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Primed-site Probing of Papain-like Cysteine Proteases

  • Bruce Merrifield Commemorative Issue
  • Published:
International Journal of Peptide Research and Therapeutics Aims and scope Submit manuscript

A general concept is presented for the development and enhancement of novel molecular probes for papain-like cysteine proteases. To achieve an increase in affinity and selectivity of such probes for the target enzymes, the specificity of the primed binding subsites of the active-site clefts were investigated by the use of partially randomized dipeptide libraries containing Ep-460, a trans-epoxysuccinyl thiol-reactive unit. Once covalently grafted to the catalytic cysteine residue, the anchor ensured proper alignment of the peptide moiety to the primed sites of the enzymes. After determining the inhibition potencies of these libraries, the best candidates were deconvoluted in a second cycle of synthesis and kinetic measurements. Proof of concept was demonstrated by application of this strategy to cathepsins B and L as well as to μ-calpain. By this approach, compounds of markedly enhanced inhibitory potency were obtained for cathepsin B and L.

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Fig. 1.
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Abbreviations

AMC:

7–amino-4-methylcumarine

Suc:

succinyl

tEps:

trans-(2S,3S)-epoxysuccinic acid (oxirane-2,3-dicarboxylic acid)

Dabcyl-:

4-[4′-(dimethylamino)phenylazo]benzoyl-

DIEA:

diisopropylethylamine

EDANS:

5-[(2′-aminoethyl)amino]naphthalene sulphonic acid

Abz:

2-aminobenzoic acid

Dnp:

2,4-dinitrophenyl ester

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Acknowledgments

The study was partly supported by the SFB 469 of the Ludwig-Maximilians-University of Munich (grants A2 and A6) and by the Deutsche Forschungsgemeinschaft (grant SCHA 1012/1). The authors thank Mrs. Elisabeth Weyher-Stingl for the MS analyses, Mrs. Barbara Meisel and Mrs. Rita Zauner for their technical assistance, and Dr. Dusica Gabrijelcic-Geiger for supervision of the preparation of calpain.

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Author notes

  1. Norbert Schaschke

    Present address: Universität Bielefeld, Universitätsstraße 25, Bielefeld, D-33615, Germany

Authors and Affiliations

  1. Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, Martinsried, D-82152, Germany

    José Maria Pfizer, Luis Moroder & Norbert Schaschke

  2. Abteilung für Klinische Chemie und Klinische Biochemie, Chirurgische Klinik, Ludwig-Maximilians-Universität München, Nußbaumstr. 20, München, 80336, Germany

    Irmgard Assfalg-Machleidt

  3. Adolf-Butenandt-Institut, Ludwig-Maximilians-Universität, Schillerstr. 42, München, D-80336, Germany

    Werner Machleidt

Authors
  1. José Maria Pfizer
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  2. Irmgard Assfalg-Machleidt
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  3. Werner Machleidt
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  4. Luis Moroder
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  5. Norbert Schaschke
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Corresponding author

Correspondence to Luis Moroder.

Additional information

This work is dedicated in memory of Professor R. Bruce Merrifield for his ingenious development of solid phase synthesis, that has been efficiently applied herein. All amino acids and related derivatives are of the L-configuration. Standard abbreviations are used for amino acids, protecting groups and reagents. Additional abbreviations are given.

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Pfizer, J.M., Assfalg-Machleidt, I., Machleidt, W. et al. Primed-site Probing of Papain-like Cysteine Proteases. Int J Pept Res Ther 13, 93–104 (2007). https://doi.org/10.1007/s10989-006-9050-3

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  • DOI: https://doi.org/10.1007/s10989-006-9050-3

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