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February 01, 2009 (Vol. 29, No. 3)

Accelerating Protease Drug Development

Use of the REPLi FRET Peptide Library Facilitates Identification of Substrates

  • Protease Substrate Identification

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    Figure 1. Principle of the REPLi of FRET peptides

    Various methods have been employed to characterize protease specificity, generally consisting of the screening of chemically or biologically produced peptide libraries.  The early mixture-based peptide libraries provided both general applicability and speed, with digestion of the library mixtures followed by N-terminal sequencing giving specificity information C-terminal to the scissile bond (P' sites; Figure 1).

    Such methods, however, preclude the continuous monitoring of proteolytic activity and do not provide information on the protease specificity N-terminal to the scissile bond (P sites). Libraries generated using solid-phase synthesis where peptides are tagged with a fluorogenic or chromogenic group that fluoresces/absorbs light after cleavage, allows the proteolytic activity to be monitored in real time. The lack of information on the primed subsite specificity (P' sites) hinders its application to endoproteases that recognize residues C-terminal to the scissile bond.

    To overcome issues such as feasibility and deconvolution, associated with large peptide libraries, Mimotopes has developed and launched, in collaboration with GlaxoSmithKline and the University of Leeds a generic fluorescence resonance energy transfer (FRET) rapid endopeptidase profiling library (REPLi) as a tool for rapidly identifying protease substrates.

    To keep the number of peptides relatively small while still representing the residue requirements for the largest number of proteases, similar amino acids are paired within a tripeptide core giving rise to a relatively small library of 3,375 peptides divided into 512 distinct pools, each containing only eight peptides. The variable central core is flanked with multiple Gly residues and an additional two Lys residues are added at the C-terminus to confer adequate solubility to peptides bearing hydrophobic variable sequences (Figure 1). 

    Potentially problematic amino acids that have been removed from the selection set include Cys (potential for introducing disulphide bonds), His (not generally observed within substrates at sites of protease cleavage), Met (due to both its hydrophobicity and bulky nature being shared by Leu and Ile and its propensity to being oxidized), and Trp (interference with fluorescence signal due to abs.l280nm/em.l320nm). Gly is also omitted since functionalized amino acids furnish comparatively more information and because cleavage around flanking Gly residues can be detected.

    The remaining 15 amino acids are grouped in matching pairs (Ala + Val, Arg + Lys, Asp + Glu, Asn + Gln, Leu + Ile, Ser + Thr, Phe + Tyr), while Pro is left as a single residue to allow access to any potential conformational information. The judicious choice of amino acid partners ensures that maximum SAR information can be derived from an initial result. This matching-pair design simplifies the deconvolution steps. The complete peptide library is synthesized using Mimotopes’ PepSets SynPhase Lantern technology and is provided for direct usage in 96-well plate format (Figure 2).

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    Figure 2. The SynPhase Lantern Platform

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