Peptide-Based Drug Design

134 Hilpert et al.
wt A C D E F … … W Y
MIDI AIDI CIDI
DIDI
EIDI
FIDI
WIDI
YIDI
MIDI MADI MCDI
MDDI
MEDI
MFDI
MWDI
MYDI
MIDI MIAI MICI
MIDI MIEI MIFI
MIWI
MIYI
MIDI MIDA MIDC MIDD MIDE MIDF MIDW MIDY
Fig. 1. Principle of a substitution analysis of the 4 mer peptide MIDI. The first column
represents the parent or wild-type (wt) peptide, which is used to compare the effects of
the substitution variants. All other columns describe the substituted amino acids at each
position of the peptide. All underlined positions show the actual substituted amino acid
in the sequence. The wild-type sequence is bolded.
Based on the substitution analysis of Bac2A (RLARIVVIRVAR-NH2), different
12mer peptides with superior activity against different human pathogen were
developed (35). For example, peptide Sub3 showed MIC values against P. aeruginosa
and S. aureus of 2 �g/mL, E.coliof 0.5 �g/mL, and Staphylococcus
epidermidis of 0.2 �g/mL. The data of the complete substitution analysis were
also used to develop shorter peptides with strong antimicrobial activity. One
8mer peptide was described showing MIC values of 2 �g/mL against E. coli and
S. aureus. Results also showed that changes in any single position of the peptide
may affect other residues at all other positions in the peptide; therefore, each
single substituted peptide variant may lead to different preferred substitutions at
any other position (36).
To investigate the flexibility of amino acid arrangements for creating active
peptides, the Bac2A sequence was scrambled (36). Consequently 49 peptide
variants of Bac2A were created, all of which were composed of the same amino
acids and had the same length, net charge, and amount of hydrophilic and
hydrophobic amino acids, and thus were ideally suited for testing the importance
of the primary amino acid sequence for activity. It has been demonstrated
that scrambling of a sequence destroys sequence-specific interactions,
as observed, e.g., with antibodies recognizing linear epitopes. In this case,
scrambled peptides are normally used as a negative control to proof the sequence
specificity. Conversely, if amino acid composition was the sole determinant
of killing activity, all scrambled peptides should be active. Experiments using
49 scrambled Bac2A peptides revealed peptides ranging in activity from superior
antimicrobial activity up to inactive, indicating that activity was not solely
dependent on the composition of amino acids or the overall charge or hydrophobicity,
but rather required particular linear sequence patterns (36). Therefore,
suitable amino acid composition including charge and ratio of hydrophobic
to hydrophilic amino acids is necessary but not sufficient for antimicrobial
activity. In the case of the scrambled peptide set, it was shown that the inactive
peptides were not able to form similar structures in lipids compared to the active