DOI: 10.1073/pnas.140132697. Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries

Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries

10.1073/pnas.140132697

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors.

Bibliography

Harris, J. L., Backes, B. J., Leonetti, F., Mahrus, S., Ellman, J. A., & Craik, C. S. (2000). Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proceedings of the National Academy of Sciences, 97(14), 7754â7759.

Authors 6

Jennifer L. Harris (first)
Bradley J. Backes (additional)
Francesco Leonetti (additional)
Sami Mahrus (additional)
Jonathan A. Ellman (additional)
Charles S. Craik (additional)

References 30 Referenced 469

A J Barrett, N D Rawlings, J F Woessner Handbook of Proteolytic Enzymes (Academic, London, 1998). / Handbook of Proteolytic Enzymes by Barrett A J (1998)
10.1126/science.8493554
10.1073/pnas.92.17.7627
K S Lam, M Lebl Methods Mol Biol 87, 1–6 (1998). / Methods Mol Biol by Lam K S (1998)
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10.1016/S0960-894X(99)00260-7
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10.1021/jo981990y
B A Bunin The Combinatorial Index (Academic, San Diego, 1998). / The Combinatorial Index by Bunin B A (1998)
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10.1016/0076-6879(94)44034-4

Dates

Type	When
Created	23 years, 1 month ago (July 26, 2002, 10:41 a.m.)
Deposited	3 years, 4 months ago (April 12, 2022, 11:27 p.m.)
Indexed	4 days, 16 hours ago (Aug. 27, 2025, 11:39 a.m.)
Issued	25 years, 2 months ago (June 27, 2000)
Published	25 years, 2 months ago (June 27, 2000)
Published Online	25 years, 2 months ago (June 27, 2000)
Published Print	25 years, 1 month ago (July 5, 2000)

Funders 0

None

BibTeX

@article{Harris_2000, title={Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries}, volume={97}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.140132697}, DOI={10.1073/pnas.140132697}, number={14}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Harris, Jennifer L. and Backes, Bradley J. and Leonetti, Francesco and Mahrus, Sami and Ellman, Jonathan A. and Craik, Charles S.}, year={2000}, month=jun, pages={7754–7759} }

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