Identification of Nile red as a fluorescent substrate of the Candida albicans ATP-binding cassette transporters Cdr1p and Cdr2p and the major facilitator superfamily transporter Mdr1p

Ivnitski-Steele, Irena; Holmes, Ann R.; Lamping, Erwin; Monk, Brian C.; Cannon, Richard D.; Sklar, Larry A.

doi:10.1016/j.ab.2009.07.001

Cited by 110 publications

(97 citation statements)

References 38 publications

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“…To circumvent this problem, several groups have studied the ability of C. albicans or of Saccharomyces cerevisiae cells expressing the C. albicans genes of interest to pump fluorescent marker compounds out of the cell. These studies have provided important insights into the energetics and kinetics of these pumps, but the fluorescent compounds used in most of these studies are unrelated structurally or functionally to the azole antifungals (14,19,23,36,38,50,52). Moreover, the fact that C. albicans translates the codon CTG as leucine rather than serine complicates the interpretation of results obtained by heterologous expression of CTG-containing C. albicans genes in S. cerevisiae or other convenient hosts (17,21,39,45,48,50).…”

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confidence: 99%

Fluconazole Transport into Candida albicans Secretory Vesicles by the Membrane Proteins Cdr1p, Cdr2p, and Mdr1p

et al. 2010

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A major cause of azole resistance in Candida albicans is overexpression of CDR1, CDR2, and/or MDR1, which encode plasma membrane efflux pumps. To analyze the catalytic properties of these pumps, we used ACT1-and GAL1-regulated expression plasmids to overexpress CDR1, CDR2, or MDR1 in a C. albicans cdr1 cdr2 mdr1-null mutant. When the genes of interest were expressed, the resulting transformants were more resistant to multiple azole antifungals, and accumulated less

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confidence: 99%

Fluconazole Transport into Candida albicans Secretory Vesicles by the Membrane Proteins Cdr1p, Cdr2p, and Mdr1p

et al. 2010

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“…As there are several fluorescent substrates of efflux pumps, fluorescence-based assays of activity are applicable to measuring the activity of ABC transporters (16,21,44). A dif-ferential signal can be obtained between cells which are actively effluxing a previously loaded fluorescent substrate (low signal at endpoint) and cells for which efflux is inhibited by a test compound (high signal at endpoint).…”

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confidence: 99%

“…A dif-ferential signal can be obtained between cells which are actively effluxing a previously loaded fluorescent substrate (low signal at endpoint) and cells for which efflux is inhibited by a test compound (high signal at endpoint). We have recently developed a flow cytometry-based HTS assay suitable for the identification of inhibitors of fungal efflux pumps, using a yeast-based assay of accumulation of fluorescent pump substrates rhodamine 6G (R6G) or Nile Red (16). The screening assay employed recombinant strains of the model yeast Saccharomyces cerevisiae in which the individual transporter genes were cloned and functionally expressed (23).…”

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confidence: 99%

“…Furthermore, the gene encoding the transporter of interest is integrated at the S. cerevisiae PDR5 genomic locus downstream of a promoter under the control of a mutant transcriptional regulator, Pdr1-3p, producing constitutive high-level expression of functional heterologous proteins in the plasma membrane of the derived recombinant strain. A panel of S. cerevisiae strains has been created that express a range of pumps (ABC and MFS) from important fungal pathogens including C. albicans, C. glabrata, and C. krusei (16).…”

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confidence: 99%

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The Monoamine Oxidase A Inhibitor Clorgyline Is a Broad-Spectrum Inhibitor of Fungal ABC and MFS Transporter Efflux Pump Activities Which Reverses the Azole Resistance of Candida albicans and Candida glabrata Clinical Isolates

