Bicyclic RGD peptides enhance nerve growth in synthetic PEG-based Anisogels.

In total, 23 plant plant medicined containing oligopeptides were cited in Chinese Pharmacopoeia (1 part) of 2015 version including Rubia cordifolia, Linum usitatissimum, Aster tataricus, Psammosilene tunicoides, Pseudostellaria heterophylla, Stellaria dichotoma, Vaccaria segetalis, Dianthus superbus, Celosia argentea, Lycii Cortex, Citrus medica, C. aurantium, Panax ginseng, Parmx notoginseng, Schisandra chinensis, Sparganium stoloniferum, Euryale ferox, Ophiopogon japonicas, Pinellia ternate, Achyranthes bidentata, Physalis alkekengi, Polygonatum odoratum, and Leonuri Fructus. There were 187 oligopeptides in plant medicines above as reported. Oligopeptides consisted mainly of linear peptides and cyclic peptides. The linear peptides included dipeptides, tripeptides and pentapeptides, and cyclic peptides included cyclic, bicyclic and tricyclic peptides. The number of residues of single cyclic peptides ranged from two to twelve. Bicyclic peptides were isolated mainly from R. cordifolia and C. argentea. Modern pharmacological study showed that oligopeptides had many pharmacological effects, including antitumor, anticoagulant, antibacterial, immune suppression and so on.

Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-A165 (VEGF-A165) in endothelial cells. To define the role of NP-1 in the biological functions of VEGF, we developed a specific peptide antagonist of VEGF binding to NP-1 based on the NP-1 binding site located in the exon 7- and 8-encoded VEGF-A165 domain. The bicyclic peptide, EG3287, potently (K(i) 1.2 microM) and effectively (>95% inhibition at 100 microM) inhibited VEGF-A165 binding to porcine aortic endothelial cells expressing NP-1 (PAE/NP-1) and breast carcinoma cells expressing only NP-1 receptors for VEGF-A, but had no effect on binding to PAE/KDR or PAE/Flt-1. Molecular dynamics calculations, a nuclear magnetic resonance structure of EG3287, and determination of stability in media, indicated that it constitutes a stable subdomain very similar to the corresponding region of native VEGF-A165. The C terminus encoded by exon 8 and the three-dimensional structure were both critical for EG3287 inhibition of NP-1 binding, whereas modifications at the N terminus had little effect. Although EG3287 had no direct effect on VEGF-A165 binding to KDR receptors, it inhibited cross-linking of VEGF-A165 to KDR in human umbilical vein endothelial cells co-expressing NP-1, and inhibited stimulation of KDR and PLC-gamma tyrosine phosphorylation, activation of ERKs1/2 and prostanoid production. These findings characterize the first specific antagonist of VEGF-A165 binding to NP-1 and demonstrate that NP-1 is essential for optimum KDR activation and intracellular signaling. The results also identify a key role for the C-terminal exon 8 domain in VEGF-A165 binding to NP-1.

Macrocyclic peptides are promising scaffolds for drug discovery due to their structural rigidity and high target specificity. Here, we report a strategy for in vitro ribosomal translation of thioisoindole‐bridged bicyclic peptides. Central to this approach is a newly developed flexizyme substrate, Ac‐Ala(NtBA) Sc ‐CME, which features a semicarbazone‐masked 2‐nicotinoyl benzaldehyde sidechain. We show that this amino acid can be efficiently charged onto tRNA with flexizyme and incorporated into ribosomal peptides using a customized flexible in vitro translation (FIT) system. The semicarbazone group can be post‐translationally removed under mild conditions, triggering spontaneous intramolecular cyclization to cysteine and lysine sidechains in the same substrate to yield thioisoindole‐bridged bicyclic (TiB) peptides. This strategy was leveraged to synthesize structurally diverse bicyclic peptides with varying sequences and ring sizes. The method maintains the integrity of mRNA and is therefore compatible with mRNA display, which opens the possibility of constructing topologically defined bicyclic peptide libraries for therapeutic peptide discovery.

