Discovery of Compound A--a selective activator of the glucocorticoid receptor with anti-inflammatory and anti-cancer activity.

Glucocorticoids are among the most effective anti-inflammatory drugs, and are widely used for cancer therapy. Unfortunately, chronic treatment with glucocorticoids results in multiple side effects. Thus, there was an intensive search for selective glucocorticoid receptor (GR) activators (SEGRA), which retain therapeutic potential of glucocorticoids, but with fewer adverse effects. GR regulates gene expression by transactivation (TA), by binding as homodimer to gene promoters, or transrepression (TR), via diverse mechanisms including negative interaction between monomeric GR and other transcription factors. It is well accepted that metabolic and atrophogenic effects of glucocorticoids are mediated by GR TA. Here we summarized the results of extensive international collaboration that led to discovery and characterization of Compound A (CpdA), a unique SEGRA with a proven "dissociating" GR ligand profile, preventing GR dimerization and shifting GR activity towards TR both in vitro and in vivo. We outlined here the unusual story of compound's discovery, and presented a comprehensive overview of CpdA ligand properties, its anti-inflammatory effects in numerous animal models of inflammation and autoimmune diseases, as well as its anti-cancer effects. Finally, we presented mechanistic analysis of CpdA and glucocorticoid effects in skin, muscle, bone, and regulation of glucose and fat metabolism to explain decreased CpdA side effects compared to glucocorticoids. Overall, the results obtained by our and other laboratories underline translational potential of CpdA and its derivatives for treatment of inflammation, autoimmune diseases and cancer.


Colony formation in soft agar
The colony-forming assay was performed as described previously [45]. Forty-eight hours after plating, cells were treated with CpdA, or vehicle (0.1% ethanol) for 2 wk. Complete medium with CpdA was changed twice weekly. Stably infected LNCaP-GR and LNCaP-V cells were cultured in the presence of 6 μg/μL blasticidin to maintain the selection. Images of entire wells were taken and colonies with diameter of > 50 μm were counted using AxioVision LE Rel. 4.5 software (Carl Zeiss MicroImaging, Inc.). Each experimental group consisted of six wells.

RNA extraction
RNA was isolated from Granta and LNCaP cells treated with Dex, CpdA, CpdA, or vehicle (0.1% ethanol and/or DMSO) for 8-16 h using TRIzol reagent according to the manufacturer's protocol. 1 ul aliquot was used for RNA fluorometric quantification (Qubit, Invitrogen) and remainder stored at −80°C until further analysis.

Gene profile analysis
The Illumina Human HT12 Expression BeadChips (DNA microarray) were utilized to profile gene expression in Granta-519 and LNCaP-GR cells. The Illumina BeadArray Reader and BeadScan software were used to scan and extract raw intensity values. The complete array results and experimental details were deposited to NCBI, the access number is GSM1815466 (Granta 519) and GSM1823672 (for LNCaP-GR).

Quantitative RT-PCR
First-strand cDNA was synthesized from 1 μg total RNA using random hexamer primers and 1 μL Superscript II Reverse Transcriptase (Invitrogen). Samples were stored at -20°C until analysis. All gene specific primers were designed using Oligo 7.0 and synthesized by Integrated DNA Technologies (Coralville, IA, USA) and are listed in Supplementary Table 3. Primers were optimized for appropriate primer concentration using a concentration gradient (150,200,250,300, and 350 nM) and validated using a 10-log dilution curve. A working solution of cDNA was prepared by diluting samples 1:10 with DEPCtreated water. Five microliters of cDNA working solution was added to a 25 μL Power SYBR Green Master Mix (Applied Biosciences), 1 μg of cDNA and 500 nM of each specific forward and reverse primer (Supplementary Table 3). Quantitative real-time PCR analysis was carried out using Bio-Rad iQ5 detection system and software (Bio-Rad Laboratories, Hercules, CA, USA). Standard thermocycler conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 30 sec, and 72°C for 30 sec. Relative fold change in target mRNAs was quantified using the ΔΔCt method where ΔΔCt was determined by subtracting the average control ΔCt from the ΔCt of the sample. All reverse-transcribed cDNA samples were assayed in triplicate for each gene, and melt curve analyses were performed to ensure specificity of amplification. Melt curve analysis was carried out for 81 cycles with 0.5°C temperature increase from 55°C to 95°C. RPL27 gene was used as the reference gene for the experiments.

Mouse studies
Animals were maintained under the protocols approved by the Northwestern University and Blokhin Cancer Research Center Institutional Animal Care and Use Committees (IACUC). Female athymic nude mice (Charles River, Wilmington, Massachusetts) at 6-8 weeks of age were injected subcutaneously with 1.2 × 10 6 PC3 or 1 × 10 7 Granta cells in 200 ul BD Matrigel in the right flank to generate solid human prostate cancer or lymphoma xenografts. When tumors reached 50 mm3, mice received CpdA, Dex or PBS intraperitoneally (i.p.) 3 times/week for 35 days. Tumor volume was determined twice per week by measuring tumor diameter with electronic calipers. Reverse primer 5′-aacaccgaaatgtatcagaccc -3′ 5′-tgtccagcttaacggaaacca -3′