Oral Presentation 8th Venoms to Drugs 2023

Chemical Synthesis of Venom Proteins. (#1)

Stephen Kent 1 2
  1. Department of Chemistry, University of Chicago, Chicago, USA
  2. Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago, Chicago, USA

Proteins are the dominant ‘natural product’ molecules of the 21st century. It is estimated that there are more than a million distinct protein toxins in venomous species worldwide (especially in Australia!), and many millions more predicted protein molecules are discovered throughout the biosphere as open reading frames by genomic and metagenomic DNA sequencing. Venom-derived cysteine-rich protein toxins typically contain multiple disulfide bonds, and their expression by recombinant DNA methods is challenging. Furthermore, venom protein toxins often contain one or more of a wide range of post-translational modifications to their molecular structure.

Chemical synthesis has been shown to be a useful method to prepare venom proteins, incorporate post-translational modifications, validate chemical structures, determine X-ray structures, study structure-activity relationships, and for precision labeling for biophysical and biological studies. Disulfide-rich venom proteins are most effectively synthesized by stepwise SPPS of high purity unprotected peptide segments followed by condensation using chemical ligation,[1, 2] to yield full length polypeptide chains that are folded to give the functional venom protein molecules.

I will illustrate and comment on chemical synthesis methods used for the preparation of venom proteins in studies of sea anemone SHK toxin (35 aa; 4 Cys/2 disulfides),[3] the Brazilian scorpion toxins Ts 1 (61 aa; 8 Cys/4 disulfides),[4] and Ts3 (64 aa; 8 Cys/4 disulfides),[5] together with examples chosen from the current literature.[6-8]

  1. Constructing proteins by dovetailing unprotected synthetic peptides: backbone engineered HIV protease. M. Schnölzer, S.B.H. Kent Science, 256, 221-225 (1992).
  2. Synthesis of proteins by native chemical ligation. Philip E. Dawson, Tom W. Muir, Ian Clark-Lewis, Stephen B.H. Kent, Science, 266, 776-779 (1994).
  3. Native chemical ligation at Asx-Cys, Glx-Cys: chemical synthesis and high resolution X-ray structure of ShK toxin by racemic crystallography. Bobo Dang, Tomoya Kubota, Kalyaneswar Mandal, Francisco Bezanilla, Stephen B.H. Kent, J. Am. Chem. Soc., 135, 11911-9 (2013).
  4. Total chemical synthesis of biologically active fluorescent dye-labeled Ts1 toxin. Bobo Dang, Tomoya Kubota, Ana M. Correa, Francisco Bezanilla, Stephen B. H. Kent, Angewandte Chem Int Ed, 53, 8970-4 (2014).
  5. Elucidation of the covalent and tertiary structures of biologically active Ts3 toxin. Bobo Dang, Tomoya Kubota, Kalyaneswar Mandal, Ana M. Correa, Francisco Bezanilla, Stephen B. H. Kent, Angewandte Chemie Int. Ed., 55, 8639-42 (2016).
  6. Cystine knot peptides with tuneable activity and mechanism. Choi Yi Li+, Fabian B. H. Rehm+, Kuok Yap, Christina N. Zdenek, Maxim D. Harding, Bryan G. Fry, Thomas Durek, David J. Craik, Simon J. de Veer. Angew. Chem. Int. Ed. 2022, 61, e202200951
  7. (Efficient thermodynamic folding of disulfide-containing proteins on a solid support). Wu, Y., Cui, Z., Huang, YH. et al. Towards a generic prototyping approach for therapeutically-relevant peptides and proteins in a cell-free translation system. Nat Commun 13, 260 (2022). https://doi.org/10.1038/s41467-021-27854-9
  8. Evaluation of peptide ligation strategies for the synthesis of the bivalent acid-sensing ion channel inhibitor Hi1a. Hue N. T. Tran, Elena Budusan, Natalie J. Saez, Alexander Norman, Isaac J. Tucker, Glenn F. King, Richard J. Payne, Lachlan D. Rash, Irina Vetter, Christina I. Schroeder . Org. Lett., 25, 4439−4444 (2023).