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Development of β-amino-carbonyl compounds as androgen receptor antagonists.

Zhang ZY, Zhu YH, Zhou CH, Liu Q, Lu HL, Ge YJ, Wang MW - Acta Pharmacol. Sin. (2014)

Bottom Line: The therapeutic effects on tumor growth in vivo were observed in male SCID mice bearing LNCaP xenografts.Further structural modifications led to the discovery of a androgen receptor antagonist (compound 6012), which blocked androgen receptor nuclear translocation, androgen-responsive gene expression and androgen-dependent LNCaP cell proliferation.The pharmacological effects of 6012, including AR binding, androgen-induced AR translocation, NH2- and COOH-terminal interaction, growth inhibition of LNCaP cells in vitro and LNCaP xenograft growth in nude mice, were mainly restricted to isomer 6012-4 (1R, 3S).

View Article: PubMed Central - PubMed

Affiliation: The National Center for Drug Screening and the CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai 201203, China.

ABSTRACT

Aim: Androgen receptor (AR) antagonists have proven to be useful in the early control of prostate cancer. The aim of this study was to identify and characterize a novel β-amino-carbonyl-based androgen receptor antagonist.

Methods: Different isomers of the β-amino-carbonyl compounds were obtained by chiral separation. The bioactivities of the isomers were evaluated by AR nuclear translocation, mammalian two-hybrid, competitive receptor binding and cell proliferation assays. The expression of genes downstream of AR was analyzed with real-time PCR. The therapeutic effects on tumor growth in vivo were observed in male SCID mice bearing LNCaP xenografts.

Results: Compound 21 was previously identified as an AR modulator by the high-throughput screening of a diverse compound library. In the present study, the two isomers of compound 21, termed compounds 21-1 and 21-2, were characterized as partial AR agonists in terms of androgen-induced AR nuclear translocation, prostate-specific antigen expression and cell proliferation. Further structural modifications led to the discovery of a androgen receptor antagonist (compound 6012), which blocked androgen receptor nuclear translocation, androgen-responsive gene expression and androgen-dependent LNCaP cell proliferation. Four stereoisomers of compound 6012 were isolated, and their bioactivities were assessed. The pharmacological effects of 6012, including AR binding, androgen-induced AR translocation, NH2- and COOH-terminal interaction, growth inhibition of LNCaP cells in vitro and LNCaP xenograft growth in nude mice, were mainly restricted to isomer 6012-4 (1R, 3S).

Conclusion: Compound 6012-4 was determined to be a novel androgen receptor antagonist with prostate cancer inhibitory activities comparable to bicalutamide both in vitro and in vivo.

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LNCaP cell proliferation and DU 145 cell cytotoxicity assays. (A, B, and C) LNCaP cells were seeded into poly-L-lysine-pretreated 96-well plates (1×105 cells per well) and incubated for 24 h at 37 °C. Compounds (from left to right: 100 nmol/L, 1 μmol/L, and 10 μmol/L) were added to the cells together with 10 nmol/L DHT, followed by incubation for another 5 d. Cell viability was measured using the CCK-8 kit. (D) DU 145 cells were seeded into 96-well plates (1×105 cells per well) and incubated for 48 h at 37 °C. Compounds (from left to right: 100 nmol/L, 1 μmol/L, and 10 μmol/L) were added and incubated with the cells for 3 more days. Cell viability was measured using the CCK-8 kit. The values are the relative cell growth compared with that of the cells treated with DMSO only. The data are representative of at least three independent experiments and are expressed as the mean±SEM. bP<0.05 vs 10 nmol/L DHT (t-test). Casodex: bicalutamide.
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fig2: LNCaP cell proliferation and DU 145 cell cytotoxicity assays. (A, B, and C) LNCaP cells were seeded into poly-L-lysine-pretreated 96-well plates (1×105 cells per well) and incubated for 24 h at 37 °C. Compounds (from left to right: 100 nmol/L, 1 μmol/L, and 10 μmol/L) were added to the cells together with 10 nmol/L DHT, followed by incubation for another 5 d. Cell viability was measured using the CCK-8 kit. (D) DU 145 cells were seeded into 96-well plates (1×105 cells per well) and incubated for 48 h at 37 °C. Compounds (from left to right: 100 nmol/L, 1 μmol/L, and 10 μmol/L) were added and incubated with the cells for 3 more days. Cell viability was measured using the CCK-8 kit. The values are the relative cell growth compared with that of the cells treated with DMSO only. The data are representative of at least three independent experiments and are expressed as the mean±SEM. bP<0.05 vs 10 nmol/L DHT (t-test). Casodex: bicalutamide.

