Oral Presentation 14th Australian Peptide Conference 2022

Cyclic and bicyclic peptides as inhibitors of viral proteases (#5)

Christoph Nitsche 1
  1. Australian National University, Canberra, ACT, Australia

Proteases are the Achilles heel of replication for many viruses. Our research analyses and targets proteases from pathogenic flavi-, alpha- and coronaviruses and aims to develop peptide-based drug candidates. We have particular interest in peptide modifications that can (i) enhance metabolic stability by greater resistance towards proteolysis, (ii) promote biological uptake across cell membranes, (iii) decrease the entropic penalty of binding by locking the peptide in the active conformation, and (iv) enable covalent interactions between peptide and protease.

After initial studies with linear and covalently binding peptides of limited stability and selectivity (ref. 1-2), we explored macrocyclic peptides as promising alternatives. We developed different in vitro peptide cyclisation and stapling strategies using our biocompatible nitrile-aminothiol Click reaction (ref. 3-5) and identified various highly active cyclic peptides with improved proteolytic stability. We also discovered macrocyclic peptides with novel binding modes using mRNA display screenings (ref. 6).

Bicyclic peptides offer even greater conformational rigidity, metabolic stability, and antibody-like affinity and specificity. We introduced a fundamentally new method to generate bicyclic peptides that uses bismuth as a selective, stable, rigid and green reagent for peptide modification (ref. 7). Bismuth represents the smallest “scaffold” ever explored and allows in-situ access to bicyclic peptides for biochemical screening assays. Using this technology, we were able to identify bicyclic inhibitors of viral proteases that were up to 130 times more active and almost 20 times more proteolytically stable than their linear peptide analogues.

In summary, we developed a large variety of high-affinity and proteolytically stable peptide inhibitors of viral proteases using innovative chemical approaches and mRNA display. Our chemical strategies proceed under biocompatible conditions, enabling in-situ access to constrained peptide ligands in presence of proteases and other drug targets. Cyclic and bicyclic peptides display extraordinarily high affinities that are similar to covalently binding inhibitors.

  1. Nitsche, C., Zhang, L., Weigel, L., Schilz, J., Graf, D., Bartenschlager, R., Hilgenfeld, R., Klein, C. D., Peptide-Boronic Acid Inhibitors of Flaviviral Proteases: Medicinal Chemistry and Structural Biology, J. Med. Chem. 2017, 60 (1), 511-516.
  2. Lei, J., Hansen, G., Nitsche, C., Klein, C. D., Zhang, L., Hilgenfeld, R., Crystal Structure of Zika Virus NS2B-NS3 Protease in Complex with a Boronate Inhibitor, Science 2016, 353 (6298), 503-505.
  3. Nitsche, C., Onagi, H., Quek, J.-P., Otting, G., Luo, D., Huber, T., Biocompatible Macrocyclization between Cysteine and 2-Cyanopyridine Generates Stable Peptide Inhibitors, Org. Lett. 2019, 21 (12), 4709-4712.
  4. Morewood, R., Nitsche, C., A Biocompatible Stapling Reaction for in situ Generation of Constrained Peptides, Chem. Sci. 2021, 12 (2), 669-674.
  5. Patil, N. A., Quek, J.-P., Schroeder, B., Morewood, R., Rademann, J., Luo, D., Nitsche, C., 2-Cyanoisonicotinamide Conjugation: A Facile Approach to Generate Potent Peptide Inhibitors of the Zika Virus Protease, ACS Med. Chem. Lett. 2021, 12 (5), 732-737.
  6. Nitsche, C., Passioura, T., Varava, P., Mahawaththa, M. C., Leuthold, M. M., Klein, C. D., Suga, H., Otting, G., De Novo Discovery of Nonstandard Macrocyclic Peptides as Noncompetitive Inhibitors of the Zika Virus NS2B-NS3 Protease, ACS Med. Chem. Lett. 2019, 10 (2), 168-174.
  7. Voss, S., Rademann, J., Nitsche, C., Peptide-Bismuth Bicycles: In Situ Access to Stable Constrained Peptides with Superior Bioactivity, Angew. Chem. Int. Ed. 2022, 61 (4), e202113857.