CyclicMPNN: Stable Cyclic Peptide Sequence Generation

Sunflower trypsin inhibitor-1 (SFTI-1) and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) are potent protease inhibitors comprising a cyclic backbone. Elucidation of structure-activity relationships for SFTI-1 and MCoTI-II was used to design inhibitors with enhanced inhibitory activity. An analog of MCoTI-II is one of the most potent inhibitors of matriptase. These results provide a solid basis for the design of selective peptide inhibitors of matriptase with therapeutic potential. The type II transmembrane serine protease matriptase is a key activator of multiple signaling pathways associated with cell proliferation and modification of the extracellular matrix. Deregulated matriptase activity correlates with a number of diseases, including cancer and hence highly selective matriptase inhibitors may have therapeutic potential. The plant-derived cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), is a promising drug scaffold with potent matriptase inhibitory activity. In the current study we have analyzed the structure-activity relationships of SFTI-1 and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II), a structurally divergent trypsin inhibitor from Momordica cochinchinensis that also contains a cyclic backbone. We show that MCoTI-II is a significantly more potent matriptase inhibitor than SFTI-1 and that all alanine mutants of both peptides, generated using positional scanning mutagenesis, have decreased trypsin affinity, whereas several mutations either maintain or result in enhanced matriptase inhibitory activity. These intriguing results were used to design one of the most potent matriptase inhibitors known to date with a 290 pm equilibrium dissociation constant, and provide the first indication on how to modulate affinity for matriptase over trypsin in cyclic peptides. This information might be useful for the design of more selective and therapeutically relevant inhibitors of matriptase.

Identification of an unknown substance without any information remains a daunting challenge despite advances in chemistry and mass spectrometry. However, an unknown cyclic peptide in a sample with very limited volume seized at a Pennsylvania racetrack has been successfully identified. The unknown sample was determined by accurate mass measurements to contain a small unknown peptide as the major component. Collision-induced dissociation (CID) of the unknown peptide revealed the presence of Lys (not Gln, by accurate mass), Phe, and Arg residues, and absence of any y-type product ion. The latter, together with the tryptic digestion results of the unusual deamidation and absence of any tryptic cleavage, suggests a cyclic structure for the peptide. Electron-transfer dissociation (ETD) of the unknown peptide indicated the presence of Gln (not Lys, by the unusual deamidation), Phe, and Arg residues and their connectivity. After all the results were pieced together, a cyclic tetrapeptide, cyclo[Arg-Lys-N(C(6)H(9))Gln-Phe], is proposed for the unknown peptide. Observations of different amino acid residues from CID and ETD experiments for the peptide were interpreted by a fragmentation pathway proposed, as was preferential CID loss of a Lys residue from the peptide. ETD was used for the first time in sequencing of a cyclic peptide; product ions resulting from ETD of the peptide identified were categorized into two types and named pseudo-b and pseudo-z ions that are important for sequencing of cyclic peptides. The ETD product ions were interpreted by fragmentation pathways proposed. Additionally, multi-stage CID mass spectrometry cannot provide complete sequence information for cyclic peptides containing adjacent Arg and Lys residues. The identified cyclic peptide has not been documented in the literature, its pharmacological effects are unknown, but it might be a "designer" drug with athletic performance-enhancing effects.

Enzyme-free biochemical production of seamlessly N-to-C cyclized peptides from natural or recombinant proteins.

β-amyloid oligomers are thought to be the most toxic species formed en route to fibril deposition in Alzheimer's disease. Transthyretin is a natural sequestering agent of β-amyloid oligomers: the binding site to β-amyloid has been traced to strands G/H of the inner β-sheet of transthyretin. A linear peptide, with the same primary sequence as the β-amyloid binding domain on transthyretin, was moderately effective at inhibiting β-amyloid fibril growth. Insertion of a β-turn template and cyclization greatly increased stability against proteolysis and improved efficacy as an amyloid inhibitor. However, the cyclic peptide still contained a significant amount of disorder. Using the Simple Cyclic Peptide Application within ROSETTA as an in silico predictor of cyclic peptide conformation and stability, we investigated putative structural enhancements, including stabilization by disulfide linkages and insertion of a second β-turn template. Several candidates were synthesized and tested for secondary structure and ability to inhibit β-amyloid aggregation. The results demonstrate that cyclization, β-sheet structure and conformational homogeneity are all preferable design features, whereas disulfide bond formation across the two β-strands is not preferable.

