G-5b

Amino Acid Sequence

3D Structure (/structures/AF-P03372-F1-model_v6.cif)

Function

Nuclear hormone receptor. The steroid hormones and their receptors are involved in the regulation of eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues. Ligand-dependent nuclear transactivation involves either direct homodimer binding to a palindromic estrogen response element (ERE) sequence or association with other DNA-binding transcription factors, such as AP-1/c-Jun, c-Fos, ATF-2, Sp1 and Sp3, to mediate ERE-independent signaling. Ligand binding induces a conformational change allowing subsequent or combinatorial association with multiprotein coactivator complexes through LXXLL motifs of their respective components. Mutual transrepression occurs between the estrogen receptor (ER) and NF-kappa-B in a cell-type specific manner. Decreases NF-kappa-B DNA-binding activity and inhibits NF-kappa-B-mediated transcription from the IL6 promoter and displace RELA/p65 and associated coregulators from the promoter. Recruited to the NF-kappa-B response element of the CCL2 and IL8 promoters and can displace CREBBP. Present with NF-kappa-B components RELA/p65 and NFKB1/p50 on ERE sequences. Can also act synergistically with NF-kappa-B to activate transcription involving respective recruitment adjacent response elements; the function involves CREBBP. Can activate the transcriptional activity of TFF1. Also mediates membrane-initiated estrogen signaling involving various kinase cascades. Essential for MTA1-mediated transcriptional regulation of BRCA1 and BCAS3. Maintains neuronal survival in response to ischemic reperfusion injury when in the presence of circulating estradiol (17-beta-estradiol/E2).

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

Since studies have indicated that the ubiquitin-proteasome system mediates ER degradation [26–28], an ER rescue assay was conducted using 1 μM MG-132, an inhibitor that blocks the proteolytic activity of the 26S proteasome complex. As anticipated, MG-132 prevented the G-5b-induced degradation of ER, leading to ER accumulation (Fig. 4B). Upon analyzing ER levels, it was evident that G-5b caused a decrease in ER in both the cytoplasm and nucleus, consistent with the observations in whole cells. While MG-132 mediated an increase in cytoplasmic ER, the nuclear content remained lower (Fig. 4B). Additionally, the RT-qPCR assay results indicated that G-5b did not significantly downregulate the ESR1 gene, which encodes ER(Fig. 4C). Subsequent co-Immunoprecipitation (Co-IP) tests confirmed that G-5b enhanced ER ubiquitination (Fig. 4D), and 1 μM MG-132 further amplified the ubiquitination level mediated by G-5b (Fig. 4E). In summary, G-5b promoted the translocation of ER from the nucleus to the cytoplasm, where the ER was swiftly degraded via the proteasome pathway, without affecting the expression of the corresponding mRNA.

Description

Following the principle of hydrophobic tags (HyTs), we attached the key parent nucleus (ligand) of GDC-9545 to a long hydrophobic side chain (HyT) at the 4-position of the 2,6-difluorophenyl ring to augment the hydrophobicity of ER (Table 1 and Table 2). Degradation assay analyzing ER levels in MCF-7 wild-type cells.

Description

G-5b (DC50 = 1.03 nM) was able to reduce the ER protein level of Y537S mutant cells (Fig. 8C)

Description

Following the principle of hydrophobic tags (HyTs), we attached the key parent nucleus (ligand) of GDC-9545 to a long hydrophobic side chain (HyT) at the 4-position of the 2,6-difluorophenyl ring to augment the hydrophobicity of ER (Table 1 and Table 2).The CellTiter-Glo assay detecting the anti-proliferative activity in MCF-7 wild-type cells.

Description

As shown in Fig. 8B, G-5b exhibited a dose-dependent growth inhibition in ESR1 mutant cells. The IC50 values of G-5b in T47DY537S and T47DD538G was 3.07 and 21.06 nM

Description

As shown in Fig. 8B, G-5b exhibited a dose-dependent growth inhibition in ESR1 mutant cells. The IC50 values of G-5b in T47DY537S and T47DD538G was 3.07 and 21.06 nM