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A High-Throughput Flow Cytometry Assay for Identification of Inhibitors of 3',5'-Cyclic Adenosine Monophosphate
Dominique Perez1, Peter C Simons1, Yelena Smagley1
1University of New Mexico Cancer Center, Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, 87131, USA.
Abstract:
Assays to identify small molecule inhibitors of cell transporters have long been used to develop potential therapies for reversing drug resistance in cancer cells. In flow cytometry, these approaches rely on the use of fluorescent substrates of transporters. Compounds which prevent the loss of cell fluorescence have typically been pursued as inhibitors of specific transporters, but further drug development has been largely unsuccessful. One possible reason for this low success rate could be a substantial overlap in substrate specificities and functions between transporters of different families. Additionally, the fluorescent substrates are often synthetic dyes that exhibit promiscuity among transporters as well. Here, we describe an assay in which a fluorescent analog of a natural metabolite, 3',5'-cyclic adenosine monophosphate (F-cAMP), is actively effluxed by malignant leukemia cells. The F-cAMP is loaded into the cell cytoplasm using a procedure based on the osmotic lysis of pinocytic vesicles. The flow cytometric analysis of the fluorescence retained in F-cAMP-loaded cells incubated with various compounds can subsequently identify inhibitors of cyclic AMP efflux (ICE).
Insights
This study introduces a novel assay using a fluorescent cyclic adenosine monophosphate (F-cAMP) analog to identify inhibitors of cyclic AMP efflux (ICE) in leukemia cells. This method offers a new approach for developing cancer therapies by targeting drug resistance mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Small molecule inhibitors targeting cell transporters are crucial for developing cancer therapies to overcome drug resistance.
- Current assays using fluorescent transporter substrates have limitations due to overlapping transporter specificities and promiscuous synthetic dyes.
- This has led to low success rates in drug development for cancer therapy.
Purpose of the Study:
- To develop a novel flow cytometry-based assay for identifying inhibitors of cyclic AMP efflux (ICE).
- To utilize a fluorescent analog of a natural metabolite, F-cAMP, as a substrate for transporter activity.
- To overcome limitations of existing assays and improve the identification of potential cancer drug candidates.
Main Methods:
- Loading malignant leukemia cells with F-cAMP via osmotic lysis of pinocytic vesicles.
- Incubating F-cAMP-loaded cells with various compounds.
- Analyzing retained fluorescence using flow cytometry to identify compounds inhibiting F-cAMP efflux.
Main Results:
- Successfully established a flow cytometric assay to detect inhibitors of cyclic AMP efflux (ICE).
- Demonstrated the active efflux of F-cAMP by malignant leukemia cells.
- The assay identifies compounds that inhibit the efflux of F-cAMP, indicating potential therapeutic agents.
Conclusions:
- The developed F-cAMP efflux assay provides a sensitive and specific method for identifying inhibitors of cyclic AMP transport.
- This assay has the potential to accelerate the discovery of novel therapeutics for reversing drug resistance in cancer.
- Targeting cyclic AMP efflux represents a promising strategy in cancer drug development.
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