Erv1 (IC50 = 900 nM); ALR (IC50 = 700 nM); Erv2 (IC50 = 1.4 μM)
Erv1 (yeast sulfhydryl oxidase) - IC50 = 900 nM [1]
ALR (mammalian homolog of Erv1, Augmenter of Liver Regeneration) - IC50 = 700 nM [1]
Erv2 (yeast endoplasmic reticulum paralog) - IC50 = 1.4 μM [1]

- MitoBloCK-6 inhibited Erv1 oxidase activity in the in vitro Amplex Red-HRP assay with an IC50 of 900 nM. It also inhibited ALR and Erv2 with IC50 values of 700 nM and 1.4 μM, respectively. [1]
- MitoBloCK-6 did not inhibit the oxidative folding activity of protein disulfide isomerase (PDI) in an insulin reduction assay. [1]
- MitoBloCK-6 did not inhibit succinate dehydrogenase activity of the mitochondrial respiratory chain in isolated mitochondria measured with a Clark-type oxygen electrode. [1]
- LC-MS analysis showed that MitoBloCK-6 was stable over pH range (3.4, 6.5, 7.4) with a similar retention time (3.03 min) and constant area under the curve. [1]
- Mass spectrometry analysis revealed that only a small fraction (<3%) of MitoBloCK-6 likely degraded to 3,5-dichlorosalicylaldehyde that covalently modified Erv1. Most of the Erv1 remained unmodified. [1]
- In a reconstituted oxidation assay, MitoBloCK-6 (at concentrations of 5, 10, 25, and 50 μM) impaired the oxidation of Tim13. After 3 hours, approximately 80% of Tim13 was oxidized in the DMSO control, whereas only 15% was oxidized in the presence of MitoBloCK-6. [1]
- MitoBloCK-6 caused a significant decrease in H2O2 production during the oxidation of Tim13 and Cmc1 in a dose-dependent manner (tested at 2.5, 5, 10, 25, and 50 μM for Cmc1 with Erv1; at 50 μM for Cmc1 with ALR). [1]
- In oxygen consumption assays using an oxygen electrode in the presence of excess DTT, MitoBloCK-6 (at 100 μM) resulted in a concentration-dependent decrease in the oxygen consumption rate by Erv1. [1]

- In zebrafish embryos: Zebrafish embryos (3 hours post-fertilization) treated with 2.5 μM MitoBloCK-6 until 72 hours post-fertilization displayed ventral curvature of the body and cardiac edema. Cardiac development was retarded; hearts failed to loop, becoming stringy and extended. Mitochondrial fluorescence (DsRed) in cardiac tissue was decreased. Heart rate was decreased by 50% compared to DMSO controls. Erythrocyte pooling along the yolk sac and absence of red blood cells in the tail were observed. These effects were reversible when MitoBloCK-6 was removed after 24 hours. Higher concentrations were toxic. [1]
- Combination treatment in zebrafish: Treatment with sub-optimal concentrations of MitoBloCK-6 (1 or 2 μM) combined with sub-optimal amounts of ALR ATG morpholino (1 or 2 ng) resulted in additive developmental defects (cardiac edema, decreased fluorescence), similar to treatment with 2.5 μM MitoBloCK-6 or 4 ng morpholino alone. [1]

