Cat. No.: HY-143346

CCW16

Name: CCW16
Brand: MedChemExpress
SKU: HY-143346
Price: 38.00 USD
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CCW16 is a non-selective cysteine-reactive covalent ligand and an RNF4 E3 ubiquitin ligase recruiter with an IC₅₀ of 1.8 μM against human RNF4^[. CCW16 covalently modifies accessible cysteine residues on RNF4, PRDX1, PRDX2, and PRDX6, attenuating the peroxide-scavenging activity of peroxiredoxins. CCW16 induces oxidative stress through upregulation of HMOX1 and NRF2, and triggers ferroptosis via lipid peroxidation and ROS signaling pathways in an RNF4-independent manner. CCW16 serves as an RNF4-recruiting moiety; it does not induce RNF4 degradation when used alone and can be used to synthesize protein degraders, such as the PROTAC compound CCW 28-3 (HY-156774). CCW16 can be used for research on acute myeloid leukemia and hepatocellular carcinoma.

For research use only. We do not sell to patients.

Purity : 98.73%
CAS No.: 2361138-33-0
Formula: C₂₂H₂₀ClNO₃
Molecular Weight:381.85

Storage:

Powder -20°C, 3 years , 4°C, 2 years ; In solvent -80°C, 6 months , -20°C, 1 month

CCW16 Chemical Structure

CAS No.: 2361138-33-0

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Top Publications Citing

Use of MCE Products

Fluorescent Dye

Strong Fluorescence Signal / Multi-Disciplinary Applications / High Fluorescence Specificity

Biological Activity
- Description
- IC₅₀ & Target
- In Vitro
Chemical Information
Solvent & Solubility
- In Vitro
Purity & Documentation
References
Help & FAQs
Classifications
Bioz

Biological Activity

Description

IC₅₀ & Target

^[4]

RNF4

1.8 μM (IC₅₀)

All Ligands for E3 Ligase Isoforms :

In Vitro

Anti-cancerOxidative StressFerroptosisOthers

Anti-cancer

CCW16 (5 μM; 6 h) in HeLa Flp-In T-REx cells with inducible RNF4 does not degrade RNF4 but induces upregulation of HMOX1, heat shock protein 70 family members, and NRF2, consistent with an oxidative stress response^[1].
CCW16 (5 μM; 6 h)-induced upregulation of HMOX1 and NRF2 occurs in both wild-type and RNF4 knockout HeLa cells, confirming that the oxidative stress response is RNF4-independent^[1].
CCW16 (6 h) alone does not degrade RNF4 protein in HepG2 cells^[3].

Oxidative Stress

CCW16 (10 μM; 2 h + overnight) in HeLa cell lysates reacts non-selectively with approximately 1200 proteins, including the catalytic cysteines of peroxiredoxins PRDX1, PRDX2, and PRDX6, identifying CCW16 as a non-selective covalent ligand that disrupts redox homeostasis^[1].

Ferroptosis

CCW16 (2.5 μM; 2.5 h) induces robust lipid peroxidation in OCI-AML2 cells within 2.5 h, a key hallmark of ferroptosis^[1].
CCW16 (1 μM; 6 h) reduces viability of OCI-AML2 cells after 6 h via induction of ferroptosis, which is rescued by the ferroptosis inhibitor Ferrostatin-1 (HY-100579)^[1].

Ferroptosis

Oxidative Stress

CCW16 (5 μM; 6 h)-induced ferroptotic cell death in HT-1080 cells is fully rescued by GPX4 overexpression, demonstrating that CCW16 exerts its ferroptotic effect through a GPX4-sensitive pathway^[1].

Others

CCW16 (10 µM; 2 h at 4 °C plus overnight at 4 °C) covalently modifies a broad spectrum of over 2000 proteins, including the catalytic cysteines of peroxiredoxins PRDX1, PRDX2, and PRDX6, indicating low target selectivity^[2].
CCW16 binds covalently to RNF4 at C132 or C135 with a low micromolar affinity of 1.8 μM but does not inhibit RNF4 enzymatic activity^[3].
CCW 16 covalently inhibits RNF4 with an IC₅₀ of 1.8 μM by modifying zinc-coordinating cysteines C132 and C135 in the RING domain^[4].

Oxidative Stress

Ferroptosis

CCW16 (5 µM; 6 h) does not degrade RNF4 but induces a strong RNF4-independent oxidative stress response marked by upregulation of HMOX1, NRF2, and heat shock proteins, with enrichment of the ferroptosis pathway^[2].
CCW16 (2.5 µM; 2.5 h) induces robust lipid peroxidation in OCI-AML2 cells^[2].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Western Blot Analysis^[1]

Cell Line:

Wild-type and RNF4 knockout HeLa cells
Concentration:

5 μM
Incubation Time:

6 h
Result:

Induced upregulation of HMOX1 in both wild-type and RNF4 knockout HeLa cells, demonstrating the stress response is independent of RNF4.
Increased NRF2 protein levels in both wild-type and RNF4 knockout HeLa cells, consistent with activation of the NRF2-mediated oxidative stress response pathway.

