SARD279

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, consistent with our initial report on protein hydrophobic-tagging,[10] degradation of the AR is dependent on the UPS—pretreatment/co-treatment of LNCaP cells with the proteasome-specific inhibitor, epoxomicin, prevents SARD-mediated degradation of the AR. To further explore the mechanism of SARD-mediated AR degradation, we investigated the possible involvement of heat shock proteins (HSPs), given their known role in stabilizing misfolded proteins or targeting them for degradation by the UPS. We found that incubating cells with the potent Hsp90 inhibitor geldanamycin, at concentrations that did not affect AR levels, enhanced AR degradation at sub-DC50 concentrations of SARD279 (Figure S3). Immunoblotting revealed a geldanamycin-dependent increase in Hsp70 levels, consistent with the finding that Hsp90 inhibition leads to activation of heat shock factor 1(HSF1) and its target genes, including HSP70.[25] Hsp70 and the associated E3 ubiquitin ligase CHIP play a key role in targeting intractably misfolded proteins to the UPS,[26] and we have shown that labeling HaloTag with hydrophobic tags leads to enhanced association with Hsp70.[27] Together, this suggests that the Hsp70/CHIP complex mediates SARD induced AR degradation and that elevated Hsp70 levels underlie the increase in SARD279 activity in the context of Hsp90 inhibition.

Description

SARD279, in which the adamantyl moiety is coupled to RU59063 through an 8-atom ester linkage reduced AR protein levels by 50 % at 1 mm (DC50) (Figure 1B), while no degradation was detected in cells treated with the parental AR ligand. B) Immunoblot analyses of LNCaP human prostate tumor cells incubated with SARDs or parent ligand for 24 h

Description

Given that both SARDs reduced expression of the AR target genes, we directly evaluated their pharmacological properties in androgen responsive element (ARE)-driven luciferase reporter assay in 293 cells. We determined that, despite their construction from an AR agonist, both SARDs blocked transactivation of the androgen receptor by the agonist R1881 (Figure S5). That the IC50 values of the SARDs in reporter assay—156 nm for SARD279 and 293 nm for SARD033—were 10-fold lower than their DC50 values to degrade AR suggests that their ability to block expression of AR target genes may be multimodal: partly due to pharmacological antagonism of AR signaling and partly due to degradation of the AR itself