Production of cyclic peptides and proteins in vivo

2019

A number of cyclic peptides have emerged as valuable pharmaceutical templates due to their resistance to chemical or enzymatic hydrolysis and high selectivity. In 2001, the celogentin family of bicyclic peptides was isolated from the seeds of Celosia argentea. The celogentins have been shown to be potent anti-mitotic agents that inhibit tubulin polymerization, with celogentin C exhibiting four-fold higher activity than the clinically used anti-cancer drug vinblastine. The celogentins have two unusual side-chain to side-chain cross-links; one between the β-position of Leu2 and the indole C-6 of Trp5 and the other between the indole C-2 of Trp5 and the imidazole of the histidine side chain. These unusual cross-links make the chemical synthesis of the celogentins very tedious, lengthy, and low yielding. We have developed new protocols for the formation of the CC and C-N cross-links on solid phase to generate monocyclic celogentin precursors. However, attempts to combine these approaches to generate the celogentin bicyclic have been unsuccessful. We have also designed and synthesized simplified celogentin mimetics, including analogues with improved biological activity. (i.e. increased inhibition of tubulin polymerization camper to vinblastine) Cyclic peptides can be formed by a variety of methods such as head to tail cyclization, side-chain to side-chain cyclization, and side-chain to head/tail cyclization. The most common route to cyclic peptides is head-to-tail cyclization. Chemical synthesis of head-to-tail cyclic peptides is challenging when using standard peptide coupling methods due to slow reactions and the propensity for side reactions such as epimerization and cyclodimerization. Efforts to overcome the intrinsic limitations of head-to-tail cyclization include metal ion templating, the use of 'capture' auxiliaries and ring expansion/contraction approaches. We have developed a new head to tail cyclization method that exploits the chemoselective reaction of peptide thioamides with Ag(I). In the presence of Ag(I) a peptide possessing an N-terminal thioamide IX

Angewandte Chemie, 2014

Heterocycle-containing cyclic peptides are promising scaffolds for the pharmaceutical industry but their chemical synthesis is very challenging. A new universal method has been devised to prepare these compounds by using a set of engineered marine-derived enzymes and substrates obtained from a family of ribosomally produced and post-translationally modified peptides called the cyanobactins. The substrate precursor peptide is engineered to have a non-native protease cleavage site that can be rapidly cleaved. The other enzymes used are heterocyclases that convert Cys or Cys/Ser/Thr into their corresponding azolines. A macrocycle is formed using a macrocyclase enzyme, followed by oxidation of the azolines to azoles with a specific oxidase. The work is exemplified by the production of 17 macrocycles containing 6-9 residues representing 11 out of the 20 canonical amino acids.

ChemBioChem, 2007

The cyclotide MCoTI-II is a powerful trypsin inhibitor recently isolated from the seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family. We report for the first time the in vivo biosynthesis of natively-folded MCoTI-II inside live E. coli cells. Our biomimetic approach involves the intracellular backbone cyclization of a linear cylotide-intein fusion precursor mediated by a modified protein splicing domain. The cyclized peptide then spontaneously folds into its native conformation. The use of genetically engineered E. coli cells containing mutations in the glutathione and thioredoxin reductase genes considerably improves the production of folded MCoTI-II in vivo. Biolchemical and structural characterization of the recombinant MCoTI-II confirmed its identity. Biosynthetic access to correctly-folded cyclotides allows the possibility of generating cell-based combinatorial libraries that can be screened inside living cells for their ability to modulate or inhibit cellular processes.

Organic Letters, 2013

Cyclic peptide architectures can be easily synthesized from cysteine-containing peptides with appending maleimides, free or protected, through an intramolecular Michael-type reaction. After peptide assembly, the peptide can cyclize either during the trifluoroacetic acid treatment, if the maleimide is not protected, or upon deprotection of the maleimide. The combination of free and protected maleimide moieties and two orthogonally protected cysteines gives access to structurally different bicyclic peptides with isolated or fused cycles. Competitividad (grants CTQ2007-68014-C02-01 and CTQ2010-21567-C02-01, and the project RNAREG, grant CSD2009-00080, funded under the programme CONSOLIDER INGENIO 2010), and the Generalitat de Catalunya (2009SGR-208). X. E. was a recipient fellow of the MINECO. Supporting Information Available. Experimental procedures, compound characterization data and spectra, and HPLC profiles. This material is available free of charge via the Internet at http://pubc.acs.org.