Cell Viability Assay^[1]

Cell Line:

OCI-AML2 acute myeloid leukemia cells
Concentration:

1 μM
Incubation Time:

6 h; 1 h (ferrostatin-1 pretreatment)
Result:

Caused a significant reduction in OCI-AML2 cell viability.
Induced cell death that was substantially rescued by pretreatment with the ferroptosis inhibitor ferrostatin-1, confirming cell death occurs via ferroptosis.

Cell Viability Assay^[1]

Cell Line:

HT-1080 fibrosarcoma cells
Concentration:

5 μM
Incubation Time:

6 h; 1 h (ferrostatin-1 pretreatment)
Result:

Induced cell death in parental HT-1080 cells carrying an empty vector.
Did not induce cell death in cells stably overexpressing GPX4, which were fully protected, indicating ferroptotic activity is linked to GPX4 function.

Related Protocols :

Chemical Information

CAS No. 2361138-33-0
Appearance Solid
Molecular Weight 381.85
Formula C₂₂H₂₀ClNO₃
Color Light yellow to yellow
SMILES

O=C(N(CC1=CC=CC=C1)C2=CC=C(OC3=CC=C(OC)C=C3)C=C2)CCl
Shipping

Room temperature in continental US; may vary elsewhere.

Storage

Powder	-20°C	3 years
	4°C	2 years
In solvent	-80°C	6 months
	-20°C	1 month

Solvent & Solubility

In Vitro:

DMSO : 25 mg/mL (65.47 mM; ultrasonic and warming and heat to 60°C; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

View the Complete Stock Solution Preparation Table

Preparing Stock Solutions

View the Complete Stock Solution Preparation Table

Concentration Solvent Mass

1 mg

5 mg

10 mg

Concentration

Solvent

Mass

1 mM

2.6188 mL

13.0941 mL

26.1883 mL

5 mM

0.5238 mL

2.6188 mL

5.2377 mL

10 mM

0.2619 mL

1.3094 mL

2.6188 mL

Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.

Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month. When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

Molarity Calculator

Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

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Dilution Calculator

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Purity & Documentation

Select Batch:

Purity: 99.98%

Assay: 99.98%

ee.: 99.98%

Data Sheet (283 KB)
SDS (458 KB)
- English - EN (458 KB)
- Français - FR (458 KB)
- Deutsch - DE (458 KB)
- Norwegian - NO (458 KB)
- Español - ES (458 KB)
- Swedish - SV (458 KB)
- Italian - IT (458 KB)
- Korean - KR (458 KB)
- Portuguese - PT (458 KB)
COA (2659 KB)
Elemental Analysis Report (2659 KB)
HNMR (2659 KB)
CNMR (2659 KB)
RP-HPLC (2659 KB)
NP-HPLC (2659 KB)
MS (2659 KB)
LCMS (2659 KB)
IR (2659 KB)
GC (2659 KB)
OR (2659 KB)
SDS-PAGE (2659 KB)
SEC-HPLC (2659 KB)
Other Report (2659 KB)
Handling Instructions (2659 KB)

References

[1]. Gotthardt G, et al. Cysteine-reactive covalent chloro-N-acetamide ligands induce ferroptosis mediated cell death. EMBO reports. 2025 Nov;26(22):5501-5532.

[2]. Gina Gotthardt, et al. The cysteine-reactive covalent RNF4 ligand CCW16 induces ferroptosis in AML cells by activation of ROS signaling. bioRxiv. December 19, 2024.

[3]. Wan H, et al. Development of the first-in-class RNF4 PROTAC degrader as potential therapeutics for hepatocellular carcinoma. European journal of medicinal chemistry. 2026 Apr 05;307:118668. [Content Brief]

[4]. Kiely-Collins H, et al. The role of reversible and irreversible covalent chemistry in targeted protein degradation. Cell Chem Biol. 2021 Jul 15;28(7):952-968. [Content Brief]

Help & FAQs

How should lyophilized recombinant proteins be reconstituted and stored?

1. Before opening the cap, centrifuge the vial at 13000 rpm for 20-30 seconds. This step will ensure that any lyophilized powder that may have adhered to the cap or walls is collected at the bottom of the vial, minimizing the risk of product loss.

Complete Stock Solution Preparation Table

Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month. When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

Optional Solvent	Concentration Solvent Mass	1 mg	5 mg	10 mg	25 mg
DMSO	1 mM	2.6188 mL	13.0941 mL	26.1883 mL	65.4707 mL
	5 mM	0.5238 mL	2.6188 mL	5.2377 mL	13.0941 mL
	10 mM	0.2619 mL	1.3094 mL	2.6188 mL	6.5471 mL
	15 mM	0.1746 mL	0.8729 mL	1.7459 mL	4.3647 mL
	20 mM	0.1309 mL	0.6547 mL	1.3094 mL	3.2735 mL
	25 mM	0.1048 mL	0.5238 mL	1.0475 mL	2.6188 mL
	30 mM	0.0873 mL	0.4365 mL	0.8729 mL	2.1824 mL
	40 mM	0.0655 mL	0.3274 mL	0.6547 mL	1.6368 mL
	50 mM	0.0524 mL	0.2619 mL	0.5238 mL	1.3094 mL
	60 mM	0.0436 mL	0.2182 mL	0.4365 mL	1.0912 mL

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CCW16

All Ligands for E3 Ligase Isoforms