Ac-IEPD-AFC
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Ac-IEPD-AFC

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Ac-IEPD-AFC is a peptide whose sequence is preferred recognition motif for the serine protease granzyme B. It is a fluorogenic substrate.

Category
Peptide Inhibitors
Catalog number
BAT-010421
CAS number
1135417-31-0
Molecular Formula
C32H38F3N5O11
Molecular Weight
725.67
Ac-IEPD-AFC
IUPAC Name
(4S)-4-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-5-[(2S)-2-[[(2S)-3-carboxy-1-oxo-1-[[2-oxo-4-(trifluoromethyl)chromen-7-yl]amino]propan-2-yl]carbamoyl]pyrrolidin-1-yl]-5-oxopentanoic acid
Synonyms
IEPD; N-Acetyl-Ile-Glu-Pro-Asp-(7-amino-4-trifluoromethylcoumarin); Ac-Ile-Glu-Pro-Asp-7-Amino-4-trifluoromethylcoumarin
Appearance
White Powder
Purity
≥95%
Density
1.438±0.06 g/cm3 (Predicted)
Boiling Point
1073.9±65.0°C (Predicted)
Sequence
Ac-Ile-Glu-Pro-Asp-AFC
Storage
Store at -20°C
Solubility
Soluble in DMF, DMSO
InChI
InChI=1S/C32H38F3N5O11/c1-4-15(2)27(36-16(3)41)30(49)38-20(9-10-24(42)43)31(50)40-11-5-6-22(40)29(48)39-21(14-25(44)45)28(47)37-17-7-8-18-19(32(33,34)35)13-26(46)51-23(18)12-17/h7-8,12-13,15,20-22,27H,4-6,9-11,14H2,1-3H3,(H,36,41)(H,37,47)(H,38,49)(H,39,48)(H,42,43)(H,44,45)/t15-,20-,21-,22-,27-/m0/s1
InChI Key
HWXKKVLJPWMVLL-FGGUPADCSA-N
Canonical SMILES
CCC(C)C(C(=O)NC(CCC(=O)O)C(=O)N1CCCC1C(=O)NC(CC(=O)O)C(=O)NC2=CC3=C(C=C2)C(=CC(=O)O3)C(F)(F)F)NC(=O)C
1. A Microfluidic Platform for Single Cell Fluorometric Granzyme B Profiling
JeongHoon Park, Hiroyuki Yoshikawa, Shohei Koyama, Eiichi Tamiya, Yujiro Naito, Jonathan C Briones, Masato Saito, Atsushi Kumanogoh, Hyota Takamatsu, Wilfred V Espulgar Theranostics . 2020 Jan 1;10(1):123-132. doi: 10.7150/thno.37728.
Granzyme B (GrB) is an essential cytotoxic effector in cancer immunotherapy as it can be a potential biomarker to predict the efficacy of immunotherapies including checkpoint inhibitors. Monitoring the Granzyme B activity in cells would help determine a patient's clinical response to treatment and lead to better treatment strategies by preventing administration of ineffective therapies and avoid adverse events resulting in a delay in subsequent treatment.Methods: A microfluidic device with hydrodynamic traps and pneumatic valving system was fabricated using photo and soft lithography. Single cell Granzyme B (GrB) activity was detected and measured fluorometrically using a commercial assay kit with a peptide substrate containing GrB recognition sequence (Ac-IEPD-AFC) and AFC (7-Amino-4-trifluoromethylcoumarin) label. Fluorescence was observed and measured using a confocal microscope with CSU-W1 scanner unit and CCD camera as well as an inverted microscope with photodetector. Model cells (NK-92, GrB-transduced Jurkat, and THP1 cells) and human PBMCs from healthy donor and lung cancer patients including an anti-PD-1 antibody treated patient were profiled of its GrB activity as proof of concept.Results: GrB expression from the model cells was found to be markedly different. NK-92 cells were found to have higher GrB activity than the GrB-transduced Jurkat cells. THP-1 was found to have relatively no significant activity. A marked increase in GrB expression was also observed in anti-PD-1 treated lung cancer patient sample in comparison to PBMC from a healthy donor. TCR+ Ig-G4+ PBMC cells were found to have high activity which signifies a clear response to PD-1 blockade.Conclusion: As proof of concept, we have shown the capability of a microfluidic platform to measure GrB production through a single cell enzymatic activity assay. Our platform might be a promising tool for evaluating the sensitivity of immunotherapies and identifying specific T cell subset responsible for the anti-tumor response.
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