Discovery of novel amide derivatives as potent quorum sensing inhibitors of Pseudomonas aeruginosa.

With the increasing reports of antibiotic resistance in this species, Pseudomonas aeruginosa is a common human pathogen with important implications for public health. Bacterial quorum sensing (QS) systems are potentially broad and versatile targets for developing new antimicrobial compounds. While previous reports have demonstrated that certain amide compounds can inhibit bacterial growth, there are few reports on the specific inhibitory effects of these compounds on bacterial quorum sensing systems. In this study, thirty-one amide derivatives were synthesized. The results of the biological activity assessment indicated that A9 and B6 could significantly inhibit the expression of lasB, rhlA, and pqsA, effectively reducing several virulence factors regulated by the QS systems of PAO1. Additionally, compound A9 attenuated the pathogenicity of PAO1 to Galleria mellonella larvae. Meanwhile, RT-qPCR, SPR, and molecular docking studies were conducted to explore the mechanism of these compounds, which suggests that compound A9 inhibited the QS systems by binding with LasR and PqsR, especially PqsR. In conclusion, amide derivatives A9 and B6 exhibit promising potential for further development as novel QS inhibitors in P. aeruginosa.

Design and Synthesis of Mandelic Acid Derivatives for Suppression of Virulence via T3SS against Citrus Canker.

Citrus canker, a highly contagious bacterial disease caused by Xanthomonas citri subsp. citri (Xcc), poses a substantial threat to citrus crops, leading to serious reductions in fruit yield and economic losses. Most commonly used bactericides against Xcc lead to the rapid development of resistant subpopulations. Therefore, it is imperative to create novel drugs, such as type III secretion system (T3SS) inhibitors, that specifically target bacterial virulence factors rather than bacterial viability. In our study, we designed and synthesized a series of mandelic acid derivatives including 2-mercapto-1,3,4-thiazole. Seven substances were found to reduce the level of transcription of hpa1 without affecting bacterial viability. In vivo bioassays indicated that compound F9 significantly inhibited hypersensitive response and pathogenicity. RT-qPCR assays showed that compound F9 visibly suppressed the expression of Xcc T3SS-related genes as well as citrus canker susceptibility gene CsLOB1. Furthermore, the combination with compound F9 and quorum-quenching bacteria HN-8 can also obviously alleviate canker symptoms.

Application and Mechanism of Cryptolepine and Neocryptolepine Derivatives as T3SS Inhibitors for Control of Bacterial Leaf Blight on Rice.

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv oryzae (Xoo) is extremely harmful to rice production. The traditional control approach is to use bactericides that target key bacterial growth factors, but the selection pressure on the pathogen makes resistant strains the dominant bacterial strains, leading to a decline in bactericidal efficacy. Type III secretion system (T3SS) is a conserved and critical virulence factor in most Gram-negative bacteria, and its expression or absence does not affect bacterial growth, rendering it an ideal target for creating drugs against Gram-negative pathogens. In this work, we synthesized a range of derivatives from cryptolepine and neocryptolepine. We found that compound Z-8 could inhibit the expression of Xoo T3SS-related genes without affecting the growth of bacteria. an in vivo bioassay showed that compound Z-8 could effectively reduce the hypersensitive response (HR) induced by Xoo in tobacco and reduce the pathogenicity of Xoo in rice. Furthermore, it exhibited synergy in control of bacterial leaf blight when combined with the quorum quenching bacterial F20.

High-Throughput, Quantitative Screening of Quorum-Sensing Inhibitors Based on a Bacterial Biosensor.

Quorum sensing (QS) is a cell-cell communication mechanism by which bacteria synchronize social behaviors such as biofilm formation and virulence factor secretion by producing and sensing small molecular signals. Quorum quenching (QQ) by degrading signals or blocking signal transmissions has become a promising strategy for disrupting QS and preventing bacterial infection and biofilm formation. However, studies of high-throughput screening and identification approaches for quorum-sensing inhibitors (QSIs) are still inadequate. In this work, we developed a sensitive, high-throughput approach for screening QSIs based on the bacterial biosensor strain Agrobacterium tumefaciens N5 (pBA7P), which contains a traG gene promoter induced by QS signals fused with a promoterless ß-lactamase gene reporter. Using this approach, we identified 31 QQ bacteria from ∼2000 soil bacterial isolates, some belonging to the genera Bosea, Cupriavidus, and Flavobacterium that have not been reported previously as QQ bacteria. We also identified four QS inhibitory compounds and one QS signal analogue from ∼5000 small-molecule compounds, which profoundly affected the expression of QS-regulated genes and phenotypes of the pathogenic bacteria. This high-throughput screening system is effective and sensitive for screening of both QQ microbes and small molecules, enabling the discovery of a wide variety of biocompatible compounds.

