Teshome Aboye

Cyclotides, a Novel Ultrastable Polypeptide Scaffold for Drug Discovery

Cyclotides are a unique and growing family of backbone cyclized peptides that also contain a cystine knot motif built from six conserved cysteine residues. This unique circular backbone topology and knotted arrangement of three disulfide bonds makes them exceptionally stable to thermal, chemical, and enzymatic degradation compared to other peptides of similar size. Aside from the conserved residues forming the cystine knot, cyclotides have been shown to have high variability in their sequences. Consisting of over 160 known members, cyclotides have many biological activities, ranging from anti-HIV, antimicrobial, hemolytic, and uterotonic capabilities; additionally, some cyclotides have been shown to have cell penetrating properties. Originally discovered and isolated from plants, cyclotides can also be produced synthetically and recombinantly. The high sequence variability, stability, and cell penetrating properties of cyclotides make them potential scaffolds to be used to graft known active peptides or engineer peptide-based drug design. The present review reports recent findings in the biological diversity and therapeutic potential of natural and engineered cyclotides.

Design of a novel cyclotide-based CXCR4 antagonist with anti-human immunodeficiency virus (HIV)-1 activity

Herein, we report for the first time the design and synthesis of a novel cyclotide able to efficiently inhibit HIV-1 viral replication by selectively targeting cytokine receptor CXCR4. This was accomplished by grafting a series of topologically modified CVX15 based peptides onto the loop 6 of cyclotide MCoTI-I. The most active compound produced in this study was a potent CXCR4 antagonist (EC50≈20 nM) and an efficient HIV-1 cell-entry blocker (EC50≈2 nM). This cyclotide also showed high stability in human serum, thereby providing a promising lead compound for the design of a novel type of peptide-based anticancer and anti-HIV-1 therapeutics.

Biological Synthesis of Circular Polypeptides

Here, we review the use of different biochemical approaches for biological synthesis of circular or backbone-cyclized proteins and peptides. These methods allow the production of circular polypeptides either in vitro or in vivo using standard recombinant DNA expression techniques. Protein circularization can significantly impact protein engineering and research in protein folding. Basic polymer theory predicts that circularization should lead to a net thermodynamic stabilization of a folded protein by reducing the entropy associated with the unfolded state. Protein cyclization also provides a valuable tool for exploring the effects of topology on protein folding kinetics. Furthermore, the biological production of cyclic polypeptides makes possible the production of cyclic polypeptide libraries. The generation of such libraries, which was previously restricted to the domain of synthetic chemists, now offers biologists access to highly diverse and stable molecular libraries for probing protein structure and function.

Efficient one-pot cyclization/folding of Rhesus θ-defensin-1 (RTD-1).

We report an efficient approach for the chemical synthesis of Rhesus θ-defensin-1 (RTD-1) using Fmoc-based solid-phase peptide synthesis in combination with an intramolecular version of native chemical ligation. The corresponding linear thioester precursor was cyclized and folded in a one-pot reaction using reduced glutathione. The reaction was extremely efficiently yielding natively folded RTD-1 with minimal or no purification at all. This approach is fully compatible with the high throughput production of chemical libraries using this peptide scaffold.

Design of a MCoTI-Based Cyclotide with Angiotensin (1-7)-Like Activity

We report for the first time the design and synthesis of a novel cyclotide able to activate the unique receptor of angiotensin (1-7) (AT1-7), the MAS1 receptor. This was accomplished by grafting an AT1-7 peptide analog onto loop 6 of cyclotide MCoTI-I using isopeptide bonds to preserve the α-amino and C-terminal carboxylate groups of AT1-7, which are required for activity. The resulting cyclotide construct was able to adopt a cyclotide-like conformation and showed similar activity to that of AT1-7. This cyclotide also showed high stability in human serum thereby providing a promising lead compound for the design of a novel type of peptide-based in the treatment of cancer and myocardial infarction.

Rapid Parallel Synthesis of Bioactive Folded Cyclotides by Using a Tea‐Bag Approach

We report here the first rapid parallel production of bioactive folded cyclotides by using Fmoc‐based solid‐phase peptide synthesis in combination with a “tea‐bag” approach. Using this approach, we efficiently synthesized 15 analogues of the CXCR4 antagonist cyclotide MCo‐CVX‐5c. Cyclotides were synthesized in a single‐pot, cyclization/folding reaction in the presence of reduced glutathione. Natively folded cyclotides were quickly purified from the cyclization/folding crude mixture by activated thiol Sepharose‐based chromatography. The different folded cyclotide analogues were then tested for their ability to inhibit the CXCR4 receptor in a cell‐based assay. The results indicated that this approach can be used for the efficient chemical synthesis of libraries of cyclotides with improved biological properties that can be easily interfaced with solution or cell‐based assays for rapid screening.

Efficient recombinant expression of SFTI‐1 in bacterial cells using intein‐mediated protein trans‐splicing

We report for the first time the recombinant expression of bioactive wild‐type sunflower trypsin inhibitor 1 (SFTI‐1) inside E. coli cells by making use of intracellular protein trans‐splicing in combination with a high efficient split‐intein. SFTI‐1 is a small backbone‐cyclized polypeptide with a single disulfide bridge and potent trypsin inhibitory activity. Recombinantly produced SFTI‐1 was fully characterized by NMR and was observed to actively inhibit trypsin. The in‐cell expression of SFTI‐1 was very efficient reaching intracellular concentration ≈ 40 µM. This study clearly demonstrates the possibility of generating genetically encoded SFTI‐based peptide libraries in live E. coli cells, and is a critical first step for developing in‐cell screening and directed evolution technologies using the cyclic peptide SFTI‐1 as a molecular scaffold.

In vivo Evaluation of an Engineered Cyclotide as Specific CXCR4 Imaging Reagent

The CXCR4 chemokine receptor plays a key regulatory role in many biological functions, including embryonic development and controlling leukocyte functions during inflammation and immunity. CXCR4 has been also associated with multiple types of cancers where its overexpression/activation promotes metastasis, angiogenesis, and tumor growth and/or survival. Furthermore, CXCR4 is involved in HIV replication, as it is a co-receptor for viral entry into host cells. Altogether, these features make CXCR4 a very attractive target for the development of imaging and therapeutic agents. Here, the in vivo evaluation of the MCoTI-based cyclotide, MCo-CVX-5c, for the development of imaging agents that target CXCR4 is reported. Cyclotide MCo-CVX-5c is a potent CXCR4 antagonist with a remarkable in vivo resistance to biological degradation in serum. A [64 Cu]-DOTA-labeled version of this cyclotide demonstrated high and significant uptake in U87-stb-CXCR4 tumors compared to the control U87 tumors. Furthermore, protracted imaging studies demonstrated radiotracer retention in the U87-stb-CXCR4 tumor at 24 h post injection. Uptake in U87-stb-CXCR4 tumors could be blocked by unlabeled MCo-CVX-5c, showing high in vivo specificity. These results demonstrate the in vivo specificity and retention of a bioactive molecularly targeted cyclotide and highlight the potential of bioactive cyclotides for the development of new imaging agents that target CXCR4.