A High-Content Assay Strategy for the Identification and Profiling of ABCG2 Modulators in Live Cells

Antczak, Christophe; Wee, Boyoung; Radu, Constantin; Bhinder, Bhavneet; Holland, Eric C.; Djaballah, Hakim

doi:10.1089/adt.2013.521

Cited by 12 publications

(4 citation statements)

References 39 publications

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“…The library screened combines 6,208 chemicals obtained from MicroSource, Prestwick, Tocris, and other commercial sources as previously described [18]. The MicroSource library contains 2,000 known drugs, natural products, and other bioactive components, such as enzyme inhibitors, receptor blockers, membrane-active compounds, and cellular toxins.…”

Section: Methodsmentioning

confidence: 99%

A 1536-Well Fluorescence Polarization Assay to Screen for Modulators of the MUSASHI Family of RNA-Binding Proteins

Minuesa¹,

Antczak²,

Shum³

et al. 2014

CCHTS

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RNA-binding proteins (RBPs) can act as stem cell modulators and oncogenic drivers, but have been largely ignored by the pharmaceutical industry as potential therapeutic targets for cancer. The MUSASHI (MSI) family has recently been demonstrated to be an attractive clinical target in the most aggressive cancers. Therefore, the discovery and development of small molecule inhibitors could provide a novel therapeutic strategy. In order to find novel compounds with MSI RNA binding inhibitory activity, we have developed a fluorescence polarization (FP) assay and optimized it for high throughput screening (HTS) in a 1536-well microtiter plate format. Using a chemical library of 6,208 compounds, we performed pilot screens, against both MSI1 and MSI2, leading to the identification of 7 molecules for MSI1, 15 for MSI2 and 5 that inhibited both. A secondary FP dose-response screen validated 3 MSI inhibitors with IC50 below 10μM. Out of the 25 compounds retested in the secondary screen only 8 demonstrated optical interference due to high fluorescence. Utilizing a SYBR-based RNA electrophoresis mobility shift assay (EMSA), we further verified MSI inhibition of the top 3 compounds. Surprisingly, even though several aminoglycosides were present in the library, they failed to demonstrate MSI inhibitor activity challenging the concept that these compounds are pan-active against RBPs. In summary, we have developed an in vitro strategy to identify MSI specific inhibitors using an FP HTS platform, which will facilitate novel drug discovery for this class of RBPs.

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Section: Methodsmentioning

confidence: 99%

A 1536-Well Fluorescence Polarization Assay to Screen for Modulators of the MUSASHI Family of RNA-Binding Proteins

Minuesa¹,

Antczak²,

Shum³

et al. 2014

CCHTS

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“…We investigated the sensitivities of the human glioblastoma cell line U87MG and a derived line expressing the ABCG2 transporter U87MG-ABCG2, 25–26 against HHT, DHT, DHHT, and BDHT ( Table 1 ). We found U87MG cells to be sensitive to HHT, DHT, DHHT, and BDHT with IC 50 values of 40, 30, 40, and 90 nM, respectively; similarly, the U87MG-ABCG2 cells were found to as sensitive to the same compounds with IC 50 values of 50, 30, 40, and 90 nM, respectively.…”

Section: Resultsmentioning

confidence: 99%

Antiproliferative Activity of Cephalotaxus Esters: Overcoming Chemoresistance in Cancer Therapy

