Use Of Cyclic Peptides Research Articles

Self-assembly mechanisms of cyclic peptide nanotubes in the presence of water molecules; study of hydrogen bonding, aggregation and Mir-210 gene delivery

Self-assembling cyclic peptides are a type of bioinspired supramolecular building blocks. They have the ability to stack together, forming supramolecular cyclic peptide nanotubes. This stacking is driven by β-sheet-like hydrogen bonding. In general, a single hydrogen bond is not strong enough to form ordered supramolecular structures. However, when multiple hydrogen bonds are aligned and built into arrays, they exhibit increased strength and directionality. This enables the formation of well-defined and organized supramolecular structures. The continuous advancements in understanding cyclic peptides and their unique properties open new doors for their application in functional materials, ion transport channels, and drug delivery systems, including gene therapy. When it comes to gene therapy, which involves introducing genetic material into cells to treat or prevent diseases, delivery systems play a crucial role. The use of cyclic peptides in these delivery systems can enhance the stability, specificity, and cellular uptake of the therapeutic cargo, thereby improving the overall success of gene therapy treatments. Hydrogen bond formation makes the cyclic peptide nanotube more stable and has an important role in its formation. The number of hydrogen bonds between cyclic peptides is 117 on average during the simulation time of 200 ns. Also, hydrogen bonds formation between Mir-210 and cyclic peptide nanotube during the simulation time observed which is an average of 17 bonds.

Synthetic Receptors Based on Abiotic Cyclo(pseudo)peptides.

Work on the use of cyclic peptides or pseudopeptides as synthetic receptors started even before the field of supramolecular chemistry was firmly established. Research initially focused on the development of synthetic ionophores and involved the use of macrocycles with a repeating sequence of subunits along the ring to facilitate the correlation between structure, conformation, and binding properties. Later, nonnatural amino acids as building blocks were also considered. With growing research in this area, cyclopeptides and related macrocycles developed into an important and structurally diverse receptor family. This review provides an overview of these developments, starting from the early years. The presented systems are classified according to characteristic structural elements present along the ring. Wherever possible, structural aspects are correlated with binding properties to illustrate how natural or nonnatural amino acids affect binding properties.

RNA-Binding Macrocyclic Peptides.

Being able to effectively target RNA with potent ligands will open up a large number of potential therapeutic options. The knowledge on how to achieve this is ever expanding but an important question that remains open is what chemical matter is suitable to achieve this goal. The high flexibility of an RNA as well as its more limited chemical diversity and featureless binding sites can be difficult to target selectively but can be addressed by well-designed cyclic peptides. In this review we will provide an overview of reported cyclic peptide ligands for therapeutically relevant RNA targets and discuss the methods used to discover them. We will also provide critical insights into the properties required for potent and selective interaction and suggestions on how to assess these parameters. The use of cyclic peptides to target RNA is still in its infancy but the lessons learned from past examples can be adopted for the development of novel potent and selective ligands.

Novel Approaches in the Immunotherapy of Multiple Sclerosis: Cyclization of Myelin Epitope Peptides and Conjugation with Mannan.

Multiple Sclerosis (MS) is a serious autoimmune disease. The patient in an advanced state of the disease has restrained mobility and remains handicapped. It is therefore understandable that there is a great need for novel drugs and vaccines for the treatment of MS. Herein we summarise two major approaches applied for the treatment of the disease using peptide molecules alone or conjugated with mannan. The first approach focuses on selective myelin epitope peptide or peptide mimetic therapy alone or conjugated with mannan, and the second on immune-therapy by preventing or controlling disease through the release of appropriate cytokines. In both approaches the use of cyclic peptides offers the advantage of increased stability from proteolytic enzymes. In these approaches, the synthesis of myelin epitope peptides conjugated to mannan is of particular interest as this was found to protect mice against experimental autoimmune encephalomyelitis, an animal model of MS, in prophylactic and therapeutic protocols. Protection was peptide-specific and associated with reduced antigen-specific T cell proliferation. The aim of the studies of these peptide epitope analogs is to understand their molecular basis of interactions with human autoimmune T-cell receptor and a MS-associated human leucocyte antigen (HLA)-DR2b. This knowledge will lead the rational design to new beneficial non-peptide mimetic analogs for the treatment of MS. Some issues of the use of nanotechnology will also be addressed as a future trend to tackle the disease. We highlight novel immunomodulation and vaccine-based research against MS based on myelin epitope peptides and strategies developed in our laboratories.