Mentions: As shown above, compound 21 and its isomers modulated the function of AR at several steps in the signaling pathway. However, our efforts in developing novel AR antagonists are limited by the agonist properties of compounds 21 and its isomer with an S configuration (21-2). Therefore, we propose that a full AR antagonist should bind to the cognate receptor at a reasonably high affinity and inhibit DHT-induced LNCaP cell growth without obvious cytotoxicity in non-androgen-dependent DU 145 cells. Based on structure-activity relationship (SAR) information obtained previously10, we designed and synthesized 72 new β-amino-carbonyl analogs and determined their binding affinities to AR (Supplemental Table 1). There were 30 compounds that displayed relatively high binding affinities were evaluated for their effects on LNCaP cell proliferation (Figures 2A–2C) and cytotoxicity measurements in DU 145 cells. These assays were performed for compounds 6012, 6014, 6015, 6022, 6023, 6024, 6027, and 6033 (Figure 2D). DU 145 is a human prostate cancer cell line that lacks measurable AR expression and serves as a control for specificity and potential cytotoxicity. Although compounds 6050, 6043, 6042, and 6049 inhibited DHT-induced LNCaP cell proliferation in a concentration-dependent manner, they all contain a ketoxime group (Figures 2A–2C). Other synthesized compounds with a ketoxime group also showed a similar inhibitory activity with this assay (data not shown), indicating a common cytotoxic nature. Therefore, these compounds were excluded from further studies.


Development of β-amino-carbonyl compounds as androgen receptor antagonists.

Zhang ZY, Zhu YH, Zhou CH, Liu Q, Lu HL, Ge YJ, Wang MW - Acta Pharmacol. Sin. (2014)

LNCaP cell proliferation and DU 145 cell cytotoxicity assays. (A, B, and C) LNCaP cells were seeded into poly-L-lysine-pretreated 96-well plates (1×105 cells per well) and incubated for 24 h at 37 °C. Compounds (from left to right: 100 nmol/L, 1 μmol/L, and 10 μmol/L) were added to the cells together with 10 nmol/L DHT, followed by incubation for another 5 d. Cell viability was measured using the CCK-8 kit. (D) DU 145 cells were seeded into 96-well plates (1×105 cells per well) and incubated for 48 h at 37 °C. Compounds (from left to right: 100 nmol/L, 1 μmol/L, and 10 μmol/L) were added and incubated with the cells for 3 more days. Cell viability was measured using the CCK-8 kit. The values are the relative cell growth compared with that of the cells treated with DMSO only. The data are representative of at least three independent experiments and are expressed as the mean±SEM. bP<0.05 vs 10 nmol/L DHT (t-test). Casodex: bicalutamide.
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Related In: Results  -  Collection

