5 research outputs found
An Expanded Set of Amino Acid Analogs for the Ribosomal Translation of Unnatural Peptides
BACKGROUND: The application of in vitro translation to the synthesis of unnatural peptides may allow the production of extremely large libraries of highly modified peptides, which are a potential source of lead compounds in the search for new pharmaceutical agents. The specificity of the translation apparatus, however, limits the diversity of unnatural amino acids that can be incorporated into peptides by ribosomal translation. We have previously shown that over 90 unnatural amino acids can be enzymatically loaded onto tRNA. METHODOLOGY/PRINCIPAL FINDINGS: We have now used a competition assay to assess the efficiency of tRNA-aminoacylation of these analogs. We have also used a series of peptide translation assays to measure the efficiency with which these analogs are incorporated into peptides. The translation apparatus tolerates most side chain derivatives, a few alpha,alpha disubstituted, N-methyl and alpha-hydroxy derivatives, but no beta-amino acids. We show that over 50 unnatural amino acids can be incorporated into peptides by ribosomal translation. Using a set of analogs that are efficiently charged and translated we were able to prepare individual peptides containing up to 13 different unnatural amino acids. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that a diverse array of unnatural building blocks can be translationally incorporated into peptides. These building blocks provide new opportunities for in vitro selections with highly modified drug-like peptides
<i>In Vitro</i> Selection of Highly Modified Cyclic Peptides That Act as Tight Binding Inhibitors
There is a great demand for the discovery of new therapeutic
molecules
that combine the high specificity and affinity of biologic drugs with
the bioavailability and lower cost of small molecules. Small, natural-product-like
peptides hold great promise in bridging this gap; however, access
to libraries of these compounds has been a limitation. Since ribosomal
peptides may be subjected to <i>in vitro</i> selection techniques,
the generation of extremely large libraries (>10<sup>13</sup>)
of
highly modified macrocyclic peptides may provide a powerful alternative
for the generation and selection of new useful bioactive molecules.
Moreover, the incorporation of many non-proteinogenic amino acids
into ribosomal peptides in conjunction with macrocyclization should
enhance the drug-like features of these libraries. Here we show that
mRNA-display, a technique that allows the <i>in vitro</i> selection of peptides, can be applied to the evolution of macrocyclic
peptides that contain a majority of unnatural amino acids. We describe
the isolation and characterization of two such unnatural cyclic peptides
that bind the protease thrombin with low nanomolar affinity, and we
show that the unnatural residues in these peptides are essential for
the observed high-affinity binding. We demonstrate that the selected
peptides are tight-binding inhibitors of thrombin, with <i>K</i><sub>i</sub><sup>app</sup> values in the low nanomolar range. The
ability to evolve highly modified macrocyclic peptides in the laboratory
is the first crucial step toward the facile generation of useful molecular
reagents and therapeutic lead molecules that combine the advantageous
features of biologics with those of small-molecule drugs