Holmes

Keniya

Ivnitski-Steele

et al. 2012

Antimicrob Agents Chemother

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Resistance to the commonly used azole antifungal fluconazole (FLC) can develop due to overexpression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) plasma membrane transporters. An approach to overcoming this resistance is to identify inhibitors of these efflux pumps. We have developed a pump assay suitable for high-throughput screening (HTS) that uses recombinant Saccharomyces cerevisiae strains hyperexpressing individual transporters from the opportunistic fungal pathogen Candida albicans. The recombinant strains possess greater resistance to azoles and other pump substrates than the parental host strain. A flow cytometry-based HTS, which measured increased intracellular retention of the fluorescent pump substrate rhodamine 6G (R6G) within yeast cells, was used to screen the Prestwick Chemical Library (PCL) of 1,200 marketed drugs. Nine compounds were identified as hits, and the monoamine oxidase A inhibitor (MAOI) clorgyline was identified as an inhibitor of two C. albicans ABC efflux pumps, CaCdr1p and CaCdr2p. Secondary in vitro assays confirmed inhibition of pumpmediated efflux by clorgyline. Clorgyline also reversed the FLC resistance of S. cerevisiae strains expressing other individual fungal ABC transporters (Candida glabrata Cdr1p or Candida krusei Abc1p) or the C. albicans MFS transporter Mdr1p. Recombinant strains were also chemosensitized by clorgyline to other azoles (itraconazole and miconazole). Importantly, clorgyline showed synergy with FLC against FLC-resistant C. albicans clinical isolates and a C. glabrata strain and inhibited R6G efflux from a FLC-resistant C. albicans clinical isolate. Clorgyline is a novel broad-spectrum inhibitor of two classes of fungal efflux pumps that acts synergistically with azoles against azole-resistant C. albicans and C. glabrata strains.

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“…From those five compounds 1 showed a strong inhibition (Figure 1) of the enzymatic activity of the multidrug efflux transporter Pdr5p of S. cerevisiae. Its IC50 value was lower than those reported for other natural compounds such as enniatin (Ivnitski-Steele et al, 2009) or curcumin (Sharma et al, 2009) two well-known inhibitors of multidrug efflux transporters. BRP-1 was not toxic to the yeast cells used in this work even at high concentrations (50 µM).…”

Section: Effect Of Brp-1 On the Intracellular Accumulation And The Efmentioning

confidence: 46%

Inhibition of Saccharomyces cerevisiae Pdr5p by a natural compound extracted from Brazilian Red Propolis

Lotti¹,

Castro²,

Sá³

et al. 2011

Rev. bras. farmacogn.

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Multidrug resistance of cancer cells and pathogenic microorganisms leading to the treatment failure of some forms of cancer or life-threatening bacterial or fungal infections is often caused by the overexpression of multidrug efflux pumps belonging to the ATP-binding cassette transporters superfamily. The multidrug resistance of fungal cells often involves the overexpression of efflux pumps belonging to the pleiotropic drug resistance (PDR) family of ABC transporters. Possibly the best-studied fungal PDR transporter is the multidrug resistance transporter Pdr5p of Saccharomyces cerevisiae. Some research groups have been searching for new inhibitors of these efflux pumps in order to alleviate resistance. Natural products are a great source for the discovery of new compounds with biological activity. Propolis is a complex resinous material collected by honeybees from exudates and buds of certain plant sources and this material is thought to serve as a defense substance for bee hives. Propolis is widely used in traditional medicine and is reported to have a broad spectrum of pharmacological properties. Literature reported some biological functionalities of propolis, such as antibacterial, antiviral, fungicidal, antiinflammatory and anti-carcinogenic activities. The chemical composition of propolis is qualitatively and quantitatively variable. Components isolated from methanolic extract of red Brazilian propolis (Alagoas, Northeast of Brazil) are isoflavonoids (including pterocarpans, isoflavans, isoflavones), flavanones and polyprenylated benzophenones. In this work we demonstrated the effects of five different isolated compounds on the ATPase activity of Pdr5p. Out of all five substances tested, only BRP-1 was able to completely abolish the enzymatic activity while others worked as positive modulators of the enzyme activity. BRP-1also inhibited the efflux of Rhodamine 6G from yeast cells overexpressing Pdr5p. Taken together, these results demonstrate that Brazilian propolis could be a source of promising compounds that can alleviate the MDR phenomenon, particularly in some fungi, where it could be used as an adjuvant for the treatment with azoles.

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Identification of Nile red as a fluorescent substrate of the Candida albicans ATP-binding cassette transporters Cdr1p and Cdr2p and the major facilitator superfamily transporter Mdr1p

Cited by 110 publications

References 38 publications

Fluconazole Transport into Candida albicans Secretory Vesicles by the Membrane Proteins Cdr1p, Cdr2p, and Mdr1p

Fluconazole Transport into Candida albicans Secretory Vesicles by the Membrane Proteins Cdr1p, Cdr2p, and Mdr1p

The Monoamine Oxidase A Inhibitor Clorgyline Is a Broad-Spectrum Inhibitor of Fungal ABC and MFS Transporter Efflux Pump Activities Which Reverses the Azole Resistance of Candida albicans and Candida glabrata Clinical Isolates

Inhibition of Saccharomyces cerevisiae Pdr5p by a natural compound extracted from Brazilian Red Propolis

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