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Structure Prediction of CPPs and Iterative Development of Novel CPPs 189
Furthermore, retro, enantio, and retro-inverso forms of penetratin can also efficiently
enter live cells. 22 Therefore, the ability of penetratins to cross biological membranes
should rely only on their physico-chemical properties. The only residue identified
so far as critical for the translocation of penetratins through biological membranes
is a Trp corresponding to the well-conserved position 48 in most of the homeodomains.
17,21,22 Indeed, substituting this residue by the otherwise hydrophobic phenylalanine
appeared to be deleterious for crossing the peptide through lipid bilayers.
It has been suggested that penetratin might cross the cellular membrane by the
transient formation of inverted micelles. 23,21 According to this model, the interaction
between the peptide and membrane lipids should be critical for the internalization
process. Several recent reports have addressed the question of physico-chemical
parameters’ conditioning the membrane crossing behavior of penetratins. They
revealed that there is only a partial relationship between the lipid-binding affinity
of penetratin variants and their capacity to cross biological membranes. Also, no
correlation exists between the helical amphipathy of the variant peptides and their
propensity to be internalized by cells. 24,25
Although the mechanisms and determinants underlying translocation of penetratin
through membranes remain to be characterized, it is obvious that, in its natural
configuration — i.e., as an α-helix constitutive of a transcription factor — this short
sequence promotes the cellular entry of large molecules. Furthermore, according to
the different homeoproteins now shown to enter live cells, the penetratin sequence
is functional even when located in the middle of the primary sequence of a long
protein. However, since the only structural data available so far for homeoproteins
are basically limited to the isolated homeodomain, it cannot be stated whether the
penetratin α-helix must be solvent accessible to be functional. Nonetheless, in the
different homeodomains or homeodomain–DNA complexes characterized so far,
charges harbored by the polar amino acid residues of the third α-helix are exposed
to the solvent or to the phosphate groups of the DNA. Strikingly, the tryptophan
residue at position 48 in the homeodomain shown to be critical for crossing biological
membranes takes part in the hydrophobic core of the homeodomain globular structure.
The recent discovery of cell-permeant peptides like penetratins, and others like the
HIV Tat peptide or transportan 21,26 (see also other chapters in this book), has opened
the perspective of using such peptides as carriers to allow intracellular delivery of
different classes of molecules of pharmacological interest, in particular of hydrophilic
compounds that hardly access the intracellular environment. 27-30 However, to evaluate
the degree of freedom in designing new cell-permeant peptide derivatives or peptide–cargo
combinations with the view of modulating cytological functions for therapeutic
purposes, for example, it is important to elaborate predictive methods, taking
into account constraints that condition the addressing of peptide vectors into live cells.
In order to get insight into molecular interactions between biological membranes
and the numerous and unrelated classes of membrane proteins or membrane interacting
proteins, modeling procedures have been developed and optimized.
To study peptide–bilayer interactions, we adopt a simulation approach using the
Monte Carlo (MC) method, in which peptide–bilayer interactions are approached
by the sum of different energy functions that mimic lipid perturbation, hydrophobicity,
and electrostatic effects. All of these energies are named restraints because