Southern King Crab (SKC) represents an important fishery resource that has the potential to be a natural source of chitosan (CS) production. In tissue engineering, CS is very useful to generate biomaterials. However, CS has a lack of signaling molecules that facilitate cell–substrate interaction. Therefore, RGD (arginine–glycine–aspartic acid) peptides corresponding to the main integrin recognition site in extracellular matrix proteins have been used to improve the CS surface. The aim of this study was to evaluate in vitro cell adhesion and proliferation of CS films synthesized from SKC shell wastes functionalized with RGD peptides. The FTIR spectrum of CS isolated from SKC shells (SKC-CS) was comparable to commercial CS. Thermal properties of films showed similar endothermic peaks at 53.4 and 53.0 °C in commercial CS and SKC-CS, respectively. The purification and molecular masses of the synthesized RGD peptides were confirmed using HPLC and ESI-MS mass spectrometry, respectively. Mouse embryonic fibroblast cells showed higher adhesion on SKC-CS (1% w/v) film when it was functionalized with linear RGD peptides. In contrast, a cyclic RGD peptide showed similar adhesion to control peptide (RDG), but the highest cell proliferation was after 48 h of culture. This study shows that functionalization of SKC-CS films with linear or cyclic RGD peptides are useful to improve effects on cell adhesion or cell proliferation. Furthermore, our work contributes to knowledge of a new source of CS to synthesize constructs for tissue engineering applications.

Cyclic peptides can bind to protein targets with high affinities and selectivities, which makes them an attractive modality for the development of research reagents and therapeutics. Additional properties, including low inherent toxicity, efficient chemical synthesis, and facile modification with labels or immobilization reagents, increase their attractiveness. Cyclic peptide ligands against a wide range of protein targets have been isolated from natural sources such as bacteria, fungi, plants, and animals. Many of them are currently used as research tools, and several have found application as therapeutics, such as the peptide hormones oxytocin and vasopressin and the antibiotics vancomycin and daptomycin, proving the utility of cyclic peptides in research and medicine. With the advent of phage display and other in vitro evolution techniques, it has become possible to generate cyclic peptide binders to diverse protein targets for which no natural peptides have been discovered. A highly robust and widely applied approach is based on the cyclization of peptides displayed on phage via a disulfide bridge. Disulfide-cyclized peptide ligands to more than a hundred different proteins have been reported in the literature. Technology advances achieved over the last three decades, including methods for generating larger phage display libraries, improved phage panning protocols, new cyclic peptide formats, and high-throughput sequencing, have enabled the generation of cyclic peptides with ever better binding affinities to more challenging targets. A relatively new cyclic peptide format developed using phage display involves bicyclic peptides. These molecules consist of two macrocyclic peptide rings cyclized through a chemical linker. Compared to monocyclic peptides of comparable molecular mass, bicyclic peptides are more constrained in their conformation. As a result, they can bind to their targets with a higher affinity and are more resistant to proteolytic degradation. Phage-encoded bicyclic peptides are generated by chemically cyclizing random peptide libraries on phage. Binders are identified by conventional phage panning and DNA sequencing. Next-generation sequencing and new sequence alignment tools have enabled the rapid identification of bicyclic peptides. Bicyclic peptide ligands were developed against a range of diverse target classes including enzymes, receptors, and cytokines. Most ligands bind with nanomolar affinities, with some reaching the picomolar range. To date, several bicyclic peptides have been positively evaluated in preclinical studies, and the first clinical tests are in sight. While bicyclic peptide phage display was developed with therapeutic applications in mind, these peptides are increasingly used as research tools for target evaluation or as basic research probes as well. Given the efficient development method, the ease of synthesis and handling, and the favorable binding and biophysical properties, bicyclic peptides are being developed against more and more targets, ever increasing their potential applications in research and medicine.

