AR-mediated Transcriptional Activation Research Articles

Lipid nanoparticles to silence androgen receptor variants for prostate cancer therapy.

Advanced-stage prostate cancer remains an incurable disease with poor patient prognosis. There is an unmet clinical need to target androgen receptor (AR) splice variants, which are key drivers of the disease. Some AR splice variants are insensitive to conventional hormonal or androgen deprivation therapy due to loss of the androgen ligand binding domain at the C-terminus and are constitutively active. Here we explore the use of RNA interference (RNAi) to target a universally conserved region of all AR splice variants for cleavage and degradation, thereby eliminating protein level resistance mechanisms. To this end, we tested five siRNA sequences designed against exon 1 of the AR mRNA and identified several that induced potent knockdown of full-length and truncated variant ARs in the 22Rv1 human prostate cancer cell line. We then demonstrated that 2'O methyl modification of the top candidate siRNA (siARvm) enhanced AR and AR-V7 mRNA silencing potency in both 22Rv1 and LNCaP cells, which represent two different prostate cancer models. For downstream in vivo delivery, we formulated siARvm-LNPs and functionally validated these in vitro by demonstrating knockdown of AR and AR-V7 mRNA in prostate cancer cells and loss of AR-mediated transcriptional activation of the PSA gene in both cell lines following treatment. We also observed that siARvm-LNP induced cell viability inhibition was more potent compared to LNP containing siRNA targeting full-length AR mRNA (siARfl-LNP) in 22Rv1 cells as their proliferation is more dependent on AR splice variants than LNCaP and PC3 cells. The in vivo biodistribution of siARvm-LNPs was determined in 22Rv1 tumor-bearing mice by incorporating 14C-radiolabelled DSPC in LNP formulation, and we observed a 4.4% ID/g tumor accumulation following intravenous administration. Finally, treatment of 22Rv1 tumor bearing mice with siARvm-LNP resulted in significant tumor growth inhibition and survival benefit compared to siARfl-LNP or the siLUC-LNP control. To best of our knowledge, this is the first report demonstrating therapeutic effects of LNP-siRNA targeting AR splice variants in prostate cancer.

A Multifunctional LNA Oligonucleotide-Based Strategy Blocks AR Expression and Transactivation Activity in PCa Cells

The androgen receptor (AR) plays a critical role in the development of prostate cancer (PCa) through the activation of androgen-induced cellular proliferation genes. Thus, blocking AR-mediated transcriptional activation is expected to inhibit the growth and spread of PCa. Using tailor-made splice-switching locked nucleic acid (LNA) oligonucleotides (SSOs), we successfully redirected splicing of the AR precursor (pre-)mRNA and destabilized the transcripts via the introduction of premature stop codons. Furthermore, the SSOs simultaneously favored production of the AR45 mRNA in lieu of the full-length AR. AR45 is an AR isoform that can attenuate the activity of both full-length and oncogenic forms of AR by binding to their common N-terminal domain (NTD), thereby blocking their transactivation potential. A large screen was subsequently used to identify individual SSOs that could best perform this dual function. The selected SSOs powerfully silence AR expression and modulate the expression of AR-responsive cellular genes. This bi-functional strategy that uses a single therapeutic molecule can be the basis for novel PCa treatments. It might also be customized to other types of therapies that require the silencing of one gene and the simultaneous expression of a different isoform.

Regulation of protein kinase C-related kinase (PRK) signalling by the TPα and TPβ isoforms of the human thromboxane A2 receptor: Implications for thromboxane- and androgen- dependent neoplastic and epigenetic responses in prostate cancer