Paclitaxel and naringenin-loaded solid lipid nanoparticles surface modified with cyclic peptides with improved tumor targeting ability in glioblastoma multiforme

The potential of peptides as drug candidates is limited by their poor pharmacokinetic properties. Many peptides have a short half-life in vivo and a lack of oral availability. Inspired by the excellent pharmacokinetic profile of cyclosporine, a natural, multiply N-methylated cyclic peptide, we envisioned multiple N-methylation as a promising way to rationally improve key pharmacokinetic characteristics. In this Account, we summarize our efforts toward modulating the properties of peptides by multiple N-methylation. As a first step, we simplified the synthesis of N-methylated amino acids in solution, by employing very mild conditions that could be tolerated by the diverse protecting groups required when working with naturally occurring amino acids. We also report the rapid and inexpensive syntheses of N-methylated peptides on a solid support; this facilitated the N-methyl scanning of bioactive peptides. Because of a lack of information regarding the conformational behavior of multiply N-methylated peptides, a complete library of N-methylated cyclic alanine pentapeptides was synthesized. The library provided valuable insight into the conformational modulation of cyclic peptides by N-methylation. This information is extremely valuable for the design of bioactive peptides and spatial screening of cyclic N-methylated peptides. To demonstrate the applicability of N-methylation to highly active but poorly bioavailable peptides, we performed a full N-methyl scan of the cyclopeptidic somatostatin analog cyclo(-PFwKTF-), known as the Veber-Hirschmann peptide. We show here for the first time that the simple approach of multiple N-methylation can drastically improve the metabolic stability and intestinal permeability of peptides, for example, resulting in 10% oral bioavailability for a tri-N-methylated Veber-Hirschmann peptide analog. In addition, we also describe a designed approach to N-methylated peptide library synthesis, which can accelerate the screening of N-methylated bioactive peptides. Finally, we find that multiple N-methylation of a cyclic hexapeptide integrin antagonist of GPIIb-IIIa (alphaIIb beta3 integrin), cyclo(-GRGDfL-), increases the selectivity of this peptide toward different integrin subtypes. This result demonstrates the utility of multiple N-methylation in elucidating the bioactive conformation of peptides.

Studies have been conducted on the construction of a bioactive proteolysis-resistant peptide sequence of the CDF-11 protein followed by the synthesis of a plasmid encoding its expression in E.coli. The prediction of biological activity, resistance to proteolysis and bioavailability of the peptide sequence was carried out on the ADMET platform. The synthesis of plasmids of the peptide sequence of a new recombinant protein was carried out in the Russian biotechnology company “Eurogen” (Moscow) by cyclic assembly of oligonucleotides. The molecular weight distribution of the peptide sequence was evaluated by mass spectrometry. The peptide sequence was identified by the MALDI-TOF MS Ultraflex method. The peptide sequence of the GDF-11 LQIIYGKIPR protein was cyclized by introducing and crosslinking three cysteine residues and a new LQIIYGKIPR sequence was obtained. The predicted values of the bioavailability and clinical use of the new peptide indicate the effectiveness of its proposed modification and the possibility of oral administration. For the expression of a recombinant protein with increased stability to proteolysis and bioavailability containing a cyclic peptide sequence, a plasmid was obtained and synthesized, and the sequence of amino acids in it was determined. Further research will focus on the expression of the modified GDF 11 mut protein in E.coli.

Copper(I) is able to catalyze Huisgen 1,3-dipolar cycloaddition in a "click" fashion. This copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction presents excellent chemoselectivity and occurs over a wide-range of reaction conditions. It shows tolerance to variation in both pH and solvent polarity, thereby facilitating the ligation of peptides and proteins to produce peptidomimetics and synthetic proteins. In addition, the only product formed is a 1,4-disubstituted-1,2,3-triazole moiety, in many aspects resembling the natural peptide bond, including hydrogen-bonding capability, planarity, distance between the 1 and 4 substituents, and conformational restriction of the peptide backbone; thus the triazole-backbone-modified peptide, in which a triazole replaces the amide bond, may be anticipated to present a secondary structure similar to that of its natural counterpart. This Focus Review describes the scope and applications of copper(I)-catalyzed alkyne–azide cycloaddition in synthetic peptide/protein chemistry.

Structure of the YSPTSPS repeat containing two SPXX motifs in the CTD of RNA polymerase II: NMR studies of cyclic model peptides reveal that the SPTS turn is more stable than SPSY in water

This work describes the synthesis and activity of a novel backbone cyclic (BC) peptide library based on the sequence of the HIV-1 Rev arginine-rich motif (ARM). All the peptides in the library possess the same sequence but differ in their ring-moiety properties. The BC peptides were synthesized using simultaneous multiple-peptide synthesis and were fully assembled using bis(trichloromethyl)carbonate as a coupling agent. All the peptides in the library had inhibitory effects on the binding of Rev-GFP to importin beta in vitro. Studies performed with one of the BC Rev-ARM analogues, Rev-13, demonstrated that, like its parental linear peptide, it is karyophilic; i.e., it is able to mediate the nuclear import of conjugated bovine serum albumin (BSA) molecules. The cell penetrating properties of the BC peptides were assessed utilizing an ELISA-based system. This assay provides a quantitative evaluation of cell penetration. Most of the peptides from the library were able to penetrate intact Colo-205 cells to varying degrees. Furthermore, these BC peptides were able to carry BSA into intact Colo-205 cells. In addition to its cell penetrating and binding properties, the BC Rev-13 analogue inhibited Rev-induced gene expression in HeLa cells by 60-70% in the low micromolar range and exhibited no cell toxicity. The potential of BC peptides bearing ARM domains as lead compounds for the production of anti-HIV drugs is discussed.