Yong-Gonzalez,

Radu,

Calder

et al. 2023

Preprint

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Omacetaxine, a semisynthetic form of Homoharringtonine (HHT), was approved for the treatment of chronic myeloid leukemia (CML). Previously, we published the synthesis of this natural alkaloid and three of its derivatives: deoxyharringtonine (DHT), deoxyhomoharringtonine (DHHT), and bis(demethyl)-deoxyharringtonine (BDHT); and reported on its refractory activity against the HL-60/RV+ cells over-expressing P-glycoprotein 1 (MDR1). In this study, we explored the extent of this resistance by first expanding the panel of established cell lines and second, using a panel of 21 leukemia patient derived primary cells. Here, we report a consistent resistance to HTT in K562 derived cells and in MES-SA/MX2 derived cells resistant to mitoxanthrone; all of them over-express MDR1, while we found U87MG-ABCG2 and H69AR cells to be very sensitive to HTT. In contrast, DHT, DHHT, and BDHT seemingly overcome this resistance due to the changes made to the acyl chain of HTT rendering the derivatives less susceptible to efflux. Surprisingly, the leukemia primary cells were very sensitive to HHT and its derivatives with low nanomolar potencies, followed by a new class of CDC7 kinase inhibitors, the anthracycline class of topoisomerase inhibitors, the DNA intercalator actinomycin-D, and the vinca alkaloid class of microtubule inhibitors. The mechanism of cell death induced by HTT and DHHT was found to be mediated via Caspase 3 cleavage leading to apoptosis. Taken together, our results confirm that HHT is a substrate for MDR1. It opens the door to a new opportunity to clinically evaluate HHT and its derivatives for the treatment of AML and other cancers.

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“…Previous studies have shown that the expression of the ABCG2 protein in various cancer cell lines is mediated by the activation of c-jun N-terminal kinase, mitogen-activated protein kinases, phosphate and tensin homolog, epidermal growth factor receptor and human epidermal growth factor (EGFR) [ 35 ]. The in vitro expression of ABCG2, at the transcription and at post-translational level, was significantly decreased in isogenic U87MG human glioblastoma cell lines by PD153035 (an EGFR antagonist) [ 36 ]. The cariprazine-induced decrease in ABCG2 protein levels could result from the rapid internalization and degradation via lysosomes and/or proteasomes.…”

Section: Discussionmentioning

confidence: 99%

Cariprazine, A Dopamine D2/D3 Receptor Partial Agonist, Modulates ABCG2-Mediated Multidrug Resistance in Cancer

Hussein

Ashby

Amawi

et al. 2018

Cancers

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Multidrug resistance (MDR) is a continuing clinical problem that limits the efficacy of chemotherapy in cancer. The over expression of the ATP-binding cassette (ABC) family G2 (ABCG2) transporter is one of the main mechanisms that mediates MDR in cancer. Molecular modeling data indicated that cariprazine, a dopamine D2/D3 receptor partial agonist, had a significant binding affinity for ABCG2 transporter with a Glide XP score of −6.515. Therefore, in this in vitro study, we determined the effect of cariprazine on MDR resulting from the overexpression of ABCG2 transporters. Alone, cariprazine, at concentrations up to 20 μM, did not significantly decrease cell viability. Cariprazine, at concentrations ranging from 1 to 10 μM, did not significantly alter the cytotoxicity of mitoxantrone (MX) in the parental non-small cell cancer cell line, H460 and colon cancer cell S1. However, cariprazine (1–20 μM) significantly enhanced the efficacy of ABCG2 substrate antineoplastic drug MX in the ABCG2-overexpressing MDR cell line, H460-MX20 and S1M1-80, by reducing the resistance fold from 28 to 1 and from 93 to 1.33, respectively. Cariprazine, in a concentration-dependent (1–20 μM), significantly increased the intracellular accumulation of Rhodamine 123 in S1M1-80. Interestingly, 10 or 20 μM of cariprazine significantly decreased the expression levels of the ABCG2 protein in the colon and lung cancer cell lines, suggesting that cariprazine inhibits both the function and expression of ABCG2 transporters at nontoxic concentrations. Overall, our results suggest that cariprazine, via several distinct mechanisms, can resensitize resistant cancer cells to mitoxantrone.

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A High-Content Assay Strategy for the Identification and Profiling of ABCG2 Modulators in Live Cells

Cited by 12 publications

References 39 publications

A 1536-Well Fluorescence Polarization Assay to Screen for Modulators of the MUSASHI Family of RNA-Binding Proteins

A 1536-Well Fluorescence Polarization Assay to Screen for Modulators of the MUSASHI Family of RNA-Binding Proteins

Antiproliferative Activity of Cephalotaxus Esters: Overcoming Chemoresistance in Cancer Therapy

Cariprazine, A Dopamine D2/D3 Receptor Partial Agonist, Modulates ABCG2-Mediated Multidrug Resistance in Cancer

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