhibitors against uveal melanoma.

Uveal melanoma (UM) is an aggressive malignancy with high mortality in adults and lacks effective systemic therapies. Activating gene mutations related to the Gαq/11 signaling pathway are prevalent in UM, and Gαq/11 inhibitors have shown anti-UM activity in vitro and in vivo. In this study, we designed and synthesized a series of imidazo[1,2-a]pyrazine derivatives as Gαq/11 inhibitors, and discovered GQ352 with the selective antiproliferative activity against UM cells. Importantly, GQ352 directly binds to the Gαq and inhibits the dissociation of Gαßγ heterotrimers with the IC50 value of 8.9 µM. GQ352 inhibits UM tumorigenesis by suppressing Gαq/11 downstream ERK phosphorylation and YAP dephosphorylation, as shown in Western blot analysis. In addition, GQ352 displayed reasonable physiochemical properties and human liver microsome stability, indicating the potential application in UM treatment.

Fabrication of copper nanoparticle composite nanogel for high-efficiency management of Pseudomonas syringae pv. tabaci on tobacco.

BACKGROUND: Copper nanoparticles (CuNPs) can release copper ions (Cu2+ ) to control bacterial diseases on crops. However, the high concentration of the CuNPs applied in disease controlling can highly limit their application. In this work, by in situ reducing CuNPs in alginate nanogels and coated with cetyl trimethyl ammonium chloride (CTAC), a CuNP composite nanogel was fabricated as a new nanopesticide with low copper content. RESULTS: Data showed that the CTAC coating would affect the antibacterial activity and leaf surface adhesion of the nanogel, while CuNP content could also influence the membrane damage ability of the gel. The nanogel could depress the growth of bacteria by rupturing its membrane and show a minimum inhibitory concentration (MIC) as low as 500 µg mL-1 , which only contain 58 µg mL-1 CuNP, and achieve a 64% of therapeutic efficiency (with 1000 µg mL-1 nanogel) in in vivo experiments, higher than that of commercial bactericide thiodiazole copper. Furthermore, the application of the nanogel can also perform a growth-promoting effect on the plant, which may be due to the supplement of copper element provided by CuNP. CONCLUSION: The CuNP composite nanogel fabricated in this work performed high leaf disease controllability and safety compared to the commercial bactericide thiodiazole copper. We hope this nanogel can provide a potential high-efficiency nano-bactericide that can be used in the leaf bacterial disease control.

Nicotiana benthamiana asparagine synthetase associates with IP-L and confers resistance against tobacco mosaic virus via the asparagine-induced salicylic acid signalling pathway.

Asparagine synthetase is a key enzyme that catalyses the conversion of amide groups from glutamine or ammonium to aspartate, which leads to the generation of asparagine. However, the role of asparagine synthetase in plant immunity remains largely unknown. Here, we identified a Nicotiana benthamiana asparagine synthetase B (NbAS-B) that associates with tomato mosaic virus coat protein-interacting protein L (IP-L) using the yeast two-hybrid assay and examined its role in tobacco mosaic virus (TMV) resistance. The association of IP-L with NbAS-B was further confirmed by in vivo co-immunoprecipitation, luciferase complementation imaging, and bimolecular fluorescence complementation assays. IP-L and NbAS-B interact in the nucleus and cytosol and IP-L apparently stabilizes NbAS-B, thus enhancing its accumulation. The expressions of IP-L and NbAS-B are continuously induced on TMV-green fluorescent protein (GFP) infection. Co-silencing of IP-L and NbAS-B facilitates TMV-GFP infection. Overexpression of NbAS-B in tobacco reduces TMV-GFP infection by significantly improving the synthesis of asparagine. Furthermore, the external application of asparagine significantly inhibits the infection of TMV-GFP by activating the salicylic acid signalling pathway. These findings hold the potential for the future application of asparagine in the control of TMV.

Targeting mutated GTPase KRAS in tumor therapies.

Kirsten rat sarcoma virus oncogene (KRAS) mutation accounts for approximately 85% of RAS-driven cancers, and participates in multiple signaling pathways and mediates cell proliferation, differentiation and metabolism. KRAS has been considered as an "undruggable" target due to the lack of effective direct inhibitors, although high frequency of KRAS mutations have been identified in multiple carcinomas in the past decades. Encouragingly, the KRASG12C inhibitor AMG510 (sotorasib), which has been approved for treating NSCLC and CRC recently, makes directly targeting KRAS the most promising strategy for cancer therapy. To better understand the current state of KRAS inhibitors, this review summarizes the biological functions of KRAS, the structure-activity relationship studies of the small-molecule inhibitors that directly target KRAS, and highlights the therapeutic agents with improved selectivity, bioavailability and physicochemical properties. Furthermore, the combined medication that can enhance efficacy and overcome drug resistance of KRAS covalent inhibitors is also reviewed.

The photocatalytic antibacterial molecular mechanisms towards Pseudomonas syringae pv. tabaci by g-C3 N4 nanosheets: insights from the cytomembrane, biofilm and motility disruption.

