A. Wako's Unique Products - 1. Inhibitors

b. Non-competitive PSA Inhibitor

PAQ-22 [3-(2,6-Diethylphenyl)-2,4-(1H, 3H)-quinazolinedione]

Wako Catalog No. 165-23581 (10 mg) <for Cellbiology> Keep at RT*

Puromycin-sensitive aminopeptidase (PSA), which is a neutral aminopeptidase with a substrate specificity similar to that of aminopeptidase N (APN), is distributed mainly in the brain and neurons. Although PSA regarded as an enkephalin-degrading enzyme, the physical roles/functions of PSA remain unclear. PSA is possibly related to cell division and Apoptosis. PSA was reported as a potent modifier of tau-induced pathology and was suggested as a possible tau-degrading enzyme in an unknown mechanism in Alzheimer's disease. PSA activity is inhibited by Puromycin but Puromycin also inhibits APN. Wako has launched a non-peptide, small-molecular, non-competitive PSA Inhibitor, PAQ-22 and the structurally modified fluorescent bioprobe, DAMPAQ-22.

*RT: room temperature

Line weaver-Burk plot anlysis of PSA inhibition by PAQ-22 and Puromycin

image
Using living human monocytic cell MOLT-4, which is known to express PSA, PSA inhibitory activity of PAQ-22 and Puromycin was determined with an indicator, which is fluorescence generated from a fluorescent substrate Ala-MCA broken down by PSA. Lineweaver-Burk plot indicated that PAQ-22 acts as specific non-competitive inhibitor. On the other hand, Puromycin acts a competitive inhibitor. In addition, PAQ-22 and DAMPAQ-22 are easily incorporated into MOLT-4 under the general cell culture conditions.

APN-Inhibitory Activity of PAQ-22 and Puromycin

  PSA IC50 (μmol/L) APN IC50 (μmol/L)
PAQ-22
3.8
>100
DAMPAQ-22
4.6
N/A
Puromycin
0.6
4.8
PSA- and APN-inhibitory activities were assayed by the use of L-Ala-MCA with MOLT-4. PAQ-22 is inactive toward APN, indicating that PAQ-22 is specific to PSA.

( These data were provided by Dr. Yuichi Hashimoto, Institute of Molecular and Cellular Biosciences, The University of Tokyo)
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Fluorescent Bioprobe for Visualization of PSA in Living Cells

DAMPAQ-22 [3-(2,6-Diethyl-4-dansylaminophenyl)-1-methyl-2,4-(1H, 3H)-quinazolinedione]

Wako Catalog No. 049-30761 (2 mg) <for Cellbiology> Keep at RT
C31H32O4N4S=556.68; CAS No. 519183-48-3; λex: 320 nm*; λem: 510 nm*

DAMPAQ-22, which is structurally modified fluorescent bioprobe of PAQ-22 is also available. The cellular localization of PSA within the living body could be specifically visualized by the use of DAMPAQ-222).

PSA IC50 : 4.6 μmol/L (Please see the above mentioned table of PAQ-22)
No inhibitory activity toward APN and LAP.

*: Excitation wavelength and fluorescence wavelength were not optimized. Please adjust them in accordance with the intended use or the experimental condition based on those wavelengths.

Visualization of PSA with DAMPAQ-22 in MOLT-4 cells

image
Fluorescent microscopic images of MOLT-4 cells treated with 10 µmol/L DAMPAQ-22 for 10 minutes.

( These data were provided by Dr. Yuichi Hashimoto, Institute of Molecular and Cellular Biosciences, The University of Tokyo)
[References]
1) Kakuta, H., Tanatani, A., Nagasawa, K., Hashimoto, Y.: "(1H,3H)-quinazolinedione skeleton", Chem. Pharm. Bull, 51, 1273-82 (2003).
2) Kakuta, H., Koiso, Y., Nagasawa, K., Hashimoto, Y.: “Fluorescent Bioprobes for Visualization of Puromycin-Sensitive Aminopeptidase in Living Cells”, Bioorg. Med. Chemd. Lett., 13, 83-6 (2003)
3) Bukowska, A., Tadje, J., Arndt, M., Wolke, C., Kähne, T., Bartsch, J., Faust, J., Neubert, K., Hashimoto, Y., Lendeckel, U.: “Transcriptional regulation of cytosol and membrane alanyl-aminopeptidase in human T cell subsets”, Biol Chem., 384, 657-65 (2003).
4) Sánchez-Morán, E., Jones, G.H., Franklin, F. C. and Santos, J. L. : Plant Cell, 16, 2895 (2004).
5) Thielitz, A., Bukowska, A., Wolke, C., Vetter, R., Lendeckel, U., Wrenger, S., Hashimoto, Y., Ansorge, S., Gollnick, H. and Reinhold, D. : Biochem. Biophys. Res. Commun., 321, 795 (2004).
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Wako Product Update Bio-No.3 [ page. 4 ]