A high throughput flow cytometric assay platform targeting transporter inhibition

Tegos, George P.; Evangelisti, Annette M.; Strouse, J. Jacob; Ursu, Oleg; Bologa, Cristian; Sklar, Larry A.

doi:10.1016/j.ddtec.2014.03.010

Cited by 21 publications

(17 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Various flow cytometry assays have already been developed for these clinically important multidrug transporters by using transporter positive and negative cell line pairs and fluorescent indicator substrates . Our results here show that DCV is an excellent indicator in this regard and we assume that it can also be applied for screening transporter inhibitors.…”

Section: Discussionsupporting

confidence: 52%

Application of fluorescent dye substrates for functional characterization of ABC multidrug transporters at a single cell level

et al. 2016

View full text Add to dashboard Cite

ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2-and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. V C 2016 International Society for Advancement of Cytometry Key terms functional transporter assay; multidrug transporters; ABCG2; ABCB1; DyeCycle Violet; Hoechts 33342; high content screening and analysis MULTIDRUG resistance is a major problem in chemotherapy of cancer or in prolonged viral infections. An important cause of the emergence of cellular multidrug resistance is the increased expression of ABC multidrug transporters (1,2). Three human ABC proteins, ABCB1, ABCG2, and ABCC1 are the key membrane proteins responsible for an increased outward drug transport in most multidrug-resistant cells. These transporters are members of the ancient ABC transporter protein family, performing primary, ATP-dependent active transport processes. The human multidrug transporters consist of conserved cytoplasmic ABC domains, responsible for the ATP binding and hydrolysis, and multipass membrane region(s) involved in drug substrate binding and outward drug transport (3,4). The above mentioned three human multidrug transporters show promiscuous drug binding characteristics and have partially overlapping substrate specificities. Prediction of interactions between ABC multidrug transporters and drugs is a challenging task, because the structure of these proteins is not known at an atomic level, and the drug-protein interaction site is probably a large and versatile interface (5).Both in vitro and in vivo studies clearly indicate that in multidrug-resistant cells the therapeutically effective drug concentrations strongly depend on the actual, func-

show abstract

Section: Discussionsupporting

confidence: 52%

Application of fluorescent dye substrates for functional characterization of ABC multidrug transporters at a single cell level

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Additionally, such assays are only applicable to yeast cells, not hyphal cells, which aggregate, cannot be held in suspension, and have variable intracellular volumes. Systems used for HTS include: the HyperCyt ® HT flow cytometry platform [100][101][102]; or a combination of multidrop delivery systems, liquid handling workstations and fluorimeters using 384 microtiter plates to reduce reagent volumes [103]. Our group has used both systems in HTS screens for inhibitors of C. albicans efflux pumps [54,76] and the C. glabrata Cdr1 efflux pump High-throughput primary screens using recombinant S. cerevisiae strains…”

Section: Low-medium Throughput Primary Screensmentioning

confidence: 99%

Targeting Efflux Pumps to Overcome Antifungal Drug Resistance

et al. 2016

View full text Add to dashboard Cite

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.

show abstract

“…This instrument is based in significant measure on developments by Sklar and colleagues (e.g. [109][110][111]), and uses segmented flow [112] to sample from 96-or 384-well U-or V-bottom plates prior to their analysis. The iQue Plus contains three excitation sources (405nm, 488nm, 640nm) and 7 fixed filter detectors (with a midpoint/range in nm of 445/45, 530/30, 572/28, 585/40, 615/24, 675/30, 780/60, giving 13 fluorescence channels) whose outputs are stored as both 'height' and area, using the FCS3.0 data file standard [113].…”

Section: Flow Cytometrymentioning

confidence: 99%

Very rapid flow cytometric assessment of antimicrobial susceptibility during the apparent lag phase of microbial (re)growth

Jindal

Thampy

Day

et al. 2018

Preprint

View full text Add to dashboard Cite

Cells of E. coli were grown in LB medium, taken from a stationary phase of 2-4h, and reinoculated into fresh media at a concentration (10 5 .mL -1 or lower) characteristic of bacteriuria. Flow cytometry was used to assess how quickly we could detect changes in cell size, number, membrane energisation (using a carbocyanine dye) and DNA distribution. It turned out that while the lag phase observable macroscopically via bulk OD measurements could be as long as 4h, the true lag phase could be less than 15-20 min, and was accompanied by many observable biochemical changes. Antibiotics to which the cells were sensitive affected these changes within 20 min of reinoculation, providing the possibility of a very rapid antibiotic susceptibility test, on a timescale compatible with a visit to a GP clinic. The strategy was applied successfully to genuine potential Urinary Tract Infection (UTI) samples taken from a doctor's surgery. The methods developed could prove of considerable value in ensuring the correct prescription and thereby lowering the spread of antimicrobial resistance.

show abstract

A high throughput flow cytometric assay platform targeting transporter inhibition

Cited by 21 publications

References 48 publications

Application of fluorescent dye substrates for functional characterization of ABC multidrug transporters at a single cell level

Application of fluorescent dye substrates for functional characterization of ABC multidrug transporters at a single cell level

Targeting Efflux Pumps to Overcome Antifungal Drug Resistance

Very rapid flow cytometric assessment of antimicrobial susceptibility during the apparent lag phase of microbial (re)growth

Contact Info

Product

Resources

About