Caspase

Apoptosis is a pathway of cell death orchestrated by a family of proteases called caspases. Reactive oxygen species (ROS) are produced when TNFα is activated, but Boc-D-fmk prevents this. With an IC50 of 39 µM[1], Boc-D-FMK blocks TNFα-induced apoptosis. Genistein-induced apoptosis of p815 cells is stopped by BocD-fmk at a concentration of 50 µM. The release of mitochondrial apoptotic factors is inhibited by BocD-fmk, according to confocal microscopy[2].

In bile duct-ligated rats, Boc-D-FMK-fmk significantly reduces hepatocyte apoptosis and may increase survival after endotoxin challenge[3]. After receiving a single injection of Boc-D-FMK, MNs are long-term protected from root avulsion-induced death for more than 8 weeks, and the Boc-D-FMK-treated MNs are able to regenerate their axons into a PN graft that has been implanted and reinnervate the target muscle[4].

In most cell types constitutive and ligand-induced apoptosis is a caspase-dependent process. In neutrophils, however, the broad-spectrum caspase inhibitor z-VAD-fmk enhances tumor necrosis factor-alpha (TNF alpha)-induced cell death, and this has been interpreted as evidence for caspase-dependent and -independent cell death pathways. Our aim was to determine the specificity of the effect of z-VAD-fmk in neutrophils and define the potential mechanism of action. While confirming that z-VAD-fmk (> 100 microM) enhances TNF alpha-induced neutrophil apoptosis, lower concentrations (1-30 microM) completely blocked TNF alpha-stimulated apoptosis. Boc-D-fmk, a similar broad-spectrum caspase inhibitor, and z-IETD-fmk, a selective caspase-8 inhibitor, caused a concentration-dependent inhibition of only TNF alpha-stimulated apoptosis. Moreover, the caspase-9 inhibitor, Ac-LEHD-cmk, had no effect on TNF alpha-induced apoptosis, and z-VAD-fmk and Boc-D-fmk inhibited TNF alpha-stimulated reactive oxygen species (ROS) generation. These data suggest that TNF alpha-induced apoptosis in neutrophils is fully caspase dependent and uses a mitochondrial-independent pathway and that the proapoptotic effects of z-VAD-fmk are compound specific and ROS independent.[1]

In a preliminary study, we found that benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD- fmk), unlike Boc-aspartyl(OMe)-fluoromethylketone (BocD-fmk), at usual dosage could not prevent genistein-induced apoptosis of p815 mastocytoma cells. This study was undertaken to reveal the mechanism underlying the incapability of zVAD-fmk in preventing this type of apoptosis. We observed that 14-3-3 protein level was reduced in genistein-treated cells and that BocD-fmk but not zVAD-fmk prevented the reduction of 14-3-3 protein level and the release of Bad from 14-3-3. We also demonstrated that truncated Bad to Bcl-xL interaction in genistein- treated cells was prevented by BocD-fmk but not by zVAD-fmk treatment. Our data indicate that BocD- fmk, compared to zVAD-fmk, has a certain preference for inhibiting 14-3-3/Bad signalling pathway. We also elucidated that this differential efficacy of BocD-fmk and zVAD-fmk resulted from the different effect in inhibiting caspase-6 and that co-treatment of zVAD-fmk and caspase-6 specific inhibitor substantially prevented genistein-induced apoptosis. Our data shows that caspase-6 plays a role on Bad/14-3-3 pathway in genistein-induced apoptosis of p815 cells, and that the usual dose of zVAD-fmk, in contrast to BocD-fmk, did not prevent caspase-6 acting on 14-3-3/Bad-mediated event.[2]

Male Sprague-Dawley rats, weighing 280-300 g were randomized to three groups of eight rats each. Group 1 (OBBOC-D) underwent common bile duct ligation and simultaneous treatment with Boc-D-FMK-fmk (dissolved in dimethylsulfoxide [DMSO]). Group 2 (OBZFA) underwent common bile duct ligation and simultaneous treatment with ZFA-fmk (dissolved in DMSO). Group 3 (SHAM) underwent sham operation and simultaneous treatment with the same amount of dimethylsulfoxide (DMSO, n = 4) or the same amount of normal saline (n = 4). After 3 days, liver tissue was harvested for histopathological analysis and measurements of apoptosis. Survival rates were measured in a separate experiment in which animals underwent the same protocol. The animals received endotoxin (15 mg/kg) in the afternoon of the third postoperative day. Animals were observed for 48 h and the survival rates were recorded. Results: When compared with sham operation, common bile duct ligation with ZFA-fmk (placebo) significantly increased hepatocyte apoptosis (P < 0.001). When compared with the OBZFA group, Boc-D-FMK significantly diminished the increased hepatocyte apoptosis in the OBBOC-D group (P < 0.001). There is no difference in hepatocyte apoptosis (P = 0.05) between OBBOC-D and SHAM groups. After endotoxin challenge, the 48 h survival rates were 100%, 87.5% and 62.5% for the SHAM, OBBOC-D and OBZFA groups, respectively. Conclusions: Boc-D-FMK-fmk effectively attenuated the hepatocyte apoptosis in bile duct-ligated rats and may improve the survival rates after endotoxin challenge.[3]

