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a hyperacute rejection xenotransplantation model [77]; blocked the
E. coli
-induced oxidative burst of granulocytes and monocytes [78];
and inhibited complement activation by cell lines SH-SY5Y, U-937,
THP-1, and ECV304 [79]. Compstatin was stable in biotranformation
studies in vitro in human blood, normal human plasma, and serum,
with increased stability upon N-terminal acetylation [72]. Compstatin
showed little or low toxicity, and no adverse effects when these were
measured [75-77]. Finally, compstatin showed species specificity and is
active only with human and primate C3 [80].
In Silico Sequence Selection
The first stage of the design approach involves the selection of sequences
compatible with the backbone template through the solution of the ILP
problem. The formulation relies only on the alpha-carbon coordinates of
the backbone residues, which were taken from the NMR-average solu-
tion structure of compstatin [71].
A full computational design study from compstatin would result in
a combinatorial search of 20
13
10
16
sequences. However, in light of
the results of the experimental studies of the rationally designed pep-
tides, a directed, rather than full, set of computational design studies
was performed. First, since the disulfide bridge was found to be essen-
tial for aiding in the formation of the hydrophobic cluster and
prohibiting the termini from drifting apart, both residues Cys
2
and
Cys
12
were maintained. In addition, because the structure of the type-I
b turn was not found to be a sufficient condition for activity, the turn
residues were fixed to be those of the parent compstatin sequence,
namely, Gln
5
-Asp
6
-Trp
7
-Gly
8
. In fact, when stronger type I b sequences
were constructed, which was supported by NMR data indicating that
these sequences provided higher b-turn populations than compstatin,
these sequences resulted in lower or no activity [73]. Therefore, the fur-
ther stabilization of the turn residues, which would likely be a
consequence of the computational peptide design procedure, may not
enhance compstatin activity. This is especially true for Trp
7
, which was
found to be a likely candidate for direct interaction with C3. For simi-
lar reasons, Val
3
≈
8
×
was maintained throughout the computational
experiments.
After designing the compstatin system to be consistent with those
features found to be essential for compstatin activity, six residue posi-
tions were selected for optimization. Of these six residues, positions 1,
4, and 13 have been shown to be structurally involved in the formation
of a hydrophobic cluster involving residues at positions 1, 2, 3, 4, 12,
and 13, a necessary but not sufficient component for compstatin bind-
ing and activity. The remaining residues, namely those at positions 9,
10, and 11, span the three positions between the turn residues and
the C-terminal cystine. For the wild-type sequence these positions are
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