Biomedical Engineering Reference
In-Depth Information
9.3 Inhibitors Designed Based on Bioscaffolds
Bioscaffolds are natural template structures that can be modified to become
highly specific inhibitors. Modification is carried out either by recombinant
DNA technology approaches or by conventional peptide chemical synthesis, in
which case a small inhibitory fraction is selected for synthesis.
9.3.1 Engineered Sunflower Trypsin Inhibitor (SFTI)
Sunflower trypsin inhibitor (SFTI) is a member of the Bowman-Birk serine
protease family. SFTI is a 14-amino-acid residue cyclic peptide (Gly-Arg-Cys-
Thr-Lys-Ser-Ile-Pro-Pro-Ile-Cys-Phe-Pro-Asp cyclized between the N-terminal
Gly and the C-terminal Asp) isolated from sunflower seeds (Helianthus annuus)
that was characterized and shown to exhibit a low K
i
of 150 pM for trypsin.
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This short peptide inhibitor can be produced in large quantities by standard
chemical synthesis protocols. Chemical synthesis also facilitates the develop-
ment of new variants that can be screened against novel protease targets for
specific inhibition. In addition, Austin et al.
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reported the expression in E. coli
of a library of cyclic SFTI variants based on the intramolecular native chemical
ligation method. These procedures can be viewed as complementary to che-
mical synthesis protocols.
Recently, SFTI was used as a bioscaffold for the design and synthesis of a
novel KLK4-specific inhibitor.
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Initially, a tetrapeptide phage display
library was screened in order to identify the best substrate for KLK4 which
had the sequence Phe-Val-Gln-Arg (FVQR). Then, a variant SFTI molecule
was designed by replacing the P1 Lys residue of the substrate tetrapeptide
with Arg, the P2 Thr with Gln, and the P4 Arg with Phe. The Cys at P3
position was not replaced by Val, as cysteine residues are important for
structural integrity. The K
i
wascalculatedtobewithin the low-nanomolar
range (3.6 nM) which is by 500-fold more selective for KLK4 than the closely
related KLK14.
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9.3.2 Mutant Serpins
Serpin bioscaffolds are amenable to engineering for the generation of highly
specific inhibitors of certain serine proteases. Essential to serpins is the reactive
center loop (RCL) that participates in the inhibition mechanism acting as a
substrate for the targeted protease. Changes in the RCL sequence can modulate
serpin specificity for specific proteases. Indeed, mutant serpins have been
exploited for the development of KLK inhibitors.
9.3.2.1 Mutant Serpins Targeting KLK2
KLK2 reacts with a
1
-antichymotrypsin (a
1
-ACT) at a slow rate (12-16 h) due
to the presence of Leu-Ser at P
1
-P
1
0
positions of the inhibitor, which
