Apoptosome

There is a dose-dependent inhibition of DEVDase activity, DFF45/ICAD and PARP cleavage, and caspase 9, 3, and 7 processing when cell extracts are stimulated with cytochrome c and dATP in the presence of NS3694 (10-100 μM; 120 min)[1].
NS3694 (10-100 μM) inhibits TNF-induced effector caspase activation and apoptosis in MCF-casp3 cells[1].
Inhibiting mitochondria-mediated apoptosis, NS3694 specifically inhibits the formation of the apoptosome complex and the activation of caspase-9 caused by cytochrome C. By preventing the initiator caspase-9's activation, NS3694 prevents the formation of the apoptosome Apaf-1[1].

---

NS3694 inhibits the coimmunoprecipitation of caspase 9 and Apaf-1 from HeLa cell cytosol stimulated with cytochrome c and dATP. Next, we tested whether NS3694 interferes with the cytochrome c- and dATP-induced formation of the apoptosome complex. Cytosolic extracts of HeLa cells were incubated with cytochrome c and dATP in the presence or absence of 100 μM NS3694 or 1 μM zVAD-fmk for 1 h prior to the immunoprecipitation of caspase 9. Caspase 9 antibody precipitated practically all caspase 9 present in both unactivated and cytochrome c- and dATP-activated extracts. As predicted, Apaf-1 coimmunoprecipitated only with caspase 9 in activated extracts, and this was also the case in the presence of zVAD-fmk (Fig. 4). Thus, the caspase 9 antibody immunoprecipitates Apaf-1 only under conditions when the apoptosome holoenzyme complex is formed. NS3694 did not affect the ability of the antibody to bind caspase 9, but it significantly inhibited the coimmunoprecipitation of Apaf-1, indicating that NS3694 alters the cytochrome c- and dATP-triggered association between Apaf-1 and caspase 9. A reverse immunoprecipitation assay using an Apaf-1 antibody was also performed, but unfortunately, activation of the apoptosome by cytochrome c and dATP inhibited the immunoprecipitation of Apaf-1, suggesting that the epitope recognized by the anti-Apaf-1 antibody was covered by the apoptosome complex (data not shown).

---

NS3694 inhibits the formation of the active ∼700-kDa apoptosome complex. [1]
We wished to determine how NS3694 disrupted the formation of the apoptosome complex, and for this purpose, we investigated the effects of the inhibitor in dATP-stimulated THP.1 cell lysates (whole-cell lysates that contain mitochondrial proteins including cytochrome c). Apoptosome formation has been well-characterized in this model system, and NS3694 also inhibited the activation and processing of caspases in a concentration-dependent manner (Fig. 5A). To analyze whether NS3694 interfered with the assembly of the apoptosome complex, we fractionated control THP.1 cell lysates and THP.1 cell lysates stimulated for 30 min with dATP in the absence or presence of NS3694 on an analytical Superose 6 HR 10/30 column. In control lysates, Apaf-1 eluted as a monomer, whereas in dATP-stimulated lysates, it was mainly oligomerized to the active ∼700-kDa complex and to a lesser extent to the inactive 1.4-MDa complex (Fig. 5B). In the presence of NS3694, dATP failed to trigger the formation of the 700-kDa complex, and all Apaf-1 was found in the 1.4-MDa complex. In control lysates, caspase 9 eluted as a monomeric proform, whereas in dATP-stimulated lysates, it was processed to the 35- or 37-kDa form and distributed between the 700-kDa complex and the free form. In the presence of NS3694, dATP-induced processing of procaspase 9 to the 35- or 37-kDa form was blocked, and it eluted with the 1.4-MDa complex.

