Biology Reference
In-Depth Information
interface makes the reproduction of such a functional domain into a
small non-immunogenic molecule a challenge, and highlights the dif-
ficulty in developing high-affinity CD4 mimics. However, despite of
the large number of residues present in gp120-CD4 interaction, stud-
ies on hormone-receptor systems have shown that few residues might
dominate the binding energy at protein-protein interface.
158
Thus,
the design of a minimal CD4 mimics may be possible.
The transfer of functional sites to small proteins acting as struc-
tural scaffolds has been proposed as a useful strategy to reproduce the
structure and function of the target protein in small molecular sys-
tems.
159,160
This approach has led to the discovery of scorpion toxin
Scyllatoxin fragment as an effective mimic of CD4. A mini-protein,
CD4M3, was chemically synthesized, folded efficiently, and presented
a circular dichroism spectrum similar to that of native Scyllatoxin. In
competitive ELISA, CD4M33 was able to specifically bind gp120 at
an IC50 of 40
M, which is four orders of magnitude higher than
that of sCD4. This strategy has been recently applied to the engi-
neering of a mini-protein that mimics the core of the CD4 protein
surface that interacts with the gp120 envelope glycoprotein of HIV-
1 and, hence, inhibits virus attachment to cells and infection.
161
The biological performance of this mini-protein was improved
using “rational” structure design. In total, five substitutions were
introduced (Gln20Ala, Thr25Ala, Leu18Lys, Ser9Arg, and Pro28)
and the resulting mini-protein (CD4M9) bound to gp120 at 400 nM,
induced conformational changes in Env, and inhibited infection of
CD4-expressing cells by different virus isolates. So far, an improved
CD4M33 has an affinity for gp120 similar to CD4,
162
and induces a
conformational change in gp120 similar to that induced by sCD4X.
162
In an earlier study, Fouts and colleagues have shown that covalently
cross-linked complexes of CD4 and HIV Env IIIB induced antibod-
ies that neutralized a wide range of primary isolates.
163
Ig with neutral-
izing activity was recovered by affinity chromatography using Env/
CD4M9 single-chain polypeptide. More recently, using CD4M9, an
earlier version of the CD4 mini-protein developed by Vita and col-
leagues, Fouts
et al
. prepared single-chain constructs of SCBal/M9
and performed immunogenicity studies in rabbits.
164
µ
They showed