Bicyclic peptides are an attractive molecule format for the development of therapeutics and research tools. Our laboratory is specialized on the development of bicyclic peptide ligands by phage display. In brief, libraries of randompeptides each containing three cysteines are displayed on the surface of a phage and cyclized with a trivalent thiol-reactive chemical reagent. Ligands of a target of interest are isolated by affinity selection and their sequence identified by DNA sequencing. The first goal of my thesis was to test if more diverse bicyclic peptide libraries can be generated if linear peptides on phage are cyclized in parallel with multiple different chemical cyclization linkers. Towards this end, we cyclized peptide libraries of the format XCXnCXnCX (X = random amino acid, C = cysteine, n = 3 or 4) with three different chemicals and panned the library against the protease urokinase-type plasminogen activator (uPA). Interestingly, different peptide sequences were enriched when the phage peptide library was cyclized with the different chemical linkers, indicating that a larger diversity of bicyclic peptides is generated. Screening the larger library generated binders with a higher binding affinity. In the second project I carried out, I isolated bicyclic peptide ligands to a protein of therapeutic interest, b-catenin. b-catenin is the main translational co-activator of theWnt cell signaling pathway. Pathological upregulation of b-catenin is associated with the majority of colorectal cancer. Blocking b-catenin activity is thus an attractive therapeutic approach. However, so far the development of b-catenin inhibitors has been challenging due to the flat and featureless surface of the protein, lacking binding pockets for synthetic ligands. Screening a phage display library comprising >12 billion bicyclic peptides yielded binders for different protein epitopes. The binding site of four peptides was identified to correspond to that of ICAT (inhibitor of b-catenin and Tcf), which is a prime target site on b-catenin for therapeutic intervention and to which no synthetic ligands could be isolated so far. In the third project, we isolated and characterized bicyclic peptides binding to globular actin (G-actin). G-actin constitutes the fundamental building block of microfilaments (also named F-actin) in eukaryotic cells. Beyond this structural function, G-actin has a role in gene transcription and chromatin remodeling. While specific F-actin probes are available for in vitro applications, G-actin probes are lacking. To fulfil this demand, we screened a library of bicyclic peptides for G-actin binders and isolated ligands with dissociation constants in the low nanomolar range. The isolated peptides were found to bind to the same surface region as thymosin b4 and a range or marine toxins. The bicyclic peptide G-actin ligands allowed measurement of the binding affinity of natural G-actin ligands in fluorescence anisotropy competition assays.

Kappa opioid receptor (KOR) antagonists have potential therapeutic applications in the treatment of stress-induced relapse to substance abuse and mood disorders. The dynorphin A analog arodyn (Ac[Phe1,2,3,Arg4,D-Ala8]dynorphin A-(1-11)-NH2) exhibits potent and selective kappa opioid receptor antagonism. Multiple cyclizations in longer peptides, such as dynorphin and its analogs, can extend the conformational constraint to additional regions of the peptide beyond what is typically constrained by a single cyclization. Here, we report the design, synthesis, and pharmacological evaluation of a bicyclic arodyn analog with two constraints in the opioid peptide sequence. The peptide, designed based on structure-activity relationships of monocyclic arodyn analogs, was synthesized by solid-phase peptide synthesis and cyclized by sequential ring-closing metathesis (RCM) in the C- and N-terminal sequences. Molecular modeling studies suggest similar interactions of key aromatic and basic residues in the bicyclic peptide with KOR as found in the cryoEM structure of KOR-bound dynorphin, despite substantial differences in the backbone conformations of the two peptides. The bicyclic peptide's affinities at KOR and mu opioid receptors (MOR) were determined in radioligand binding assays, and its KOR antagonism was determined in the [35S]GTPγS assay in KOR-expressing cells. The bicyclic analog retains KOR affinity and selectivity (Ki = 26 nM, 97-fold selectivity over MOR) similar to arodyn and exhibits potent KOR antagonism in the dynorphin-stimulated [35S]GTPγS assay. This bicyclic peptide represents a promising advance in preparing cyclic opioid peptide ligands and opens avenues for the rational design of additional bicyclic opioid peptide analogs.

