tightening molecular knots

 

Tightening Molecular Knots

Figure 1. Acyclic inhibitor cystine knot oMCoTI-II which is often used as a molecular scaffold in our research
Figure 1. Acyclic inhibitor cystine knot oMCoTI-II which is often used as a molecular scaffold in our research

Natural polypeptides comprising multiple disulfide bonds with a mechanically interlocked topology are found in diverse organisms, among them arthropoda, mollusca, porifera, vertebrata, fungi, and plantae. These biomolecules usually possess a peptidic backbone of about 30 amino acid residues and can be divided into three major subclasses: inhibitor cystine knots (ICK), cyclic cystine knots (CCK) and growth factor cystine knots (GFCK). While ICK peptides are most often linear protease inhibitors with inhibition constants of a low nanomolar to picomolar range, CCK peptides are defined through a head-to-tail backbone cyclization motif. Being rather small, they demonstrate the properties of full-size proteins due to their unique architecture defined by three β-strands that are interconnected by three disulfide bonds, where CysI of the sequence is connected to CysIV, CysII to CysV, and CysIII to CysVI (Figure 1). The interlocked topology is formed by two disulfide bonds that together with the connecting loops form a ring through which the third disulfide bond is threaded. This results in a rigid structural core with exceptional thermal and biological stability. Cystine-knot peptides Knottins can be endowed with novel bioactivities by variation of surface-exposed loops and are therefore considered as promising peptidic scaffolds for the generation of tailor-made bioactive compounds for various diagnostic and therapeutic applications. Several miniproteins have already been applied as frameworks for the development of peptide-based pharmaceuticals, among them cyclotides, spider toxins, scyllatoxin and squash trypsin inhibitors.

 

In our group, we conduct chemical synthesis of these interesting molecular constructs, which includes assembly of their amide backbone using manual or microwave-assisted Fmoc-SPPS followed by oxidative folding towards a bioactive isomer. Depending on the ultimate goal, we introduce desired non-natural elements both in the backbone and in the side-chains. Although to date SPPS of large cysteine-rich peptides is still not a labor routine, increasing availability of resources, resins and microwave assistance allow for overcoming typical synthetic problems and obtaining peptides that comprise more than 30 amino acid residues in good yields and enantiomeric purity. From a synthetic point of view, the most challenging issues in the synthesis of cystine-rich miniproteins are associated with their backbone macrocyclization and folding. In our lab, we have developed several methods for the oxidative formation of disulfide connectivities and for the macrocyclization through the non-natural head-to-tail connection.

 

Related Publications

  • 1. M. Reinwarth, D. Nasu, H. Kolmar, O. Avrutina, Chemical synthesis of cystine knots – promising scaffolds for applications in drug design, Molecules, 2012, 17 (11), 12533-12552.
  • 2. M. Reinwarth, B. Glotzbach, M. Tomaszowski, S. Fabritz, O. Avrutina, H. Kolmar, Oxidative folding of peptides with cystine-knot architectures: kinetic studies and optimization of folding conditions, ChemBioChem, 2013, 14 (1), 137-146.
  • 3. O. Avrutina, H.-U. Schmoldt, D. Gabrijelcic-Geiger, A. Wentzel, H. Frauendorf, C. P. Sommerhoff, U. Diederichsen, H. Kolmar, Head-to-tail cyclized cystine-knot peptides by a combined recombinant and chemical route of synthesis, ChemBioChem, 2008, 9 (1), 33-37.
  • 4. O. Avrutina, H.-U. Schmoldt, H. Kolmar, U. Diederichsen, Fmoc-assisted synthesis of a 29-residue cystine-knot trypsin inhibitor containing a guaninyl amino acid at the P1 position, Eur. J. Org. Chem, 2004, 23, 4931-4935.
  • 5. O. Avrutina, H.-U. Schmoldt, D. Gabrijelcic-Geiger, D. Le Nguyen, C. P. Sommerhoff, U. Diederichsen, H. Kolmar, Trypsin inhibition by macrocyclic and open-chain variants of the squash inhibitor MCoTI II, Biol. Chem., 2005, 386 (12), 1301-1306.