Irena Ivnitski-Steele

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

Clinically relevant azole resistance in the fungal pathogen Candida albicans is most often associated with the increased expression of plasma membrane efflux pumps, specifically the ATP-binding cassette (ABC) transporters CaCdr1p and CaCdr2p and the major facilitator superfamily (MFS) transporter CaMdr1p. Development of potent pump inhibitors that chemosensitize cells to azoles is a promising approach to overcome antifungal resistance. Here we identify Nile red as a new fluorescent substrate for CaCdr1p, CaCdr2p, and CaMdr1p. Nile red was effluxed efficiently from Saccharomyces cerevisiae cells heterologously expressing these transporters. Enniatin selectively inhibited the efflux of Nile red from S. cerevisiae cells expressing CaCdr1p or CaMdr1p but not from cells expressing CaCdr2p. This indicates that Nile red can be used for the identification of inhibitors specific for particular transporters mediating antifungal resistance in pathogenic yeast.

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

ABSTRACT 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 pump-mediated 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.

Fluorescent substrates for flow cytometric evaluation of efflux inhibition in ABCB1, ABCC1, and ABCG2 transporters

ATP binding cassette (ABC) transmembrane efflux pumps such as P-glycoprotein (ABCB1), multidrug resistance protein 1 (ABCC1), and breast cancer resistance protein (ABCG2) play an important role in anticancer drug resistance. A large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps. In vitro assessment of the affinity of drug candidates for multidrug resistance proteins is central to predict in vivo pharmacokinetics and drug-drug interactions. The objective of this study was to identify and characterize new substrates for these transporters. As part of a collaborative project with Life Technologies, 102 fluorescent probes were investigated in a flow cytometric screen of ABC transporters. The primary screen compared substrate efflux activity in parental cell lines with their corresponding highly expressing resistant counterparts. The fluorescent compound library included a range of excitation/emission profiles and required dual laser excitation as well as multiple fluorescence detection channels. A total of 31 substrates with active efflux in one or more pumps and practical fluorescence response ranges were identified and tested for interaction with eight known inhibitors. This screening approach provides an efficient tool for identification and characterization of new fluorescent substrates for ABCB1, ABCC1, and ABCG2.

Inhibition of neovascularization by environmental agents

The formation of new blood vessels, neovascularization, occurs by two unique processes: vasculogenesis, the de novo assembly of blood vessels from angioblast precursors, and angiogenesis, the formation of new capillary sprouts from pre-existing vessels. There are many potential targets by which environmental pollutants may inhibit neovascularization and thus there are many possible phenotypic outcomes. Two examples of environmental pollutants that have been demonstrated to inhibit neovascularization include 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototypical halogenated aromatic hydrocarbon, and constituents found in environmental tobacco smoke. Studies have shown that TCDD disrupts neoangiogenesis by inhibiting the expression of angiogenic stimuli as well as by reducing the responsiveness of endothelial cells to those stimuli. Additionally, studies have shown that constituents of environmental tobacco smoke, including pyradine and pyrazine derivatives, can potently inhibit the angiogenic process of branching as well as the vasculogenic process involved in capillary plexus formation. Further, the inhibition of neovascularization by either TCDD or environmental tobacco smoke constituents is associated with reduced endothelial cell proliferation and altered expression of extracellular matrix proteins. Future research that identifies the specific angiogenic signaling pathways that are disrupted by these pollutants will improve our ability to assess their risk to human health. Finally, it is likely that many other environmental pollutants impact neovascularization; however, very few have been studied in sufficient detail. Thus, additional research also is needed to identify those environmental agents that mediate their toxicity by disrupting neovascularization.

A Selective ATP-Binding Cassette Subfamily G Member 2 Efflux Inhibitor Revealed via High-Throughput Flow Cytometry

Chemotherapeutics tumor resistance is a principal reason for treatment failure, and clinical and experimental data indicate that multidrug transporters such as ATP-binding cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate, we identified a piperazine-substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused structure-activity relationship (SAR)–driven chemistry effort, we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2-overexpressing tumor model. At least two analogues significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.

High‐Throughput Cytotoxicity Screening by Propidium Iodide Staining

This unit describes a system for the automated high‐throughput analysis of cell cytotoxicity in 96‐well and 384‐well microplates. Discrete cell cultures are analyzed at rates of 40/min (∼2.5 min/96 wells, ∼10 min/384 wells) and cytotoxicity is quantified on the basis of a combination of propidium iodide (PI) fluorescence analysis and cell counting performed by the flow cytometer. Only 2 µl is aspirated from a culture for analysis so that assays can be performed in small volumes to minimize reagent cost and usage.

Targeting efflux pumps to overcome antifungal drug resistance

Resistance to antifungal drugs is an increasingly significant clinical problem. The most common antifungal resistance encountered is efflux pump-mediated resistance of Candida species to azole drugs. One approach to overcome this resistance is to inhibit the pumps and chemosensitize resistant strains to azole drugs. Drug discovery targeting fungal efflux pumps could thus result in the development of azole-enhancing combination therapy. Heterologous expression of fungal efflux pumps in Saccharomyces cerevisiae provides a versatile system for screening for pump inhibitors. Fungal efflux pumps transport a range of xenobiotics including fluorescent compounds. This enables the use of fluorescence-based detection, as well as growth inhibition assays, in screens to discover compounds targeting efflux-mediated antifungal drug resistance. A variety of medium- and high-throughput screens have been used to identify a number of chemical entities that inhibit fungal efflux pumps.