A modular synthesis of some cyclic peptides related to a class of histone deacetylase inhibitors is reported. The synthesis utilizes a late-stage functionalization of a cyclic tetrapeptide scaffold to link the pendant alkyl chain through cross metathesis. It is observed that while the cross-metathesis reaction of a terminal olefin having a β-substituent is marginally successful with the scaffold, the corresponding reaction with an α,β-unsaturated ketone/ester is more promising. The utility of the protocol is demonstrated through the preparation of three cyclic tetrapeptides structurally related to the novel histone deacetylase inhibitors FR-225497 and trapoxin B.

Amino Acids, 2010

We present the in-vivo biosynthesis of wild-type sunflower trypsin inhibitor 1 (SFTI-1) inside E. coli cells using an intramolecular native chemical ligation in combination with a modified protein splicing unit. SFTI-1 is a small backbone cyclized polypeptide with a single disulfide bridge. A small library containing multiple Ala mutants was also biosynthesized and its activity was assayed using a trypsin-binding assay. This study clearly demonstrates the exciting possibility of generating large cyclic peptide libraries in live E. coli cells, and is a critical first step for developing in-vivo screening and directed evolution technologies using the cyclic peptide SFTI-1 as a molecular scaffold.

Biopolymers, 2013

Here we review the different biochemical approaches available for the expression of backbone-cyclized polypeptides, including peptides and proteins. These methods allow for the production of circular polypeptides either in vitro or in vivo using standard recombinant DNA expression techniques. Polypeptide circularization provides a valuable tool to study the effects of topology on protein stability and folding kinetics. Furthermore, having biosynthetic access to backbone-cyclized polypeptides makes the production of genetically encoded libraries of cyclic polypeptides possible. The production of such libraries, which was previously restricted to the domain of synthetic chemistry, now offers biologists access to highly diverse and stable molecular libraries that can be screened using high-throughput methods for the rapid selection of novel cyclic polypeptide sequences with new biological activities.

Angew. Chem. Int. Ed., 2018

Increased tolerance of enzymes towards thermal and chemical stress is required for many applications and can be achieved by macrocyclization of the enzyme resulting in the stabilizing of its tertiary structure. Thus far, macrocyclization approaches utilize a very limited structural diversity, which complicates the design process. Herein, we report an approach that enables cyclization through the installation of modular crosslinks into native proteins composed entirely of proteinogenic amino acids. Our stabilization procedure involves the introduction of three surface-exposed cysteine residues, which are reacted with a triselectrophile, resulting in the in situ cyclization of the protein (INCYPRO). A bicyclic version of sortase A was designed that exhibits increased tolerance towards thermal as well as chemical denaturation, and proved to be efficient in protein labeling under denaturing conditions. In addition, we applied INCYPRO to the KIX domain, resulting in up to 24°C increased thermal stability.

The Journal of Organic Chemistry, 2001

Cyclic peptides are appealing targets in the drug-discovery process. Unfortunately, there currently exist no robust solid-phase strategies that allow the synthesis of large arrays of discrete cyclic peptides. Existing strategies are complicated, when synthesizing large libraries, by the extensive workup that is required to extract the cyclic product from the deprotection/cleavage mixture. To overcome this, we have developed a new safety-catch linker. The safety-catch concept described here involves the use of a protected catechol derivative in which one of the hydroxyls is masked with a benzyl group during peptide synthesis, thus making the linker deactivated to aminolysis. This masked derivative of the linker allows BOC solid-phase peptide assembly of the linear precursor. Prior to cyclization, the linker is activated and the linear peptide deprotected using conditions commonly employed (TFMSA), resulting in deprotected peptide attached to the activated form of the linker. Scavengers and deprotection adducts are removed by simple washing and filtration. Upon neutralization of the N-terminal amine, cyclization with concomitant cleavage from the resin yields the cyclic peptide in DMF solution. Workup is simple solvent removal. To exemplify this strategy, several cyclic peptides were synthesized targeted toward the somatostatin and integrin receptors. From this initial study and to show the strength of this method, we were able to synthesize a cyclic-peptide library containing over 400 members. This linker technology provides a new solidphase avenue to access large arrays of cyclic peptides.

Chemical Communications, 2009

Installation of reversible, metathesis-active tethers into linear peptide sequences can be used to promote formation of cyclic peptide amides and esters.