Peptides typically have poor biostabilities and natural sequences cannot easily be converted into drug-like molecules without extensive medicinal chemistry. We have adapted mRNA display to evolve highly stable cyclic peptides while preserving target affinity. To do this, we incorporated an unnatural 21st amino acid in an mRNA display library which was subjected to proteolysis prior to selection for function. The resulting “SUPR peptide” (Scanning Unnatural Protease Resistant) showed ∼500-fold improvement in serum stability (t1/2 = 160 hours) and up to 3,700-fold improvement in protease resistance versus the parent sequence. We extended this approach by carrying out SUPR peptide selections against Her2-positive cells in culture. The resulting SUPR4 peptide showed low nanomolar affinity toward Her2, excellent specificity, and selective tumor uptake in vivo. These results argue that this is a general method to design potent and stable peptides for in vivo imaging and therapy.
In a large AF kindred, we have identified a novel AF locus on chromosome 5p15 and shown that affected individuals with AF and mutation carriers can be identified by a prolonged SA P-wave duration. Importantly, identification of an endophenotype in this kindred not only aided ascertainment of additional family members but also increased the LOD score, providing increased support for linkage at this locus. Identification of the causal gene, mapped to chromosome 5p15, will advance our understanding of the molecular basis of AF.
Radiolabeling of substrates with 2-[F]fluoroethylazide exploits the rapid kinetics, chemical selectivity, and mild conditions of the copper-catalyzed azide-alkyne cycloaddition reaction. While this methodology has proven to result in near-quantitative labeling of alkyne-tagged precursors, the relatively small size of the fluoroethylazide group makes separation of the F-labeled radiotracer and the unreacted precursor challenging, particularly with precursors>500 Da (e.g., peptides). We have developed an inexpensive azide-functionalized resin to rapidly remove unreacted alkyne precursor following the fluoroethylazide labeling reaction and integrated it into a fully automated radiosynthesis platform. We have carried out 2-[F]fluoroethylazide labeling of four different alkynes ranging from <300 Da to >1700 Da and found that >98% of the unreacted alkyne was removed in less than 20 min at room temperature to afford the final radiotracers at >99% radiochemical purity with specific activities up to >200 GBq/μmol. We have applied this technique to label a novel cyclic peptide previously evolved to bind the Her2 receptor with high affinity, and demonstrated tumor-specific uptake and low nonspecific background by PET/CT. This resin-based methodology is automated, rapid, mild, and general allowing peptide-based fluorine-18 radiotracers to be obtained with clinically relevant specific activities without chromatographic separation and with only a minimal increase in total synthesis time.
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