A9

Amino Acid Sequence

3D Structure (/structures/AF-P10275-F1-model_v6.cif)

Function

Steroid hormone receptors are ligand-activated transcription factors that regulate eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues. Transcription factor activity is modulated by bound coactivator and corepressor proteins like ZBTB7A that recruits NCOR1 and NCOR2 to the androgen response elements/ARE on target genes, negatively regulating androgen receptor signaling and androgen-induced cell proliferation. Transcription activation is also down-regulated by NR0B2. Activated, but not phosphorylated, by HIPK3 and ZIPK/DAPK3

Mechanism

The ubiquitin-proteasome pathway (UPP) constitutes the principal pathway for protein degradation in eukaryotic cells, mediating the breakdown of target proteins through ubiquitin tagging and subsequent proteasomal degradation. In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation.

Description

Furthermore, to investigated the specific mechanism of action of AR degradation mediated by compound A9, we pre-treated LNCaP cells with the proteasome inhibitor MG132, the lysosomal inhibitor E-64 (Fig. 6D). Our data show that AR degradation was blocked by MG132, but was unaffected by E-64, it suggested that compound A9 induces the degradation of AR in cells via the proteasome. (D) A9-mediated AR degradation (10 µM) at 24 h was blocked by 3h pretreatment with proteasome inhibitor MG132 (10 µM) and was unaffected by 3h pretreatment with lysosomal inhibitor E-64 (10 µM)

Description

Then we evaluated the kinetics of A9 in induction of AR degradation at 10 µM in the LNCaP cell lines, our data showed that A9 effectively reduced the AR protein level and achieved near-complete AR depletion with a 72 h treatment (Fig 6B)

Description

To evaluate their inhibition of prostate cancer cell proliferation, we estimated the anti-proliferative activity of these compounds in LNCaP (AR-positive, dependent), C4-2B (AR-positive, independent), PC-3 (AR-negative) and WPMY-1 (human normal prostate stromal cell) cell lines, and the cytotoxic activity of target compounds were detected by SRB (Sulforhodanime B) assay. The IC50 values (concentration required to inhibit tumor cell proliferation by 50%) were listed in Table 2, with Enzalutamide used as positive control.

Description

To evaluate their inhibition of prostate cancer cell proliferation, we estimated the anti-proliferative activity of these compounds in LNCaP (AR-positive, dependent), C4-2B (AR-positive, independent), PC-3 (AR-negative) and WPMY-1 (human normal prostate stromal cell) cell lines, and the cytotoxic activity of target compounds were detected by SRB (Sulforhodanime B) assay. The IC50 values (concentration required to inhibit tumor cell proliferation by 50%) were listed in Table 2, with Enzalutamide used as positive control.

Description

As can be seen in Table 3, some compounds with better growth inhibitory effects were chosen to evaluate for their cytotoxic activity against another three cancer cell lines in SGC-7901 (human stomach cancer line, AR positive), MCF-7(human breast cancer cell line, AR positive) and GES-1 (human normal gastric cells, AR negative). To our delight, four selected products exerted good inhibitory activity against SGC-7901 cells with the IC50 values of 4.11, 7.79, 6.93 and 4.98 μM, respectively.

Description

As can be seen in Table 3, some compounds with better growth inhibitory effects were chosen to evaluate for their cytotoxic activity against another three cancer cell lines in SGC-7901 (human stomach cancer line, AR positive), MCF-7(human breast cancer cell line, AR positive) and GES-1 (human normal gastric cells, AR negative). In contrast, compounds A7, A8, A9 and A14 were less potent against MCF-7 cells with the IC50 values of 9.12, 13.33, 50.50 and 7.23 μM, respectively.

Description

We also found that these four compounds had no cytotoxicity against normal gastric GES-1 cells. Predictably, target cytotoxcity by the SARDs is selective for the AR.