Macrocyclic peptides are promising scaffolds for drug discovery due to their structural rigidity and high target specificity. Here, we report a strategy for in vitro ribosomal translation of thioisoindole‐bridged bicyclic peptides. Central to this approach is a newly developed flexizyme substrate, Ac‐Ala(NtBA)Sc‐CME, which features a semicarbazone‐masked 2‐nicotinoyl benzaldehyde sidechain. We show that this amino acid can be efficiently charged onto tRNA with flexizyme and incorporated into ribosomal peptides using a customized flexible in vitro translation (FIT) system. The semicarbazone group can be post‐translationally removed under mild conditions, triggering spontaneous intramolecular cyclization to cysteine and lysine sidechains in the same substrate to yield thioisoindole‐bridged bicyclic (TiB) peptides. This strategy was leveraged to synthesize structurally diverse bicyclic peptides with varying sequences and ring sizes. The method maintains the integrity of mRNA and is therefore compatible with mRNA display, which opens the possibility of constructing topologically defined bicyclic peptide libraries for therapeutic peptide discovery.

The objective of this study was to evaluate the relationship between conformational flexibility and solution stability of a linear RGD peptide (Arg-Gly-Asp-Phe-OH; 1) and a cyclic RGD peptide (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH2; 2); as a function of pH. Previously, it was found that cyclic peptide 2 was 30-fold more stable than linear peptide 1. Therefore, this study was performed to explain the increase in chemical stability based on the preferred conformation of the peptides. Molecular dynamics simulations and energy minimizations were conducted to evaluate the backbone flexibility of both peptides under simulated pH conditions of 3, 7 and 10 in the presence of water. The reactive sites for degradation for both molecules were also followed during the simulations. The backbone of linear peptide 1 exhibited more flexibility than that of cyclic peptide 2, which was reflected in the rotation about the phi and psi dihedral angles. This was further supported by the low r.m.s. deviations of the backbone atoms for peptide 2 compared with those of peptide 1 that were observed among structures sampled during the molecular dynamics simulations. The presence of a salt bridge between the side chain groups of the Arg and Asp residues was also indicated for the cyclic peptide under simulated conditions of neutral pH. The increase in stability of the cyclic peptide 2 compared with the linear peptide 1, especially at neutral pH, is due to decreased structural flexibility imposed by the ring, as well as salt bridge formation between the side chains of the Arg and Asp residues in cyclic peptide 2. This rigidity would prevent the Asp side chain carboxylic acid from orienting itself in the appropriate position for attack on the peptide backbone.

Arg-Gly-Asp (RGD) peptides contain an aspartic acid residue that is highly susceptible to chemical degradation and leads to the loss of biological activity. Our hypothesis is that cyclization of RGD peptides via disulphide bond linkage can induce structural rigidity, thereby preventing degradation mediated by the aspartic acid residue. In this paper, we compared the solution stability of a linear peptide (Arg-Gly-Asp-Phe-OH; 1) and a cyclic peptide (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH2; 2) as a function of pH and buffer concentration. The decomposition of both peptides was studied in buffers ranging from pH 2-12 at 50 degrees C. Reversed-phase HPLC was used as the main tool in determining the degradation rates and pathways of both peptides. Fast atom bombardment mass spectrometry (FAB-MS), electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, liquid chromatography-mass spectrometry (LC-MS), and one- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) were used to characterize peptides 1 and 2 and their degradation products. In addition, co-elution with authentic samples was used to identify degradation products. Both peptides displayed pseudo-first-order kinetics at all pH values studied. The cyclic peptide 2 appeared to be 30-fold more stable than the linear peptide 1 at pH 7. The degradation mechanisms of linear (1) and cyclic (2) peptides primarily involved the aspartic acid residue. However, above pH 8 the stability of the cyclic peptide decreased dramatically due to disulphide bond degradation. Both peptides also exhibited a change in degradation mechanism upon an increase in pH. The increase in stability of cyclic peptide 2 compared to linear peptide 1, especially at neutral pH, may be due to decreased structural flexibility imposed by the ring. This rigidity would prevent the Asp side chain carboxylic acid from orientating itself in the appropriate position for attack on the peptide backbone.

