Sequencing Of Cyclic Peptides Research Articles

Leveraging High-Resolution Ion Mobility-Mass Spectrometry for Cyclic Peptide Soft Spot Identification.

Cyclic peptides are an important class of molecules that gained significant attention in the field of drug discovery due to their unique pharmacological characteristics and enhanced proteolytic stability. Yet, gastrointestinal degradation remains a major hurdle in the discovery of orally bioavailable cyclic peptides. Soft spot identification (SSID) of the regions in the cyclic peptide sequence susceptible to amide hydrolysis by proteases is used in the discovery stage to guide medicinal chemistry design. SSID can be an arduous task, traditionally performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), often resulting in complex and time-consuming manual analysis, particularly when isomeric linear peptide metabolites chromatographically coelute. Here, we present an alternative orthogonal approach that entails a high-resolution ion mobility (HRIM) system based on Structures for Lossless Ion Manipulation (SLIM) technology interfaced with quadrupole time-of-flight (QTOF) mass spectrometry to address some of the challenges associated with SSID. Two strategies were used to resolve linear isomeric peptide metabolites: labeled and label-free, both utilizing the HRIM platform. The label-free strategy leverages negative polarity to ionize the isomers which achieves better separation of the gas phase ions in the ion mobility (IM) dimension as compared to positive polarity, which is a more conventional approach when studying proteins and peptides. The second approach uses an isotope-labeled dimethyl tag on the terminal amine group, acting as a "shift reagent" to influence the mobility of isomers in the positive mode. This method resulted in baseline separation for the isomers of interest and produced unique product ions in the fragmentation spectra for unambiguous soft spot identification. Both label-free and labeled strategies demonstrated the ability to solve the challenges associated with SSID for cyclic peptides.

Tetrazine cyclized peptides for one-bead-one-compound library: Synthesis and sequencing.

While most FDA-approved peptide drugs are cyclic, robust cyclization chemistry of peptides and the deconvolution of the cyclic peptide sequences using tandem mass spectrometry render cyclic peptide drug discovery difficult. In this chapter, the protocol for the successful synthesis of tetrazine-linked cyclic peptide library in solid phase, which shows both robust cyclization and easy sequence deconvolution, is described. The protocol for the linearization and cleavage of cyclic peptides from the solid phase by simple UV light irradiation, followed by accurate sequencing using tandem mass spectrometry, is described. We describe the troubleshooting for this dithiol bis-arylation reaction and for the successful cleavage of the aryl cyclic peptide into linear form. This method for efficient solid-phase macrocyclization can be used for the rapid production of loop-based peptides and screening for inhibition of protein-protein interactions, by using the covalent inverse electron-demand Diels Alder reaction to supplement the non-covalent interaction between a protein and its peptide binder, isolating highly selective peptides in the process.

Enhanced Neuron Growth and Electrical Activity by a Supramolecular Netrin-1 Mimetic Nanofiber.

Neurotrophic factors are essential not only for guiding the organization of the developing nervous system but also for supporting the survival and growth of neurons after traumatic injury. In the central nervous system (CNS), inhibitory factors and the formation of a glial scar after injury hinder the functional recovery of neurons, requiring exogenous therapies to promote regeneration. Netrin-1, a neurotrophic factor, can initiate axon guidance, outgrowth, and branching, as well as synaptogenesis, through activation of deleted in colorectal cancer (DCC) receptors. We report here the development of a nanofiber-shaped supramolecular mimetic of netrin-1 with monomers that incorporate a cyclic peptide sequence as the bioactive component. The mimetic structure was found to activate the DCC receptor in primary cortical neurons using low molar ratios of the bioactive comonomer. The supramolecular nanofibers enhanced neurite outgrowth and upregulated maturation as well as pre- and postsynaptic markers over time, resulting in differences in electrical activity similar to neurons treated with the recombinant netrin-1 protein. The results suggest the possibility of using the supramolecular structure as a therapeutic to promote regenerative bioactivity in CNS injuries.

Design of Cyclic Peptides Targeting Protein-Protein Interactions Using AlphaFold.

More than 930,000 protein-protein interactions (PPIs) have been identified in recent years, but their physicochemical properties differ from conventional drug targets, complicating the use of conventional small molecules as modalities. Cyclic peptides are a promising modality for targeting PPIs, but it is difficult to predict the structure of a target protein-cyclic peptide complex or to design a cyclic peptide sequence that binds to the target protein using computational methods. Recently, AlphaFold with a cyclic offset has enabled predicting the structure of cyclic peptides, thereby enabling de novo cyclic peptide designs. We developed a cyclic peptide complex offset to enable the structural prediction of target proteins and cyclic peptide complexes and found AlphaFold2 with a cyclic peptide complex offset can predict structures with high accuracy. We also applied the cyclic peptide complex offset to the binder hallucination protocol of AfDesign, a de novo protein design method using AlphaFold, and we could design a high predicted local-distance difference test and lower separated binding energy per unit interface area than the native MDM2/p53 structure. Furthermore, the method was applied to 12 other protein-peptide complexes and one protein-protein complex. Our approach shows that it is possible to design putative cyclic peptide sequences targeting PPI.