---

Apoptosome activation is essential for TNF-induced caspase activation in tumor cells. [1]
Ectopic expression of Bcl-2 or Bcl-XL effectively inhibits TNF-induced caspase activation and death in MCF-7 breast carcinoma and ME-180 cervix carcinoma cells, suggesting that MOMP is essential for death signaling in these cells (9, 27). Since MOMP can lead to caspase activation and death via either cytochrome c-induced apoptosome formation or IAP antagonism mediated by Smac/Diablo or Omi/htra2 (6, 12), we next used NS3694 to investigate the role of the apoptosome in this process. Supporting the requirement of cytochrome c release for this death pathway, the decreased viability of MCF-casp3 cells induced by 1 ng of TNF per ml was completely blocked by pretreatment of cells with 50 μM NS3694, and the protection was still evident following treatment with 5 ng of TNF per ml (Fig. 6A). The level of protection was comparable to that observed by the inhibition of effector caspases by 200 μM DEVD-CHO but was somewhat weaker than that conferred by 5 μM zVAD-fmk, which inhibits the activity of all known caspases, including the TNF-activated initiator caspase, caspase 8 (Fig. 6A).

---

NS3694 has no effect on TNF-induced caspase-independent death of WEHI-S cells. [1]
TNF triggers caspase activation and cell death in WEHI-S murine fibrosarcoma cells (8). The activated caspases are, however, not responsible for cell death, as the complete inhibition of activated caspases by 1 to 10 μM zVAD-fmk results in increased TNF-induced death (8). Accordingly, NS3694 (10 to 50 μM) treatment effectively inhibited TNF-induced caspase activation without affecting TNF-induced cell death (Fig. 8). As we have shown earlier, high concentrations of zVAD-fmk (>30 μM) that also inhibit other cysteine proteases (i.e., cathepsins and calpains [8, 20]) confer complete protection in this model system (Fig. 8).

---

NS3694 does not inhibit FasL-induced caspase activation or death in type I cells. [1]
In SKW6.4 cells, FasL triggers the extrinsic pathway mediated by the direct activation of caspase 3 by caspase 8 (27). To test the ability of NS3694 to interfere with the extrinsic death pathway, we treated SKW6.4 cells with NS3694 at concentrations up to 50 μM prior to treatment with FasL and measured cell viability and effector caspase activity (DEVDase) after 16 h. NS3694 failed to inhibit FasL-induced cell death and caspase activation in SKW6.4 cells (Fig. 9).

In vitro apoptosome assay and caspase activity measurements. [1]
Subconfluent cultures of HeLa cells were harvested by scraping on ice, washed in ice-cold phosphate-buffered saline (PBS), and resuspended in equal volume of ice-cold isotonic lysis buffer (20 mM HEPES-KOH [pH 7.5], 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 1 mM dithiothreitol [DTT], 10 μg of aprotinin per ml, 1 μg of leupeptin per ml, 1 μg of pepstatin A per ml, 100 μg of pefabloc per ml). After 30-min incubation on ice, the cells were lysed by 30 strokes of a Dounce homogenizer and centrifuged at 750 × g for 10 min. The supernatant obtained was further centrifuged at 10,000 × g for 10 min and at 20,000 × g for 30 min. The clarified supernatant was stored in aliquots at −80°C and used at protein concentrations ranging from 5 to 10 mg/ml. The apoptosome was activated by the addition of 1 mM dATP and 1 μM horse heart cytochrome c to the cytosolic HeLa cell extract (protein concentration, 5 to 10 mg/ml) containing 100 μM DEVD-7-amino-4-(trimethyl-fluoromethyl) coumarin (AFC). When screening the molecular library, the compounds were added at a concentration of 100 μM prior to the addition of cytochrome c and dATP. After 30-min incubation at 37°C, the Vmax of the liberation of AFC (excitation wavelength, 400 nm; emission wavelength, 489 nm) was measured for 30 to 45 min at 37°C with a Spectramax Gemini fluorometer.
To measure the total cellular DEVDase activity, cells were treated as indicated in the figure legends, scraped on ice, washed in PBS, and resuspended in ice-cold caspase lysis buffer (25 mM HEPES, 5 mM MgCl2, 1 mM EGTA, 0,5% Triton X-100, 5 mM DTT, 10 μg of aprotinin per ml, 1 μg of leupeptin per ml, 1 μg of pepstatin A per ml, 1 μM pefabloc [pH 7.5]) and left on ice for 30 min. Lysates were then centrifuged at 20,000 × g for 10 min, and the supernatant was analyzed for protein content by using the Bio-Rad protein assay kit. The enzyme activities were estimated by adding cell extracts (10 μl) to caspase reaction buffer (40 μl) (100 mM HEPES, 20% glycerol, 0.5 mM EDTA, 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 5 mM DTT, 1 mM pefabloc [pH 7.5]) in the presence of 100 μM DEVD-AFC. The Vmax of liberation of AFC was measured as described above and corrected for the protein concentration of the untreated sample.
The activities of recombinant caspases were measured as described above by mixing the indicated compounds with recombinant caspase 3 (60 ng/ml) or caspase 9 (12 μg/ml; Calbiochem) in 25 μl of caspase lysis buffer and adding 200 μM DEVD-AFC or LEHD-AFC, respectively, in 25 μl of caspase reaction buffer. The fluorogenic chromophore AFC served as a control.
To measure DEVDase activity in SKW6.4 cells, 300,000 cells/1.5 ml of complete medium were incubated with 25% FasL supernatant for 16 h. Cells were pelleted and permeabilized for 15 min on ice in an extraction buffer (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA) containing 1 mM pefabloc and 200 μg of digitonin per ml. Cells were then pelleted, and DEVDase activity, and lactate dehydrogenase (LDH) activity was measured in 50 μl of supernatant. DEVDase activity was measured as described above, and LDH activity was measured according to the manufacturer's instructions.

