Laurie Smith

In Vitro Selection of Highly Modified Cyclic Peptides That Act as Tight Binding Inhibitors

Blog Post created by Laurie Smith on Mar 13, 2014

Yollete V. Guillen Schlippe , Matthew C. T. Hartman , Kristopher Josephson , and Jack W. Szostak *

Howard Hughes Medical Institute, Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital

Journal of the American Chemical Society 2012, 134 (25) pp 10469-10477, doi:  10.1021/ja301017y

Highlight by: Ms. Zinaida Polonskaya, The Scripps Research Institute


Peptide-based drugs have the potential to combine the advantages of small molecule drugs (size, low production costs, ease of storage and handling) with those of biologics (high selectivity and specificity, low toxicity). Large peptide libraries (>106 members) can be screened using molecular evolution techniques, which facilitates lead identification. But until recently peptides have been considered relatively poor drug candidates due to their susceptibility to protease degradation and low bioavailability. While inclusion of unnatural amino acids and cyclization of the peptide backbone can increase both peptide stability and affinity, such modifications are not readily incorporated using standard ribosomal peptide synthesis.


The authors of this paper combined two previously reported techniques to perform in vitro selection with unnatural amino acid-containing peptides. They used mRNA display technology to chemically attach peptides to the mRNA that encodes them, effectively linking “genotype” to “phenotype” and enabling selection. To diversify the pool of amino acids the authors applied Protein Synthesis Using Recombinant Elements (PURE) system that reconstitutes translational machinery of the cell in vitro. In the absence of natural substrates some E. coli aminoacyl-tRNA synthetases will charge tRNAs with certain structurally similar unnatural amino acids. 12 out of 20 natural amino acids were replaced with unusual amino acids displaying various functional groups not found in standard proteins, such as alkynes, aryl halides, etc. A DNA library encoding 10 random amino acids flanked by two cysteines (to enable cyclization) was designed, transcribed and translated with unnatural amino acids to yield ~1013 unique members. A control library was made using only natural amino acids. Both libraries were subjected to in vitro selection for members that bind to the protein thrombin. After 7 selection cycles the “natural library” converged on a single motif that was previously found in a similar screen. In contrast, the “unnatural library” yielded a diverse set of winners after 10 cycles, all of them having 4 to 7 unnatural amino acids. 2 representative unnatural peptides had binding affinities of 4.5 and 20 nM, and inhibited thrombin activity at 23 and 35 nM, respectively. These parameters are comparable to those of the natural peptide (affinity of 1.5 nM, inhibition at 6.3 nM), however,  “unnatural” peptides may have other beneficial characteristics, such as improved stability.


This work demonstrates that in vitro selection techniques can be successfully applied to highly modified peptides, putting the powerful tools of molecular biology in hands of peptide designers. It is a great first step towards generating more chemically diverse peptides with higher stability and bioavailability and, ultimately, better drug-like properties.


Recommended reading:

- mRNA display:  (a) “RNA-peptide fusions for the in vitro selection of peptides and proteins” Roberts R.W.; Szostak, J.W., PNAS 1997, 94(23), 12297-12302. (b) “In-vitro protein evolution by ribosome display and mRNA display” Lipovsek, D.; Plückthun, A., J. Immunol. Meth. 2004, 290, 51-67.

- PURE: (a) “Cell-free translation reconstituted with purified components” Shimizu, Y.; Inoue, A.; Tomari, Y.; Suzuki, T.; Yokogawa, T.; Nishikawa, K.; Ueda, T., Nat. Biotechnol. 2001, 19, 751 -755. (b) “Ribosomal Synthesis of Unnatural Peptides”  Josephson, K.; Hartman, M. C. T.; Szostak, J. W. J. Am. Chem. Soc. 2005, 127, 11727-11735.