The prostanoid thromboxane (TX) A2 and its T Prostanoid receptor (the TP) are increasingly implicated in prostate cancer (PCa). Mechanistically, we recently discovered that both TPα and TPβ form functional signalling complexes with members of the protein kinase C-related kinase (PRK) family, AGC- kinases essential for the epigenetic regulation of androgen receptor (AR)-dependent transcription and promising therapeutic targets for treatment of castrate-resistant prostate cancer (CRPC). Critically, similar to androgens, activation of the PRKs through the TXA2/TP signalling axis induces phosphorylation of histone H3 at Thr11 (H3Thr11), a marker of androgen-induced chromatin remodelling and transcriptional activation, raising the possibility that TXA2-TP signalling can mimic and/or enhance AR-induced cellular changes even in the absence of circulating androgens such as in CRPC. Hence the aim of the current study was to investigate whether TXA2/TP-induced PRK activation can mimic and/or enhance AR-mediated cellular responses in the model androgen-responsive prostate adenocarcinoma LNCaP cell line. We reveal that TXA2/TP signalling can act as a neoplastic- and epigenetic-regulator, promoting and enhancing both AR-associated chromatin remodelling (H3Thr11 phosphorylation, WDR5 recruitment and acetylation of histone H4 at lysine 16) and AR-mediated transcriptional activation (e.g of the KLK3/prostate-specific antigen and TMPRSS2 genes) through mechanisms involving TPα/TPβ mediated-PRK1 and PRK2, but not PRK3, signalling complexes. Overall, these data demonstrate that TPα/TPβ can act as neoplastic and epigenetic regulators by mimicking and/or enhancing the actions of androgens within the prostate and provides further mechanistic insights into the role of the TXA2/TP signalling axis in PCa, including potentially in CRPC.

Development and pre-validation of an in vitro transactivation assay for detection of (anti)androgenic potential compounds using 22Rv1/MMTV cells.

The endocrine-disrupting effects of androgenic signaling play crucial roles in several androgen-related diseases. In attempting to develop an in vitro cell line to be used in androgen receptor (AR)-mediated reporter gene assays, we developed a stable 22Rv1/MMTV cell line, which is a human prostate cancer cell line that endogenously expresses functional AR, to evaluate AR-mediated transcriptional activation (TA). Using 22Rv1/MMTV cells, we established and optimized a test protocol for the AR-TA assay and validated the proposed assay using 20 compounds recommended by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). All the performance parameters for agonist and antagonist assays were 91-100% comparable between the 22Rv1/MMTV assay and the ICCVAM report. In conclusion, the AR-TA assay using 22Rv1/MMTV cells might be a quick and relatively inexpensive method for screening large numbers of chemicals for their potential to activate or inhibit AR-mediated gene transcription.

Abstract 616: Transcription-regulating kinases CDK8 and CDK19 as novel therapeutic targets for advanced prostate cancer

Abstract Most of castration-refractory prostate cancers (CRPC) continue to express androgen receptor (AR) that maintains its activity at low levels or even in the absence of androgen, driving tumor cell proliferation. Androgen-depleting drugs and androgen antagonists have little or no effect against AR+ cancers that are fully androgen-independent (AI). On the other hand, experimental compounds that cause AR degradation prevent AR-mediated repression of tumor-promoting genes, a tumor-suppressive effect of AR. We report a novel druggable target for CRPC, which mediates the induction but not the repression of gene expression by AR and inhibits the growth of AI prostate cancer (PCa) cells. This target is two closely related transcription-regulating kinases CDK8/CDK19 that unlike some other members of the CDK family are not required for cell cycle progression but play critical roles in several transcriptional signaling pathways. CDK8 and CDK19 are highly expressed in AR+ PCa cells and elevated under the conditions of androgen deprivation; CDK19 is overexpressed in AR+ PCa cell lines and in metastatic clinical PCa. A novel selective CDK8/19 kinase inhibitor (Senexin B) blocks ligand-induced AR-mediated transcriptional activation in androgen-dependent (AD) PCa cells but does not affect the AR-mediated gene repression. The CDK8/19 inhibitor also inhibits ligand-independent AR activation and AI cell growth in AI-PCa cells resistant to the anti-androgenic drug enzalutamide, both in vitro and in a mouse xenograft model. We have also discovered that CDK8/19 inhibition decreases NFκB mediated transcriptional activation of a group of tumor-promoting chemokines, such as CXCL1, CXCL2 and IL8, suggesting that CDK8/19 may promote prostate cancer chemoresistance and metastasis through the NFκB pathway. The above results suggest that CDK8/19 is an especially attractive therapeutic target for advanced PCa, which controls both AR-driven cell proliferation and other oncogenic pathways. Citation Format: Mengqian Chen, Vimala Kaza, Jiaxin Liang, Martina McDermott, Igor Roninson. Transcription-regulating kinases CDK8 and CDK19 as novel therapeutic targets for advanced prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 616. doi:10.1158/1538-7445.AM2014-616

