Imaging reversal of multidrug resistance in living mice with bioluminescence:
            <i>MDR1</i>
            P-glycoprotein transports coelenterazine

Pichler, Andrea; Prior, Julie L.; Piwnica‐Worms, David

doi:10.1073/pnas.0304326101

Cited by 129 publications

(103 citation statements)

References 33 publications

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“…Overall, there was no significant difference between the two Hsp90 isoforms (a and h) in determining the sensitivity to both geldanamycin-based and purine-scaffold Hsp90 inhibitors. There was no significant differences in the expression of MDR1 or the efflux of rhodamine 123 in PU-H71 or carrier control-treated 293T stable cells (data not shown); thus, the decrease in complemented RL activity was not due to up-regulation/activation of MDR1 that reduces accumulation of coelenterazine (35).…”

Section: Resultsmentioning

confidence: 94%

Molecular Imaging of the Efficacy of Heat Shock Protein 90 Inhibitors in Living Subjects

et al. 2008

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Heat shock protein 90A (Hsp90A)/p23 and Hsp90B/p23 interactions are crucial for proper folding of proteins involved in cancer and neurodegenerative diseases. Small molecule Hsp90 inhibitors block Hsp90A/p23 and Hsp90B/p23 interactions in part by preventing ATP binding to Hsp90. The importance of isoform-selective Hsp90A/p23 and Hsp90B/p23 interactions in determining the sensitivity to Hsp90 was examined using 293T human kidney cancer cells stably expressing split Renilla luciferase (RL) reporters. Interactions between Hsp90A/p23 and Hsp90B/p23 in the split RL reporters led to complementation of RL activity, which was determined by bioluminescence imaging of intact cells in cell culture and living mice using a cooled charge-coupled device camera. The three geldanamycin-based and seven purinescaffold Hsp90 inhibitors led to different levels of inhibition of complemented RL activities (10-70%). However, there was no isoform selectivity to both classes of Hsp90 inhibitors in cell culture conditions. The most potent Hsp90 inhibitor, PU-H71, however, led to a 60% and 30% decrease in RL activity (14 hr) in 293T xenografts expressing Hsp90A/p23 and Hsp90B/p23 split reporters respectively, relative to carrier control-treated mice. Molecular imaging of isoform-specific Hsp90A/p23 and Hsp90B/p23 interactions and efficacy of different classes of Hsp90 inhibitors in living subjects have been achieved with a novel genetically encoded reporter gene strategy that should help in accelerating development of potent and isoformselective Hsp90 inhibitors. [Cancer Res 2008;68(1):216-26]

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Section: Resultsmentioning

confidence: 94%

Molecular Imaging of the Efficacy of Heat Shock Protein 90 Inhibitors in Living Subjects

et al. 2008

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“…Up-regulation of the multidrug-resistant P-glycoprotein by ER ligands and associated change in the signal by coelenterazine (11) were ruled out by different experiments in cell lysates and intact cells (Fig. 8, which is published as supporting information on the PNAS web site).…”

Section: Rluc Complementation As Sensors Of Er Ligand-induced Intramomentioning

confidence: 99%

An intramolecular folding sensor for imaging estrogen receptor–ligand interactions

Paulmurugan

Gambhir

2006

Proc. Natl. Acad. Sci. U.S.A.

