Method of treating disease and selectively modulating transcriptional regulation by a glucocorticoid receptor by administering aclacinomycin and dexamethasone

#Not Applicable

#GR is a nuclear receptor (NR), a family of intracellular ligand-regulated transcription factors (McMaster, A. et al., Exp Physiol 92:299-309 (2007)). GR is expressed ubiquitously in humans. Activation of GR by hormones such as cortisol causes nuclear translocation, interaction with co-regulators, and binding to specific genomic sites to regulate transcription (Yamamoto, K. R. et al., Cold Spring Harbor Symposia on Quantitative Biology 63:587-598 (1998); So, A. Y. et al., PLoS Genet 3:e94 (2007)). This mediates the broad systemic effects of GR signaling and underlies glucocorticoid treatment of diverse immune-mediated diseases such as asthma and rheumatoid arthritis. However, severe dose-limiting side effects occur, including osteoporosis, muscle wasting, and diabetes (Schacke, H. et al., Pharmacology & Therapeutics 96:23-43 (2002)). No existing drugs selectively induce beneficial effects of GR.

#Beneficial and harmful effects of glucocorticoids are due to selective activation or repression of particular genes by GR. This selectivity is in part based on tissue-specific factors and cross-talk pathways (Kassel, O. et al., Molecular and Cellular Endocrinology 275:13-29 (2007)). For example, GR reduces the expression of certain inflammatory cytokines by inhibiting other transcription factors such as AP-1 and NF-Kb (Smoak, K. A. et al., Mech Ageing Dev 125:697-706 (2004)). Conversely, GR increases expression of RANKL, a gene co-regulated by the Vitamin D receptor that activates bone resorption by osteoclasts (Hofbauer, L. C. et al., Endocrinology 140:4382-4389 (1999); Kim, S. et al., Mol. Cell. Biol. 26:6469-6486 (2006)). Controlling GR activity at certain tissues or promoters has profound therapeutic implications.

#FIG. 1 shows measurement of activity from four promoters simultaneously with fluorescent reporters.

#A-B. Fluorescence microscopy of A549 cells transfected with pFKBP5-OFP and treated with dex as indicated.

#C. The peak fluorescent output of A549 cells transfected with the indicated reporter constructs and treated with dex as indicated was measured using a monochrometer-based plate reader.

#We have developed an assay in which simultaneous measurement of four promoter-fluorescent protein (FP) reporter constructs quantifies promoter activity. In a high-throughput screen of a library of natural products and FDA-approved compounds, we identified multiple classes of compounds that modulate GR signaling at endogenous target genes. Importantly, we discovered several compounds that tuned GR responses, i.e. those that caused GR to regulate only a subset of its normal spectrum of target genes. We also identified molecules that act on GR signaling via heterologous pathways, and a compound with cell specific effects. This work has important implications for drug discovery and for genetic studies to elucidate factors and signaling pathways that regulate GR transcription.

#The Power of a Multi-Promoter Readout

#Transcriptional regulation is usually measured in high throughput with a single experimental promoter driving an enzyme-based reporter or FP (Necela, B. M. et al., Steroids 68:341-350 (2003); Suzuki, T. et al., Phytomedicine 13:401-411 (2006); Sonneveld, E. et al., Toxicol. Sci. 83:136-148 (2005)). Although laser-based systems and high content microscopy have been proposed as general tools to measure multiple cellular processes, they have not been widely applied for multiplexed measurement of transcription (Auld, D. S. et al., Methods in Enzymology (Academic Press, Vol. Volume 414, pp. 566-589 (2006)). The multiplexed GR reporter assay facilitates the identification of compounds (e.g. forskolin) with relatively modest effects on NR signaling. Refinement of weak hits from screening small, biologically active libraries is an attractive alternative to large-scale screens. The system can also be adapted to use mixtures of cell types harboring distinguishable FP-reporters and thereby identify cell-selective modulators such as Emc. At this time, the ease of measurement and low variability of FP expression present advantages over screening systems that directly measure mRNA levels (Hieronymus, H. et al., Cancer Cell 10:321-330 (2006)).