Discovery and Characterization of NK13650s, Naturally Occurring p300-Selective Histone Acetyltransferase Inhibitors

The histone acetyltransferase (HAT) activity of p300 is essential for androgen receptor (AR) function. Androgen-independent prostate cancer cells require AR-mediated transcriptional activation for their growth. These observations indicate that p300 HAT is a promising target to overcome such hormone-resistant cancer cells. We sought p300 HAT inhibitors among microbial metabolites. By culturing a production strain belonging to Penicillium, we identified two new compounds, NK13650A and NK13650B, which were obtained as specific p300 HAT inhibitors. Structural analyses of these compounds elucidated that NK13650s have novel chemical structures comprising several amino acids and citrate. We applied a newly developed biosynthesis-based method to reveal the absolute configuration at the citrate quaternary carbon. This was accomplished by feeding a (13)C-labeled biosynthetic precursor of citrate. NK13650s selectively inhibited the activity of p300 HAT but not that of Tip60 HAT. NK13650s showed inhibitory activity against agonist-induced AR transcriptional activation, and NK13650A treatment inhibited hormone-dependent and -independent growth of prostate cancer cells.

Long Terminal Repeats Act as Androgen-Responsive Enhancers for the PSA-Kallikrein Locus

The androgen receptor (AR) signaling pathway is a common therapeutic target for prostate cancer, because it is critical for the survival of both hormone-responsive and castrate-resistant tumor cells. Most of the detailed understanding that we have of AR transcriptional activation has been gained by studying classical target genes. For more than two decades, Kallikrein 3 (KLK3) (prostate-specific antigen) has been used as a prototypical AR target gene, because it is highly androgen responsive in prostate cancer cells. Three regions upstream of the KLK3 gene, including the distal enhancer, are known to contain consensus androgen-responsive elements required for AR-mediated transcriptional activation. Here, we show that KLK3 is one of a specific cluster of androgen-regulated genes at the centromeric end of the kallikrein locus with enhancers that evolved from the long terminal repeat (LTR) (LTR40a) of an endogenous retrovirus. Ligand-dependent recruitment of the AR to individual LTR-derived enhancers results in concurrent up-regulation of endogenous KLK2, KLK3, and KLKP1 expression in LNCaP prostate cancer cells. At the molecular level, a kallikrein-specific duplication within the LTR is required for maximal androgen responsiveness. Therefore, KLK3 represents a subset of target genes regulated by repetitive elements but is not typical of the whole spectrum of androgen-responsive transcripts. These data provide a novel and more detailed understanding of AR transcriptional activation and emphasize the importance of repetitive elements as functional regulatory units.

Multivalent Peptidomimetic Conjugates: A Versatile Platform for Modulating Androgen Receptor Activity

We introduce a family of multivalent peptidomimetic conjugates that modulate the activity of the androgen receptor (AR). Bioactive ethisterone ligands were conjugated to a set of sequence-specific peptoid oligomers. Certain multivalent peptoid conjugates enhance AR-mediated transcriptional activation. We identify a linear and a cyclic conjugate that exhibit potent anti-proliferative activity in LNCaP-abl cells, a model of therapy-resistant prostate cancer. The linear conjugate blocks AR action by competing for ligand binding. In contrast, the cyclic conjugate is active despite its inability to compete against endogenous ligand for binding to AR in vitro, suggesting a non-competitive mode of action. These results establish a versatile platform to design competitive and non-competitive AR modulators with potential therapeutic significance.

