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142 Cell-Penetrating Peptides: Processes and Applications
ABSTRACT
The goal of this review is to summarize previously published work on the design
and biological activity of a family of guanidinium peptoid transporters that differ in
the length of their side chains and to compare these results with a more recently
designed family of transporters containing non-α-amino acids, i.e., variations along
the backbone. The latter group of peptides differs by the spacing of the arginine
subunits along the peptide backbone. When these analogs were assayed for cellular
uptake, the most important feature was shown to be not the nature of the substituted
amino acid, but rather the distance between each of the arginines.
When results of the two studies were compared a very similar pattern was
observed. By increasing the conformational freedom of either the side chains of the
peptoids or the backbone of peptides by the addition of methylene units, a significant
enhancement in the rate of cellular uptake of the transporter was seen. Even though
the conformational flexibility of peptoids and peptides is very different, addition of
methylenes in either the backbone or the side chain results in enhanced cellular
uptake, arguing that the flexibility of the guanidine headgroups can be accomplished
in either of these ways. Typically, the biological activity of most biological ligands,
particularly those functioning as inhibitors, increases with conformational restriction
as preorganization in the form of the bound conformer favors tighter binding. The
enhanced activity associated with increased conformational mobility observed for
molecular transporters is in agreement with a dynamic transport system in which
turnover rather than tightness of binding is critical for function.
7.1 INTRODUCTION
Biological barriers have evolved to prevent entry of xenobiotics into tissues and
cells. However, these same barriers often limit or preclude uptake, and therefore the
therapeutic benefit, of a variety of drugs. Consequently, most drugs must be restricted
in physical properties to allow cellular and tissue uptake. This requirement greatly
limits the universe of potential therapeutics to a very small number of compounds.
The studies reviewed below were directed toward the goal of enhancing or enabling
delivery of drugs through biological barriers by conjugation to transporter molecules.
Oligomers of arginine composed of six or more amino acids, either alone or when
covalently attached to a variety of small molecules, cross biological membranes very
effectively by an as yet undefined mechanism. 1 The chirality of the amino acid subunits
in the arginine oligomers does not affect transport activity. In addition, a variety of other
nonpeptide, guanidine-rich molecular transporters that do not share the composition or
the spacing of a peptide have been shown to exhibit transport into cells.
The guanidine headgroup of arginine appears to be the key structural unit for
cellular uptake. 1,2 The rate of uptake is dependent on both the concentration of the
oligomers and their guanidine content, with a greater rate observed as the length of the
peptide is increased up to approximately 15 residues. Oligomers beyond this length
enter cells less effectively, precipitate serum proteins, and exhibit cellular cytotoxicity
at high micromolar concentrations. Detailed kinetic analyses demonstrated that nonamers
of arginine were significantly more effective at crossing biological membranes than