- High-throughput screening Amplex Red-HRP assay: Recombinant Erv1 (10 μM) was aliquoted into 384-well plates. Compounds (at ~12.5 μM final concentration) or DMSO were added and incubated for 1 hour at 25°C. Amplex Red (46 μM final) and horseradish peroxidase (0.092 U/ml final) were then added, followed by incubation for 10 minutes. The reaction was initiated by adding DTT (20 μM final). After 12 minutes of incubation (to achieve maximal signal-to-noise ratio in the kinetic linear range), fluorescence was measured at an excitation wavelength of 545 nm and an emission wavelength of 590 nm. A counter screen was performed by reacting H2O2 (800 nM) with Amplex Red-HRP in the presence of compounds to eliminate false positives that directly inhibited the detection system. [1]
- IC50 determination: Serial dilutions of compounds in 100% DMSO were pinned into assay plate wells containing 10 μM Erv1, Erv2, or ALR. The assay was performed similarly to the HTS method, and IC50 values were calculated from dose-response curves. [1]
- PDI insulin reduction assay: The ability of PDI to reduce insulin was assessed in the presence of MitoBloCK-6 to test for general impairment of redox-active enzymes. [1]
- Succinate dehydrogenase activity assay: Isolated mitochondria were incubated in a Clark-type oxygen electrode, and oxygen consumption was measured after succinate addition. MitoBloCK-6 or DMSO was added to assess effects on succinate dehydrogenase. Malonate was used as a control inhibitor, and CCCP was used to uncouple respiration. [1]
- LC-MS stability analysis: MitoBloCK-6 stability was assessed at pH 6.5, 7.4, and 3.4 using liquid chromatography-mass spectrometry. Retention time and area under the curve were analyzed. [1]
- Mass spectrometry for covalent modification: Erv1 (25 μM) was incubated with MitoBloCK-6 (75 μM) or 3,5-dichlorosalicylaldehyde (75 μM). Mass spectrometry was performed to detect covalent adducts. Lysozyme was used as a control protein, and formaldehyde was used as a positive control for covalent modification. [1]
- Reconstituted Tim13 oxidation assay: Reduced Tim13 (15 μM) was incubated with catalytic amounts of Erv1 (1 μM) and Mia40 (1 μM) in an aerobic environment, with or without MitoBloCK-6. At indicated time points, aliquots were removed and free thiols on Tim13 were modified by addition of 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid (AMS). Oxidized and reduced Tim13 were detected by non-reducing SDS-PAGE and immunoblotting with anti-Tim13 antibodies. [1]
- Reconstituted H2O2 production assay: The same reconstitution assay as for Tim13 or Cmc1 oxidation was performed, and H2O2 production was monitored over a 30-minute period using the Amplex Red-HRP assay. [1]
- Oxygen consumption assay for Erv1: Erv1 oxidase activity was measured using a Clark-type oxygen electrode in the presence of excess DTT. The oxygen consumption rate was recorded after addition of Erv1 alone, Erv1 with DMSO, or Erv1 with varying concentrations of MitoBloCK-6. [1]