Therapeutic targeting of angiotensin II receptor type 1 to regulate androgen receptor in prostate cancer

With the limited strategies for curative treatment of castration-resistant prostate cancer (CRPC), public interest has focused on the potential prevention of prostate cancer. Recent studies have demonstrated that an angiotensin II receptor blocker (ARB) has the potential to decrease serum prostate-specific antigen (PSA) level and improve performance status in CRPC patients. These facts prompted us to investigate the direct effects of ARBs on prostate cancer growth and progression. Transgenic rat for adenocarcinoma of prostate (TRAP) model established in our laboratory was used. TRAP rats of 3 weeks of age received ARB (telmisartan or candesartan) at the concentration of 2 or 10 mg/kg/day in drinking water for 12 weeks. In vitro analyses for cell growth, ubiquitylation or reporter gene assay were performed using LNCaP cells. We found that both telmisartan and candesartan attenuated prostate carcinogenesis in TRAP rats by augmentation of apoptosis resulting from activation of caspases, inactivation of p38 MAPK and down-regulation of the androgen receptor (AR). Further, microarray analysis demonstrated up-regulation of estrogen receptor β (ERβ) by ARB treatment. In both parental and androgen-independent LNCaP cells, ARB inhibited both cell growth and AR-mediated transcriptional activity. ARB also exerted a mild additional effect on AR-mediated transcriptional activation by the ERβ up-regulation. An intervention study revealed that PSA progression was prolonged in prostate cancer patients given an ARB compared with placebo control. These data provide a new concept that ARBs are promising potential chemopreventive and chemotherapeutic agents for prostate cancer.

Green tea polyphenol EGCG blunts androgen receptor function in prostate cancer

Androgen deprivation therapy is the major treatment for advanced prostate cancer (PCa). However, it is a temporary remission, and the patients almost inevitably develop hormone refractory prostate cancer (HRPC). HRPC is almost incurable, although most HRPC cells still express androgen receptor (AR) and depend on the AR for growth, making AR a prime drug target. Here, we provide evidence that epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, is a direct antagonist of androgen action. In silico modeling and FRET-based competition assay showed that EGCG physically interacts with the ligand-binding domain of AR by replacing a high-affinity labeled ligand (IC(50) 0.4 μM). The functional consequence of this interaction was a decrease in AR-mediated transcriptional activation, which was due to EGCG mediated inhibition of interdomain N-C termini interaction of AR. Treatment with EGCG also repressed the transcriptional activation by a hotspot mutant AR (T877A) expressed ectopically as well as the endogenous AR mutant. As the physiological consequence of AR antagonism, EGCG repressed R1881-induced PCa cell growth. In a xenograft model, EGCG was found to inhibit AR nuclear translocation and protein expression. We also observed a significant down-regulation of androgen-regulated miRNA-21 and up-regulation of a tumor suppressor, miRNA-330, in tumors of mice treated with EGCG. Taken together, we provide evidence that EGCG functionally antagonizes androgen action at multiple levels, resulting in inhibition of PCa growth.

Tat-Binding Protein-1 (TBP-1), an ATPase of 19S Regulatory Particles of the 26S Proteasome, Enhances Androgen Receptor Function in Cooperation with TBP-1-Interacting Protein/Hop2

The 26S proteasome, which degrades ubiquitinated proteins, appears to contribute to the cyclical loading of androgen receptor (AR) to androgen response elements of target gene promoters; however, the mechanism whereby the 26S proteasome modulates AR recruitment remains unknown. Using yeast two-hybrid screening, we previously identified Tat-binding protein-1 (TBP-1), an adenosine triphosphatase of 19S regulatory particles of the 26S proteasome, as a transcriptional coactivator of thyroid hormone receptor. Independently, TBP-1-interacting protein (TBPIP) was also identified as a coactivator of several nuclear receptors, including AR. Here, we investigated whether TBP-1 could interact with and modulate transcriptional activation by AR cooperatively with TBPIP. TBP-1 mRNA was ubiquitously expressed in human tissues, including the testis and prostate, as well as in LNCaP cells. TBP-1 directly bound TBPIP through the amino-terminal domain possessing the leucine zipper structure. AR is physically associated with TBP-1 and TBPIP in vitro and in LNCaP cells. TBP-1 similarly and additively augmented AR-mediated transcription upon coexpression with TBPIP, and the ATPase domain, as well as leucine zipper structure in TBP-1, was essential for transcriptional enhancement. Overexpression of TBP-1 did not alter AR protein and mRNA levels. In the chromatin immunoprecipitation assay, TBP-1 was transiently recruited to the proximal androgen response element of the prostate-specific antigen gene promoter in a ligand-dependent manner in LNCaP cells. These findings suggest that a component of 19S regulatory particles directly binds AR and might participate in AR-mediated transcriptional activation in cooperation with TBPIP.