BACKGROUND: Antibacterial photocatalytic therapy has been employed as a promising strategy to combat antibiotic-resistant bacteria in the water disinfection field, especially some non-metal inorganic nanomaterials. However, their antibacterial activities on plant phytopathogens are poorly understood. Here, the photocatalytic antibacterial mechanism of the urea-synthesized graphitic carbon nitride nanosheets (g-C3 N4 nanosheets) against Pseudomonas syringae pv. tabaci was systematically investigated in vitro and in vivo. RESULTS: The g-C3 N4 nanosheets exhibited remarkable concentration-dependent and irradiation-time-dependent antibacterial properties, and the 0.5 mg mL-1 concentration ameliorated tobacco wildfire disease in host plants. Specifically, under visible irradiation, g-C3 N4 nanosheets produced numerous reactive oxygen species (ROS), supplementing the plentiful extracellular and intracellular ROS in bacteria. After exposing light-induced g-C3 N4 nanosheets for 1 h, 500 genes were differentially expressed, according to transcriptome analyses. Notably, the expression of genes related 'antioxidant activity' and 'membrane transport' was sharply upregulated, and those related to 'bacterial chemotaxis', 'biofilm formation', 'energy metabolism' and 'cell motility' were downregulated. After exposure for over 2 h, the longer-time pressure on the target bacteria cause the decreased biofilm formation and flagellum motility, further injuring the cell membranes leading to cytoplasm leakage and damaged DNA, eventually resulting in the bacterial death. Concomitantly, the attachment of g-C3 N4 nanosheets was a synergistic physical antibacterial pathway. The infection capacity assessment also supported the earlier supposition. CONCLUSION: These results provide novel insights into the photocatalytic antibacterial mechanisms of g-C3 N4 nanosheets at the transcriptome level, which are expected to be useful for dissecting the response pathways in antibacterial activities and for improving g-C3 N4 -based photocatalysts practices in plant disease control. © 2021 Society of Chemical Industry.

Cytidine-to-Uridine RNA Editing Factor NbMORF8 Negatively Regulates Plant Immunity to Phytophthora Pathogens.

Mitochondria and chloroplasts play key roles in plant-pathogen interactions. Cytidine-to-uridine (C-to-U) RNA editing is a critical posttranscriptional modification in mitochondria and chloroplasts that is specific to flowering plants. Multiple organellar RNA-editing factors (MORFs) form a protein family that participates in C-to-U RNA editing, but little is known regarding their immune functions. Here, we report the identification of NbMORF8, a negative regulator of plant immunity to Phytophthora pathogens. Using virus-induced gene silencing and transient expression in Nicotiana benthamiana, we show that NbMORF8 functions through the regulation of reactive oxygen species production, salicylic acid signaling, and accumulation of multiple Arg-X-Leu-Arg effectors of Phytophthora pathogens. NbMORF8 is localized to mitochondria and chloroplasts, and its immune function requires mitochondrial targeting. The conserved MORF box domain is not required for its immune function. Furthermore, we show that the preferentially mitochondrion-localized NbMORF proteins negatively regulate plant resistance against Phytophthora, whereas the preferentially chloroplast-localized ones are positive immune regulators. Our study reveals that the C-to-U RNA-editing factor NbMORF8 negatively regulates plant immunity to the oomycete pathogen Phytophthora and that mitochondrion- and chloroplast-localized NbMORF family members exert opposing effects on immune regulation.

An RXLR effector secreted by Phytophthora parasitica is a virulence factor and triggers cell death in various plants.

RXLR effectors encoded by Phytophthora species play a central role in pathogen-plant interactions. An understanding of the biological functions of RXLR effectors is conducive to the illumination of the pathogenic mechanisms and the development of disease control strategies. However, the virulence function of Phytophthora parasitica RXLR effectors is poorly understood. Here, we describe the identification of a P. parasitica RXLR effector gene, PPTG00121 (PpE4), which is highly transcribed during the early stages of infection. Live cell imaging of P. parasitica transformants expressing a full-length PpE4 (E4FL)-mCherry protein indicated that PpE4 is secreted and accumulates around haustoria during plant infection. Silencing of PpE4 in P. parasitica resulted in significantly reduced virulence on Nicotiana benthamiana. Transient expression of PpE4 in N. benthamiana in turn restored the pathogenicity of the PpE4-silenced lines. Furthermore, the expression of PpE4 in both N. benthamiana and Arabidopsis thaliana consistently enhanced plant susceptibility to P. parasitica. These results indicate that PpE4 contributes to pathogen infection. Finally, heterologous expression experiments showed that PpE4 triggers non-specific cell death in a variety of plants, including tobacco, tomato, potato and A. thaliana. Virus-induced gene silencing assays revealed that PpE4-induced cell death is dependent on HSP90, NPK and SGT1, suggesting that PpE4 is recognized by the plant immune system. In conclusion, PpE4 is an important virulence RXLR effector of P. parasitica and recognized by a wide range of host plants.