High-Throughput Screening with HyperCyt® Flow Cytometry to Detect Small Molecule Formylpeptide Receptor Ligands

Young, Susan; Bologa, Cristian; Prossnitz, Eric R.; Oprea, Tudor I.; Sklar, Larry A.; Edwards, Bruce S.

doi:10.1177/1087057105274532

Cited by 62 publications

(54 citation statements)

References 16 publications

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“…In order to find agonists with this potency, we screened a library of small compounds and several ligands for FPR2 with varying EC 50 values were identified. The identification of small-molecule ligands represent an attractive approach to analyze structure and function of FPRs, and in line with this several research articles using the same approach have been published during the last couple of years [16,[38][39][40]. Of the compounds that we used and found to activate human neutrophils to produce/release superoxide anions, 5 (4 unique and one enantiomer) have been described earlier.…”

Section: Discussionmentioning

confidence: 80%

Stable formyl peptide receptor agonists that activate the neutrophil NADPH-oxidase identified through screening of a compound library

Forsman

Kalderén

Nordin

et al. 2011

Biochemical Pharmacology

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The neutrophil formyl peptide receptors (FPR1 and FPR2) are G-protein coupled receptors that can induce pro-inflammatory as well as anti-inflammatory activities when activated. Accordingly, these receptors may become therapeutic targets for the development of novel drugs to be used for reducing the inflammation induced injuries in asthma, rheumatoid arthritis, Alzheimer's disease, cardiovascular diseases and traumatic shock. We screened a library of more then 50 K small compounds for an ability of the compounds to induce a transient rise in intracellular Ca 2+ in cells transfected to express FPR2 (earlier called FPRL1 or the lipoxin A 4 receptor). Ten agonists hits were selected for further analysis representing different chemical series and five new together with five earlier described molecules were further profiled.. Compounds 1 -10 gave rise to a calcium response in the FPR2 transfectants with EC 50 values ranging from 4 x 10 -9 M to 2 x 10 -7 M. All 10 compounds activated human neutrophils to release superoxide , and based on the potency of their activity, the three most potent activators of the neutrophil NADPH-oxidase were further characterized. These three agonists were largely resistant to inactivation by neutrophil produced reactive oxygen species and shown to trigger the same functional repertoire in neutrophils as earlier described peptide agonists. Accordingly they induced chemotaxis, granule mobilization and secretion of superoxide. Interestingly, the oxidase activity was largely inhibited by cyclosporine H, an FPR1 selective antagonist, but not by PBP10, an FPR2 selective inhibitor, suggesting that FPR1 is the preferred receptor in neutrophils for all three agonists.

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Section: Discussionmentioning

confidence: 80%

Stable formyl peptide receptor agonists that activate the neutrophil NADPH-oxidase identified through screening of a compound library

Forsman

Kalderén

Nordin

et al. 2011

Biochemical Pharmacology

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“…Chemical libraries that are composed of established drugs can be used to screen compounds for new uses, purposes, and indications. 20 Using a novel approach to HTS, we modified the JC-1 efflux assay 8,9 for implementation in HyperCyt 7 and, within the Prestwick Chemical Library, measured ABCB1-mediated efflux. Our high frequency of "hits," 2.15% of the library (19 of 880 compounds), is much higher than expected from HTS of random chemical structures.…”

Section: Discussionmentioning

confidence: 99%

“…7 The HTS response data were measured in the FL1 green fluorescence emission channel (530-540 nm), and the FL3 red fluorescence emission channel (> 650 nm) was used for detection of Cytoplex L9 or L10 beads (Duke Scientific, Palo Alto, CA). The beads were used as an internal control to facilitate the proper registration of flow cytometry data with source wells.…”

