Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Dec 25;284(52):36367-36376.
doi: 10.1074/jbc.M109.049734. Epub 2009 Oct 21.

Identification of anabolic selective androgen receptor modulators with reduced activities in reproductive tissues and sebaceous glands

Azriel Schmidt  1 , Shun-Ichi Harada  2 , Donald B Kimmel  2 , Chang Bai  2 , Fang Chen  2 , Su Jane Rutledge  2 , Robert L Vogel  2 , Angela Scafonas  2 , Michael A Gentile  2 , Pascale V Nantermet  2 , Sheila McElwee-Witmer  2 , Brenda Pennypacker  2 , Patricia Masarachia  2 , Soumya P Sahoo  3 , Yuntae Kim  4 , Robert S Meissner  4 , George D Hartman  4 , Mark E Duggan  4 , Gideon A Rodan  2 , Dwight A Towler  2 , William J Ray  2
Affiliations

Identification of anabolic selective androgen receptor modulators with reduced activities in reproductive tissues and sebaceous glands

Azriel Schmidt et al. J Biol Chem. .

Abstract

Androgen replacement therapy is a promising strategy for the treatment of frailty; however, androgens pose risks for unwanted effects including virilization and hypertrophy of reproductive organs. Selective Androgen Receptor Modulators (SARMs) retain the anabolic properties of androgens in bone and muscle while having reduced effects in other tissues. We describe two structurally similar 4-aza-steroidal androgen receptor (AR) ligands, Cl-4AS-1, a full agonist, and TFM-4AS-1, which is a SARM. TFM-4AS-1 is a potent AR ligand (IC(50), 38 nm) that partially activates an AR-dependent MMTV promoter (55% of maximal response) while antagonizing the N-terminal/C-terminal interaction within AR that is required for full receptor activation. Microarray analyses of MDA-MB-453 cells show that whereas Cl-4AS-1 behaves like 5alpha-dihydrotestosterone (DHT), TFM-4AS-1 acts as a gene-selective agonist, inducing some genes as effectively as DHT and others to a lesser extent or not at all. This gene-selective agonism manifests as tissue-selectivity: in ovariectomized rats, Cl-4AS-1 mimics DHT while TFM-4AS-1 promotes the accrual of bone and muscle mass while having reduced effects on reproductive organs and sebaceous glands. Moreover, TFM-4AS-1 does not promote prostate growth and antagonizes DHT in seminal vesicles. To confirm that the biochemical properties of TFM-4AS-1 confer tissue selectivity, we identified a structurally unrelated compound, FTBU-1, with partial agonist activity coupled with antagonism of the N-terminal/C-terminal interaction and found that it also behaves as a SARM. TFM-4AS-1 and FTBU-1 represent two new classes of SARMs and will allow for comparative studies aimed at understanding the biophysical and physiological basis of tissue-selective effects of nuclear receptor ligands.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Structure and binding affinities of Cl-4AS-1 and TFM-4AS-1. A, structure and 5α-reductase type I, type II, and AR binding IC50 values (43). B, ligand displacement assays with native AR (apo-AR) and truncated rhARLBD from yeast (AR-LBD). Binding activities from reactions in the absence of unlabeled ligand competition were identified as maximal binding. The nonspecific binding was determined in the presence of 500-fold excess unlabeled ligand (35).
FIGURE 2.
FIGURE 2.
