RA3

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

MCF-7 cells were preincubated with VER-155008, an Hsp70 inhibitor (Hsp70i), for 2 h prior to RA3 treatment for 12 h. As depicted in Figure 2A,B, the inclusion of VER-155008 notably increased ERα levels from 23.5% to 71.9%, suggesting the involvement of Hsp70 in the degradation process. Similarly, the addition of 17-AAG, an Hsp90 inhibitor, produced comparable results, while the combined treatment with VER-155008 and 17-AAG led to an even more pronounced restoration of cellular ERα levels (91%). Furthermore, we assessed the role of the ubiquitination-proteasome system in this degradation process by employing MG132, a peptide aldehyde that effectively blocks the proteolytic activity of the 26S proteasome, and MLN4924, a potent and selective inhibitor of NEDD8-activating enzyme, which are involved in the ubiquitination pathway. Our findings reveal that pretreatment with MG132 and/or MLN4924 for 2 h attenuated the ability of RA3 to induce ERα degradation (Figures 2C and S4). These results underscore the pivotal role played by the ubiquitination proteasome system in mediating the degradation of ERα induced by RA3

Description

The efficacy of the conjugates in degrading ERαin breast cancer cells was rigorously investigated through immunoblot ting assays. MCF-7 cells, a well-established model for studying ERα-positive breast cancer, were treated with the compounds for 24 h.21 The results, as presented in Figure 1A, revealed a notable decrease in ERα levels across all four compounds, with compound RA3 demonstrating the highest potency, achieving a degradation efficacy of 83.1%. Figure 1. (A) ERα levels of MCF-7 cells treated with raloxifene, D-ring-contracted artemisinin, andRA2−RA6(10μM) for 24 h

Description

The antiproliferation effects of the compounds against MCF-7 cells are depicted in Figure 3A. Notably, RA3 exhibited a remarkable inhibitory potential toward the proliferation of MCF-7 cells, achieving an IC50 value of 2.28μM (Table S1).

Description

The remarkable therapeutic potential of RA3 observed in vitro prompted a deeper investigation into its antitumor capabilities utilizing an MCF-7 breast cancer xenograft mouse model. The experimental design entailed dividing the tumor-bearing mice into five distinct groups: (I) a vehicle control group, (II) a group treated with the ERα inhibitor raloxifene at 5 mpk, (III) a group administered the commercial ERα degrader Faslodex at 5 mpk, (IV) a group receiving RA3 at 5 mpk, and (V) a group treated with a higher dose of RA3 at 10 mpk.32 Upon reaching a tumor volume of approximately 160 mm3 , the mice in each group underwent intravenous injections of the respective compounds. These injections were repeated every 3 days, and the tumor volume was meticulously monitored over a period of 21 days (Figure 5A). The findings were noteworthy, as even the low dosage of RA3 (5 mpk) effectively stymied tumor growth, achieving a tumor growth inhibition (TGI) of 93.3%, surpassing both raloxifene (TGI = 68.8%) and Faslodex (TGI = 87.0%). Remarkably, when the RA3 dosage was escalated to 10 mpk, the tumor volume not only ceased to grow but regressed from 165.5 to 65.1 mm3 , resulting in a TGI of 112.4%