Section: Flow Cytometric Detection Of Abcb1 Reversal (Hypercyt Assay)mentioning

confidence: 99%

“…To address the need to identify target compounds that block ABCB1-mediated drug transport, we employed HyperCyt 7 to screen a library of more than 880 off-patent drugs for potential use as chemosensitizing agents. We developed a chemoresistant T-ALL cell line that could be tested for ABCB1-reversal agents using J-aggregate-forming lipophilic cation 5,5,′6,6′-tetrachloro-1,1, ′3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1), 8,9 a cationic dye that exhibits differential fluorescence based on its intracellular concentrations.…”

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confidence: 99%

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High-Throughput Screening for Daunorubicin-Mediated Drug Resistance Identifies Mometasone Furoate as a Novel ABCB1-Reversal Agent

Winter¹,

Lovato²,

Khawaja³

et al. 2008

SLAS Discovery

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The overexpression of P-glycoprotein, encoded by the ATP Binding Cassette B1 (ABCB1) gene, contributes to multidrug resistance (MDR) and is considered one of the major obstacles to successful cancer chemotherapy. The authors previously developed a T-lineage acute lymphoblastic leukemia (T-ALL) cell line that overexpresses ABCB1 and exhibits MDR to daunorubicin (DNR), prednisolone, and vincristine. Using this cell line and the fluorescent probe JC-1, they developed a flow cytometrybased, high-throughput screening (HTS) assay that quantifies ABCB1 efflux. They screened a library of 880 off-patent drugs for their ability to inhibit ABCB1 efflux and then measured the ability of 11 lead compounds to reverse in vitro DNR-mediated drug resistance and the toxic doses for each agent. Seven of the 11 drugs were able to reverse drug resistance at a concentration significantly below its toxic dose. Of the remaining 7, only 1 compound, mometasone furoate, has not been previously described as an ABCB1 antagonist to DNR-mediated drug resistance. On the basis of its high ABC modulator activity and relatively large in vitro therapeutic window, this drug warrants further investigation. In addition, the approach used in this study is useful for identifying off-patent drugs that may be repurposed for novel clinical indications. (Journal of Biomolecular Screening 2008:185-193)

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“…The introduction of high-throughput flow cytometry 1 has addressed some of the issues associated with this method; however, the development of homogeneous flow cytometry assays can still present a challenge due to high background fluorescence. Fluorometric microvolume assay technology (FMAT), described in 1999, 2,3 presented researchers with a homogeneous, mix-and-read assay solution for measurement of binding to cells or beads.…”

Section: Introductionmentioning

confidence: 99%

Application of the Mirrorball High-Sensitivity Cytometer to Multiplexed Assays for Antibody Drug Discovery

et al. 2015

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Highly sensitive, high-throughput assay technologies are required for the identification of antibody therapeutics. Multiplexed assay systems are particularly advantageous because they allow evaluation of several parameters within 1 well, increasing throughput and reducing hands-on laboratory time.The mirrorball (TTP Labtech), using high-throughput fluorometric microvolume assay technology, offers simultaneous scanning with up to 3 lasers as well as laser scatter detection. This makes the mirrorball especially suitable for the development of highly sensitive and multiplexed assays.We have developed bead-and cell-based binding assays that demonstrate how the multilaser capability of the mirrorball can be exploited to enhance assay sensitivity. In addition, using the multilaser simultaneous scanning capability, we have established multiplexed cytokine quantitation assays and antibody-cell binding assays.Our results demonstrate the potential utility of this technology to improve the sensitivity and efficiency of biologics screening, resulting in streamlining of the lead antibody selection process.

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High-Throughput Screening with HyperCyt® Flow Cytometry to Detect Small Molecule Formylpeptide Receptor Ligands

Cited by 62 publications

References 16 publications

Stable formyl peptide receptor agonists that activate the neutrophil NADPH-oxidase identified through screening of a compound library

Stable formyl peptide receptor agonists that activate the neutrophil NADPH-oxidase identified through screening of a compound library

High-Throughput Screening for Daunorubicin-Mediated Drug Resistance Identifies Mometasone Furoate as a Novel ABCB1-Reversal Agent

Application of the Mirrorball High-Sensitivity Cytometer to Multiplexed Assays for Antibody Drug Discovery

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