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Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin

Nature Biotechnology volume 18, pages 187–193 (2000)Cite this article

An Erratum to this article was published on 01 May 2000

Abstract

We have developed a strategy for the synthesis of positional-scanning synthetic combinatorial libraries (PS-SCL) that does not depend on the identity of the P1 substituent. To demonstrate the strategy, we synthesized a tetrapeptide positional library in which the P1 amino acid is held constant as a lysine and the P4-P3-P2 positions are positionally randomized. The 6,859 members of the library were synthesized on solid support with an alkane sulfonamide linker, and then displaced from the solid support by condensation with a fluorogenic 7-amino-4-methylcoumarin-derivatized lysine. This library was used to determine the extended substrate specificities of two trypsin-like enzymes, plasmin and thrombin, which are involved in the blood coagulation pathway. The optimal P4 to P2 substrate specificity for plasmin was P4-Lys/Nle (norleucine)/Val/Ile/Phe, P3-Xaa, and P2-Tyr/Phe/Trp. This cleavage sequence has recently been identified in some of plasmin's physiological substrates. The optimal P4 to P2 extended substrate sequence determined for thrombin was P4-Nle/Leu/Ile/Phe/Val, P3-Xaa, and P2-Pro, a sequence found in many of the physiological substrates of thrombin. Single-substrate kinetic analysis of plasmin and thrombin was used to validate the substrate preferences resulting from the PS-SCL. By three-dimensional structural modeling of the substrates into the active sites of plasmin and thrombin, we identified potential determinants of the defined substrate specificity. This method is amenable to the incorporation of diverse substituents at the P1 position for exploring molecular recognition elements in proteolytic enzymes.

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Figure 1: Three sublibraries (P2, P3, P4), each made up of 19 wells containing 361 compounds, are prepared by a segment condensation reaction with a P1 fluorogenic amino acid substrate.
Figure 2: (A) Activity of plasmin in a P1-Lys positional-scanning synthetic combinatorial library.
Figure 3: (A) Three-dimensional model of plasmin bound to the tetrapeptide substrate Lys-Thr-Phe-Lys.
Figure 4
Figure 5

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Acknowledgements

We thank Shaun R. Coughlin and Mark Lipton for insightful discussions. We extend our appreciation to Keith W. Burdick for helpful discussion and assistance with the figures and to Matthew Trammel for assistance with the preparation and analysis of single-substrate kinetics. This work was supported in part by National Science Foundation Grant MCB9604379 and National Institutes of Health Grant CA72006 (to C.S.C.), National Institutes of Health Grant GM 54051 (to J.A.E.) and National Institutes of Health Biotechnology Training Grant Fellowship (to J.L.H.).

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  1. Bradley J. Backes

    Present address: Chemistry Department, Genomics Institute of the Novartis Research Foundation, 3015 Merryfield Row, San Diego, CA, 92121

  2. Bradley J. Backes and Jennifer L. Harris: These authors contributed equally to this work.

Authors and Affiliations

  1. Chemistry Department, University of California Berkeley, Berkeley, 94720, CA

    Bradley J. Backes, Francesco Leonetti & Jonathan A. Ellman

  2. Department of Pharmaceutical Chemistry, Program in Chemistry and Chemical Biology, University of California San Francisco, San Francisco, 94143, CA

    Jennifer L. Harris & Charles S. Craik

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Backes, B., Harris, J., Leonetti, F. et al. Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin. Nat Biotechnol 18, 187–193 (2000). https://doi.org/10.1038/72642

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