Innovative Strategy for the Control of Citrus Canker: Inhibitors Targeting the Type III Secretion System of Xanthomonas citri Subsp. citri.

To find potential type III secretion system (T3SS) inhibitors against citrus canker caused by Xanthomonas citri subsp. citri (Xcc), a new series of 5-phenyl-2-furan carboxylic acid derivatives stitched with 2-mercapto-1,3,4-thiadiazole were designed and synthesized. Among the 30 compounds synthesized, 14 compounds significantly inhibited the promoter activity of a harpin gene hpa1. Eight of the 14 compounds did not affect the growth of Xcc, but significantly reduced the hypersensitive response (HR) of tobacco and decreased the pathogenicity of Xcc on citrus plants. Subsequent studies have demonstrated that these inhibitory molecules effectively suppress the T3SS of Xcc and significantly impair the pathogen's ability to subvert citrus immunity, resulting in a reduction in the level of disease progression. As a result, our work has identified a series of potentially attractive agents for the control of citrus canker.

Derivative of cinnamic acid inhibits T3SS of Xanthomonas oryzae pv. oryzae through the HrpG-HrpX regulatory cascade.

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) has a significant impact on rice yield and quality worldwide. Traditionally, bactericide application has been commonly used to control this devastating disease. However, the overuse of fungicides has led to a number of problems such as the development of resistance and environmental pollution. Therefore, the development of new methods and approaches for disease control are still urgent. In this paper, a series of cinnamic acid derivatives were designed and synthesized, and three novel T3SS inhibitors A10, A12 and A20 were discovered. Novel T3SS inhibitors A10, A12 and A20 significantly inhibited the hpa1 promoter activity without affecting Xoo growth. Further studies revealed that the title compounds A10, A12 and A20 significantly impaired hypersensitivity in non-host plant tobacco leaves, while applications on rice significantly reduced symptoms of bacterial leaf blight. RT-PCR showed that compound A20 inhibited the expression of T3SS-related genes. In summary, this work exemplifies the potential of the title compound as an inhibitor of T3SS and its efficacy in the control of bacterial leaf blight.

A novel strategy to control Pseudomonas syringae through inhibition of type III secretion system.

Pseudomonas syringae (P. syringae) is a highly prevalent Gram-negative pathogen with over 60 pathogenic variants that cause yield losses of up to 80% in various crops. Traditional control methods mainly involve the application of antibiotics to inactivate pathogenic bacteria, but large-scale application of antibiotics has led to the development of bacterial resistance. Gram-negative pathogens including P. syringae commonly use the type III secretion system (T3SS) as a transport channel to deliver effector proteins into host cells, disrupting host defences and facilitating virulence, providing a novel target for antibacterial drug development. In this study, we constructed a high-throughput screening reporter system based on our previous work to screen for imidazole, oxazole and thiazole compounds. The screening indicated that the three compounds (II-14, II-15 and II-24) significantly inhibited hrpW and hrpL gene promoter activity without influencing the growth of P. syringae, and the inhibitory activity was better than that of the positive control sulforaphane (4-methylsulfinylbutyl isothiocyanate, SFN) at 50 µM. Three compounds suppressed the transcript levels of representative T3SS genes to different degrees, suggesting that the compounds may suppress the expression of T3SS by modulating the HrpR/S-HrpL regulatory pathway. Inoculation experiments indicated that all three compounds suppressed the pathogenicity of Pseudomonas syringae pv. tomato DC3000 in tomato and Pseudomonas syringae pv. phaseolicola 1448A in bean to varying degrees. One representative compound, II-15, significantly inhibited the secretion of the Pst DC3000 AvrPto effector protein. These findings provide a theoretical basis for the development of novel P. syringae T3SS inhibitors for application in disease prevention and control.

Novel mandelic acid derivatives suppress virulence of Ralstonia solanacearum via type III secretion system.