The structural complexity of many natural products sets them apart from common synthetic drugs, allowing them to access a biological target space that lies beyond the enzyme active sites and receptors targeted by conventional small molecule drugs. Naturally occurring cyclic peptides, in particular, exhibit a wide variety of unusual and potent biological activities. Many of these compounds penetrate cells by passive diffusion and some, like the clinically important drug cyclosporine A, are orally bioavailable. These natural products tend to have molecular weights and polar group counts that put them outside the norm based on classic predictors of "drug-likeness". Because of their size and complexity, cyclic peptides occupy a chemical "middle space" in drug discovery that may provide useful scaffolds for modulating more challenging biological targets such as protein-protein interactions and allosteric binding sites. However, the relationship between structure and pharmacokinetic (PK) behavior, especially cell permeability and metabolic clearance, in cyclic peptides has not been studied systematically, and the generality of cyclic peptides as orally bioavailable scaffolds remains an open question. This review focuses on cyclic peptide natural products from a "structure-PK" perspective, outlining what we know and don't know about their properties in the hope of uncovering trends that might be useful in the design of novel "rule-breaking" molecules.

Backbone cyclization has a profound impact on the biological activity and thermal and proteolytic stability of proteins and peptides. Chemical methods for cyclization are not always feasible, especially for large peptides or proteins. Recombinant Staphylococcus aureus sortase A shows potential as a new tool for the cyclization of both proteins and peptides. In this review, the scope and background of the sortase-mediated cyclization are discussed. High efficiency, versatility, and easy access make sortase A a promising cyclization tool, both for recombinant and chemo-enzymatic production methods.

AEP-mediated backbone cyclization of cyclotides-A chemoenzymatic method for cyclotide production.

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14 amino acid cyclic peptide from sunflower seeds, which possesses exceptionally potent trypsin-inhibitory activity, and has promise as a stable peptide-based drug template. Within its compact structure, SFTI-1 combines a head-to-tail cyclized backbone and a disulfide bond. In this study, we synthesized a range of acyclic and disulfide-deficient analogs of SFTI-1 to investigate enzyme-assisted cyclization of the peptide backbone and proteolytic degradation that occurs as a result of incubation with trypsin. Electrospray and matrix-assisted laser desorption ionization mass spectrometry allowed the characterization of a range of novel degradation products and elucidation of the time-course for cyclization and/or proteolysis. Trypsin displayed the ability to resynthesize the scissile bond(s) and hence cyclize two of the linear permutants, whereas irreversible degradation was observed for another two permutants. An interesting ring contraction mediated by trypsin was observed, supporting a role for protease catalyzed splicing as a way of increasing the combinatorial diversity of cyclic peptides in nature. Disulfide-deficient mutants were degraded within minutes, emphasizing the critical role of the cysteine bridge in maintaining proteolytic stability of SFTI-1. Overall, the study provides additional support for the proposal that naturally occurring cyclic peptides like SFTI-1 are biosynthesized by proteolytic enzymes effectively catalyzing the reverse of their normal reaction to make, rather than break peptide bonds.

Matrix metallopeptidase 7 (MMP7) plays a crucial role in cancer metastasis and progression, making it an attractive target for therapeutic development. However, the development of selective MMP7 inhibitors is challenging due to the conservation of active sites across various matrix metalloproteinases (MMPs). Here, we have developed mirror‐image random nonstandard peptides integrated discovery (MI‐RaPID) technology to discover innate protease‐resistant macrocyclic peptides that specifically bind to and inhibit human MMP7. One identified macrocyclic peptide against D‐MMP7, termed D20, was synthesized in its mirror‐image form, D’20, consisting of 12 D‐amino acids, one cyclic β‐amino acid, and a thioether bond. Notably, it potently inhibited MMP7 with an IC50 value of 90 nM, and showed excellent selectivity over other MMPs with similar substrate specificity. Moreover, D’20 inhibited the migration of pancreatic cell line CFPAC‐1, but had no effect on the cell proliferation and viability. D’20 exhibited excellent stability in human serum, as well as in simulated gastric and intestinal fluids. This study highlights that MI‐RaPID technology can serve as a powerful tool to develop in vivo stable macrocyclic peptides for therapeutic applications.