Final Report Summary - PEPTIDE DENDRIMERS (Peptide Dendrimers - A Chemical Platform for Functional Diversity) Peptides have emerged as a therapeutically important class of drugs, offering the advantage of great specificity, potency, and low toxicity. However, short circulation half-life, and poor proteolytic stability and bioavailability have prevented them from becoming a mainstream source of drug candidates. Dendrimers are versatile, derivatisable, well-defined branched synthetic macromolecules with promising properties for a variety of applications in technology and medicine. Combining therapeutic peptides with dendrimers represents a promising approach to overcome bioavailability limitations, while at the same time introducing novel pharmacological and biophysical properties. The project objectives were to develop a synthetic method to access such peptide dendrimers and to characterise them by biophysical and pharmacological means. Oxime ligation, a chemoselective reaction of an amino-oxy group with an aldehyde, in combination with solid-phase peptide synthesis for the production of the individual building blocks, became the strategy of choice for the assembly of the peptide dendrimers. A standardised and robust protocol for the assembly and purification was established. Using this protocol, a wide range of peptide dendrimers including varying degrees of multivalency (2x, 4x, 8x, 16x), different sizes of linkers (lysine, PEG2000, PEG20000, PEG40000), and a diverse set of functional peptides (glutamate, poly-arginine, substance P, oxytocin, and alpha-conotoxin RgIA and MI) were synthesised. A synthetic strategy was devised to equip these dendrimers with imaging tags, chelating agents, biotin, additional functional groups or PEG units. These peptide dendrimers were characterised by mass spectrometry, nuclear magnetic resonance (NMR) and gel electrophoresis. No cell toxicity was observed up to concentrations of 50 μM. The peptide dendrimers were pharmacologically characterised on their corresponding receptors/targets: glutamate - AMPA receptor; poly-arginine - cellular qDot delivery; substance P - tachykinin receptors and TRPV1 channel; oxytocin - oxytocin and vasopressin receptors; and alpha-conotoxins - nicotinic acetylcholine receptor. NMR studies and in-depth pharmacological characterization of the peptide dendrimers showed that various linker lengths can be used to tune potency and selectivity. Peptide dendrimers with long PEG linkers interacted with the receptor without any binding restrictions and displayed similar bioactivity and selectivity as their endogenous monomers. In the case of oxytocin dendrimers, we identified linker lengths that activated the oxytocin receptor at a ~1000-fold lower concentration and with enhanced selectivity. Molecular modelling and further pharmacological studies showed that this increase in activity is due to the presence of receptor homodimers that are simultaneously activated as a result of the multivalent architecture of the dendrimers. Regarding the quantum dot delivery studies using poly-arginine dendrimers, a clear trend was observed, with dendrimers of increased branching exhibiting a higher degree of cell uptake.In summary, we have established an efficient and robust chemical method that allows the production of diverse non-toxic peptide dendrimers. We have shown that these peptide dendrimers can be assembled without compromising bioactivity and that the dendritic structure allows for further modifications, including imaging tags, chelating groups, PEG units, biotin or other functional molecules. Various linker lengths can be chosen to tune potency and selectivity. Ongoing stability and bioavailability studies will reveal whether this novel class of macromolecules offers additional benefits in terms of enzymatic degradation and renal clearance. We believe that the results of this work will have a significant impact on the design of next-generation molecular probes and peptide therapeutics, particularly given the therapeutic significance of G protein-coupled receptors.
