R8

Amino Acid Sequence

3D Structure (/structures/struct-20261022-174618-4e0e78.cif)

Function

Essential antioxidant peroxidase that directly reduces phospholipid hydroperoxide even if they are incorporated in membranes and lipoproteins. Can also reduce cholesterol hydroperoxide and thymine hydroperoxide (By similarity). Plays a key role in protecting cells from oxidative damage by preventing membrane lipid peroxidation. Required to prevent cells from ferroptosis, a non-apoptotic cell death resulting from an iron-dependent accumulation of lipid reactive oxygen species. The presence of selenocysteine (Sec) versus Cys at the active site is essential for life: it provides resistance to overoxidation and prevents cells against ferroptosis (By similarity). The presence of Sec at the active site is also essential for the survival of a specific type of parvalbumin-positive interneurons, thereby preventing against fatal epileptic seizures (By similarity). May be required to protect cells from the toxicity of ingested lipid hydroperoxides (By similarity). Required for normal sperm development and male fertility (By similarity). Essential for maturation and survival of photoreceptor cells (By similarity). Plays a role in a primary T-cell response to viral and parasitic infection by protecting T-cells from ferroptosis and by supporting T-cell expansion (By similarity). Plays a role of glutathione peroxidase in platelets in the arachidonic acid metabolism. Reduces hydroperoxy ester lipids formed by a 15-lipoxygenase that may play a role as down-regulator of the cellular 15-lipoxygenase pathway (By similarity). Can reduce fatty acid-derived hydroperoxides. Can also reduce small soluble hydroperoxides such as H2O2, cumene hydroperoxide and tert-butyl hydroperoxide.

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

Considering the strong degradation potency of R8, it was selected for further investigation of the degradation mechanism of GPX4 in HT1080 cells. Cycloheximide (CHX) has been known as a protein synthesis inhibitor, which can be employed to block the protein synthesis in eukaryotic cells. The degradation of GPX4 was only observed with 0.05 μM R8 alone for 7 h (Fig. 3C), whereas the significant degradation of GPX4 merely occurred in 4 h through the co-treatment of CHX andR8 (Fig. 4A and B). The acceleration of degrading GPX4 by compound R8 with the facilitation of CHX, which could impede protein translation, indicated that R8 may induce GPX4 degradation via the ubiquitin-proteasome system (UPS) pathway in a time-dependent manner. In view of the inherent capability of RSL3 to decrease GPX4 protein [46], the inhibitor competitive assays by using RSL3 or E3 ligand may be unfavorable to carry out. To gain deeper envision into the mechanism of compound R8, we performed the comparative experiment of RSL3 and R8 with the co-treatment of MG132 (a proteasome inhibitor) and Chloroquine (CQ, lysosomal inhibitor) respectively. As shown in Fig. 4C-F and S4, the GPX4 degradation of compound R8 was entirely obstructed when treated with 0.3 μM MG132 for 12 h in HT1080 cells. However, the protein level of GPX4 was not affected by R8, when cells were pretreated with CQ. These results underscore the main involvement of UPS pathway in the degradation of GPX4 induced by R8.

Description

We further evaluated the degradation efficacy of compound R8 at different concentrations (Fig. 3A and 3B). The result revealed that R8 degraded GPX4 in a dose-dependent manner, with a DC50(half-maximal degradation concentration) value of 0.019 μM.

Description

In this study, we reported the design, synthesis and biological evaluation of a series of HyT molecules for degrading GPX4. Through the study of structure-activity relationships, we successfully discovered the highly active compound R8 as the first RSL3-based GPX4 HyT degrader. Compound R8 exhibited superior in vitro antitumor potency against HT1080 and MDA-MB-231 cell lines with IC50 values of 24 nM and 32 nM respectively, which is 4 times more potent than parental compound RSL3.

Description

In this study, we reported the design, synthesis and biological evaluation of a series of HyT molecules for degrading GPX4. Through the study of structure-activity relationships, we successfully discovered the highly active compound R8 as the first RSL3-based GPX4 HyT degrader. Compound R8 exhibited superior in vitro antitumor potency against HT1080 and MDA-MB-231 cell lines with IC50 values of 24 nM and 32 nM respectively, which is 4 times more potent than parental compound RSL3.