Ac-WEHD-AFC is a fluorogenic substrate that can be cleaved by group I caspases (caspase-1, -4, and -5).1 Caspase activity can be quantified by fluorescent detection of free AFC (also known as 7-amino-4-trifluoromethylcoumarin), which is excited at 400 nm and emits at 505 nm.
CAT No: R1870
CAS No:210344-99-3
Synonyms/Alias:Ac-WEHD-AFC;210344-99-3;(4S)-4-[[(2S)-2-acetamido-3-(1H-indol-3-yl)propanoyl]amino]-5-[[(2S)-1-[[(2S)-3-carboxy-1-oxo-1-[[2-oxo-4-(trifluoromethyl)chromen-7-yl]amino]propan-2-yl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid;Ac-Trp-Glu-His-Asp-AFC trifluoroacetate salt;N-Acetyl-L-tryptophyl-L-alpha-glutamyl-L-histidyl-N-[2-oxo-4-(trifluoromethyl)-2H-1-benzopyran-7-yl]-L-alpha-asparagine;MFCD01862607;NEGOLAGPKZGIKJ-DZUOILHNSA-N;HY-P2617;EX-A11091;NCGC00485377-01;DA-50205;FA110614;CS-0136652;N-Acetyl-Trp-Glu-His-Asp-7-amino-4-(trifluoromethyl)coumarin;(4S,7S,10S,13S)-10-((1H-Imidazol-4-yl)methyl)-4-((1H-indol-3-yl)methyl)-7-(2-carboxyethyl)-2,5,8,11-tetraoxo-13-((2-oxo-4-(trifluoromethyl)-2H-chromen-7-yl)carbamoyl)-3,6,9,12-tetraazapentadecan-15-oic acid;N-Acetyl-Trp-Glu-His-Asp-7-amido-4-trifluoromethylcoumarin trifluoroacetate salt, >=97% (HPLC), powder;
Chemical Name:(4S)-4-[[(2S)-2-acetamido-3-(1H-indol-3-yl)propanoyl]amino]-5-[[(2S)-1-[[(2S)-3-carboxy-1-oxo-1-[[2-oxo-4-(trifluoromethyl)chromen-7-yl]amino]propan-2-yl]amino]-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid
Ac-WEHD-AFC is a synthetic peptide substrate widely utilized in the study of protease activity, particularly for the detection and quantification of caspase-1 (ICE) and related cysteine proteases. Structurally, it comprises the tetrapeptide sequence WEHD, which mimics natural cleavage sites recognized by inflammatory caspases, conjugated at the C-terminus to 7-amino-4-trifluoromethylcoumarin (AFC), a fluorogenic leaving group. Upon enzymatic cleavage, AFC is released, producing a measurable fluorescent signal. This molecular design enables precise monitoring of proteolytic processes in real-time, making Ac-WEHD-AFC a valuable biochemical reagent in apoptosis, inflammation, and cell death research.
Enzyme Activity Assays: Ac-WEHD-AFC is extensively employed in in vitro fluorometric assays to measure the activity of caspase-1 and related proteases. Its peptide sequence is specifically recognized and hydrolyzed by these enzymes, resulting in the liberation of AFC, which emits a strong fluorescent signal upon excitation. This property facilitates quantitative assessment of enzyme kinetics, substrate specificity, and inhibitor screening in biochemical and cell-based assay formats. Researchers rely on this substrate to accurately monitor caspase function under various experimental conditions, supporting investigations into the molecular mechanisms of programmed cell death and inflammation.
Inflammasome Research: The substrate's WEHD recognition motif aligns with the cleavage preferences of inflammatory caspases, making it a critical tool for dissecting inflammasome activation pathways. By enabling sensitive detection of caspase-1 activity, it allows for the characterization of inflammasome assembly, regulation, and downstream signaling events in immune cells. This application is particularly relevant in studies exploring innate immune responses, cytokine maturation, and the cellular consequences of pathogen recognition.
Drug Discovery and Inhibitor Profiling: In pharmaceutical and academic research, Ac-WEHD-AFC serves as a reliable substrate for high-throughput screening of small-molecule inhibitors targeting caspase-1 and related enzymes. The robust and quantifiable fluorescence output upon substrate cleavage allows for rapid evaluation of compound efficacy, mechanism of action, and selectivity. This accelerates the identification and optimization of novel modulators of caspase activity, supporting the development of research tools and potential therapeutic leads for inflammation-related disorders.
Cell Death Pathway Elucidation: The use of this fluorogenic peptide substrate extends to the detailed investigation of apoptosis and pyroptosis pathways. By tracking caspase-1 activation in cell lysates or intact cells, researchers can delineate the sequence of molecular events that govern cell fate decisions. The ability to monitor protease activity in real time provides insights into the regulation, timing, and interplay of cell death mechanisms, facilitating a deeper understanding of cellular stress responses and immune defense strategies.
Biochemical Characterization of Proteases: Ac-WEHD-AFC is also instrumental in the detailed biochemical characterization of caspase-1 and related cysteine proteases. It enables determination of kinetic parameters such as Km and Vmax, substrate preferences, and the influence of cofactors or post-translational modifications on enzyme function. This information is vital for elucidating protease regulation, substrate recognition, and the broader roles of these enzymes in cellular physiology and pathophysiology. Through these diverse applications, the substrate remains an essential component of experimental workflows in enzymology, immunology, and cell biology research.
1. Adipose tissue is a key organ for the beneficial effects of GLP-2 metabolic function
2. SERS spectrum of the peptide thymosin‐β4 obtained with Ag nanorod substrate
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