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fig2: LNCaP cell proliferation and DU 145 cell cytotoxicity assays. (A, B, and C) LNCaP cells were seeded into poly-L-lysine-pretreated 96-well plates (1×105 cells per well) and incubated for 24 h at 37 °C. Compounds (from left to right: 100 nmol/L, 1 μmol/L, and 10 μmol/L) were added to the cells together with 10 nmol/L DHT, followed by incubation for another 5 d. Cell viability was measured using the CCK-8 kit. (D) DU 145 cells were seeded into 96-well plates (1×105 cells per well) and incubated for 48 h at 37 °C. Compounds (from left to right: 100 nmol/L, 1 μmol/L, and 10 μmol/L) were added and incubated with the cells for 3 more days. Cell viability was measured using the CCK-8 kit. The values are the relative cell growth compared with that of the cells treated with DMSO only. The data are representative of at least three independent experiments and are expressed as the mean±SEM. bP<0.05 vs 10 nmol/L DHT (t-test). Casodex: bicalutamide.
Mentions: As shown above, compound 21 and its isomers modulated the function of AR at several steps in the signaling pathway. However, our efforts in developing novel AR antagonists are limited by the agonist properties of compounds 21 and its isomer with an S configuration (21-2). Therefore, we propose that a full AR antagonist should bind to the cognate receptor at a reasonably high affinity and inhibit DHT-induced LNCaP cell growth without obvious cytotoxicity in non-androgen-dependent DU 145 cells. Based on structure-activity relationship (SAR) information obtained previously10, we designed and synthesized 72 new β-amino-carbonyl analogs and determined their binding affinities to AR (Supplemental Table 1). There were 30 compounds that displayed relatively high binding affinities were evaluated for their effects on LNCaP cell proliferation (Figures 2A–2C) and cytotoxicity measurements in DU 145 cells. These assays were performed for compounds 6012, 6014, 6015, 6022, 6023, 6024, 6027, and 6033 (Figure 2D). DU 145 is a human prostate cancer cell line that lacks measurable AR expression and serves as a control for specificity and potential cytotoxicity. Although compounds 6050, 6043, 6042, and 6049 inhibited DHT-induced LNCaP cell proliferation in a concentration-dependent manner, they all contain a ketoxime group (Figures 2A–2C). Other synthesized compounds with a ketoxime group also showed a similar inhibitory activity with this assay (data not shown), indicating a common cytotoxic nature. Therefore, these compounds were excluded from further studies.

Bottom Line: The therapeutic effects on tumor growth in vivo were observed in male SCID mice bearing LNCaP xenografts.Further structural modifications led to the discovery of a androgen receptor antagonist (compound 6012), which blocked androgen receptor nuclear translocation, androgen-responsive gene expression and androgen-dependent LNCaP cell proliferation.The pharmacological effects of 6012, including AR binding, androgen-induced AR translocation, NH2- and COOH-terminal interaction, growth inhibition of LNCaP cells in vitro and LNCaP xenograft growth in nude mice, were mainly restricted to isomer 6012-4 (1R, 3S).

View Article: PubMed Central - PubMed

Affiliation: The National Center for Drug Screening and the CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), Shanghai 201203, China.

ABSTRACT

Aim: Androgen receptor (AR) antagonists have proven to be useful in the early control of prostate cancer. The aim of this study was to identify and characterize a novel β-amino-carbonyl-based androgen receptor antagonist.

Methods: Different isomers of the β-amino-carbonyl compounds were obtained by chiral separation. The bioactivities of the isomers were evaluated by AR nuclear translocation, mammalian two-hybrid, competitive receptor binding and cell proliferation assays. The expression of genes downstream of AR was analyzed with real-time PCR. The therapeutic effects on tumor growth in vivo were observed in male SCID mice bearing LNCaP xenografts.

Results: Compound 21 was previously identified as an AR modulator by the high-throughput screening of a diverse compound library. In the present study, the two isomers of compound 21, termed compounds 21-1 and 21-2, were characterized as partial AR agonists in terms of androgen-induced AR nuclear translocation, prostate-specific antigen expression and cell proliferation. Further structural modifications led to the discovery of a androgen receptor antagonist (compound 6012), which blocked androgen receptor nuclear translocation, androgen-responsive gene expression and androgen-dependent LNCaP cell proliferation. Four stereoisomers of compound 6012 were isolated, and their bioactivities were assessed. The pharmacological effects of 6012, including AR binding, androgen-induced AR translocation, NH2- and COOH-terminal interaction, growth inhibition of LNCaP cells in vitro and LNCaP xenograft growth in nude mice, were mainly restricted to isomer 6012-4 (1R, 3S).

Conclusion: Compound 6012-4 was determined to be a novel androgen receptor antagonist with prostate cancer inhibitory activities comparable to bicalutamide both in vitro and in vivo.

Show MeSH
Related in: MedlinePlus