VI-10h

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

Notably, co-treatment with the N-degron inhibitor alanine-arginine (Ala-Arg) [29]and VI10h failed to block ERα degradation (Figure 2C), whereas co-treatment with the proteasome inhibitor bortezomib and VI-10h completely abolished ERα degradation. These findings suggest that VI-10h-induced ERα degradation may rely on the proteasome pathway. Although MG132 inhibits 20S proteasome activity [30] and blocks the proteolytic function of the 26S proteasome complex, it did not fully restore ERα protein levels, likely due to its potential autophagy-activating properties, which may promote compensatory activation of autophagy-related degradation pathways (Figure 2D). Furthermore, treatment with the autophagy inhibitors [31] 3-methyladenine (3-MA) [32]and ammonium chloride (NH4Cl) [33] also failed to inhibit VI10h-induced ERα degradation (Figure 2E), further indicating that autophagy is not the primary mechanism underlying VI10h-induced ERα degradation. Taken together, VI-10h induced ERα degradation in MCF-7 cells via a proteasome-dependent pathway, while the partial inhibitory effect of MG132 may stem from its nonspecific induction of autophagic upregulation. For the control OBHSA, we verified its degradation mechanism to exclude parent scaffold interference (Figure S2D–F), confirming UPS-dependent ERα degradation consistent with our previous work.

Description

VI10h (DC50 = 1.08 nM, Figure 1G,J)and Ful (DC50 = 1.45 nM, Figure 1H,J) induced dose-dependent ERα degradation in MCF-7 cells with significantly stronger potency than OBHSA (DC50 = 0.73 μM, Figure 1I,J), while VI-10j (DC50 =20.0 nM, Figure S1C,E) and VI-10m (DC50 = 31.6 nM, Figure S1D,F) also mediated dose-dependent ERα degradation

Description

Notably, VI-10h significantly suppressed the colony-forming capacity of MCF-7 cells at 0.005 to 5 μM(Figure 1C,D), consistent with its antiproliferative activity measured by the CCK-8 assay (Figure 1B). (B) The antiproliferative effects of VI-10h, VI-10j, Ful, and OBHSA in MCF-7 cells for 96 h.

Description

Given the superior antiproliferative and degradation activities of VI-10h, its ER transcriptional regulatory activity was assessed in HEK-293T cells via dual-luciferase reporter assays with Ful and 4-hydroxytamoxifen (4-OHT) as controls. The results showed that VI-10h effectively antagonized ERα transcriptional activity (EC50 = 0.82 nM, Figure S1I) while significantly activating ERβ transcriptional activity (EC50 = 0.089 nM, Figure S1J) without exerting an antagonistic effect on ERβ

Description

Given the superior antiproliferative and degradation activities of VI-10h, its ER transcriptional regulatory activity was assessed in HEK-293T cells via dual-luciferase reporter assays with Ful and 4-hydroxytamoxifen (4-OHT) as controls. The results showed that VI-10h effectively antagonized ERα transcriptional activity (EC50 = 0.82 nM, Figure S1I) while significantly activating ERβ transcriptional activity (EC50 = 0.089 nM, Figure S1J) without exerting an antagonistic effect on ERβ