Facile de Novo Sequencing of Tetrazine-Cyclized Peptides through UV-Induced Ring-Opening and Cleavage from the Solid Phase.

While most FDA-approved peptide drugs are cyclic, the robust cyclization chemistry of peptides and the deconvolution of cyclic peptide sequences by using tandem mass spectrometry render cyclic peptide drug discovery difficult. Here we present the successful design of cyclic peptides on solid phase that addresses both of these problems. We demonstrate that this peptide cyclization method using dichloro-s-tetrazine on solid phase allows successful cyclization of a panel of random peptide sequences with various charges and hydrophobicities. The cyclic peptides can be linearized and cleaved from the solid phase by simple UV light irradiation, and we demonstrate that accurate sequence information can be obtained for the UV-cleaved linearized peptides by using tandem mass spectrometry. The tetrazine linker used in the cyclic peptides can further be explored for inverse electron-demand Diels-Alder (IEDDA) reactions for screening or bioconjugation applications in the future.

The flax genome reveals orbitide diversity

BackgroundRibosomally-synthesized cyclic peptides are widely found in plants and exhibit useful bioactivities for humans. The identification of cyclic peptide sequences and their precursor proteins is facilitated by the growing number of sequenced genomes. While previous research largely focused on the chemical diversity of these peptides across various species, there is little attention to a broader range of potential peptides that are not chemically identified.ResultsA pioneering study was initiated to explore the genetic diversity of linusorbs, a group of cyclic peptides uniquely occurring in cultivated flax (Linum usitatissimum). Phylogenetic analysis clustered the 5 known linusorb precursor proteins into two clades and one singleton. Preliminary tBLASTn search of the published flax genome using the whole protein sequence as query could only retrieve its homologues within the same clade. This limitation was overcome using a profile-based mining strategy. After genome reannotation, a hidden Markov Model (HMM)-based approach identified 58 repeats homologous to the linusorb-embedded repeats in 8 novel proteins, implying that they share common ancestry with the linusorb-embedded repeats. Subsequently, we developed a customized profile composed of a random linusorb-like domain (LLD) flanked by 5 conserved sites and used it for string search of the proteome, which extracted 281 LLD-containing repeats (LLDRs) in 25 proteins. Comparative analysis of different repeat categories suggested that the 5 conserved flanking sites among the non-homologous repeats have undergone convergent evolution driven by functional selection.ConclusionsThe profile-based mining approach is suitable for analyzing repetitive sequences. The 25 LLDR proteins identified herein represent the potential diversity of cyclic peptides within the flax genome and lay a foundation for further studies on the functions and evolution of these protein tandem repeats.

Continuous Flow Bioconjugations of NIR-AZA Fluorophores via Strained Alkyne Cycloadditions with Intra-Chip Fluorogenic Monitoring.

The importance of bioconjugation reactions continues to grow for cell specific targeting and dual therapeutic plus diagnostic medical applications. This necessitates the development of new bioconjugation chemistries, in‐flow synthetic and analytical methods. With this goal, continuous flow bioconjugations were readily achieved with short residence times for strained alkyne substituted carbohydrate and therapeutic peptide biomolecules in reaction with azide and tetrazine substituted fluorophores. The strained alkyne substrates included substituted 2‐amino‐2‐deoxy‐α‐D‐glucopyranose, and the linear and cyclic peptide sequences QIRQQPRDPPTETLELEVSPDPAS‐OH and c(RGDfK) respectively. The catalyst and reagent‐free inverse electron demand tetrazine cycloadditions proved more favourable than the azide 1,3‐dipolar cycloadditions. Reaction completion was achieved with residence times of 5 min at 40 °C for tetrazine versus 10 min at 80 °C for azide cycloadditions. The use of a fluorogenic tetrazine fluorophore, in a glass channelled reactor chip, allowed for intra‐chip reaction monitoring by recording fluorescence intensities at various positions throughout the chip. As the Diels‐Alder reactions proceeded through the chip, the fluorescence intensity increased accordingly in real‐time. The application of continuous flow fluorogenic bioconjugations could offer an efficient translational access to theranostic agents.