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We examined whether (1) a pan-caspase inhibitor, Boc-D-FMK, exerts long-term neuroprotective effects on spinal motoneurons (MNs) after root avulsion in neonatal rats and (2) whether the rescued spinal MNs regenerate their axons into a peripheral nerve (PN) graft and reinnervate a previously denervated target muscle. Eight weeks after root avulsion, 67% of spinal MNs remained in the Boc-D-FMK-treated group, whereas all MNs died in the sham control group. By 12 weeks postinjury, however, all Boc-D-FMK treated MNs died. In the regeneration experiment, a PN graft was implanted at different times after injury. The animals were allowed to survive for 4 weeks following the operation. Without caspase inhibition, MNs did not regenerate at any time point. In animals treated with Ac-DEVD-CHO, a caspase-3-specific inhibitor, and Boc-D-FMK, 44 and 62% of MNs, respectively, were found to regenerate their axons into a PN graft implanted immediately after root avulsion. When the PN graft was implanted 2 weeks after injury, however, MNs failed to regenerate following Ac-DEVD-CHO treatment, whereas 53% of MNs regenerated their axons into the graft after treatment with Boc-D-FMK. No regeneration was observed when a PN graft was implanted later than 2 weeks after injury. In the reinnervation study, injured MNs and the target biceps muscle were reconnected by a PN bridge implanted 2 weeks after root avulsion with administration of Boc-D-FMK. Eight weeks following the operation, 39% of MNs reinnervated the biceps muscle. Morphologically normal synapses and motor endplates were reformed in the muscle fibers. Collectively, these data provide evidence that injured neonatal motoneurons can survive and reinnervate peripheral muscle targets following inhibition of caspases.[4]

[1]. z-VAD-fmk augmentation of TNF alpha-stimulated neutrophil apoptosis is compound specific and does not involve the generation of reactive oxygen species.

[2]. zVAD-fmk, unlike BocD-fmk, does not inhibit caspase-6 acting on 14-3-3/Bad pathway in apoptosis of p815 mastocytoma cells. Exp Mol Med. 2006 Dec 31;38(6):634-42.

[3]. Effect of Boc-D-Fmk on hepatocyte apoptosis after bile duct ligation in rat and survival rate after endotoxin challenge. J Gastroenterol Hepatol. 2008 Aug;23(8 Pt 1):1276-9.

[4]. Inhibition of caspases promotes long-term survival and reinnervation by axotomized spinal motoneurons of denervated muscle in newborn rats. Exp Neurol. 2003 Jun;181(2):190-203.

Surgical procedures[4]
On the day of birth, newborn female Spraque–Dawley rats were anesthetized under deep hypothermia. Under a surgical microscope, a dorsal laminectomy was carried out and the spinal root of the seventh cervical (C7) segment was identified. The C7 ventral root together with the dorsal root were avulsed by a pair of microhemostatic forceps. To study the long-term neuroprotective effect of Boc-D-FMK, animals were divided into two groups. There were six rats in each group at each time point. The first...

Long-term neuroprotective effect of Boc-D-FMK[4]
Motoneurons were identified and counted as described previously (Clarke and Oppenheim, 1995). In brief, only MNs with a large nucleus containing clearly visible nucleoli and a largely distinct cytoplasm were counted. Because the number of MNs on the contralateral intact side of the experimental animals was not significantly different from normal control animals (data not shown), the contralateral side served as an internal control. We have previously reported that by 7 days postlesion, there...

Discussion[4]
The present results indicate that caspases play a key role in the death of spinal MNs after injury in neonates. Inhibition of caspases led to long-term neuroprotection as well as axonal regeneration of avulsed spinal MNs. With a PN bridge between the spinal cord and the denervated muscle target, the caspase inhibitor-treated MNs were able to reinnervate the neuromuscular junction and muscular atrophy was reduced. These results suggest that the inhibition of caspases may be a potent strategy for ...

Conclusion[4]
The experiments presented here provide evidence that following root avulsion, neonatal spinal MNs can survive and reinnervate target muscle if appropriate treatment is provided. A single injection of Boc-D-FMK results in long-term protection of MNs against root avulsion-induced death for more than 8 weeks and the Boc-D-FMK-treated MNs are able to regenerate their axons into an implanted PN graft and reinnervate the target muscle. Taken together, these data suggest that local administration of...

C11H18FNO5

263.26272726059

263.12

C, 50.19; H, 6.89; F, 7.22; N, 5.32; O, 30.39

634911-80-1

634911-80-1

16760348

Boc-DL-Asp(OMe)-CH2F

Solid if <31.2°C; Liquid if >31.2°C; Light yellow to yellow color

1.150

0.9

1

6

8

18

324

0

FCC(C(CC(=O)OC)NC(=O)OC(C)(C)C)=O

MXOOUCRHWJYCAL-UHFFFAOYSA-N

InChI=1S/C11H18FNO5/c1-11(2,3)18-10(16)13-7(8(14)6-12)5-9(15)17-4/h7H,5-6H2,1-4H3,(H,13,16)

methyl 5-fluoro-3-[(2-methylpropan-2-yl)oxycarbonylamino]-4-oxopentanoate

BOC-D-FMK; 634911-80-1; Caspase Inhibitor III; 3-[[(tert-Butoxy)carbonyl]amino]-5-fluoro-4-oxopentanoic acid methyl ester; methyl 5-fluoro-3-[(2-methylpropan-2-yl)oxycarbonylamino]-4-oxopentanoate; Caspase3-Inhibitor BOC-D-FMK; BOC-D-FMK?; C11H18FNO5;

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: ~100 mg/mL (~379.9 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.50 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (9.50 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (9.50 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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