BACKGROUND: Bacterial wilt induced by Ralstonia solanacearum is regarded as one of the most devastating diseases. However, excessive and repeated use of the same bactericides has resulted in development of bacterial resistance. Targeting bacterial virulence factors, such as type III secretion system (T3SS), without inhibiting bacterial growth is a possible assay to discover new antimicrobial agents. RESULTS: In this work, identifying new T3SS inhibitors, a series of mandelic acid derivatives with 2-mercapto-1,3,4-thiazole moiety was synthesized. One of them, F-24, inhibited the transcription of hrpY gene significantly. The presence of this compound obviously attenuated hypersensitive response (HR) without inhibiting bacterial growth of R. solanacearum. The transcription levels of those typical T3SS genes were reduced to various degrees. The test of the ability of F-24 in protecting plants demonstrated that F-24 protected tomato plants against bacterial wilt without restricting the multiplication of R. solanacearum. The mechanism of this T3SS inhibition is through the PhcR-PhcA-PrhG-HrpB pathway. CONCULSION: The screened F-24 could inhibit R. solanacearum T3SS and showed better inhibitory activity than previously reported inhibitors without affecting the growth of the strain, and F-24 is a compound with good potential in the control of R. solanacearum. © 2023 Society of Chemical Industry.

Novel Ethyl-3-Aryl-2-Nitroacrylate Derivatives as Potential T3SS Inhibitors against Xanthomonas oryzae pv. oryzae on Rice.

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a highly destructive bacterial disease. Traditional prevention methods have utilized antibiotics to target bacterial growth, which has accelerated the emergence of resistant strains. New prevention techniques are developing agents such as type III secretion system (T3SS) inhibitors that target bacterial virulence factors without affecting bacterial growth. To explore novel T3SS inhibitors, a series of ethyl-3-aryl-2-nitroacrylate derivatives were designed and synthesized. Preliminary screening of T3SS inhibitors was based on the inhibition of the hpa1 gene promoter and showed no effect on bacterial growth. Compounds B9 and B10, obtained in the primary screening, significantly inhibited the hypersensitive response (HR) in tobacco and the expression of T3SS genes in the hrp cluster including key regulatory genes. In vivo bioassays showed that T3SS inhibitors obviously inhibited BLB and appeared to be more effective when combined with quorum quenching bacteria F20.

Novel pyrimidin-4-one derivatives as potential T3SS inhibitors against Xanthomonas campestris pv. campestris.

BACKGROUND: Cruciferous black rot is caused by Xanthomonas campestris pv. campestris (Xcc) infection and is a widespread disease worldwide. Excessive and repeated use of bactericide is an important cause of the development of bacterial resistance. It is imperative to take new approaches to screening compounds that target virulence factors rather than kill bacterial pathogens. The type III secretion system (T3SS) invades a variety of cells by transporting virulence effector factors into the cytoplasm and is an attractive antitoxic target. Toward the search of new T3SS inhibitors, an alternative series of novel pyrimidin-4-one derivatives were designed and synthesized and assessed for their effect in blocking the virulence. RESULTS: All of the target compounds were characterized by proton (1 H) nuclear magnetic resonance (NMR), carbon-13 (13 C) NMR, fluorine-19 (19 F) NMR and high-resolution mass spectrometry (HRMS). All compounds were evaluated using high-throughput screening systems against Xcc. The results of the biological activity test revealed that the compound SPF-9 could highly inhibit the activity of xopN gene promoter and the hypersensitivity (HR) of tobacco without affecting bacterial growth. Moreover, messenger RNA (mRNA) level measurements showed that compound SPF-9 inhibited the expression of some representative genes (hrp/hrc genes). Compound SPF-9 weakened the pathogenicity of Xcc to Raphanus sativus L. CONCLUSION: Compound SPF-9 has good potential for further development as a novel T3SS inhibitor against Xcc. © 2023 Society of Chemical Industry.

Discovery of 2,5-disubstituted furan derivatives featuring a benzamide motif for overcoming P-glycoprotein mediated multidrug resistance in MCF-7/ADR cell.

P-glycoprotein (P-gp) is one of the drug efflux transporters that triggers multidrug resistance (MDR) in cells. Herein, by utilizing the strategies of active skeleton splicing and structural optimization on the lead compound 5 m, a total of 50 novel 2,5-disubstituted furan derivatives were designed, synthesized, and screened for P-gp inhibitory activity. The structure-activity relationship analysis enabled the identification of an important pharmacophore N-phenylbenzamide, which resulted in the discovery of a promising drug lead compound Ⅲ-8. Ⅲ-8 possesses broad-spectrum reversal activity and low toxicity in MCF-7/ADR cells. Western blot and Rh123 accumulation assay demonstrated that Ⅲ-8 displayed the reversal activity by inhibiting P-gp efflux. Molecular docking analysis indicated a potent affinity of Ⅲ-8 to P-gp by forming H-bond interactions with residues Asn 721 and Met 986. Ⅲ-8 was determined to be a highly effective and safe P-gp inhibitor in an MCF-7/ADR xenograft mouse model.

Synthesis and biological evaluation of 2,5-disubstituted furan derivatives containing 1,3-thiazole moiety as potential α-glucosidase inhibitors.

