Identification of Anabolic Selective Androgen Receptor Modulators with Reduced Activities in Reproductive Tissues and Sebaceous Glands

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 5␣-dihydrotestosterone (DHT), TFM-4AS-1 acts as a geneselective 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.Androgens, primarily testosterone (T) 7 and its more potent derivative, 5␣-dihydrotestosterone (DHT), induce male reproductive physiology and secondary sexual traits such as facial hair and deepened voice. Additionally, in both genders androgens regulate bone and muscle anabolism, adipose mass, lipoprotein metabolism, and behavior (1-3). Androgens decline with age in both men and women (4), which contributes to age-related bone and muscle loss and increases in fat mass (5). Several studies report low testosterone as a risk factor for age-related diseases including osteoporosis (6), sarcopenia (7), atherosclerosis (8), type II diabetes/metabolic syndrome and obesity (9), cognitive impairment (10), and depression (11). Restoring androgens to youthful levels could thus slow unfavorable changes in body composition and improve mood, motivation, and general health. Unfortunately, current androgens induce male secondary sexual traits such as acne and hirsutism, an effect known as virilization, (12) and pose concerns related to unwanted effects in the prostate and other reproductive organs (13-15). Therefore, androgens are limited by concerns over safety and tolerability.Androgens exert their physiological effects by activating the androgen receptor ...

Discovery of the Selective Androgen Receptor Modulator MK-0773 Using a Rational Development Strategy Based on Differential Transcriptional Requirements for Androgenic Anabolism Versus Reproductive Physiology

“…Female rats were ovariectomized (OVX) at 12 weeks, and test compound administration began at least 8 weeks post-surgery for 24 days (20). Animals were randomized into groups (n ϭ 10 -16), and compounds dissolved in propylene glycol were administered by subcutaneous injection.…”

“…Measurements of sebaceous gland size were assessed by using a Nikon Eclipse E1000M microscope/digital camera system and BioQuant software. Tibiae were collected for RNA processing and histological studies (20). Data were analyzed by one way analysis of variance, Fisher's protected least significant difference (Statview, Version 5.0).…”

“…Double calcein labeling was used to identify regions of periosteum undergoing mineralization in aged, long term post-OVX rats that were also treated with alendronate (5.6 g/week) to reduce bone turnover. Bone formation rate (BFR), the most reliable osteoanabolic endpoint, is calculated by multiplying the extent of periosteal bone surface with double label by the mineral apposition rate (distance between labels/time between labels) (20).…”

“…Chemistry design targeted the generation of compounds that had high affinity binding to AR natively expressed in human MDA-MB-453 breast cancer cells (20,28). Compounds that exhibited apparent binding affinity Ͻ300 nM with little or no displacement of radioligands from related steroid hormone receptors (estrogen receptor ␣, progesterone receptor, or glucocorticoid receptor at 2 M) were tested in the TAMAR assay for their ability to stimulate transcription from the transiently transfected modified MMTV reporter gene (20,28). This assay was chosen because in pilot experiments, TAMAR activities accurately represented the levels of agonism or antagonism of known AR ligands that matched their activities in animal studies or the clinic.…”

“…Preclinical proof of concept for an anabolic SARM with reduced androgenization was established in animal models by several SARMs such as TZP-4238, S-40503, S-1, S-4, LGD2226, LGD2941, JNJ-28330835, and JNJ-37654032 (12)(13)(14)(15)(16)(17)(18)(19). We developed a proof-of-concept SARM, TFM-4AS-1, that increases periosteal bone formation, cortical bone mass, and lean body mass as effectively as DHT but with markedly reduced effects on uterus, prostate, and the pilosebaceous unit (PSU) (20). However, as each SARM has a unique in vivo profile that is rarely linked to a specific in vitro activity profile, the relationship between transcriptional activity and physiological effect has not been established.…”