Engineered gene over-expression as a method of drug target identification

Sugden, Christopher; Roper, Janine R.; Williams, Jeffrey

doi:10.1016/j.bbrc.2005.06.117

Cited by 17 publications

(11 citation statements)

References 7 publications

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“…FabH Overexpression Increases Resistance to AMY. It seemed likely that AMY targets an enzyme involved in fatty acid biosynthesis; therefore, we manipulated expression levels of fatty acid biosynthetic enzymes, an approach that has previously been used to identify antibiotic targets (42)(43)(44)(45). As a control, we cloned the 3-oxoacyl-[acyl-carrier-protein] synthase 2 gene (fabF) into a plasmid under a constitutive promoter; tested its effect on the susceptibility of S. aureus to platensimycin, a well-characterized FabF inhibitor (40); and observed a fourfold increase in resistance to platensimycin (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

Amycomicin is a potent and specific antibiotic discovered with a targeted interaction screen

Pishchany

Mevers

Ndousse-Fetter

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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The rapid emergence of antibiotic-resistant pathogenic bacteria has accelerated the search for new antibiotics. Many clinically used antibacterials were discovered through culturing a single microbial species under nutrient-rich conditions, but in the environment, bacteria constantly encounter poor nutrient conditions and interact with neighboring microbial species. In an effort to recapitulate this environment, we generated a nine-strain actinomycete community and used 16S rDNA sequencing to deconvolute the stochastic production of antimicrobial activity that was not observed from any of the axenic cultures. We subsequently simplified the community to just two strains and identified sp. AA4 as the producing strain and M145 as an inducing strain. Bioassay-guided isolation identified amycomicin (AMY), a highly modified fatty acid containing an epoxide isonitrile warhead as a potent and specific inhibitor of Amycomicin targets an essential enzyme (FabH) in fatty acid biosynthesis and reduces infection in a mouse skin-infection model. The discovery of AMY demonstrates the utility of screening complex communities against specific targets to discover small-molecule antibiotics.

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Section: Resultsmentioning

confidence: 99%

Amycomicin is a potent and specific antibiotic discovered with a targeted interaction screen

Pishchany

Mevers

Ndousse-Fetter

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…Over-expression of farnesyl diphosphate synthase (FDP synthase) in social amoeba Dictyostelium resulted in resistance to aminobisphosphate inhibitors [42]. Butcher and coworkers [43] constructed a pool of Saccharomyces cerevisiae strains each over-expressing a different protein.…”

Section: Discussionmentioning

confidence: 99%

“…Candidate target proteins of rapamycin were identified [43]. While the approaches by these groups [42,43] are valuable and robust, they are limited only for use in identification of targets of antifungal compounds. In a recent report using a bacterial system (E. coli), multicopy suppressors were identified for antibacterial compounds as a method of determining the targets of the inhibitors and the mechanisms of resistance [29].…”

Section: Discussionmentioning

confidence: 99%

An array of Escherichia coli clones over-expressing essential proteins: A new strategy of identifying cellular targets of potent antibacterial compounds

Real

Bailey

2006

Biochemical and Biophysical Research Communications

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With the advancement of high throughput screening, it has become easier and faster to discover hit compounds that inhibit proliferation of bacterial cells. However, development in technologies used to identify cellular targets of potent antibacterial inhibitors has lagged behind. Here we describe a novel strategy of target identification for antibacterial inhibitors using an array of Escherichia coli clones each over-expressing one essential protein. In a proof-of-concept study, eight essential genes were cloned into pLex5BA vector under the control of an inducible promoter. Overexpression of target proteins was confirmed. For two clones, one over-expressing FabI and the other over-expressing MurA enzymes, the host cells became 17-fold and 139-fold more resistant to the specific inhibitors triclosan and phosphomycin, respectively, while the susceptibility of other clones towards these inhibitors remained unchanged after induction of gene expression. Target identification via target protein over-expression was demonstrated using both mixed clone and individual clone assay formats.

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“…The DNA fragment amplified from pLD1A15SN [26] by use of primers VIP 660 and VIP 661 encoded FDPS with the addition of the C-terminal sequence Gly-Ala-Gly-Ala. This DNA fragment was digested with HindIII and BamHI and ligated into HindIII- and BamHI-digested pGFP to give pFDPS–GFP.…”

Section: Methodsmentioning

confidence: 99%

Farnesyl diphosphate synthase, the target for nitrogen-containing bisphosphonate drugs, is a peroxisomal enzyme in the model system Dictyostelium discoideum

Nuttall

Hettema

Watts

2012

Biochemical Journal

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NBP (nitrogen-containing bisphosphonate) drugs protect against excessive osteoclast-mediated bone resorption. After binding to bone mineral, they are taken up selectively by the osteoclasts and inhibit the essential enzyme FDPS (farnesyl diphosphate synthase). NBPs inhibit also growth of amoebae of Dictyostelium discoideum in which their target is again FDPS. A fusion protein between FDPS and GFP (green fluorescent protein) was found, in D. discoideum, to localize to peroxisomes and to confer resistance to the NBP alendronate. GFP was also directed to peroxisomes by a fragment of FDPS comprising amino acids 1–22. This contains a sequence of nine amino acids that closely resembles the nonapeptide PTS2 (peroxisomal targeting signal type 2): there is only a single amino acid mismatch between the two sequences. Mutation analysis confirmed that the atypical PTS2 directs FDPS into peroxisomes. Furthermore, expression of the D. discoideum FDPS–GFP fusion protein in strains of Saccharomyces cerevisiae defective in peroxisomal protein import demonstrated that import of FDPS into peroxisomes was blocked in a strain lacking the PTS2-dependent import pathway. The peroxisomal location of FDPS in D. discoideum indicates that NBPs have to cross the peroxisomal membrane before they can bind to their target.

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Engineered gene over-expression as a method of drug target identification

Cited by 17 publications

References 7 publications

Amycomicin is a potent and specific antibiotic discovered with a targeted interaction screen

Amycomicin is a potent and specific antibiotic discovered with a targeted interaction screen

An array of Escherichia coli clones over-expressing essential proteins: A new strategy of identifying cellular targets of potent antibacterial compounds

Farnesyl diphosphate synthase, the target for nitrogen-containing bisphosphonate drugs, is a peroxisomal enzyme in the model system Dictyostelium discoideum

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