α-Glucosidase, which is involved in the hydrolysis of carbohydrates to glucose and directly mediates blood glucose elevation, is a crucial therapeutic target for type 2 diabetes. In this work, 2,5-disubstituted furan derivatives containing 1,3-thiazole-2-amino or 1,3-thiazole-2-thiol moiety (III-01 âˆ¼ III-30) were synthesized and screened for their inhibitory activity against α-glucosidase. α-Glucosidase inhibition assay demonstrated that all compounds had IC50 in the range of 0.645-94.033 µM and more potent than standard inhibitor acarbose (IC50 = 452.243 ± 54.142 µM). The most promising inhibitors of the two series were compound III-10 (IC50 = 4.120 ± 0.764 µM) and III-24 (IC50 = 0.645 ± 0.052 µM), respectively. Kinetic study and molecular docking simulation revealed that compound III-10 (Ki = 2.04 ± 0.72 µM) is a competitive inhibitor and III-24 (Ki = 0.44 ± 0.53 µM) is a noncompetitive inhibitor against α-glucosidase. Significantly, these two compounds showed nontoxicity towards HEK293, RAW264.7 and HepG2 cells, suggesting that compounds may be considered as a class of potential candidates for further developing novel antidiabetic drugs.

2,5-Disubstituted furan derivatives containing imidazole, triazole or tetrazole moiety as potent α-glucosidase inhibitors.

α-Glucosidase inhibitors (AGIs) are oral antidiabetic drugs, preferably used in treating type 2 diabetes mellitus, that delay the absorption of carbohydrates from the gastrointestinal system. In this work, 2,5-disubstituted furan derivatives containing imidazole, triazole or tetrazole moiety (III-01 âˆ¼ III-45) were synthesized and characterized by elemental analysis, HRMS, 1H NMR, 13C NMR and single crystal X-ray. Their inhibitory activity against α-glucosidase was screened. The most promising inhibitors were compound III-11 (IC50 = 6.0 ± 1.1 µM), III-16 (IC50 = 2.2 ± 0.2 µM) and III-39 (IC50 = 4.6 ± 1.9 µM), respectively. Kinetic study revealed that compounds III-11 and III-39 were uncompetitive inhibitors against α-glucosidase. Meanwhile, III-16 (Ki = 5.1 ± 0.7 µM) was a competitive inhibitor. Furthermore, molecular docking studies indicated that the existence of the azole group played a critically important role in hydrogen bond interaction with α-glucosidase. Significantly, in vivo toxicity towards HEK293 cells, RAW264.7 cells and HepG2 cells suggested that compounds III-11 and III-39 possessed non-toxicity, that could be considered as potential candidates for further development of novel antidiabetic drugs.

Diversity-oriented synthesis of fluoroalkylated amines via the palladium-catalyzed divergent fluoroalkylamination of 1,3-dienes.

Herein, we reported the first versatile and expeditious protocol for the diversity-oriented synthesis (DOS) of fluoroalkylated amines via the photoinduced palladium-catalyzed cross coupling of 1,3-dienes, amines and fluoroalkyl iodides, which features excellent 3,4- and 1,4-selectivity controlled by fluoroalkyl iodides, a broad substrate scope as well as good function group tolerance, and could be extended to the late-stage modification of bioactive molecules.

Discovery of a series of 5-phenyl-2-furan derivatives containing 1,3-thiazole moiety as potent Escherichia coli ß-glucuronidase inhibitors.

Gut microbial ß-glucuronidases have drawn much attention due to their role as a potential therapeutic target to alleviate some drugs or their metabolites-induced gastrointestinal toxicity. In this study, fifteen 5-phenyl-2-furan derivatives containing 1,3-thiazole moiety (1-15) were synthesized and evaluated for their inhibitory effects against Escherichia coli ß-glucuronidase (EcGUS). Twelve of them showed satisfactory inhibition against EcGUS with IC50 values ranging from 0.25 µM to 2.13 µM with compound 12 exhibited the best inhibition. Inhibition kinetics studies indicated that compound 12 (Ki = 0.14 ± 0.01 µM) was an uncompetitive inhibitor for EcGUS and molecular docking simulation further predicted the binding model and capability of compound 12 with EcGUS. A preliminary structure-inhibitory activity relationship study revealed that the heterocyclic backbone and bromine substitution of benzene may be essential for inhibition against EcGUS. The compounds have the potential to be applied in drug-induced gastrointestinal toxicity and the findings would help researchers to design and develop more effective 5-phenyl-2-furan type EcGUS inhibitors.