Histone deacetylases are required for androgen receptor function in hormone-sensitive and castrate-resistant prostate cancer.

Transcriptional activity of the androgen receptor (AR) is crucial for growth and survival of prostate cancer even upon development of resistance to androgen ablation and antiandrogen therapies. Therefore, novel therapies that can suppress AR transcriptional activity when conventional hormone therapies fail are needed. Here, we show that histone deacetylase (HDAC) inhibitors, including SAHA (vorinostat) and LBH589, which are currently being tested in clinic, could be such a therapy. HDAC inhibitors block the AR-mediated transcriptional activation of many genes, including the TMPRSS2 gene involved in fusion with ETS family members in a majority of prostate cancers. Genetic knockdown of either HDAC1 or HDAC3 can also suppress expression of AR-regulated genes, recapitulating the effect of HDAC inhibitor treatment. Whereas HDAC inhibitor treatment can lower androgen receptor protein levels in prostate cancer cells, we show that independent of AR protein levels, HDAC inhibitors block AR activity through inhibiting the assembly of coactivator/RNA polymerase II complex after AR binds to the enhancers of target genes. Failed complex assembly is associated with a phase shift in the cyclical wave of AR recruitment that typically occurs in response to ligand treatment. HDAC inhibitors retain the ability to block AR activity in castration-resistant prostate cancer models and, therefore, merit clinical investigation in this setting. The HDAC-regulated AR target genes defined here can serve as biomarkers to ensure sufficient levels of HDAC inhibition.

TGF‐β signaling and androgen receptor status determine apoptotic cross‐talk in human prostate cancer cells

A signaling interaction between transforming growth factor-beta (TGF-beta) and androgens promotes apoptosis in human prostate cancer cells LNCaP-TbetaRII (androgen-sensitive and TGF-beta responsive). This study investigated the contribution of androgen receptor (AR) in the combined effect of TGF-beta and dihydrotestosterone (DHT), on regulation of apoptosis and AR- and TGF-beta mediated transcriptional activity in human prostate cancer cells. Transcriptional activation in response to TGF-beta (5 ng/ml) and DHT (1 nM) was evaluated using transient transfections and luciferase assays in human prostate cancer cells, LNCaP-TbetaRII and PC-3, overexpressing the wild type AR. The apoptotic response to DHT/TGFbeta treatment was correlated with AR cellular distribution and the AR interaction with TGF-beta intracellular effector Smad4. The results revealed that TGF-beta signaling induced AR-mediated transcriptional activation in two androgen-responsive promoters [probasin and prostate specific antigen (PSA)]. TGF-beta1 induced transcriptional activity enhanced by DHT in both cell lines (LNCaP-TbetaRII and PC-3-AR) via AR-Smad4 interaction. This interaction however does not exclusively drive TGF-beta mediated apoptosis as DHT failed to enhance such an effect in PC-3 AR (wt) cells. These results demonstrate that the AR status determines the sensitivity of prostate cancer cells to the apoptotic effects of TGF-beta1, thus providing a new insight into the mechanism via which TGF-beta cross-sections the AR axis toward the functional convergence of the two pathways in the development of androgen-independent prostate cancer. This study is potentially significant in defining the contribution of AR status to the emergence of androgen-independent prostate tumors.