In vitro activities of CL-4AS-1 and TFM-4AS-1. A, TFM-4AS-1 is a partial agonist in the MMTV-luciferase transactivation assay in AR+ MDA-MB-453 cells. Left, dose response curves of R1881, TFM-4AS-1, and Cl-4AS-1 reveal the partial agonism of TFM-4AS-1. Right, TFM-4AS-1 partially antagonizes the effect of 1 nm R1881 relative to the antagonist bicalutamide. B, MMP-1 promoter repression assay in 22RV1 cells. DHT and Cl-4AS-1 repress promoter activity whereas hydroxyflutamide and TFM-4AS-1 do not. C, AR N-terminal domain/C-terminal domain mammalian two-hybrid assay in CV1 cells. Left, in an agonist mode, the graph shows the maximal activities of hydroxyflutamide, bicalutamide Cl-4AS-1 and TFM-4AS-1 at doses of 1 μm relative to 1 nm R1881; right, antagonist action of TFM-4AS-1, the graph expresses the maximum fold induction relative to DMSO. Note that TFM-4AS-1 antagonizes the effects of Cl-4AS-1. All error bars represent the S.D. of >3 measurements; data are representative of >3 experiments.
FIGURE 3.
FIGURE 3.
Microarray analyses of TFM-4AS-1 gene transcription effects in MDA-MB-453 cells. A, one-dimensional false color agglomerative cluster map of RNA expression values for genes significantly responsive to 200 nm DHT (see “Materials and Methods”) after 18 h of treatment. Magenta indicates up-regulation and cyan down-regulation relative to DMSO alone controls with color intensity proportional to fold change; log10 scale bar upper right. B, gene expression values for all 294 RNAs significantly regulated by 200 nm DHT, expressed as a percentage of 200 nm DHT values and averaged (± S.D.). EC50 values were calculated and provided for DHT and TFM-4AS-1. Emax for TFM-4AS-1 is significantly different from DHT and Cl-4AS-1 (p = 1 × 10−13 and 1 × 10−6, respectively, ANOVA). C, histogram depicting the distribution of RNA expression values that vary from the mean for 20 nm DHT expressed by S.D. D, microarray data for the RNAs encoding UGT2B7 and FGF18 illustrate the differential responsiveness of individual genes to DHT and SARM treatment. E, quantitative RT-PCR data showing the induction of UGT2B7 and FGF18 by 200 nm DHT, 200 nm DHT and bicalutamide (10 μm), 200 nm DHT (6 h treatment), or 200 nm DHT and the translation inhibitor 10 μg/ml cycloheximide (6 h of treatment).
FIGURE 4.
FIGURE 4.
TFM-4AS-1, but not Cl-4AS-1 exhibits SARM activities in OVX rats. OVX rats were treated with AR ligands at the indicated doses (mpk) plus alendronate (5.6 μg/week) and subjected to double-calcein labeling. A, periosteal bone formation measured as bone formation rate (percent double-labeled surface/total bone surface. B, mineral apposition rate measuring rate of bone growth in double-labeled regions (microns per day). C, calculated annual bone formation rate (mm2/mm/year). D, uterine wet weight as a measure of androgen effects on reproductive organs. E, anabolic effects of DHT and TFM-4AS-1 in muscle. F, effects of DHR and TFM-4AS-1 on adiposity. Lean and fat mass was measured in OVX rats. Animals were scanned by dual x-ray absorptiometry before and after 6 weeks of dosing with 3 mpk DHT or 10 mpkTFM-4AS-1. Values for lean mass (E) and fat mass (F) are expressed as mean change from baseline. Uterine wet weight was determined at the end of the experiment. All values are ± S.E., n = 10–16. *, different from ovariectomy (OVX) alone (p < 0.05, Kruskal-Wallis).
FIGURE 5.
FIGURE 5.
Anabolic doses of TFM-4AS-1 have reduced effects on the formation of sebaceous glands in skin and the growth of prostate and seminal vesicles. OVX rats were dosed for 24 days with DHT or TFM-4AS-1 at fully anabolic exposures: A, bone formation rate determined from double-calcein labeling; B, mean sebaceous gland area determined by quantitative histomorphometry of dorsal back skin (n>3 fields per specimen, 10–16 per group); and C, uterine wet weight. All values are ± S.E., n = 10–16. *, different from vehicle (p < 0.05, Kruskal-Wallis). D, effects of Cl-4AS-1 and TFM-4AS-1 on rat prostate. Intact or castrated (ORX) rats treated for 7 days with the