Target Discovery in Ralstonia solanacearum through an Activity-Based Protein Profiling Technique Based on Bioactive Oxadiazole Sulfones.

Ralstonia solanacearum is an extremely destructive and rebellious phytopathogen that can cause bacterial wilt diseases in more than 200 plant species. To explore and discover the potential targets in R. solanacearum for the purpose of developing new agrochemicals targeting this infection, here, we exploited a typical activity-based protein profiling technique for target discovery in R. solanacearum based on an activity-based probe 1 derived from bioactive oxadiazole sulfones. A total of 65 specific targets were identified with high confidence through a quantitative chemical proteomic approach. Three representative proteins (glycine cleavage system H protein, thiol peroxidase, and dihydrolipoamide S-succinyltransferase) were validated as the targets by using the immunoblotting analysis with their respective antibodies. Additionally, the in vitro interaction between the recombinant thiol peroxidase and probe 1 further confirmed that this protein was a target of oxadiazole sulfones. We anticipated that these discovered protein targets in R. solanacearum can stimulate the discovery and development of novel agrochemicals targeting bacterial infections caused by R. solanacearum.

Fabrication of Furan-Functionalized Quinazoline Hybrids: Their Antibacterial Evaluation, Quantitative Proteomics, and Induced Phytopathogen Morphological Variation Studies.

The limited number of agrochemicals targeting plant bacterial diseases has driven us to develop highly efficient, low-cost, and versatile antibacterial alternatives. Herein, a novel type of simple furan-functionalized quinazolin-4-amines was systematically fabricated and screened for their antibacterial activity. Bioassay results revealed that compounds C1 and E4 could substantially block the growth of two frequently mentioned pathogens Xanthomonas oryzae pv oryzae and X. axonopodis pv citri in vitro, displaying appreciable EC50 values of 7.13 and 10.3 mg/L, respectively. This effect was prominently improved by comparing those of mainly used agrochemicals. An in vivo experiment against bacterial blight further illustrated their viable applications as antimicrobial ingredients. Quantitative proteomics demonstrated that C1 possessed a remarkable ability to manipulate the upregulation and downregulation of expressed proteins, which probably involved d-glucose and biotin metabolic pathways. This finding was substantially verified by parallel reaction monitoring analysis. Scanning electron microscopy images and fluorescence spectra also indicated that the designed compounds had versatile capacities for destroying the integrity of bacteria. Given these remarkable characteristics, furan-functionalized quinazoline hybrids can serve as a viable platform for developing innovative antibiotic alternatives against bacterial infections.

Synthesis and In Vitro and In Vivo Biological Activity Evaluation and Quantitative Proteome Profiling of Oxadiazoles Bearing Flexible Heterocyclic Patterns.

A novel series of simple 1,3,4-oxadiazoles that bear flexible heterocyclic patterns was prepared, and their biological activities in plant pathogenic bacteria, fungi, oomycetes, and Meloidogyne incognita in vitro and in vivo were screened to explore low-cost and versatile antimicrobial agents. Screening results showed that compounds, such as A0, B0, and C4, were bioactive against Xanthomonas oryzae pv oryzae in vitro and in vivo, and such bioactivities were superior to those of commercial agents bismerthiazol and thiodiazole copper. Their antibacterial mechanisms were further investigated by quantitative proteomics and concentration-dependent scanning electron microscopy images. Antifungal results indicated that compound A0 displayed a selective and better antifungal effect on Botrytis cinerea with inhibition rate of 96.8% at 50 µg/mL. Nematocidal bioassays suggested that compound D1 had good in vitro nematocidal activity toward M. incognita at 24, 48, and 72 h, with the corresponding insecticidal efficiency of 48.7%, 64.1%, and 87.2% at 40 µg/mL. In vivo study further confirmed that compounds D1 and F2 showed nematocidal actions at 80 µg/mL with a disease index of 1.5. Given these advantages, this kind of molecular frameworks could be a suitable platform for exploring highly efficient agrochemicals.

Identification of Racemic and Chiral Carbazole Derivatives Containing an Isopropanolamine Linker as Prospective Surrogates against Plant Pathogenic Bacteria: In Vitro and In Vivo Assays and Quantitative Proteomics.

Recent observations on the emergence of drug-resistant plant pathogenic bacteria have highlighted and elicited an acute campaign to develop novel, highly efficient antibiotic surrogates for managing bacterial diseases in agriculture. Thus, a type of racemic and chiral carbazole derivative containing an isopropanolamine pattern was systematically synthesized to discover low-cost and efficient antibacterial candidates. Screening results showed that compounds 2f, 6c, and 2j could significantly suppress the growth of tested plant pathogens, namely Xanthomonas oryzae pv oryzae, X. axonopodis pv citri, and Pseudomonas syringae pv actinidiae, and provided the corresponding EC50 values of 1.27, 0.993, and 0.603 µg/mL, which were significantly better than those of existing commercial drugs. In vivo studies confirmed their prospective applications for controlling plant bacterial diseases. Label-free quantitative proteomics analysis indicated that compound 2f could dramatically induce the up- and down-regulation of a total of 247 differentially expressed proteins, which was further validated by the parallel reaction monitoring technique. Moreover, fluorescence spectra and SEM images were obtained to further explore the antibacterial mechanism.

Sulfone-Based Probes Unraveled Dihydrolipoamide S-Succinyltransferase as an Unprecedented Target in Phytopathogens.

Target validation of current drugs remains the major challenge for target-based drug discovery, especially for agrochemical discovery. The bactericide 0 represents a novel lead structure and has shown potent efficacy against those diseases that are extremely difficult to control, such as rice bacterial leaf blight. However, no detailed target analysis of this bactericide has been reported. Here, we developed a panel of 0-derived probes 1-6, in which a conservative modification (alkyne tag) was introduced to keep the antibacterial activity of 0 and provide functionality for target identification via click chemistry. With these cell-permeable probes, we were able to discover dihydrolipoamide S-succinyltransferase (DLST) as an unprecedented target in living cells. The probes showed good preference for DLST, especially probe 1, which demonstrated distinct selectivity and reactivity. Also, we reported 0 as the first covalent DLST inhibitor, which has been used to confirm the involvement of DLST in the regulation of energy production.

Current advances of carbene-mediated photoaffinity labeling in medicinal chemistry.

Photoaffinity labeling (PAL) in combination with a chemical probe to covalently bind its target upon UV irradiation has demonstrated considerable promise in drug discovery for identifying new drug targets and binding sites. In particular, carbene-mediated photoaffinity labeling (cmPAL) has been widely used in drug target identification owing to its excellent photolabeling efficiency, minimal steric interference and longer excitation wavelength. Specifically, diazirines, which are among the precursors of carbenes and have higher carbene yields and greater chemical stability than diazo compounds, have proved to be valuable photolabile reagents in a diverse range of biological systems. This review highlights current advances of cmPAL in medicinal chemistry, with a focus on structures and applications for identifying small molecule-protein and macromolecule-protein interactions and ligand-gated ion channels, coupled with advances in the discovery of targets and inhibitors using carbene precursor-based biological probes developed in recent decades.