In Vitroandin VivoStructure-Activity Relationships of Novel Androgen Receptor Ligands with Multiple Substituents in the B-Ring

We recently reported two nonsteroidal androgen receptor (AR) ligands that demonstrate tissue-selective pharmacological activity, identifying these S-3-(phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamide analogs as the first members of a new class of drugs known as selective androgen receptor modulators. The purpose of these studies was to explore additional structure-activity relationships of selective androgen receptor modulators to enhance their AR binding affinity, AR-mediated transcriptional activation, and in vivo pharmacological activity. The AR binding affinity (K(i)) of 29 novel synthetic AR ligands was determined by a radioligand competitive binding assay and ranged from 1.0-51 nM. Compounds with electron-withdrawing substituents at the para- and meta-positions of the B-ring demonstrated the highest AR binding affinity. The AR-mediated transcriptional activation was determined using a cotransfection assay in CV-1 cells. Most compounds with two substituents in the B-ring maintained or improved their functional activity in vitro. However, compounds with three halogen substituents exhibited significant regioselectivity. Fifteen compounds were selected to examine their pharmacological activity in castrated rats. In vivo pharmacological activity and selectivity were significantly changed by structural modification in the B-ring. Compounds with halogen groups at the para- and meta-positions of the B-ring displayed the highest pharmacological activity. Incorporating substituents at the ortho-position of the B-ring resulted in poor pharmacological activity. In vitro and in vivo agonist activities were partially correlated. In conclusion, novel selective androgen receptor modulators with improved in vivo pharmacological activity can be designed and synthesized based on the structure-activity relationship identified in these studies.

Serenoa repens (Permixon®) inhibits the 5α‐reductase activity of human prostate cancer cell lines without interfering with PSA expression

The phytotherapeutic agent Serenoa repens is an effective dual inhibitor of 5alpha-reductase isoenzyme activity in the prostate. Unlike other 5alpha-reductase inhibitors, Serenoa repens induces its effects without interfering with the cellular capacity to secrete PSA. Here, we focussed on the possible pathways that might differentiate the action of Permixon from that of synthetic 5alpha-reductase inhibitors. We demonstrate that Serenoa repens, unlike other 5alpha-reductase inhibitors, does not inhibit binding between activated AR and the steroid receptor-binding consensus in the promoter region of the PSA gene. This was shown by a combination of techniques: assessment of the effect of Permixon on androgen action in the LNCaP prostate cancer cell line revealed no suppression of AR and maintenance of PSA protein expression at control levels. This was consistent with reporter gene experiments showing that Permixon failed to interfere with AR-mediated transcriptional activation of PSA and that both testosterone and DHT were equally effective at maintaining this activity. Our results demonstrate that despite Serenoa repens effective inhibition of 5alpha-reductase activity in the prostate, it did not suppress PSA secretion. Therefore, we confirm the therapeutic advantage of Serenoa repens over other 5alpha-reductase inhibitors as treatment with the phytotherapeutic agent will permit the continuous use of PSA measurements as a useful biomarker for prostate cancer screening and for evaluating tumour progression.

Design, Synthesis, and Biological Characterization of Metabolically Stable Selective Androgen Receptor Modulators

A series of nonsteroidal ligands were synthesized as second-generation agonists for the androgen receptor (AR). These ligands were designed to eliminate metabolic sites identified in one of our first-generation AR agonists, which was inactive in vivo due to its rapid metabolism to inactive constituents. The binding affinity of these compounds was evaluated using AR isolated from rat ventral prostate. These second-generation compounds bound the AR in a high affinity and stereoselective manner, with K(i) values ranging from about 4 to 130 nM. The ability of these ligands to stimulate AR-mediated transcriptional activation was examined in cells transfected with the human AR and a hormone-dependent luciferase reporter gene. Although some compounds were unable to stimulate AR-mediated transcription, several demonstrated activity similar to that of dihydrotestosterone (DHT, an endogenous steroidal ligand for the AR). We also evaluated the in vivo pharmacologic activity of selected compounds in castrated male rats. Three compounds were identified as selective androgen receptor modulators (SARMs), exhibiting significant anabolic activity while having only moderate to minimal androgenic activity in vivo.

Key structural features of nonsteroidal ligands for binding and activation of the androgen receptor.