- Import assays in isolated yeast mitochondria: Energized wild-type yeast mitochondria were preincubated with 20-50 μM MitoBloCK-6 or 1% DMSO for 15 minutes, followed by addition of radiolabeled substrates (Mia40, Cmc1, Cox19, Tim8, Tim23, AAC, Su9-DHFR). A time course assay was performed, and aliquots were removed and treated with protease to remove non-imported precursors. Import was strongly decreased for twin CX9C proteins and Erv1; Tim8 import was impaired by 40%; TIM22 substrates (Tim23, AAC) were decreased by approximately 50% at 20 μM; TIM23 substrates (Su9-DHFR, cyt b2-DHFR, Hsp60) were not impaired even at 50 μM. [1]
- Blue-Native PAGE analysis of AAC import: AAC was imported into mitochondria in the presence of DMSO or 25 μM MitoBloCK-6. Aliquots were removed, mitochondria were solubilized in 1% digitonin, and samples were separated on blue-native gels followed by autoradiography or immunoblotting with anti-Tom40 antibodies. In the presence of DMSO, AAC accumulated in a 90 kDa complex (assembled dimer). In the presence of MitoBloCK-6, AAC accumulated in a 500 kDa complex with the TOM complex, and this intermediate was protease-sensitive. [1]
- Import assays in Erv1-overexpressing mitochondria: Import assays for Mia40, Cmc1, and AAC were performed in mitochondria isolated from wild-type yeast or yeast overexpressing Erv1-His ([a2up]Erv1). The concentration of MitoBloCK-6 required to inhibit import increased from 10 μM to 50 μM for Mia40 and Cmc1, and from 15 μM to 30 μM for AAC. [1]
- MIC50 determination in yeast: The MIC50 of MitoBloCK-6 was determined in a Δpdr5Δsnq2 yeast strain (lacking multi-drug resistance pumps) and in the same strain overexpressing Erv1-His from a high-copy plasmid. The MIC50 was 15.2 μM in the parental strain and increased to 28.3 μM in the Erv1-overexpressing strain. [1]
- Mitochondrial integrity assays: Isolated energized mitochondria were incubated with 1% DMSO or MitoBloCK-6, then centrifuged. Released proteins in the supernatant were analyzed by Coomassie staining and immunoblotting for marker proteins (aconitase, AAC, Tim54, Mia40, Ccp1, cyt c). No protein release was detected, indicating that MitoBloCK-6 did not alter mitochondrial membrane integrity. [1]
- Membrane potential assay: Isolated mitochondria were incubated in a Clark-type oxygen electrode, and respiration was initiated with NADH. Oxygen consumption rate was measured after addition of DMSO vehicle or MitoBloCK-6. CCCP was used as a control to uncouple mitochondria. MitoBloCK-6 did not alter the oxygen consumption rate. [1]
- Co-immunoprecipitation from isolated mitochondria: Mitochondria from an Erv1-His strain were incubated with 50 μM MitoBloCK-6 or 1% DMSO for 30 minutes at 25°C, then solubilized in 1% digitonin. Erv1-His was purified with Ni⁺²-agarose. Bound proteins were eluted and analyzed by immunoblotting for Mia40 and cyt c. In the presence of MitoBloCK-6, binding of Mia40 and cyt c to Erv1 was decreased by 75% and 95%, respectively. [1]
- HeLa cell assays: HeLa cells transiently transfected with mitochondrial matrix-targeted Su9-EGFR were treated with 50 μM MitoBloCK-6 for 12-16 hours. Mitochondrial morphology was visualized by microscopy after co-labeling with Mitotracker-Red. No disruption of the mitochondrial network was observed, even at concentrations up to 100 μM. MTT cell viability assay showed that 100 μM MitoBloCK-6 did not significantly reduce cell viability. Cyt c release assay showed that 50 μM MitoBloCK-6 treatment for 12-16 hours failed to initiate cyt c release, unlike staurosporine (positive control). [1]
- hESC (HSF1) assays: HSF1 human embryonic stem cells were treated with 20 μM MitoBloCK-6, ES-1, ES-2, or 0.1% DMSO. MitoBloCK-6 and ES-2 (but not ES-1) induced marked cell death and apoptosis. Immunofluorescence microscopy with anti-cyt c and anti-Tomm20 antibodies showed that MitoBloCK-6 and ES-2 caused cyt c release from mitochondria (diffuse staining not overlapping with Tomm20). Quantification showed that the percentage of cells with cyt c release was similar to that induced by actinomycin D (a known apoptosis inducer). Western blot analysis detected cleaved caspase-3 and PARP cleavage after MitoBloCK-6 treatment. Alkaline phosphatase activity staining showed that hESC viability started to decline after 5 hours of MitoBloCK-6 treatment. Differentiation of HSF1 cells with 10 μM retinoic acid for 4 days rendered them resistant to MitoBloCK-6-induced cell death. [1]

- Zebrafish embryo treatment: Zebrafish embryos at 3 hours post-fertilization (hpf) were placed in either 1% DMSO (vehicle control) or 2.5 μM MitoBloCK-6 in buffered water. Embryos were allowed to develop until 72 hpf, and development was visualized by microscopy. For reversibility studies, embryos were treated with MitoBloCK-6 from 3 to 24 hpf, followed by removal of the compound and further development until 72 hpf. [1]
- Zebrafish o-dianisidine staining: Embryos at 72 hpf were stained with o-dianisidine, which binds to heme, to visualize hematopoietic development and red blood cells. [1]
- Zebrafish cardiac imaging: A transgenic zebrafish line with DsRed targeted to mitochondria under control of the cardiac-specific cardiac myosin light chain promoter (cmlc2) was used. Embryos were treated with DMSO or 2.5 μM MitoBloCK-6, and cardiac development and mitochondrial fluorescence were visualized by fluorescence microscopy at 72 hpf. Heart rate was measured. [1]
- Zebrafish morpholino injections: One-cell zebrafish embryos were injected with 4 ng of an ATG morpholino targeted to ALR to prevent ALR translation. Phenotypes were compared to MitoBloCK-6-treated embryos. For combination studies, embryos were treated with sub-optimal concentrations of MitoBloCK-6 (1 or 2 μM) and injected with sub-optimal amounts of ALR ATG morpholino (1 or 2 ng) in different combinations. [1]

