RSL3

Protein target name:

GPX4

Mechanism of action:

Inhibitor

Probe Availability

MedChem Express (1219810-16-8)

Probe Information

Target and Probe Information

Target Details

Target class:

Lipid metabolism

Subclass:

Glutathione Peroxidase

HUGO ID:

HGNC:4556

Entrez gene ID:

2879

UNIPROT ID:

P36969

Target alias:

Glutathione peroxidase 4, GPx-4, GSHPx-4, MCSP, PHGPx, SMDS, snGPx, snPHGPx

Probe Details

Chemical Probes Portal ID:

CPP664

Probe alias:

1S, 3R-RSL3, SCHEMBL13402744, CS-5650

PubChem CID:

1750826

ChEMBL ID:

CHEMBL3182405

SMILES string:

O=C([C@H]1CC2=C([C@H](C3=CC=C(C(OC)=O)C=C3)N1C(CCl)=O)NC4=C2C=CC=C4)OC

InChi Key:

TXJZRSRTYPUYRW-NQIIRXRSSA-N

Physico-chemical properties

Solubility - preferred solvent:

DMSO

Solubility - concentration:

25 mM

NMR:

(1S, 3R)-RSL3
1H NMR (500 MHz, DMF) δ 8.19 (s, 1H), 8.10 (s, 2H), 7.87 (s, 2H), 7.69 (d, J = 6.9
Hz, 1H), 7.48 (d, J = 6.9 Hz, 1H), 7.22 (dd, J = 19.1, 7.2 Hz, 2H), 6.52 (s, 1H), 5.56 (s, 1H), 4.83 (d, J = 12.8 Hz, 1H), 4.51 (d, J = 12.3 Hz, 1H), 4.04 (s, 3H), 3.90 – 3.71 (m, 4H), 3.65 (d, J = 13.4 Hz, 1H). 13C NMR (75 MHz, DMF) δ 171.93, 171.27, 168.63, 167.63, 166.71, 166.34, 137.86, 137.30, 134.12, 133.31, 129.91, 129.63, 127.19, 126.82, 125.25, 122.03, 119.54, 118.37, 111.85, 110.35, 57.58, 57.00, 52.42, 51.93, 43.23, 23.75.

More NMR info is available at: http://www.cell.com/cms/attachment/2021778256/2041666892/mmc1.pdf

Storage Recommendations

Storage (dry powder):

-20 ºC

Storage (solution):

DMSO, -20 ºC

Storage sensitivities:

RLS3 generally retains potency through multiple freeze-thaw cycles and extended storage (days) at room temperature in DMSO.

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

111 nM

Potency assay:

RSL3 was evaluated for binding using Microscale thermophoresis (MST) with labelled His-tagged GPX4(U46C) protein. The Kd is defined as the concentration of ligands at which the binding site on GPX4 protein is half occupied.

Structure-activity relationship:

Only the (1S,3R) isomer was able to induce death in BJeHLT cells. The (1R,3R) isomer is considered to be inactive.

Off target activity

Probe Selectivity in Vitro:

Not available

In cell validation

On target activity in cells

Potency in cells:

EC50

100 nM

Potency assay (cells):

HT-1080 cells were treated with a serial dilution of RSL3. Viability was assayed using Alamar Blue reagent. The EC50 is defined as the concentration that results in 50% viability. Chemoproteomic affinity capture with a modified version of RSL3 identified GPX4 as a cellular protein target. In lysates from a COH BR1 breast cancer cell line overexpressing GPX4, RLS3 inhibited the reduction of 7α-cholesterol hydroperoxide to 7α-cholesterol-OH. siRNA-mediated knockdown of GPX4 sensitized cells to RSL3, whereas overexpression of GPX4 rendered cells insensitive to RSL3.

Target engagement assay (cells):

Chemoproteomic affinity capture

Off target activity in cells

Potency assay, off target (cells):

Chemoproteomic affinity capture with a modified version of RSL3 also bound to SELT and SMG8, but siRNA or shRNA knockdown of these proteins did not impact the activity of RSL3.

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

RSL3 induces lipid ROS and ferroptotic cell death.