Gold(I) Cationization Promotes Ring Opening in Lysine-Containing Cyclic Peptides.

A strategy to sequence lysine-containing cyclic peptides by MSn is presented. Doubly protonated cyclic peptides ions are transformed into gold (I) cationized peptide ions via cation switching ion/ion reaction. Gold(I) cationization facilitates the oxidation of neutral lysine residues in the gas phase, weakening the adjacent amide bond. Upon activation, facile cleavage N-terminal to the oxidized lysine residue provides a site-specific ring opening pathway that converts cyclic peptides into acyclic analogs. The ensuing ion contains a cyclic imine as the new N-terminus and an oxazolone, or structural equivalent, as the new C-terminus. Product ions are formed from subsequent fragmentation events of the linearized peptide ion. Such an approach simplifies MS/MS data interpretation as a series of fragment ions with common N- and C-termini are generated. Results are presented for two cyclic peptides, sunflower trypsin inhibitor and the model cyclic peptide, β-Loop. The power of this strategy lies in the ability to generate the oxidized peptide, which is easily identified via the loss of HAuNH3 from [M + Au]+. While some competitive processes are observed, the site of ring opening can be pinpointed to the lysine residue upon MS4 enabling the unambiguous sequencing of cyclic peptides.

Characterization of New Cyclic D,L-α-Alternate Amino Acid Peptides by Capillary Electrophoresis Coupled to Electrospray Ionization Mass Spectrometry.

The self-assembly of peptide nanotubes (PNTs) depends on the structure and chemistry of cyclic peptide (CP) monomers, impacting on their properties, which makes the choice of their monomers and their characterization a high challenge. For this purpose, we developed for the first time a capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) methodology and characterized a set of eight original CP sequences of 8, 10, and 12 D,L-α-alternate amino acids with a controlled internal diameter (from 7 to 13Å) and various properties (diameter, global surface charge, hydrophobicity). This new CE-ESI-MS methodology allows verifying the structure, the purity, as well as the stability (when stored during several months) of interesting potential precursors for PNTs that could be employed as nanoplatforms in diagnostics or pseudo sieving tools for separation purposes.

CycLS: Accurate, whole-library sequencing of cyclic peptides using tandem mass spectrometry

Cyclic peptides are of great interest as therapeutic compounds due to their potential for specificity and intracellular activity, but specific compounds can be difficult to identify from large libraries without resorting to molecular encoding techniques. Large libraries of cyclic peptides are often DNA-encoded or linearized before sequencing, but both of those deconvolution strategies constrain the chemistry, assays, and quantification methods which can be used. We developed an automated sequencing program, CycLS, to identify cyclic peptides contained within large synthetic libraries. CycLS facilitates quick and easy identification of all library-members via tandem mass spectrometry data without requiring any specific chemical moieties or modifications within the library. Validation of CycLS against a library of 400 cyclic hexapeptide peptoid hybrids (peptomers) of unique mass yielded a result of 95% accuracy when compared against a simulated library size of 234,256 compounds. CycLS was also evaluated by resynthesizing pure compounds from a separate 1800-member library of cyclic hexapeptides and hexapeptomers with high mass redundancy. Of 22 peptides resynthesized, 17 recapitulated the retention times and fragmentation patterns assigned to them from the whole-library bulk assay results. Implementing a database-matching approach, CycLS is fast and provides a robust method for sequencing cyclic peptides that is particularly applicable to the deconvolution of synthetic libraries.

Peptide ligands for targeting the extracellular domain of EGFR: Comparison between linear and cyclic peptides.

Colorectal cancer (CRC) is the third most common solid internal malignancy among cancers. Early detection of cancer is key to increasing the survival rate of colorectal cancer patients. Overexpression of the EGFR protein is associated with CRC. We have designed a series of peptides that are highly specific for the extracellular domain of EGFR, based on our earlier studies on linear peptides. The previously reported linear peptide LARLLT, known to bind to EGFR, was modified with the goals of increasing its stability and its specificity toward EGFR. Peptide modifications, including D-amino acid substitution, cyclization, and chain reversal, were investigated. In addition, to facilitate labeling of the peptide with a fluorescent dye, an additional lysine residue was introduced onto the linear (KLARLLT) and cyclic peptides cyclo(KLARLLT) (Cyclo.L1). The lysine residue was also converted into an azide group in both a linear and reversed cyclic peptide sequences cyclo(K(N3)larllt) (Cyclo.L1.1) to allow for subsequent "click" conjugation. The cyclic peptides showed enhanced binding to EGFR by SPR. NMR and molecular modeling studies suggest that the peptides acquire a β-turn structure in solution. In vitro stability studies in human serum show that the cyclic peptide is more stable than the linear peptide.

