Caspase Inhibitor VI
[CAS# 161401-82-7]

Identification

• Classification: Biochemical >> Inhibitor >> Apoptosis >> Caspase inhibitor
• Name: Caspase Inhibitor VI
• Synonyms: Z-VAD-FMK; Z-Val-Ala-Asp fluoromethyl ketone; Z-VAD(OH)-FMK; (3S)-5-fluoro-3-[[(2S)-2-[[(2S)-3-methyl-2-(phenylmethoxycarbonylamino)butanoyl]amino]propanoyl]amino]-4-oxopentanoic acid
• Protein Sequence: VAD
• Molecular Formula: C₂₁H₂₈FN₃O₇
• Molecular Weight: 453.46
• CAS Registry Number: 161401-82-7
• SMILES: C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)CF)NC(=O)[C@H](C(C)C)NC(=O)OCC1=CC=CC=C1

Properties

• Density: 1.3±0.1 g/cm³ Calc.^*
• Boiling point: 758.0±60.0 ºC 760 mmHg (Calc.)^*
• Flash point: 412.2±32.9 ºC (Calc.)^*
• Index of refraction: 1.525 (Calc.)^*

^* Calculated using Advanced Chemistry Development (ACD/Labs) Software.

Safety Data

• Hazard Symbols: GHS07 Warning
• Hazard Statements: H302-H315-H319-H335
• Precautionary Statements: P261-P305+P351+P338

Discovory and Applicatios

Caspase Inhibitor VI, also known by the chemical name Z-Val-Ala-Asp(OH)-fluoromethylketone (Z-VAD(OH)-FMK), is a synthetic pan-caspase inhibitor that was designed for research use in the study of programmed cell death, or apoptosis. This compound is a fluoromethyl ketone derivative of a tripeptide sequence and functions as an irreversible inhibitor of caspases, which are cysteine proteases essential to the execution phase of apoptosis. It belongs to a class of peptide-based inhibitors that mimic natural substrates and bind covalently to the active site cysteine of caspases, thereby halting their enzymatic activity.

The development of caspase inhibitors emerged during the 1990s when extensive research efforts were underway to delineate the molecular pathways of apoptosis. Caspases had been identified as key mediators in the apoptotic cascade, and selective inhibitors were needed to study their individual roles. Z-VAD(OH)-FMK was synthesized as a broad-spectrum tool capable of targeting multiple caspases simultaneously. Its structure incorporates a benzyloxycarbonyl-protected valine-alanine-aspartic acid sequence, with a fluoromethyl ketone moiety replacing the C-terminal carboxyl group. This reactive group forms a stable thioether bond with the nucleophilic cysteine residue in the catalytic site of caspases, leading to irreversible inactivation.

Z-VAD(OH)-FMK has been widely used in cell biology to investigate apoptosis in cultured cells. It is typically administered prior to or concurrently with an apoptotic stimulus to assess the dependence of cell death on caspase activation. For example, in human cancer cell lines, the compound has been applied to inhibit apoptosis induced by tumor necrosis factor-related ligands, DNA-damaging agents, or oxidative stress. Its efficacy in blocking caspase-dependent apoptotic features—such as DNA fragmentation, membrane blebbing, and chromatin condensation—has been documented in numerous cell types.

Beyond cell culture systems, Z-VAD(OH)-FMK has also been employed in animal models of disease to study the contribution of apoptosis to tissue injury. In rodent models, systemic administration of the compound has been used to evaluate its protective effects in settings such as myocardial infarction, ischemia-reperfusion injury, sepsis, neurodegeneration, and muscle trauma. Its ability to reduce caspase activity in tissues and attenuate cellular apoptosis has been confirmed in various organ systems, including the heart, brain, liver, and skeletal muscle.

Z-VAD(OH)-FMK is typically supplied as a lyophilized powder and is soluble in dimethyl sulfoxide (DMSO). It is stable under standard laboratory storage conditions, generally maintained at -20 $degree$C. The compound is used in micromolar concentrations, and dosing regimens vary depending on the biological system and route of administration. While effective in inhibiting caspase activity, Z-VAD(OH)-FMK does not affect non-caspase proteases and is considered selective within the cysteine-aspartic protease family.

The utility of Z-VAD(OH)-FMK is primarily confined to experimental studies due to its peptide-based nature and limited pharmacokinetic properties. However, it has contributed significantly to the understanding of apoptosis mechanisms and has helped validate caspase-dependent pathways as targets for therapeutic intervention. Its applications span cancer biology, immunology, cardiology, and neuroscience, where caspase-mediated apoptosis plays a critical role in disease progression or response to treatment.

References

1996. Requirement of an ICE-Like Protease for Induction of Apoptosis and Ceramide Generation by REAPER. Science, 271(5250).
DOI: 10.1126/science.271.5250.808

2015. PARP Inhibitors Sensitize Ewing Sarcoma Cells to Temozolomide-Induced Apoptosis via the Mitochondrial Pathway. Molecular Cancer Therapeutics, 14(12).
DOI: 10.1158/1535-7163.mct-15-0587

2023. In vivo quantitative high-throughput screening for drug discovery and comparative toxicology. Disease Models & Mechanisms, 16(3).
DOI: 10.1242/dmm.049863