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Diversifying Constrained Peptide Scaffolds: How To Truss Up a Bundle Really Tight Esra Bozkurt* and Angela M. Gronenborn* Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260, United States
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P
roteins are an endless source of fascination and inspiration. For a protein engineer, (re)constructing structures with extraordinary properties, such as extreme thermostability, protease resistance, cell penetrability, and tailored bioactivities, is clearly the ultimate goal. However, a common problem in the protein (re)design arena is the loss or decline of existing properties while trying to graft on new ones. This severely curtails fast and efficient generation of newly designed constructs for biomedical and biotechnological applications. Therefore, great interest and attention are now paid to fashion templates beyond Nature’s natural sampling space, endowed with formidable and unique functions. One emerging class of such biomolecules consists of knotted, i.e., entangled, peptides. They comprise lasso peptides1 and knottins,2 among others. The former are wellknown for their unusual interlocked architectures that maintain biological activities at elevated temperatures and are resistant to proteolytic degradation. The unique three-dimensional structures of the latter are rich in disulfide bonds that tie up the fold with multiple linkages. Such multiple constrained peptides occupy a unique niche between traditional small molecule chemical drugs and the new biologics, rendering them promising candidates for clinical trials. As a result, creating and further refining three-dimensional structures of such molecules are receiving an increased level of attention from protein designers.3 While using numerous disulfides for tying up the polypeptide has great potential, it intrinsically faces the oxidative folding problem, necessitating elaborate strategies for maximizing correctly folded molecules. This prompted protein designers to embrace chemical synthesis technologies for creating small proteins, comprising chemical compositions and folds that are inaccessible to natural proteins. Writing in Chemical Science and ChemBioChem, Zheng et al.4 and Richelle et al.,5 respectively, report experiments using multicyclization strategies for creating constrained peptides in high yield and purity. Although the experimental design approaches employed by the two groups are distinct, they share a common goal, the generation of multicyclic peptide scaffolds that are amenable to sequence randomization (Figure 1). Zheng et al. accomplish this goal by utilizing orthogonal cysteine/penicillamine (Cys/Pen) pairing technology through strategic direction of three Cys/Pen substitutions, while Richelle et al. use an enzymatic head-to-tail cyclization, followed by copper-mediated “triple-C” locking. The first approach provides flexibility in terms of peptide length, positioning of cysteine residues for Pen pairing, and number of isomers. The latter offers a straightforward route for generating tetracyclic templates bearing two cysteine and two © XXXX American Chemical Society
Figure 1. Representative multicyclic peptides, devised by the triple Clocking and Cys/Pen pairing approach illustrated on top of knotted sculptures by Jesus Pedraglio. The sculptures are reproduced with permission from the artist.
azidohomoalanine (Aha) residues. Both groups select representative examples to illustrate that the designed constructs can tolerate grafting of bioactive amino acid sequences and evaluate the bioactivity of their designed constructs toward their respective therapeutic targets. In summary, both studies represent important steps forward in engineering sophisticated compact folds that tolerate amino acid sequence manipulation. Is it envisaged that generating such molecules will permit (re)programming and fine-tuning of specific interactions with their desired therapeutic targets? Importantly, such molecules also pose intriguing challenges and questions for computational biochemists, such as whether it is possible to devise automated computational algorithms to systematically and reliably predict or guide the generation of specific constrained isomers and/or topologies. Or, are there other orthogonal pairing strategies that can be exploited to accomplish high yields and purities? Naturally, whenever doors are opened, new questions emerge, yet moving across the thresholds into these new rooms no doubt will bring forward inspiring and even more imaginative strategies for building hybrid multicyclic chemical and biological molecules, filling the drug pipelines with much needed novel promising candidates. Received: January 23, 2019
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DOI: 10.1021/acs.biochem.9b00065 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Esra Bozkurt: 0000-0001-8492-1162 Angela M. Gronenborn: 0000-0001-9072-3525 Author Contributions
E.B. conceived the topic, and E.B. and A.M.G. wrote and approved the final version of the manuscript. Funding
The work in the authors’ laboratory is funded by the National Institutes of Health (GM082251 and DK114855) and the National Science Foundation (CHE-1708773). Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Hegemann, J. D., Zimmermann, M., Xie, X., and Marahiel, M. A. (2015) Lasso Peptides: An Intriguing Class of Bacterial Natural Products. Acc. Chem. Res. 48, 1909−1919. (2) Ackerman, S. E., Currier, N. V., Bergen, J. M., and Cochran, J. R. (2014) Cystine-knot peptides: Emerging tools for cancer imaging and therapy. Expert Rev. Proteomics 11, 561−572. (3) Bhardwaj, G., Mulligan, V. K., Bahl, C. D., Gilmore, J. M., Harvey, P. J., Cheneval, O., Buchko, G. W., Pulavarti, S. V. S. R. K., Kaas, Q., Eletsky, A., Huang, P. S., Johnsen, W. A., Greisen, P. J., Rocklin, G. J., Song, Y., Linsky, T. W., Watkins, A., Rettie, S. A., Xu, X., Carter, L. P., Bonneau, R., Olson, J. M., Coutsias, E., Correnti, C. E., Szyperski, T., Craik, D. J., and Baker, D. (2016) Accurate de novo design of hyperstable constrained peptides. Nature 538, 329−335. (4) Zheng, Y., Meng, X., Wu, Y., Zhao, Y., and Wu, C. (2018) De novo design of constrained and sequence-independent peptide scaffolds with topologically-formidable disulfide connectivities. Chem. Sci. 9, 569−575. (5) Richelle, G. J. J., Schmidt, M., Ippel, H., Hackeng, T. M., van Maarseveen, J. H., Nuijens, T., and Timmerman, P. (2018) A OnePot “Triple-C” Multicyclization Methodology for the Synthesis of Highly Constrained Isomerically Pure Tetracyclic Peptides. ChemBioChem 19, 1934−1938.
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DOI: 10.1021/acs.biochem.9b00065 Biochemistry XXXX, XXX, XXX−XXX