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Strategies for high-throughput analysis of interactions between various hormones and drugs with the estrogen receptor (ER) are crucial for accelerating the understanding of ER biology and pharmacology. Through careful analyses of the crystal structures of the human ER (hER) ligand-binding domain (hER-LBD) in complex with different ligands, we hypothesized that the hER-LBD intramolecular folding pattern could be used to distinguish ER agonists from selective ER modulators and pure antiestrogens. We therefore constructed and validated intramolecular folding sensors encoding various hER-LBD fusion proteins that could lead to split Renilla͞firefly luciferase reporter complementation in the presence of the appropriate ligands. A mutant hER-LBD with low affinity for circulating estradiol was also identified for imaging in living subjects. Cells stably expressing the intramolecular folding sensors expressing wild-type and mutant hER-LBD were used for imaging ligand-induced intramolecular folding in living mice. This is the first hER-LBD intramolecular folding sensor suited for high-throughput quantitative analysis of interactions between hER with hormones and drugs using cell lysates, intact cells, and molecular imaging of small living subjects. The strategies developed can also be extended to study and image other important protein intramolecular folding systems.complementation ͉ optical imaging ͉ split reporters E strogens are responsible for the growth, development, and maintenance of the reproductive, skeletal, neuronal, and immune systems as well as and other systems. The physiological effects of these hormones are mediated by the estrogen receptor (ER), which is a ligand-inducible nuclear transcription factor (1). In the classical pathway of steroid hormone action, 17␤-estradiol (E2), hormones, and a variety of other estrogens bind to the ligandbinding domain (LBD) of ER and lead to its dimerization and subsequent binding to a specific regulatory sequence in the promoters of ER target genes known as the estrogen response elements (2, 3), which then trigger activation or repression of many downstream target genes (4). The deficiency or excess of estrogens can lead to various pathological conditions including osteoporosis and breast carcinomas (5), making ER a major cellular therapeutic target.Elegant crystallographic studies with ER-LBD have shown that conformation of helix 12 (H12) is critical in responses observed with various ER ligands (4, 6, 7). The conformation of H12 behaves as a ''molecular switch'' that either prevents or enhances ER from binding to an array of coactivator proteins, which then activates transcription of many downstream estrogen-regulated genes responsible for cell growth. Given the critical role of H12 in ER signaling, we reasoned that it may be feasible to develop an intramolecular ER folding sensor with specific split reporter complementation patterns to study ligand pharmacology based directly on the conformational changes of H12 in response to different ligands (Fig. 1).We used a spli...

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“…Renilla luciferase and, similar to Taxol, a substrate for Pgpmediated efflux (37). Coelenterazines are made up of a lipophilic, amine-containing heterocycle with physicochemical properties similar to other substrates of Pgp.…”

Section: Gate (6) Of Coelenterazine H (5) Coelenterazine H Is a Subsmentioning

confidence: 99%

Overcoming multidrug resistance of small-molecule therapeutics through conjugation with releasable octaarginine transporters

Dubikovskaya¹,

Thorne

Pillow³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

224

213

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Many cancer therapeutic agents elicit resistance that renders them ineffective and often produces cross-resistance to other drugs. One of the most common mechanisms of resistance involves P-glycoprotein (Pgp)-mediated drug efflux. To address this problem, new agents have been sought that are less prone to inducing resistance and less likely to serve as substrates for Pgp efflux. An alternative to this approach is to deliver established agents as molecular transporter conjugates into cells through a mechanism that circumvents Pgp-mediated efflux and allows for release of free drug only after cell entry. Here we report that the widely used chemotherapeutic agent Taxol, ineffective against Taxol-resistant human ovarian cancer cell lines, can be incorporated into a releasable octaarginine conjugate that is effective against the same Taxolresistant cell lines. It is significant that the ability of the Taxol conjugates to overcome Taxol resistance is observed both in cell culture and in animal models of ovarian cancer. The generality and mechanistic basis for this effect were also explored with coelenterazine, a Pgp substrate. Although coelenterazine itself does not enter cells because of Pgp efflux, its octaarginine conjugate does so readily. This approach shows generality for overcoming the multidrug resistance elicited by small-molecule cancer chemotherapeutics and could improve the prognosis for many patients with cancer and fundamentally alter search strategies for novel therapeutic agents that are effective against resistant disease.ovarian cancer ͉ Pgp ͉ Taxol ͉ imaging ͉ luciferase

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Imaging reversal of multidrug resistance in living mice with bioluminescence: MDR1 P-glycoprotein transports coelenterazine

Cited by 129 publications

References 33 publications

Molecular Imaging of the Efficacy of Heat Shock Protein 90 Inhibitors in Living Subjects

Molecular Imaging of the Efficacy of Heat Shock Protein 90 Inhibitors in Living Subjects

An intramolecular folding sensor for imaging estrogen receptor–ligand interactions

Overcoming multidrug resistance of small-molecule therapeutics through conjugation with releasable octaarginine transporters

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