In vivo validation

Organism:

Athymic Nude Mice

Dose:

100 mg/kg

Route of delivery:

Subcutaneous

Primary Reference

Reference (doi):

10.1016/j.chembiol.2008.02.010 10.1016/j.cell.2013.12.010

Reference (PMID):

18355723 24439385

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

3 review(s)

SAB Members

SAB Comments

Some intriguing RAS-dependent biology has been uncovered using this probe with respect to its induction of ferroptosis through inhibition of GPX4, which at least in certain contexts was demonstrated to be synthetic lethal with mutated RAS. The published literature suggests that in this setting, this compound's primary mode of action is indeed through GPX4 inhibition. However, there is insufficient characterization of RSL3 for it to be recommended as a widely applicable probe. Although there is LC-MS data that demonstrates that it inhibits GPX4's ability to reduce phosphatidylcholine hydroperoxide (PC-OOH, a GPX4-specific substrate), the mechanistic details (e.g., is it a covalent modifier) and quantification (IC50 value) of this inhibition remain unclear. There is no selectivity data with respect to PC-OOH reduction inhibition of other GPX subtypes, nor has any biochemical data from a broad screen been disclosed (e.g., against the kinome or other target classes). The fact that it's biological activity is strongly dependent on stereochemistry helps mitigate some of the concerns that it lacks a specific mechanism of action; however, given the inherent reactivity of chloroacetyl group that is required for its activity, there is an additional burden of proof to demonstrate inherent selectivity for it to be considered a widely useful probe. In terms of in vivo utility, it has anti-tumor activity in murine models but only with subcutanesous administration. No PK or blood stability data have been reported. When used to supplement genetic validation of GPX4, RPL3 could be a useful tool, but given the caveats above, the compound should be used with caution.

Dec 29 2016 - 1:39pm

RSL3 is a ferroptosis inducer that covalently inhibits GPX4. Literature reports claim RSL3 inhibits GPX4 by binding to the catalytic selenocysteine residue, although this has not been directly demonstrated. RSL3 does not reduce glutathione levels like other ferrptosis inducers (e.g., erastin). Only the (1S,3R) diastereomer induces ferroptosis, suggesting that GPX4 inhibition is not driven solely by the reactivity of the chloroacetamide group of RSL3. However, chemoproteomic affinity capture using an RSL3 analog also indicates additional protein targets, including other selenoproteins. The cellular effects of RLS3 can be suppressed by ferroptosis inhibitors, including iron chelators (e.g., deferoxamine) and lipophilic antioxidants (e.g., vitamin E, ferrostatin-1). At higher doses in cells (>10 micromolar) the effects of RSL3 cannot be rescued by ferroptosis inhibitors, indicating off-mechanism activity. While RSL3, administered by subcutaneous injection, has shown activity in an in vivo mouse xenograft study, no pharmacokinetic (PK) characterization has been reported. Unknown PK properties and potential off-target liabilities argue against using RSL3 in animal experiments. As a cellular probe, RSL3 can be useful if studies are conducted carefully with appropriate controls, including rescue experiments and genetic validation.

Apr 7 2017 - 1:41pm

RSL3 has been demonstrated to induce ferroptosis in certain cellular contexts. The (1S,3R) diastereomer induces ferroptosis, while the (R,R) diasteriomer does not. Chemoproteomic studies show that (an analog of) the active isomer pulls down GPX4, while an inactive analog does not. Given the structure of the molecule, which contains a highly reactive chloroacetamide and two ester groups, which may show some instability in buffer or in cells (particularly the aromatic ester), there is an additional burden of proof to show that this compound is specific, selective and stable. Limited evidence of the compounds effect on GPX4 is provided. It is good to see that RSL3 inhibits the reduction of 7a-cholesterol-OOH and the inactive isomer does not; however, no quantification of this activity (e.g., IC50) is provided, and the mode of action (presumably a covalent inhibitor) is not studied. Removal of GSH induces ferroptosis. The chloroacetamide group will react with GSH over time - from a chemistry point of view, there is little doubt about this, so it is necessary to show thoroughly that this is not the mechanism of action for the compound. As such, it is good to see that the compound does not deplete GSH in an experiment using a "lethal" 2 uM concentration of RSL3. However, the time course is not shown, and I cannot see in the experimental details what the time frame for the experiment was. In order to confirm GSH depletion is not contributing to the cellular activity, this experiment should be carried out at multiple concentrations and times. This compound will react with GSH - it's just a matter of how quickly. In addition, no work has been presented to study the chemical stability of the compound. The ester group may be prone to hydrolysis. Although the biological effect of this compound is interesting, I would like to see more evidence for how the compound interacts with GPX4, more studies on its stability in vitro (and in vivo - the lack of any pharmacokinetic data does not support the use as an in vivo probe at this time), and more studies on its selectivity. The availability of an inactive control means this molecule can be considered for use as a probe, but only with significant caution in interpretation of the results.

May 25 2017 - 1:28pm

RSL3

Probe Availability

Probe Information

Target Details

Glutathione peroxidase 4, GPx-4, GSHPx-4, MCSP, PHGPx, SMDS, snGPx, snPHGPx

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

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RSL3

Probe Availability

Probe Information

Target Details

Glutathione peroxidase 4, GPx-4, GSHPx-4, MCSP, PHGPx, SMDS, snGPx, snPHGPx

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?