Membrane and secretory proteins fold in the endoplasmic reticulum (ER), and misfolded proteins may be retained and targeted for ER-associated protein degradation (ERAD). To elucidate the mechanism by which an integral membrane protein in the ER is degraded, we studied the fate of the cystic fibrosis transmembrane conductance regulator (CFTR) in the yeast Saccharomyces cerevisiae. Our data indicate that CFTR resides in the ER and is stabilized in strains defective for proteasome activity or deleted for the ubiquitin-conjugating enzymes Ubc6p and Ubc7p, thus demonstrating that CFTR is a bona fide ERAD substrate in yeast. We also found that heat shock protein 70 (Hsp70), although not required for the degradation of soluble lumenal ERAD substrates, is required to facilitate CFTR turnover. Conversely, calnexin and binding protein (BiP), which are required for the proteolysis of ER lumenal proteins in both yeast and mammals, are dispensable for the degradation of CFTR, suggesting unique mechanisms for the disposal of at least some soluble and integral membrane ERAD substrates in yeast.

Bioluminescence imaging (BLI) is becoming indispensable to the study of transgene expression during development and, in many in vivo models of disease such as cancer, for high throughput drug screening in vitro. Because reaction of D-luciferin with firefly luciferase ( fLuc) produces photons of sufficiently long wavelength to permit imaging in intact animals, use of this substrate and enzyme pair has become the method of choice for performing BLI in vivo. We now show that expression of the ATP-binding cassette (ABC) family transporter ABCG2/BCRP affects BLI signal output from the substrate D-luciferin. In vitro studies show that D-luciferin is a substrate for ABCG2/BCRP but not for the MDR1 P-glycoprotein (ABCB1/Pgp), multidrug resistance protein 1 (MRP1/ ABCC1), or multidrug resistance protein 2 (MRP2/ABCC2). D-Luciferin uptake within cells is shown to be modulated by ABC transporter inhibitors, including the potent and selective ABCG2/BCRP inhibitor fumitremorgin C. Images of xenografts engineered to express transgenic ABCG2/BCRP, as well as xenografts derived from the human prostate cancer cell line 22Rv1 that naturally express ABCG2/BCRP, show that ABCG2/ BCRP expression and function within regions of interest substantially influence D-luciferin-dependent bioluminescent output in vivo. These findings highlight the need to consider ABCG2/BCRP effects during D-luciferin-based BLI and suggest novel high throughput methods for identifying new ABCG2/

HhAntag691 (GDC-0449), a low-molecular weight inhibitor of the tumor-promoting hedgehog (Hh) signaling pathway, has been used to treat medulloblastoma in animal models and has recently entered clinical trials for a variety of solid tumors. Here, we show that HhAntag691 inhibits multiple ATP-binding cassette (ABC) transporters. ATP-binding cassette transporters are within a family of membrane proteins, the overexpression of which is associated with multidrug resistance, a major impediment to successful cancer treatment. HhAntag691 is a potent inhibitor of two ABC transporters, ABCG2/BCRP and ABCB1/Pgp, and is a mild inhibitor of ABCC1/MRP1. In ABCG2-overexpressing HEK293 cells, HhAntag691 increased retention of the fluorescent ABCG2 substrate BODIPY-prazosin and resensitized these cells to mitoxantrone, an antineoplastic ABCG2 substrate. In Madin-Darby canine kidney II cells engineered to overexpress Pgp or MRP1, HhAntag691 increased the retention of calcein-AM and resensitized them to colchicine. HhAntag691 also resensitized human non-small cell lung carcinoma cells NCI-H460/par and NCI-H460/MX20, which overexpress ABCG2 in response to mitoxantrone, to mitoxantrone, and to topotecan or SN-38. The IC(50) values of HhAntag691 for inhibition of ABCG2 and Pgp were approximately 1.4 and approximately 3.0 microM, respectively. Because ABC transporters are highly expressed at the blood-brain barrier and on many tumor cells, they contribute significantly to treatment failure of many types of cancer, particularly of those within the neuraxis. In addition to its effect on Hh signaling, the ability of HhAntag691 and related compounds to inhibit two key ABC transporters could contribute to their effectiveness in treating malignancies.

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ABCG2 is a member of the ATP-binding cassette (ABC) family of transporters, the overexpression of which is associated with tumor resistance to a variety of chemotherapeutic agents. Accordingly, combining ABCG2 inhibitor(s) with chemotherapy has the potential to improve treatment outcome. To search for clinically useful ABCG2 inhibitors, a bioluminescence imaging (BLI)-based assay was developed to allow highthroughput compound screening. This assay exploits our finding that D-luciferin, the substrate of firefly luciferase ( fLuc), is a specific substrate of ABCG2, and ABCG2 inhibitors block the export of D-luciferin and enhance bioluminescence signal by increasing intracellular D-luciferin concentrations. HEK293 cells, engineered to express ABCG2 and fLuc, were used to screen the Hopkins Drug Library that includes drugs approved by the Food and Drug Administration (FDA) as well as drug candidates that have entered phase II clinical trials. Forty-seven compounds showed BLI enhancement, a measure of anti-ABCG2 activity, of z5-fold, the majority of which were not previously known as ABCG2 inhibitors. The assay was validated by its identification of known ABCG2 inhibitors and by confirming previously unknown ABCG2 inhibitors using established in vitro assays (e.g., mitoxantrone resensitization and BODIPY-prazosin assays). Glafenine, a potent new inhibitor, also inhibited ABCG2 activity in vivo. The BLI-based assay is an efficient method to identify new inhibitors of ABCG2. As they were derived from a FDA-approved compound library, many of the inhibitors uncovered in this study are ready for clinical testing.