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Feb 1, 2009 (Vol. 29, No. 3)

Accelerating Protease Drug Development

Use of the REPLi FRET Peptide Library Facilitates Identification of Substrates

  • Library Validation

    Click Image To Enlarge +
    Figure 3. Screening process using the REPLi to identify protease substrates.

    To validate the library, scientists at GSK and the University of Leeds profiled the REPLi library against representative members from each of the four mechanistic protease classes—trypsin, pepsin, matrix metalloprotease (MMP)-12 (macrophage elastase), MMP-13 (collagenase-3), calpain-1 (µ-calpain) and calpain-2 (m-calpain). 

    The substrate specificities of calpains-1 and -2 are almost identical and few peptide substrates for either enzyme have been reported. Thus, it was of particular interest that the REPLi returned several substrates that were preferentially cleaved by either one or other enzyme. The variable core sequence of the peptide pool that was specifically cleaved by calpain-1 was -Pro-[Ile/Leu]-[Phe/Tyr]-, while that which was specifically cleaved by calpain-2 contained -[Ile/Leu]-[Asn/Gln]-[Phe/Tyr]- in the variable core. 

    The peptide pool comprising the eight peptides with the core motif [Asn/Gln]-[Ile/Leu]-[Phe/Tyr] that returned the highest signal:background ratio with calpain-2 was subjected to full LC-MS characterization (Figure 3).

    This type of analysis enables the identification of the cleavage site within the peptide (MS measurement) and aids the determination of the preferred substrate sequence within the pool (LC data). To identify the actual peptide sequences in the pool cleaved by calpain-2, all eight peptides were synthesized as discrete candidates and monitored in real-time assays. These allowed the concomitant determination of the nature of the optimal residues for each of the three variable positions and also provided kinetic data for each substrate (Figure 3).

    As predicted, the kinetic characterization of single peptides was in complete agreement with LC-MS data obtained for the pool and showed that the activity was confined to specific sequences. Utilizing this information obtained from the REPLi, a highly sensitive and efficient fluorogenic substrate for calpain-2 was developed from a minimum number of experimental iterations. 

    The thorough validation of this peptide library with representatives from each of the four mechanistic protease classes indicates that the REPLi will be useful for the rapid identification of substrates for multiple proteases and has potential applications in high-throughput screening. 



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