Fluorogenic protease substrates

Maggiora LL, Smith CW, Zhang ZY (1992) A general method for the preparation of internally quenched fluorogenic protease substrates using solid-phase peptide synthesis. J Med Chem 35 3727-3730... 

Figure 2. Internally quenched fluorogenic protease substrate enable signal generation by liberation of the fluorophoie from close proximity with the quenching chromophoie. Efficient quenching is achieved due to the excellent spectral overlap of the Edans fluorophoie and the Dabcyl chromophoie.

Enzymatic activity within hve cells is an alternative option for measuring cell viabiUty. A cell-permeable fluorogenic protease substrate (GF-AFC) has recendy been developed to selectively detect protease activity that is restricted to viable cells [80]. After GF-AFC penetrates into liver cells, the cytoplasmic aminopepti-dase cleaves the substrate by removing amino acid residues Gly and Phe. The final product aminofluorocoumarin (AFC), is released, which generates a fluorescent signal proportional to the quantity of viable cells. ATP has been widely... 

D. J. Maly, F. Leonetti, B. J. Backes, D. S. Dauber, J. L. Harris, C. S. Craik, J. A. Ellman, Expedient solid-phase synthesis of fluorogenic protease substrates using the 7-amino-4-carba-moylmethylcoumarin (ACC) fluorophore. J. Org. Chem. 2002, 67, 910-915. 

Q. Zhu, D. B. Li, M. Uttamchandani, S. Q. Yao, Facile synthesis of 7-amino-4-carbamoyl-methylcoumarin (ACC)-containing solid supports and their corresponding fluorogenic protease substrates. Bioorg. Med. Chem. Lett. 2003,13, 1033 1036. 

Sinha, R Gossrau, R. Isoelectric focusing (IFF) and band detection with fluorogenic protease substrates. 

Scheme 13 Fluorogenic Depsipeptide Substrates of Human Hepatitis C Virus Protease...

Wang, G.T. et al. 1990. Design and synthesis of new fluorogenic HIV protease substrates based on resonance energy transfer. Tetrahedron Lett. 31, 6493-6496. 

Small molecule or peptide substrates can also be used to profile protease and other enzyme activities. Salisbury et al. described a protease-substrate microarray in which the carboxyl end of the peptide substrates was conjugated to 7-amino-4-car-bamoylmethyl coumarin, a fluorogenic compound. The conjugate was non-fluores-cent when the electron-donating group on the coumarin was attached to the peptide. Upon proteolysis, the peptide was released and the microarray spot fluoresced. Zhu et al. demonstrated that small molecule microarrays could be used to detect enzyme activities of epoxide hydrolases and phosphatases. 

Blackman MJ, Corrie JET, CroneyJC, Kelly G, Eccleston JF, and Jameson DM. Structural and Biochemical Characterization of a Fluorogenic Rhodamine-Laheled Malarial Protease Substrate. Biochemistry 2002 41 12244-12252. 

Iwanaga, S., Kato, H., Maruyama, R., Adachi, N., Ohno, Y., Takada, K., Kimura, T., and Sakaldbara, S., Fluorogenic peptide substrates for proteases in blood coagulation, kalUkrein-ldnin and fibrinolytic systems. Thromb. Haemostasis 42, 49 (abstr.) (1979). Jackson, C., and Nemerson, Y., Blood coagulation. Anna. Rev. Biochem. 49, 765-811 (1980). 

Table I. Kinetic Parameters for Fluorogenic HIV Protease Substrates ...

Caspase activity was measured using fluorogenic tetrapeptide substrates as described previously (9). The substrates (Peptide Institute, Osaka, Japan) used were Ac-YVAD-methycoumarylamide (MCA) for caspase-1-like proteases, Ac-DEVD-MCA for caspase-3-like proteases, Ac-DMQD-MCA for caspase-3, Ac-... 

Gosalia DN, Salisbury CM, Maly DJ, Ellman JA, Diamond SL (2005) Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays. Proteomics 5 1292-1298... 

Gershkovich AA, Kholodovych W (1996) Fluorogenic substrates for proteases based on intramolecular fluorescence energy transfer (IFETS). J Biochem Biophys Methods 33 135-162... 

Matayoshi, E. D., Wang, G. T., Krafft, G. A. and Erickson, J. (1990). Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science 247, 954-958. 

Note Reactions were performed under the conditions described in the text with either colorimetric peptide pNA4 (250 pM) or fluorogenic peptide F3 (35 pM) as a substrate. The IC50 values represent the inhibitor concentration required to reduce the protease activity by 50% of the control containing no inhibitor. NI, no inhibition was observed at the concentrations indicated. E64, frans-epoxysuccinyl-L-leucylamide-(4-guanidino)-butane PMSF, phenylmethylsulphonyl fluoride TLCK, tosyl-L-lysine-chloromethyl ketone. 

Bratanova, E.K. and Petkov, D.D. 1987. N-antraniloylation converts peptide p-nitroanilides into fluorogenic substrates of proteases without loss of their chromogenic properties. Anal. Biochem. 162, 213-218. 

Harris, J.L. et al. 2000. Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libaries. Proc. Natl. Acad. Sci. USA, 7754-7759. 

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