indicated compound (10 mpk) and prostate wet weights were measured and expressed as percent of body weight (mean of 8 animals, ± S.E.). Note that Cl-4AS-1 is less effective at reducing prostate wet weight, and more effective at restoring prostate weight in ORX rats than is TFM-4AS-1. E, TFM-4AS-1 and bicalutamide antagonize the stimulations of SV growth by DHT. Seminal vesicle weight in ORX or mock-castrated (SHAM) animals treated for 14 days with a DHT pellet designed to provide DHT at a constant level regardless of gonadotropins or 5α-reductase inhibition. Bicalutamide fully inhibited the effects of DHT whereas TFM-4AS-1 partially inhibited DHT at 30 mg/kg/day. All values are ± S.E., n = 9. *, indicates different from control (p < 0.05, Kruskal-Wallis). Control represents vehicle-treated ORX animals with only DHT pellets.
FIGURE 6.
FIGURE 6.
Microarray analysis of TFM-4AS-1 in rat prostate tissue. A, agglomerative 1-dimensional false-color heatmap (as in Fig. 2) showing the gene expression effects of DHT and TFM-4AS-1 in prostate tissue from castrated males 6 and 24 h after a single injection. Note that TFM-4AS-1 alters the expression of some but not all DHT-regulated genes. Gene expression values are the mean of three measurements each from pooled RNA from three specimens. B, actions of DHT and TFM-AAS-1 on gene expression in prostate was measured by quantitative RT-PCR data for four selected genes, uterocalin (lipocalin 2, Lcn2), FGFR4, IGF-1, and cyclin D1. The activities were normalized to cyclophilin. Nine days after surgery, ORX rats were daily treated with vehicle, 3 mg/kg DHT, or 10 mg/kg TFM-4AS-1 (n = 6/group). Prostate samples were collected at 0.25, 1, 4, and 7 days of dosing. Note that over time TFM-4AS-1 altered uterocalin and FGFR4 RNAs similarly to DHT, whereas it had no effect on cyclin D1 and IGF-1 at any time point. Values are normalized to cyclophilin levels measured within the same reaction and are the mean of six measurements from individual animals ± S.D.; *, different than vehicle treatment values (p < 0.05 ANOVA).
FIGURE 7.
FIGURE 7.
SARM properties of a novel non-steroidal AR ligand, FTBU-1. A, chemical structure, 5α-reductase inhibition, and AR binding IC50 values. B, percent activity relative to 1 nm R1881 in the MMTV-luciferase assay in MDA-MB-453 cells and in the AR NTD/CTD interaction assay in CV1 cells (mean of >3 independent measurements). C, summary of microarray results comparing 1 μm FTBU-1 to 200 nm DHT in MDA-MB-453 cells as in Fig. 2. Note that ∼35% of FTBU-1-regulated RNAs have changes in gene expression values more than 2 S.D. below the mean for 200 nm DHT. D, tissue-selective effects of FTBU-1 in ovariectomized rats treated for 24 days. FTBU-1 was administered at the given doses once daily, and bone formation rate was measured by double calcein labeling. Compound levels in plasma were measured in three separate animals at 0.25, 1, 2, 4, 8, and 24 h, and the area under the curve (AUC) was determined to allow comparison of 24-h exposures. Shown are mean values of 10–16 animals ± S.E.; *, significantly different than ovariectomy + vehicle alone (p < 0.05, Kruskal-Wallis).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bachmann G. A. (1999) Am. J. Obstetrics Gynecol. 180, S308–311 - PubMed
    1. Bain J. (2007) Clin. Interventions Aging 2, 567–576 - PMC - PubMed
    1. Vermeulen A. (2003) Ann. Endocrinol. 64, 109–114 - PubMed
    1. Harman S. M., Metter E. J., Tobin J. D., Pearson J., Blackman M. R. (2001) J. Clin. Endocrinol. Metab. 86, 724–731 - PubMed
    1. Valenti G., Ceresini G., Maggio M. (2007) Minerva Ginecologica 59, 43–49 - PubMed