In previous papers, we observed that dendrimers of peptide mimotopes of the nicotinic receptor ligand site are strong antidotes against the lethality of the nicotinic receptor ligand ␣-bungarotoxin. Although their in vitro activity is identical to that of dendrimers, the corresponding monomeric peptide mimotopes are not effective in vivo. Because the higher in vivo efficiency of dendrimers could not in this case be related to polyvalent interaction, the stability to blood protease activity of monomeric versus tetrabranched dendrimeric mimotope peptides was compared here by incubating three different mimotopes with human plasma and serum. Unmodified peptides and cleaved sequences were followed by high pressure liquid chromatography and mass spectrometry. Tetrabranched peptides were shown to be much more stable in plasma and also in serum. To assess the notable stability of multimeric peptides, different bioactive neuropeptides, including enkephalins, neurotensin and nociceptin, were synthesized in monomeric and tetrabranched forms and incubated with human plasma and serum and with rat brain membrane extracts. All the tetrabranched neuropeptides fully retained biological activity and generally showed much greater stability to blood and brain protease activity. Some tetrabranched peptides were also resistant to trypsin and chymotrypsin. Our findings provide new insights into the possible therapeutic use of bioactive peptides.Hundreds of peptides with potential therapeutic activities have been identified. These include naturally occurring peptide hormones and neurotransmitters, which influence and control series of vital functions, such as cell proliferation, tissue development, metabolism, immune defense, perception of pain, reproduction, behavior, and blood pressure. Selective agonists or antagonists of these natural peptides are extremely useful for the investigation of peptidergic systems and are also potential therapeutic agents (1). Moreover, several peptide fragments or mimotopes derived from potential therapeutic proteins show promising biological activity (2).However, the use of peptides as therapeutic drugs has largely been limited by their short half-life in vivo. Because peptides are mainly broken down by proteases and peptidases, peptide delivery is the bottleneck in the development of new peptide drugs. To increase peptide half-life, many strategies involving different levels of chemical modification are possible (3, 4). The introduction of D-amino acids, or pseudo amino acids, and peptide cyclization are the most common strategies to increase peptide stability. However, these modifications may profoundly alter peptide activity. Alternatively, peptidomimetic molecules can be developed by the synthesis of conformationally restricted compounds, in which the peptide is locally or globally constrained in order to reproduce the active conformation. The resulting structures are mostly non-peptide molecules, more resistant to degrading enzymes.In general, peptide molecules have the advantage of good specifici...

A large 10-mer phage peptide library was panned against whole Escherichia coli cells, and an antimicrobial peptide (QEKIRVRLSA) was selected. The peptide was synthesized in monomeric and dendrimeric tetrabranched form (multiple antigen peptide [MAP]), which generally allows a dramatic increase of peptide stability to peptidases and proteases. The antibacterial activity of the dendrimeric peptide against E. coli was much higher than that of the monomeric form. Modification of the original sequence, by residue substitution or sequence shortening, produced three different MAPs, M4 (QAKIRVRLSA), M5 (KIRVRLSA), and M6 (QKKIRVRLSA) with enhanced stability to natural degradation and antimicrobial activity against a large panel of gram-negative bacteria. The MICs of the most potent peptide, M6, were as low as 4 to 8 g/ml against recent clinical isolates of multidrug-resistant Pseudomonas aeruginosa and members of the Enterobacteriaceae. The same dendrimeric peptides showed high stability to blood proteases, low hemolytic activity, and low cytotoxic effects on eukaryotic cells, making them promising candidates for the development of new antibacterial drugs.

A synthetic antimicrobial peptide was identified as a possible candidate for the development of a new antibacterial drug. The peptide, SET-M33L, showed a MIC90 below 1.5 μM and 3 μM for Pseudomonas aeruginosa and Klebsiella pneumoniae, respectively. In in vivo models of P. aeruginosa infections, the peptide and its pegylated form (SET-M33L-PEG) enabled a survival percentage of 60–80% in sepsis and lung infections when injected twice i.v. at 5 mg/Kg, and completely healed skin infections when administered topically. Plasma clearance showed different kinetics for SET-M33L and SET-M33L-PEG, the latter having greater persistence two hours after injection. Bio-distribution in organs did not show significant differences in uptake of the two peptides. Unlike colistin, SET-M33L did not select resistant mutants in bacterial cultures and also proved non genotoxic and to have much lower in vivo toxicity than antimicrobial peptides already used in clinical practice. The characterizations reported here are part of a preclinical development plan that should bring the molecule to clinical trial in the next few years.

We found that synthetic peptides in the form of dendrimers become resistant to proteolysis. To determine the molecular basis of this resistance, different bioactive peptides were synthesized in monomeric, two-branched and tetra-branched form and incubated with human plasma and serum. Proteolytic resistance of branched multimeric sequences was compared to that of the same peptides synthesized as multimeric linear molecules. Unmodified peptides and cleaved sequences were detected by high pressure liquid chromatography and mass spectrometry. An increase in peptide copies did not increase peptide resistance in linear multimeric sequences, whereas multimericity progressively enhanced proteolytic stability of branched multimeric peptides. A structure-based hypothesis of branched peptide resistance to proteolysis by metallopeptidases is presented.

Receptors for endogenous regulatory peptides, like the neuropeptide neurotensin, are overexpressed in several human cancers and can be targets for peptide-mediated tumor-selective therapy. Peptides, however, have the main drawback of an extremely short half-life in vivo. We showed that neurotensin and other endogenous peptides, when synthesized as dendrimers, retain biological activity and become resistant to proteolysis. Here, we synthesized the neurotensin functional fragment NT(8-13) in a tetrabranched form linked to different units for tumor therapy or diagnosis. Fluorescent molecules were used to monitor receptor binding and internalization in HT29 human adenocarcinoma cells and receptor binding in HT29 tumor xenografts in nude mice. Linking of chemotherapic molecules like chlorin e6 and methotrexate to dendrimers resulted in a dramatic increase in drug selectivity, uptake of which by target cells became dependent on peptide receptor binding. When nude mice carrying human tumor xenografts were treated with branched NT(8-13)-methotrexate, a 60% reduction in tumor growth was observed with respect to mice treated with the free drug. [Mol Cancer Ther 2007;6(9):2441 -8]