Cyclic Phosphopeptides to Rationalize the Role of Phosphoamino Acids in Uranyl Binding to Biological Targets.

The specific molecular interactions responsible for uranium toxicity are not yet understood. The uranyl binding sites in high-affinity target proteins have not been identified yet and the involvement of phosphoamino acids is still an important question. Short cyclic peptide sequences, with three glutamic acids and one phosphoamino acid, are used as simple models to mimic metal binding sites in phosphoproteins and to help understand the mechanisms involved in uranium toxicity. A combination of peptide design and synthesis, analytical chemistry, extended X-ray absorption fine structure (EXAFS) spectroscopy, and DFT calculations demonstrates the involvement of the phosphate group in the uranyl coordination sphere together with the three carboxylates of the glutamate moieties. The affinity constants measured with a reliable analytical competitive approach at physiological pH are significantly enhanced owing to the presence of the phosphorous moiety. These findings corroborate the importance of phosphoamino acids in uranyl binding in proteins and the relevance of considering phosphoproteins as potential uranyl targets in vivo.

High Affinity Recognition of a Selected Amino Acid Epitope within a Protein by Cucurbit[8]uril Complexation.

Supramolecular interactions between the host cucurbit[8]uril (CB[8]) and amino acids have been widely interrogated, but recognition of specific motifs within a protein domain have never been reported. A phage display approach was herein used to select motifs with the highest binding affinity for the heteroternary complex with methyl viologen and CB[8] (MV⋅CB[8]) within a vast pool of cyclic peptide sequences. From the selected motifs, an epitope consisting of three amino acid was extrapolated and incorporated into a solvent-exposed loop of a protein domain; the protein exhibited micromolar binding affinity for the MV⋅CB[8] complex, matching that of the cyclic peptide. By achieving selective CB[8]-mediated conjugation of a small molecule to a recombinant protein scaffold we pave the way to biomedical applications of this simple ternary system.

High Affinity Recognition of a Selected Amino Acid Epitope within a Protein by Cucurbit[8]uril Complexation

AbstractSupramolecular interactions between the host cucurbit[8]uril (CB[8]) and amino acids have been widely interrogated, but recognition of specific motifs within a protein domain have never been reported. A phage display approach was herein used to select motifs with the highest binding affinity for the heteroternary complex with methyl viologen and CB[8] (MV⋅CB[8]) within a vast pool of cyclic peptide sequences. From the selected motifs, an epitope consisting of three amino acid was extrapolated and incorporated into a solvent‐exposed loop of a protein domain; the protein exhibited micromolar binding affinity for the MV⋅CB[8] complex, matching that of the cyclic peptide. By achieving selective CB[8]‐mediated conjugation of a small molecule to a recombinant protein scaffold we pave the way to biomedical applications of this simple ternary system.

Design, synthesis, and characterization of new cyclic d,l-α-alternate amino acid peptides by capillary electrophoresis coupled to electrospray ionization mass spectrometry

The self-assembly of peptide nanotubes (PNTs) depends on the structure and chemistry of cyclic peptide (CP) monomers, having an impact on their properties, making the choice of their monomers and their characterization a great challenge. We synthesized for the first time a new set of eight original CP sequences of 8, 10, and 12 d,l-α-alternate amino acids with a controlled internal diameter from 7 to 13 Å. They present various properties (e.g., diameter, global surface charge, hydrophobicity) that can open the way to new applications. Their structure and purity were determined thanks to a capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE–ESI–MS) methodology developed for the first time for this purpose. The CPs were successfully separated in a basic hydro-organic background electrolyte (BGE, pH 8.0, H2O/EtOH 50:50, v/v) and analyzed in MS positive mode. The effect of CP structure on electrophoretic mobility was studied, and the mass spectra were deeply analyzed. This methodology allowed verifying their purity and the absence of linear peptide precursors as well as their stability when stored over several months. Therefore, we have developed a new CE–ESI–MS methodology for the structure and purity control of interesting potential precursors for PNTs that could be employed as nanoplatforms in diagnostics or as pseudo sieving tools for separative purposes.