[1]. Diarylurea compounds inhibit caspase activation by preventing the formation of the active 700-kilodalton apoptosome complex. Mol Cell Biol. 2003 Nov;23(21):7829-37.

4-chloro-2-[[oxo-[3-(trifluoromethyl)anilino]methyl]amino]benzoic acid is a member of ureas.

---

The release of mitochondrial proapoptotic proteins into the cytosol is the key event in apoptosis signaling, leading to the activation of caspases. Once in the cytosol, cytochrome c triggers the formation of a caspase-activating protein complex called the apoptosome, whereas Smac/Diablo and Omi/htra2 antagonize the caspase inhibitory effect of inhibitor of apoptosis proteins (IAPs). Here, we identify diarylurea compounds as effective inhibitors of the cytochrome c-induced formation of the active, approximately 700-kDa apoptosome complex and caspase activation. Using diarylureas to inhibit the formation of the apoptosome complex, we demonstrated that cytochrome c, rather than IAP antagonists, is the major mitochondrial caspase activation factor in tumor cells treated with tumor necrosis factor. Thus, we have identified a novel class of compounds that inhibits apoptosis by blocking the activation of the initiator caspase 9 by directly inhibiting the formation of the apoptosome complex. This mechanism of action is different from that employed by the widely used tetrapeptide inhibitors of caspases or known endogenous apoptosis inhibitors, such as Bcl-2 and IAPs. Thus, these compounds provide a novel specific tool to investigate the role of the apoptosome in mitochondrion-dependent death paradigms.[1]

C15H10CLF3N2O3

358.6997

358.033

426834-38-0

426834-38-0

10109069

Solid

1.574g/cm3

378.448ºC at 760 mmHg

182.679ºC

1.638

4.847

3

6

3

24

475

0

ClC1C=CC(C(O)=O)=C(NC(NC2C=CC=C(C(F)(F)F)C=2)=O)C=1

GNCZTZCPXFDPLI-UHFFFAOYSA-N

InChI=1S/C15H10ClF3N2O3/c16-9-4-5-11(13(22)23)12(7-9)21-14(24)20-10-3-1-2-8(6-10)15(17,18)19/h1-7H,(H,22,23)(H2,20,21,24)

4-chloro-2-[[3-(trifluoromethyl)phenyl]carbamoylamino]benzoic acid

NS 3694; NS3694; 426834-38-0; Apoptosis Inhibitor II, 4-chloro-2-[[3-(trifluoromethyl)phenyl]carbamoylamino]benzoic Acid; CHEMBL154696; 4-chloro-2-(3-(3-(trifluoromethyl)phenyl)ureido)benzoic acid; 4-Chloro-2-[3-(3-trifluoromethyl-phenyl)-ureido]benzoic acid; NS-3694

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: ~250 mg/mL (~697.0 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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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