Design, synthesis and bioactivity study on 5-phenylfuran derivatives as potent reversal agents against P-glycoprotein-mediated multidrug resistance in MCF-7/ADR cell.

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is a phenomenon in which cells become resistant to structurally and mechanistically unrelated drugs resulting in low intracellular drug concentrations. It is one of the noteworthy problems in malignant tumor clinical therapeutics. So P-gp protein is one of the ideal targets to solve MDR. Based on the lead compound 5m obtained from our previous work, a series of furan derivatives featuring alkyl-substituted phenols and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline were designed and synthesized as reversal agents against P-gp in this paper. Compound 16 containing isopropoxy possessed good potency against P-gp mediated MDR in MCF-7/ADR (IC50 (doxorubicin) = 0.73 µM, RF = 69.6 with 5 µM 16 treated). Western blot results and Rh123 accumulation assays showed that 16 effectively inhibited P-gp efflux function but not its expression. The preliminary structure-activity relationship and docking studies demonstrated that compound 16 would be a potential P-gp inhibitor. Most worthy of mention is that compound 16 has achieved satisfactory results in combination with a variety of anti-tumor drugs, such as doxorubicin, paclitaxel, and vincristine. This study forwards a hopeful P-gp inhibitor for withstanding malignant tumor cell with multidrug resistance setting the basis for further studies.

2,5-Disubstituted furan derivatives containing 1,3,4-thiadiazole moiety as potent α-glucosidase and E. coli ß-glucuronidase inhibitors.

In this paper, the 2,5-disubstituted furan derivatives containing 1,3,4-thiadiazole were synthesized and screened for their inhibitory activity against α-glucosidase and ß-glucuronidases to obtain potent α-glucosidase inhibitor 9 (IC50 = 0.186 µM) and E. coli ß-glucuronidase inhibitor 26 (IC50 = 0.082 µM), respectively. The mechanisms of the compounds were studied. The kinetic study revealed that compound 9 is a competitive inhibitor against α-glucosidase (Ki = 0.05 ± 0.003 µM) and molecular docking simulation showed several key interactions between 9 and the target including hydrogen bond and p-π stacking interaction. Derivative 26 (Ki = 0.06 ± 0.005 µM) displayed uncompetitive inhibition behavior against EcGUS. Furthermore, the result of docking revealed the furan ring of 26 may be a key moiety in obstructing the active domain of EcGUS. In addition, compound 15 exhibited significant inhibitory activity against these two enzymes, with potential therapeutic effects against diabetes and against CPT-11-induced diarrhea. At the same time, their low toxicity against normal liver tissue LO2 cells lays the foundation for in vivo studies and the development of bifunctional drug.

Synthesis and bioactivity of phenyl substituted furan and oxazole carboxylic acid derivatives as potential PDE4 inhibitors.

In this present study, a series of 5-phenyl-2-furan and 4-phenyl-2-oxazole derivatives were designed and synthesized as phosphodiesterase type 4 (PDE4) inhibitors. In vitro results showed that the synthesized compounds exhibited considerable inhibitory activity against PDE4B and blockade of LPS-induced TNF-α release. Among the designed compounds, Compound 5j exhibited lower IC50 value (1.4 µM) against PDE4 than parent rolipram (2.0 µM) in in vitro enzyme assay, which also displayed good in vivo activity in animal models of asthma/COPD and sepsis induced by LPS. Docking results suggested that introduction of methoxy group at para-position of phenyl ring, demonstrated good interaction with metal binding pocket domain of PDE4B, which was helpful to enhance inhibitory activity.

Thiazolidin-2-cyanamides derivatives as novel potent Escherichia coli ß-glucuronidase inhibitors and their structure-inhibitory activity relationships.

Gut microbial ß-glucuronidases have the ability to deconjugate glucuronides of some drugs, thus have been considered as an important drug target to alleviate the drug metabolites-induced gastrointestinal toxicity. In this study, thiazolidin-2-cyanamide derivatives containing 5-phenyl-2-furan moiety (1-13) were evaluated for inhibitory activity against Escherichia coli ß-glucuronidase (EcGUS). All of them showed more potent inhibition than a commonly used positive control, d-saccharic acid 1,4-lactone, with the IC50 values ranging from 1.2 µM to 23.1 µM. Inhibition kinetics studies indicated that compound 1-3 were competitive type inhibitors for EcGUS. Molecular docking studies were performed and predicted the potential molecular determinants for their potent inhibitory effects towards EcGUS. Structure-inhibitory activity relationship study revealed that chloro substitution on the phenyl moiety was essential for EcGUS inhibition, which would help researchers to design and develop more effective thiazolidin-2-cyanamide type inhibitors against EcGUS.