Peptide-Based Drug Design

The Spot Technique 57
(see Fig. 3) (13,16,43). In general, a length between 10 and 15 amino acids
is usual. Shifting of the frame should be between one and five amino acids,
where the smaller the steps are, the more precise will be the identification of the
minimal peptide length required for activity.
A special application of the peptide scan is the hybritope scan (hybridepitope)
(44,45). In order to increase the affinity by extention of the peptide
sequence in a hybritope scan, several mixtures of amino acids at positions
flanking the related sequence of the protein are introduced. Another strategy to
optimize a peptide scan is the so-called duotope scan (duo-epitope) (46). Fora
duotope scan one generates the peptide scan twice and combines the sequence of
both arrays linked via a spacer, such as two �-alanine molecules. In this way it is
possible to avoid possible steric hindrance and to increase activity by extention
of the active sequence. The pattern of such a duotope scan resembles that of a
combinatorial library with the exception that each field represents a combination
of two sequences rather than two amino acids.
3.2.2. Substitution Analysis (Replacement Analysis,
Mutational Analysis)
Substitution analyses, developed by Geysen and coworkers for peptides
bound to polypropylene rods (28), are used for investigation of the importance
or of amino acids in a known peptide sequence. Therefore, one can generate
the sequences for synthesis by successive systematic substitution of each amino
acid by other amino acids or building blocks of interest (see Fig. 4) (for instance
d-amino acids (36,47) and peptoidic monomers (40)). Usually, only one single
amino acid should be exchanged at one spot position (43,48,49). However,
2D-substitution analysis is also possible, where two amino acids in a sequence
are simultaneously exchanged on a single peptide spot. The pattern of such a
2D-substitution analysis is similar to that of a combinatorial library (40).
Special applications of substitution analysis include the so-called amino acid
walks or scans (e.g., alanine scan, glycine walk) (50,51). In this type of screening
one replaces each single amino acid of the sequence with only one distinct amino
acid (usually alanine or glycine). The progressive amino acid scan is achieved by
a stepwise increase in the number of positions exchanged by the distinct amino
acid (52).
3.2.3. Combinatorial Peptide Library
A very powerful method for screening active peptides without knowing
the actual sequence is the combinatorial peptide library. This method was
first described by Houghten and coworkers for peptides synthesized on a
polymer resin (53). Using combinatorial libraries, screening begins in theory