High-Content Assay to Study Protein Prenylation

Simonen, Mika; Ibig-Rehm, Yvonne; Hofmann, Gabriele; Zimmermann, Johann; Albrecht, Geneviève; Magnier, Maxime; Heidinger, Valérie; Gabriel, Daniela

doi:10.1177/1087057108318757

Cited by 17 publications

(7 citation statements)

References 39 publications

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“…Because resistance to existing antibiotics and herbicides progressively limits their usefulness, screening methods such as this will be essential for future drug development and sustainable agriculture. While this manuscript was in preparation, Simonen et al (2008) reported a visual system similar to that described here that was used for the optical high-throughput screening of protein isoprenyltransferase inhibitors based on GFP, which was tagged with the isoprenylation motif of human H-Ras and transformed into human osteosarcoma (U-2 OS) cells.…”

Section: Discussionmentioning

confidence: 99%

The Plastidial 2-C-Methyl-d-Erythritol 4-Phosphate Pathway Provides the Isoprenyl Moiety for Protein Geranylgeranylation in Tobacco BY-2 Cells

et al. 2009

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Protein farnesylation and geranylgeranylation are important posttranslational modifications in eukaryotic cells. We visualized in transformed Nicotiana tabacum Bright Yellow-2 (BY-2) cells the geranylgeranylation and plasma membrane localization of GFP-BD-CVIL, which consists of green fluorescent protein (GFP) fused to the C-terminal polybasic domain (BD) and CVIL isoprenylation motif from the Oryza sativa calmodulin, CaM61. Treatment with fosmidomycin (Fos) or oxoclomazone (OC), inhibitors of the plastidial 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, caused mislocalization of the protein to the nucleus, whereas treatment with mevinolin, an inhibitor of the cytosolic mevalonate pathway, did not. The nuclear localization of GFP-BD-CVIL in the presence of MEP pathway inhibitors was completely reversed by all-transgeranylgeraniol (GGol). Furthermore, 1-deoxy-D-xylulose (DX) reversed the effects of OC, but not Fos, consistent with the hypothesis that OC blocks 1-deoxy-D-xylulose 5-phosphate synthesis, whereas Fos inhibits its conversion to 2-C-methyl-Derythritol 4-phosphate. By contrast, GGol and DX did not rescue the nuclear mislocalization of GFP-BD-CVIL in the presence of a protein geranylgeranyltransferase type 1 inhibitor. Thus, the MEP pathway has an essential role in geranylgeranyl diphosphate (GGPP) biosynthesis and protein geranylgeranylation in BY-2 cells. GFP-BD-CVIL is a versatile tool for identifying pharmaceuticals and herbicides that interfere either with GGPP biosynthesis or with protein geranylgeranylation.

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Section: Discussionmentioning

confidence: 99%

The Plastidial 2-C-Methyl-d-Erythritol 4-Phosphate Pathway Provides the Isoprenyl Moiety for Protein Geranylgeranylation in Tobacco BY-2 Cells

et al. 2009

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“…To monitor protein prenylation in C. elegans, we adapted a GFP-based method previously described for mammalian cells (41). The plasmid pGLO-1P::GFP-CAAX carries the intestinal-specific promoter glo-1 (22,42) to express a modified GFP fused to the last 12 aa of the C. elegans ras-2 gene, including the terminal prenylation motif sequence CLIS (see SI Materials and Methods).…”

Section: Methodsmentioning

confidence: 99%

Statins inhibit protein lipidation and induce the unfolded protein response in the non-sterol producing nematode Caenorhabditis elegans

Mörck¹,

Olsen

Kurth

et al. 2009

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

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Statins are compounds prescribed to lower blood cholesterol in millions of patients worldwide. They act by inhibiting HMG-CoA reductase, the rate-limiting enzyme in the mevalonate pathway that leads to the synthesis of farnesyl pyrophosphate, a precursor for cholesterol synthesis and the source of lipid moieties for protein prenylation. The nematode Caenorhabditis elegans possesses a mevalonate pathway that lacks the branch leading to cholesterol synthesis, and thus represents an ideal organism to specifically study the noncholesterol roles of the pathway. Inhibiting HMG-CoA reductase in C. elegans using statins or RNAi leads to developmental arrest and loss of membrane association of a GFP-based prenylation reporter. The unfolded protein response (UPR) is also strongly activated, suggesting that impaired prenylation of small GTPases leads to the accumulation of unfolded proteins and ER stress. UPR induction was also observed upon pharmacological inhibition of farnesyl transferases or RNAi inhibition of a specific isoprenoid transferase (M57.2) and found to be dependent on both ire-1 and xbp-1 but not on pek-1 or atf-6, which are all known regulators of the UPR. The lipid stores and fatty acid composition were unaffected in statin-treated worms, even though they showed reduced staining with Nile red. We conclude that inhibitors of HMG-CoA reductase or of farnesyl transferases induce the UPR by inhibiting the prenylation of M57.2 substrates, resulting in developmental arrest in C. elegans. These results provide a mechanism for the pleiotropic effects of statins and suggest that statins could be used clinically where UPR activation may be of therapeutic benefit.

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“…However,t hese salicylica cid derivatives were, at best, only weakly active in ac ellular plasma membrane translocation assay. [24] With an IC 50 value of 20 mm,c ompound 3i was found to be among the most active compounds in this cellular assay. The poor cellular permeability of this compound series, resulting from the carboxylic acid functionality,p robably precluded ah igher cellular potency.…”

Section: Discovery Of the Salicylic Acid Class Of Allosteric Fpps Inhmentioning

confidence: 80%

Discovery of Novel Allosteric Non‐Bisphosphonate Inhibitors of Farnesyl Pyrophosphate Synthase by Integrated Lead Finding

et al. 2015

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Farnesyl pyrophosphate synthase (FPPS) is an established target for the treatment of bone diseases, but also shows promise as an anticancer and anti-infective drug target. Currently available anti-FPPS drugs are active-site-directed bisphosphonate inhibitors, the peculiar pharmacological profile of which is inadequate for therapeutic indications beyond bone diseases. The recent discovery of an allosteric binding site has paved the way toward the development of novel non-bisphosphonate FPPS inhibitors with broader therapeutic potential, notably as immunomodulators in oncology. Herein we report the discovery, by an integrated lead finding approach, of two new chemical classes of allosteric FPPS inhibitors that belong to the salicylic acid and quinoline chemotypes. We present their synthesis, biochemical and cellular activities, structure-activity relationships, and provide X-ray structures of several representative FPPS complexes. These novel allosteric FPPS inhibitors are devoid of any affinity for bone mineral and could serve as leads to evaluate their potential in none-bone diseases.

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High-Content Assay to Study Protein Prenylation

Cited by 17 publications

References 39 publications

The Plastidial 2-C-Methyl-d-Erythritol 4-Phosphate Pathway Provides the Isoprenyl Moiety for Protein Geranylgeranylation in Tobacco BY-2 Cells

The Plastidial 2-C-Methyl-d-Erythritol 4-Phosphate Pathway Provides the Isoprenyl Moiety for Protein Geranylgeranylation in Tobacco BY-2 Cells

Statins inhibit protein lipidation and induce the unfolded protein response in the non-sterol producing nematode Caenorhabditis elegans

Discovery of Novel Allosteric Non‐Bisphosphonate Inhibitors of Farnesyl Pyrophosphate Synthase by Integrated Lead Finding

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