Compound 13

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.

Amino Acid Sequence

3D Structure (/structures/AF-P10275-3-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

A Cellular Thermal Shift Assay (CESTA) demonstrated that treatment with compound 13 significantly increased the thermal stability of AR and AR-V7, indicating that the lead compound can bind directly to both proteins in cells (Fig. 2E). qRT-PCR analysis showed that compound 13 had no effect on the mRNA levels of AR or AR-V7 (Fig. 2F). Meanwhile, we pre-treated CRPC cells with MG132 and CQ, inhibitors of the proteasome and lysosome, respectively, and found that MG132 significantly attenuated compound 13-mediated degradation of AR/AR-V7 (Fig. 2G, H). These results confirm that compound 13 primarily mediates the ubiquitin-dependent degradation of AR/AR-V7 through the proteasomal pathway. Results from cycloheximide (CHX) chase assays further showed that compound 13 significantly shortened the half-lives of both proteins (Fig. 2I, J). Subsequently, immunoprecipitation-immunoblotting experiments (IP-IB) experiments provided additional evidence that treatment of PCa cells with compound 13 significantly increased the ubiquitination levels of AR/AR-V7 (Fig. 2K). We also analyzed protein expression levels before and after compound administration using quantitative proteomics. The results showed that AR was among the proteins with the most significant downregulation post-treatment, indicating that compound 13 exhibits good selectivity for AR proteins degradation (Fig. 2L). In summary, the above experimental results fully demonstrate that the SiHyT-containing compound 13 induced the potent and selective degradation of AR and AR-V7 in PCa cells via the ubiquitin-proteasome system.

Description

To further evaluate the ability of compound 13 to degrade AR/AR-V7 proteins, the compound was incubated with two CRPC cell lines, 22Rv1 and VCaP, with different concentrations of the compound for varying durations. Western blotting was used to detect the expression of AR/AR-V7 in each group. The results showed that compound 13 degraded both oncoproteins in a dose-dependent and time-dependent manner in the two CRPC cell lines (Fig. 2A, B). The DC₅₀ values for Journal Pre-proof compound 13-mediated AR degradation were 0.6617 μM and 1.948 μM, respectively, while those for AR-V7 degradation were 0.852 μM and 1.024 μM (Fig. 2C)

Description

To further evaluate the ability of compound 13 to degrade AR/AR-V7 proteins, the compound was incubated with two CRPC cell lines, 22Rv1 and VCaP, with different concentrations of the compound for varying durations. Western blotting was used to detect the expression of AR/AR-V7 in each group. The results showed that compound 13 degraded both oncoproteins in a dose-dependent and time-dependent manner in the two CRPC cell lines (Fig. 2A, B). The DC₅₀ values for Journal Pre-proof compound 13-mediated AR degradation were 0.6617 μM and 1.948 μM, respectively, while those for AR-V7 degradation were 0.852 μM and 1.024 μM (Fig. 2C)

Description

To further evaluate the ability of compound 13 to degrade AR/AR-V7 proteins, the compound was incubated with two CRPC cell lines, 22Rv1 and VCaP, with different concentrations of the compound for varying durations. Western blotting was used to detect the expression of AR/AR-V7 in each group. The results showed that compound 13 degraded both oncoproteins in a dose-dependent and time-dependent manner in the two CRPC cell lines (Fig. 2A, B). The DC₅₀ values for Journal Pre-proof compound 13-mediated AR degradation were 0.6617 μM and 1.948 μM, respectively, while those for AR-V7 degradation were 0.852 μM and 1.024 μM (Fig. 2C)

Description

To further evaluate the ability of compound 13 to degrade AR/AR-V7 proteins, the compound was incubated with two CRPC cell lines, 22Rv1 and VCaP, with different concentrations of the compound for varying durations. Western blotting was used to detect the expression of AR/AR-V7 in each group. The results showed that compound 13 degraded both oncoproteins in a dose-dependent and time-dependent manner in the two CRPC cell lines (Fig. 2A, B). The DC₅₀ values for Journal Pre-proof compound 13-mediated AR degradation were 0.6617 μM and 1.948 μM, respectively, while those for AR-V7 degradation were 0.852 μM and 1.024 μM (Fig. 2C)

Description

Having confirmed the remarkable degradation effect of compound 13 on AR/AR-V7, the effect of the degrader on the viability of (CRPC cells were subsequently evaluated. First, cell proliferation assays showed that compared to Enzalutamide (ENZ), compound 13 more significantly inhibited the proliferation of the two CRPC cell lines, with IC₅₀ values of 3.567 μM and 3.351 μM. MTT assay was performed to evaluate the proliferative capacity of 22Rv1 and VCaP cells following treatment with compound 13 or ENZ at specified time points. IC50 values of compound 13 and ENZ were calculated using GraphPad Prism 9 software.

Description

Having confirmed the remarkable degradation effect of compound 13 on AR/AR-V7, the effect of the degrader on the viability of (CRPC cells were subsequently evaluated. First, cell proliferation assays showed that compared to Enzalutamide (ENZ), compound 13 more significantly inhibited the proliferation of the two CRPC cell lines, with IC₅₀ values of 3.567 μM and 3.351 μM. MTT assay was performed to evaluate the proliferative capacity of 22Rv1 and VCaP cells following treatment with compound 13 or ENZ at specified time points. IC50 values of compound 13 and ENZ were calculated using GraphPad Prism 9 software.