4-tert-butylphenoxy substituted phthalocyanine with RGD motif as highly selective one-photon and two-photon imaging probe for mitochondria and cancer cell

An unsymmetrical phthalocyanine based one- and two-photon fluorescence imaging probe that substituted with 4-tert-butylphenoxy and RDGyK moieties was developed and characterized by UV-vis and high-resolution MALDI-TOF/MS. The conjugate is non-aggregated in [Formula: see text],[Formula: see text]-dimethylformamide, with relatively weak fluorescence emission ([Formula: see text] 0.023) and high singlet oxygen quantum yield ([Formula: see text] 0.55). Conjugation of the cyclic peptide sequence c(RGDyK) can enhance the cellular uptake towards the DU145 and PC3 cells. While the fluorescence is greatly enhanced in mitochondria, the conjugate is non-cytotoxicity either in dark or upon exposure to red-light with dose up to 12 J.cm[Formula: see text]. The results suggest that this conjugate is a promising multifunctional imaging probe for mitochondria and cancer.

Synthesis and In Vitro Photodynamic Activities of an Integrin‐Targeting cRGD‐Conjugated Zinc(II) Phthalocyanine

A 1,4-disubstituted zinc(II) phthalocyanine conjugated with a cyclic Arg-Gly-Asp-D-Phe-Lys (cRGDfK) moiety through a triazole linker was prepared and characterized by UV/Vis spectroscopy and high-resolution ESI-MS. The conjugate showed a relatively weak fluorescence emission in N,N-dimethylformamide (ΦF =0.08), but it was a very efficient singlet oxygen generator (ΦΔ=0.80) as a result of the di-α-substituted structure. Owing to the presence of the cyclic peptide sequence cRGDfK, which is a well-known αvβ3 -integrin antagonist, this conjugate exhibited significantly higher cellular uptake toward the αvβ3(+) U87-MG cells compared with the αvβ3(-) MCF-7 cells, as determined by flow cytometry and fluorescence microscopy. The photocytotoxicity of this compound against these two cell lines, however, was comparable owing to the similar efficiency of intracellular reactive oxygen species generation. Confocal microscopic studies also revealed that this conjugate localized preferentially in the lysosomes, but not in the nucleus, endoplasmic reticulum, and mitochondria of the U87-MG cells.

Practical Ring-Opening Strategy for the Sequence Determination of Cyclic Peptides from One-Bead-One-Compound Libraries

The use of cyclic peptides in one-bead-one-compound libraries is limited by difficulties in sequencing hit compounds. Lacking a free N-terminal amine, such peptides cannot be sequenced by the Edman degradation approach, and complex fragmentation patterns are obtained by tandem mass spectrometry. To overcome this problem, we designed an alternative approach introducing a methionine residue within the macrocycle and as a linker to allow simultaneous ring-opening and release from the resin upon treatment with cyanogen bromide. The methionine linker was inverted relative to the peptide chain to allow the synthesis of cyclic peptides anchored by a lysine side chain and to avoid the presence of two C-terminal homoserine lactones on the released linear peptides. After MALDI-TOF MS/MS analysis, the peptides released from a single bead were sequenced manually and with a de novo sequencing software. The strategy described herein is compatible with commonly used amino acids and allows sequencing of cyclic peptides in one-bead-one-compound libraries, thus reducing the need for encoding.

Sequencing of cyclic peptides by NMR and MS techniques demonstrated on stylissamides A–F

The Caribbean sponge Stylissa caribica is the source of a series of six proline-rich cyclic heptapeptides, the stylissamides A–F (1–6). Here, we review the elucidation of the structures of 1–6 by a combination of NMR and mass spectrometry (MS/MS and MSn experiments). A detailed description of the NMR analysis (including semi-selective 1H,13C-HMBC and NOESY experiments) for sequencing the peptides is provided for stylissamides A (1) and E (5).

Design of Cyclic Peptides Featuring Proline Predominantly in the cis Conformation under Physiological Conditions

Turns are secondary-structure elements that are omnipresent in natively folded polypeptide chains. A large variety of four-residue β-turns exist, which differ mainly in the backbone dihedral angle values of the two central residues i+1 and i+2. The βVI-type turns are of particular biological interest because the i+2 residue is always a proline in the cis conformation and might thus serve as target of peptidyl prolyl cis/trans isomerases (PPIases). We have designed cyclic hexapeptides containing two proline residues that predominantly adopt the cis conformation in aqueous solution. NMR data and MD calculations indicated that the cyclic peptide sequences c-(-DXaa-Ser-Pro-DXaa-Lys-Pro-) result in highly symmetric backbone structures when both prolines are in the cis conformation and the D-amino acids are either alanine or phenylalanine residues. Replacement of the serine residue either by phosphoserine or by tyrosine compromises this symmetry, but further increases the cis conformation content of both prolines. As a result, we obtained a cyclic hexapeptide that exists almost exclusively as the cis-Pro/cis-Pro conformer but shows no cis/trans interconversion even in the presence of the PPIase Pin1, apparently due to an energetically quite favorable but highly restricted conformational space.