Screening for Arabidopsis mutants with altered Ca2+ signal response using aequorin-based Ca2+ reporter system

Aim and Objective: Antibiotic resistance is a serious constraint to the development of new effective antibacterials. Therefore, the discovery of the new antibacterials remains one of the main challenges in modern medicinal chemistry. This study was undertaken to identify novel molecules with antibacterial activity. Materials and Methods: Using our unique double-reporter system, in-house large-scale HTS campaign was conducted for the identification of antibacterial potency of small-molecule compounds. The construction allows us to visually assess the underlying mechanism of action. After the initial HTS and rescreen procedure, luciferase assay, C14-test, determination of MIC value and PrestoBlue test were carried out. Results: HTS rounds and rescreen campaign have revealed the antibacterial activity of a series of Nsubstituted triazolo-azetidines and their isosteric derivatives that has not been reported previously. Primary hit-molecule demonstrated a MIC value of 12.5 µg/mL against E. coli Δ tolC with signs of translation blockage and no SOS-response. Translation inhibition (26%, luciferase assay) was achieved at high concentrations up to 160 µg/mL, while no activity was found using C14-test. The compound did not demonstrate cytotoxicity in the PrestoBlue assay against a panel of eukaryotic cells. Within a series of direct structural analogues bearing the same or bioisosteric scaffold, compound 2 was found to have an improved antibacterial potency (MIC=6.25 µg/mL) close to Erythromycin (MIC=2.5-5 µg/mL) against the same strain. In contrast to the parent hit, this compound was more active and selective, and provided a robust IP position. Conclusion: N-substituted triazolo-azetidine scaffold may be used as a versatile starting point for the development of novel active and selective antibacterial compounds.

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Engineering subtilisin proteases that specifically degrade active RAS

Communications Biology ◽

10.1038/s42003-021-01818-7 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Yingwei Chen ◽

Eric A. Toth ◽

Biao Ruan ◽

Eun Jung Choi ◽

Richard Simmerman ◽

...

Keyword(s):

Active Form ◽

High Specificity ◽

Kinetic Properties ◽

Target Sequence ◽

Reporter System ◽

Human Cell Culture ◽

Conserved Sequence ◽

Cofactor Binding ◽

Selective Proteolysis

AbstractWe describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

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Arabidopsis mutants deficient in polyunsaturated fatty acid synthesis. Biochemical and genetic characterization of a plant oleoyl-phosphatidylcholine desaturase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)45974-1 ◽

1992 ◽

Vol 267 (3) ◽

pp. 1502-1509 ◽

Cited By ~ 3

Author(s):

M Miquel ◽

J Browse

Keyword(s):

Fatty Acid ◽

Polyunsaturated Fatty Acid ◽

Fatty Acid Synthesis ◽

Genetic Characterization ◽

Acid Synthesis ◽

Arabidopsis Mutants

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Isolation and Characterization of Broad-Spectrum Disease-Resistant Arabidopsis Mutants

Genetics ◽

10.1093/genetics/160.4.1661 ◽

2002 ◽

Vol 160 (4) ◽

pp. 1661-1671

Author(s):

Klaus Maleck ◽

Urs Neuenschwander ◽

Rebecca M Cade ◽

Robert A Dietrich ◽

Jeffery L Dangl ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Systemic Acquired Resistance ◽

Acquired Resistance ◽

Constitutive Expression ◽

Firefly Luciferase ◽

Marker Genes ◽

Arabidopsis Mutants ◽

Isolation And Characterization ◽

Firefly Luciferase Gene

Abstract To identify Arabidopsis mutants that constitutively express systemic acquired resistance (SAR), we constructed reporter lines expressing the firefly luciferase gene under the control of the SAR-inducible PR-1 promoter (PR-1/luc). After EMS mutagenesis of a well-characterized transgenic line, we screened 250,000 M2 plants for constitutive expression of the reporter gene in vivo. From a mutant collection containing several hundred putative mutants, we concentrated on 16 mutants lacking spontaneous hypersensitive response (HR) cell death. We mapped 4 of these constitutive immunity (cim) mutants to chromosome arms. Constitutive expression of disease resistance was established by analyzing responses to virulent Peronospora parasitica and Pseudomonas syringae strains, by RNA blot analysis for endogenous marker genes, and by determination of salicylic acid levels in the mutants. The variety of the cim phenotypes allowed us to define distinct steps in both the canonical SAR signaling pathway and a separate pathway for resistance to Erysiphe cichoracearum, active in only a subset of the mutants.

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