The study notes that MitoBloCK-6 was stable over a pH range of 3.4 to 7.4 based on LC-MS analysis. [1]

- In HeLa cells, MitoBloCK-6 at concentrations up to 100 μM did not significantly reduce cell viability (MTT assay) and did not disrupt mitochondrial morphology or cause cyt c release at 50 μM for 12-16 hours. [1]
- In zebrafish embryos, higher concentrations of MitoBloCK-6 than 2.5 μM were toxic. [1]
- MitoBloCK-6 did not alter mitochondrial membrane integrity in isolated mitochondria, as no release of marker proteins (aconitase, AAC, Tim54, Mia40, Ccp1, cyt c) was detected. [1]
- MitoBloCK-6 did not dissipate the mitochondrial membrane potential (Δψ) or disrupt oxidative phosphorylation, as measured by oxygen consumption with a Clark-type electrode. [1]

[1]. A small molecule inhibitor of redox-regulated protein translocation into mitochondria. Dev Cell. 2013 Apr 15;25(1):81-92.

- MitoBloCK-6 (2,4-dichloro-6-(((phenylamino)phenyl)imino)methyl)phenol) was identified from a high-throughput screen of approximately 50,000 compounds from Chembridge, Kwon, and Asinex libraries at 10 μM concentration. The screen yielded 184 primary candidate inhibitors, and MitoBloCK-6 was selected for further characterization as a "MitoBloCK" (Mitochondrial protein import Blocker from the Carla Koehler lab) compound. [1]
- The mechanism of action involves inhibition of Erv1/ALR oxidase activity, which impairs the Mia40/Erv1 disulfide relay system. MitoBloCK-6 disrupts the interaction between Erv1 and its partner proteins Mia40 and cytochrome c (decreased binding by 75% and 95%, respectively). [1]
- MitoBloCK-6 specifically inhibits the import of substrates of the Mia40/Erv1 pathway and the TIM22 import pathway, but not the TIM23 import pathway. [1]
- The SAR compound ES-2, but not ES-1, inhibited Erv1 function similarly to MitoBloCK-6 (IC50 of 2.2 μM for ES-2). [1]
- MitoBloCK-6 did not inhibit differentiated cells (HeLa, HEK293, NHDF) but specifically induced apoptosis in human embryonic stem cells (hESCs), suggesting a distinct role for ALR in pluripotent stem cell maintenance. Differentiated HSF1 cells (treated with retinoic acid) were resistant to MitoBloCK-6. [1]
- In zebrafish, MitoBloCK-6 treatment phenocopied ALR knockdown by ATG morpholino, causing cardiac defects, edema, and impaired erythropoiesis. The effects were additive when sub-optimal concentrations of MitoBloCK-6 and morpholino were combined. [1]

C19H14CL2N2O

357.234

356.048

C, 63.88; H, 3.95; Cl, 19.85; N, 7.84; O, 4.48

303215-67-0

303215-67-0

1035235

Yellow to orange solid

1.3±0.1 g/cm3

535.1±50.0 °C at 760 mmHg

277.4±30.1 °C

0.0±1.5 mmHg at 25°C

1.627

6.34

2

3

4

24

406

0

ClC1=C([H])C(=C([H])C(/C(/[H])=N/C2C([H])=C([H])C(=C([H])C=2[H])N([H])C2C([H])=C([H])C([H])=C([H])C=2[H])=C1O[H])Cl

DBYOPSAQYAKIQV-UHFFFAOYSA-N

InChI=1S/C19H14Cl2N2O/c20-14-10-13(19(24)18(21)11-14)12-22-15-6-8-17(9-7-15)23-16-4-2-1-3-5-16/h1-12,23-24H

2-[(4-anilinophenyl)iminomethyl]-4,6-dichlorophenol

MitoBlock6; MitoBlock-6; Mito BloCK-6; 303215-67-0; 2-[[(4-Anilinophenyl)imino]methyl]-4,6-dichlorophenol; 2-{[(4-anilinophenyl)imino]methyl}-4,6-dichlorophenol; 2-[(4-anilinophenyl)iminomethyl]-4,6-dichlorophenol; MitoBlock 6

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~50 mg/mL (~140.0 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)