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Identification of Active Sequences
The beads from the library that demonstrated activity were
removed using tweezers. Each bead contains approximately 30 pmoles of peptide,
which is sufficient for micro-analytical methods to determine the sequence of
the peptide without ambiguity. Active sequences were read by automated Edman
analysis. All the active sequences were found to show a strong consensus, and
multiple copies were independently found for almost all sequences. The consensus
for chymotrypsin inhibition at the positions varied was found to be X=Threonine,
Y=Phenyl Alanine, Z=Isoleucine; and this particular sequence was found on no
less than 5 different individual beads. Re-synthesis of this most active
sequence was found to provide an extremely potent inhibitor of the target
enzyme, with a Ki value of 20 nM.
Applications and Future Directions
This work was the first time that randomisations within a specific binding
motif were used to redirect specificity using a synthetic peptide library. The
advantage of a template-based library, rather than a completely random one, is
that it is much more likely to produce potent inhibitors. In conjunction with
support from GlaxoSmithKline, we are currently extending this work to generate
inhibitors of therapeutically interesting proteases, and are examining the
molecular details of the interaction in much greater detail.
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