The purposes of the present studies were to examine the androgen receptor (AR) binding ability and in vitro functional activity of multiple series of nonsteroidal compounds derived from known antiandrogen pharmacophores and to investigate the structure-activity relationships (SARs) of these nonsteroidal compounds. The AR binding properties of sixty-five nonsteroidal compounds were assessed by a radioligand competitive binding assay with the use of cytosolic AR prepared from rat prostates. The AR agonist and antagonist activities of high-affinity ligands were determined by the ability of the ligand to regulate AR-mediated transcriptional activation in cultured CV-1 cells, using a cotransfection assay. Nonsteroidal compounds with diverse structural features demonstrated a wide range of binding affinity for the AR. Ten compounds, mainly from the bicalutamide-related series, showed a binding affinity superior to the structural pharmacophore from which they were derived. Several SARs regarding nonsteroidal AR binding were revealed from the binding data, including stereoisomeric conformation, steric effect, and electronic effect. The functional activity of high-affinity ligands ranged from antagonist to full agonist for the AR. Several structural features were found to be determinative of agonist and antagonist activities. The nonsteroidal AR agonists identified from the present studies provided a pool of candidates for further development of selective androgen receptor modulators (SARMs) for androgen therapy. Also, these studies uncovered or confirmed numerous important SARs governing AR binding and functional properties by nonsteroidal molecules, which would be valuable in the future structural optimization of SARMs.

Novel Nonsteroidal Ligands with High Binding Affinity and Potent Functional Activity for the Androgen Receptor.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Novel nonsteroidal ligands with high binding affinity and potent functional activity for the androgen receptor

While nonsteroidal androgen receptor (AR) antagonists have been known for many years, and used in the clinic for the treatment of hormone dependent prostate cancer, very little is known about nonsteroidal AR agonists. We designed and synthesized a series of chiral bicalutamide analogs, which bear electron-withdrawing groups (either a cyano or a nitro group at the 4-position and a trifluoromethyl group at the 3-position) in the aromatic A ring, and different substituents at the para position in the aromatic B ring of the parent molecule. We also synthesized a series of racemic bicalutamide analogs, which have a trifluoromethyl group instead of a methyl group at the R 2 position. We examined AR binding affinities of our compounds in a competitive binding assay with a radiolabeled high affinity AR ligand, 3H-mibolerone, and also measured their abilities to stimulate AR-mediated transcriptional activation in a cotransfection assay. These studies demonstrated that (1) nonsteroidal ligands can be structurally modified from known nonsteroidal antiandrogens to generate ligands capable of activating AR-mediated transcriptional activation. (2) R-isomer analogs exhibit higher AR binding affinity and more potent functional activity than their corresponding S-isomers in all cases. (3) All sulphide analogs show higher AR binding affinity and more potent functional activity than their corresponding sulphone analogs, with the exception of ligand R- 8. Those ligands which exhibit high AR binding affinity and potent functional activity for human AR may provide effective clinical uses for male fertility, male contraception, and hormone replacement therapy.

FXXLF and WXXLF Sequences Mediate the NH2-terminal Interaction with the Ligand Binding Domain of the Androgen Receptor

The nuclear receptor superfamily members of eukaryotic transcriptional regulators contain a highly conserved activation function 2 (AF2) in the hormone binding carboxyl-terminal domain and, for some, an additional activation function 1 in the NH(2)-terminal region which is not conserved. Recent biochemical and crystallographic studies revealed the molecular basis of AF2 is hormone-dependent recruitment of LXXLL motif-containing coactivators, including the p160 family, to a hydrophobic cleft in the ligand binding domain. Our previous studies demonstrated that AF2 in the androgen receptor (AR) binds only weakly to LXXLL motif-containing coactivators and instead mediates an androgen-dependent interaction with the AR NH(2)-terminal domain required for its physiological function. Here we demonstrate in a mammalian two-hybrid assay, glutathione S-transferase fusion protein binding studies, and functional assays that two predicted alpha-helical regions that are similar, but functionally distinct from the p160 coactivator interaction sequence, mediate the androgen-dependent, NH(2)- and carboxyl-terminal interaction. FXXLF in the AR NH(2)-terminal domain with the sequence (23)FQNLF(27) mediates interaction with AF2 and is the predominant androgen-dependent interaction site. This FXXLF sequence and a second NH(2)-terminal WXXLF sequence (433)WHTLF(437) interact with different regions of the ligand binding domain to stabilize the hormone-receptor complex and may compete with AF2 recruitment of LXXLL motif-containing coactivators. The results suggest a unique mechanism for AR-mediated transcriptional activation.