Noncanonical peptides as probes and inhibitors for flavivirus proteases — ASN Events
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  3. Noncanonical peptides as probes and inhibitors for flavivirus proteases

Noncanonical peptides as probes and inhibitors for flavivirus proteases (#184)

Christoph Nitsche 1 , Ralf Bartenschlager 2 , Rolf Hilgenfeld 3 , Thomas Huber 1 , Christian Klein 2 , Dahai Luo 4 , Gottfried Otting 1 , Toby Passioura 5 , Hiroaki Suga 5
  1. Australian National University, Canberra, ACT, Australia
  2. Heidelberg University, Heidelberg, Germany
  3. University of Lübeck, Lübeck, Germany
  4. Nanyang Technological University, Singapore
  5. The University of Tokyo, Tokyo, Japan

Infections with flaviviruses, such as dengue, West Nile or the recently emerged Zika virus are serious global health threats. Currently, no effective treatments are known. Cross-reactivities of antibodies among serotypes and flaviviruses can enhance symptoms and challenge vaccination campaigns. Our research targets flavivirus proteases as the Achilles heel of viral replication (ref. 1). Three unrelated strategies how peptides with noncanonical modifications can selectively target these viral enzymes are presented.

In order to create covalent interactions with the active protease site, we developed small boronic acid-based peptides that not only showed strong pan-flaviviral protease inhibition, but also served as valuable tools to structurally validate this important class of drug targets. The key feature of these inhibitors is a boronate moiety, which covalently binds to the highly conserved and catalytically active serine residue 135. Structural analogues without a boronic acid bind less tightly by three orders of magnitude (ref. 2).

In an orthogonal strategy capitalising on an in vitro mRNA display screening of a genetically reprogrammed library, we de novo discovered macrocyclic peptides with allosteric inhibition modes. Remarkably, not all compounds with nanomolar protease affinity displayed inhibition of the protease activity, suggesting applications in selective probing of flavivirus proteases without perturbing their activities (ref. 3).

Finally, we developed a method to spontaneously cyclise peptides under biocompatible conditions, using an N-terminal cysteine residue and the unnatural amino acid 3-(2-cyano-4-pyridyl)alanine. The reaction is orthogonal to all proteinogenic amino acids, including cysteine residues that are not at the N-terminus. Simple macrocyclization of the substrate sequence of flavivirus proteases generated high-affinity active-site inhibitors with remarkable proteolytic stability. In contrast to linear peptides, the cyclized substrate is cleaved only very slowly and the resulting linear peptide remains bound (ref. 4).

  1. Nitsche, C., Proteases from dengue, West Nile and Zika viruses as drug targets. Biophys. Rev. 2019, 11 (2), 157-165.
  2. Nitsche, C.; Zhang, L.; Weigel, L. F.; 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.
  3. 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.
  4. 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, in revision.