A gene expression-based comparison of cell adhesion to extracellular matrix and RGD-terminated monolayers

We have previously reported that star shaped poly(ethylene oxide-stat-propylene oxide) macromers with 80% EO content and isocyanate functional groups at the distal ends [NCO-sP(EO-stat-PO)] can be used to generate coatings that are non-adhesive but easily functionalized for specific cell adhesion. In the present study, we investigated whether the NCO-sP(EO-stat-PO) surfaces maintain peptide configuration-specific cell-surface interactions or if differences between dissimilar binding molecules are concealed by the coating. To this end, we have covalently immobilized both linear-RGD peptides (gRGDsc) and cyclic-RGD (RGDfK) peptides in such coatings. Subsequently, SaOS-2 or human multipotent mesenchymal stromal cells (MSC) were seeded on these substrates. Cell adhesion, spreading and survival was observed for up to 30 days. The time span for cell adherence was not different on linear and cyclic RGD peptides, but was shorter in comparison to the unmodified glass surface. MSC proliferation on cyclic RGDfK modified coatings was 4 times higher than on films functionalized by linear gRGDsc sequences, underlining that the NCO-sP(EO-stat-PO) film preserves the configuration-specific biochemical peptide properties. Under basal conditions, MSC expressed osteogenic marker genes after 14 days on cyclic RGD peptides, but not on linear RGD peptides or the unmodified glass surfaces. Our results indicate specific effects of these adhesion peptides on MSC biology and show that this coating system is useful for selective testing of cellular interactions with adhesive ligands.

The aim of our study was to generate a biofunctionalized, three-dimensional (3D) biomaterial to enhance jaw periosteal cell (JPC) adhesion and differentiation into osteogenic tissue. Therefore, open-cell polylactic acid (OPLA) scaffolds were coated covalently with different RGD peptides (a conserved recognition sequence of the most ECM proteins--arginine-glycine-asparagine) and different coating variants. The linear and cyclic RGD peptides were either applied directly or indirectly via a poly-L-lysine (PLL) spacer. JPCs were analyzed on coated constructs in 2D and 3D cultures and showed enhanced rates for indirectly coated scaffolds using the PLL spacer. By gene expression, we detected significantly increased levels of osteogenic marker genes, such as alkaline phosphatase, RUNX2, and AMELY in JPCs seeded onto PLL/linear RGD constructs compared to the otherwise-coated constructs. An analysis of the JPC mineralization capacity revealed the highest amounts of calcium-phosphate precipitates in cells growing within the PLL/linear scaffolds. Additionally, the JPC adhesion behavior on OPLA scaffolds seems to be mediated by ITGB3, ITGB1, and ITGAV, as shown by blocking assays. We concluded that coating of OPLA constructs with linear RGD peptides via PLL represents a suitable approach for functionalizing the polymer surface and enhancing adhesion, proliferation, and mineralization of JPCs.

A convenient strategy for the on-resin synthesis of macrocyclic peptides (3- to 13-mers) via intramolecular halide substitution by a diamino acid is described. The method is compatible with standard Fmoc/tBu SPPS and affords a tail-to-side-chain macrocyclic peptide featuring an endocyclic secondary amine. This functional group is still reactive toward acylation, allowing for the continuation of the synthesis. An application to the synthesis of lipidated cyclic and bicyclic antimicrobial peptides is presented.

In this work, we develop a platform for multimerizing peptides/miniproteins using various polyvalent thioester cores derived from easy-to-synthesize N-hydroxysuccinimide esters. We employed native chemical ligation to attach multiple copies of peptide/miniprotein around a fixed multi-armed thioester core in a one-pot fashion, leading to the first-generation multimers. Further, using a reverse thioether ligation strategy, we synthesized second-generation branched homo/hetero multimers. The reactions proceed under an aqueous environment that closely mimics physiological conditions, forming multimeric products with yields ranging from 30% to 90%. The general utility of this strategy is showcased by the synthesis of multimeric linear and bicyclic peptides, bicyclic peptide-cell penetrating peptide conjugates, and multimeric miniproteins which show picomolar affinity against SARS-CoV-2 receptor binding domain. We believe that this modular approach of generating multimeric molecules should find broad applicability in peptide chemistry and chemical biology.