RS-1

Amino Acid Sequence

3D Structure (/structures/struct-20261022-220722-e1bdd2.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

As a definitive experiment to validate proteasome-dependent degradation, cells were cotreated with RS-1 (100 nM) and the proteasome inhibitor MG-132. Western blot analysis confirmed that MG-132 completely rescued the RS-1−induced loss of GPX4 (as shown in Figure 3), demonstrating that the degradation is strictly dependent on a functional proteasome. In a direct comparison, the concentration of RSL3 required to achieve a level of GPX4 degradation comparable to that of RS-1 was 10-fold higher (1000 vs 100 nM). Importantly, the degradation activity of RSL3 was not attenuated by MG-132 cotreatment, in sharp contrast to the complete blockade observed for RS-1. These results collectively establish that RS 1andRSL3 operate via fundamentally distinct mechanisms, with the action of RSL3 being independent of the proteasomal pathway.

Description

GPX4 degradation efficacy was evaluated through the Western blot analysis of HT1080 cells treated with RS-1 for 24 h. Quantitative densitometry revealed a dose-dependent depletion of GPX4 protein, achieving a calculated DC 50 value of 8.9 nM

Description

Given the established role of GPX4 inhibition in triggering ferroptosis, we evaluated the ferroptosis-mediated cytotoxicity of RS-1 in HT1080, A549, and 4T1tumor cell lines using a 48 h treatment protocol. Dose−response profiling revealed sub micromolar antiproliferative activity with IC 50 values of 130 nM (HT1080),6.4μM(A549),and700nM(4T1)(Figure4A−C). Notably, RS-1 exhibited nearly twice the potency of RSL3 across all lines, consistent with its enhanced GPX4 degradation efficiency.

Description

Given the established role of GPX4 inhibition in triggering ferroptosis, we evaluated the ferroptosis-mediated cytotoxicity of RS-1 in HT1080, A549, and 4T1tumor cell lines using a 48 h treatment protocol. Dose−response profiling revealed sub micromolar antiproliferative activity with IC 50 values of 130 nM (HT1080),6.4μM(A549),and700nM(4T1)(Figure4A−C). Notably, RS-1 exhibited nearly twice the potency of RSL3 across all lines, consistent with its enhanced GPX4 degradation efficiency.

Description

Given the established role of GPX4 inhibition in triggering ferroptosis, we evaluated the ferroptosis-mediated cytotoxicity of RS-1 in HT1080, A549, and 4T1tumor cell lines using a 48 h treatment protocol. Dose−response profiling revealed sub micromolar antiproliferative activity with IC 50 values of 130 nM (HT1080),6.4μM(A549),and700nM(4T1)(Figure4A−C). Notably, RS-1 exhibited nearly twice the potency of RSL3 across all lines, consistent with its enhanced GPX4 degradation efficiency.