Use of cyclic peptides to induce crystallization: case study with prolyl hydroxylase domain 2

Crystallization is the bottleneck in macromolecular crystallography; even when a protein crystallises, crystal packing often influences ligand-binding and protein–protein interaction interfaces, which are the key points of interest for functional and drug discovery studies. The human hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) readily crystallises as a homotrimer, but with a sterically blocked active site. We explored strategies aimed at altering PHD2 crystal packing by protein modification and molecules that bind at its active site and elsewhere. Following the observation that, despite weak inhibition/binding in solution, succinamic acid derivatives readily enable PHD2 crystallization, we explored methods to induce crystallization without active site binding. Cyclic peptides obtained via mRNA display bind PHD2 tightly away from the active site. They efficiently enable PHD2 crystallization in different forms, both with/without substrates, apparently by promoting oligomerization involving binding to the C-terminal region. Although our work involves a specific case study, together with those of others, the results suggest that mRNA display-derived cyclic peptides may be useful in challenging protein crystallization cases.

Hydrogel and Organogel Formation by Hierarchical Self-Assembly of Cyclic Peptides Nanotubes.

Breaking away from the linear structure of previously reported peptide-based gelators, this study reports the first example of gel formation based on the use of cyclic peptides made of alternating d- and l-amino acids, known to self-assemble in solution to form long nanotubes. Herein, a library of cyclic peptides was systemically studied for their gelation properties in various solvents, uncovering key parameters driving both organogel and hydrogel formation. The hierarchical nature of the self-assembly process in water was characterised by a combination of electron microscopy imaging and small-angle X-ray scattering, revealing a porous network of entangled nanofibres composed by the aggregation of several cyclic peptide nanotubes. Rheology measurements then confirmed the formation of soft hydrogels.

Non-competitive cyclic peptides for targeting enzyme-substrate complexes.

Affinity reagents are of central importance for selectively identifying proteins and investigating their interactions. We report on the development and use of cyclic peptides, identified by mRNA display-based RaPID methodology, that are selective for, and tight binders of, the human hypoxia inducible factor prolyl hydroxylases (PHDs) - enzymes crucial in hypoxia sensing. Biophysical analyses reveal the cyclic peptides to bind in a distinct site, away from the enzyme active site pocket, enabling conservation of substrate binding and catalysis. A biotinylated cyclic peptide captures not only the PHDs, but also their primary substrate hypoxia inducible factor HIF1-α. Our work highlights the potential for tight, non-active site binding cyclic peptides to act as promising affinity reagents for studying protein-protein interactions.

An engineered cyclic peptide alleviates symptoms of inflammation in a murine model of inflammatory bowel disease

Inflammatory bowel diseases (IBDs) are a set of complex and debilitating diseases for which there is no satisfactory treatment. Recent studies have shown that small peptides show promise for reducing inflammation in models of IBD. However, these small peptides are likely to be unstable and rapidly cleared from the circulation, and therefore, if not modified for better stability, represent non-viable drug leads. We hypothesized that improving the stability of these peptides by grafting them into a stable cyclic peptide scaffold may enhance their therapeutic potential. Using this approach, we have designed a novel cyclic peptide that comprises a small bioactive peptide from the annexin A1 protein grafted into a sunflower trypsin inhibitor cyclic scaffold. We used native chemical ligation to synthesize the grafted cyclic peptide. This engineered cyclic peptide maintained the overall fold of the naturally occurring cyclic peptide, was more effective at reducing inflammation in a mouse model of acute colitis than the bioactive peptide alone, and showed enhanced stability in human serum. Our findings suggest that the use of cyclic peptides as structural backbones offers a promising approach for the treatment of IBD and potentially other chronic inflammatory conditions.

Accurate quantification of modified cyclic peptides without the need for authentic standards

There is a growing interest in the use of cyclic peptides as therapeutics, but their efficient production is often the bottleneck in taking them forward in the development pipeline. We have recently developed a method to synthesise azole-containing cyclic peptides using enzymes derived from different cyanobactin biosynthetic pathways. Accurate quantification is crucial for calculation of the reaction yield and for the downstream biological testing of the products. In this study, we demonstrate the development and validation of two methods to accurately quantify these compounds in the reaction mixture and after purification. The first method involves the use of a HPLC coupled in parallel to an ESMS and an ICP-MS, hence correlating the calculated sulfur content to the amount of cyclic peptide. The second method is an NMR ERETIC method for quantifying the solution concentration of cyclic peptides. These methods make the quantification of new compounds much easier as there is no need for the use of authentic standards when they are not available.

MiniAp-4: A Venom-Inspired Peptidomimetic for Brain Delivery.

Drug delivery across the blood–brain barrier (BBB) is a formidable challenge for therapies targeting the central nervous system. Although BBB shuttle peptides enhance transport into the brain non‐invasively, their application is partly limited by lability to proteases. The present study proposes the use of cyclic peptides derived from venoms as an affordable way to circumvent this drawback. Apamin, a neurotoxin from bee venom, was minimized by reducing its complexity, toxicity, and immunogenicity, while preserving brain targeting, active transport, and protease resistance. Among the analogues designed, the monocyclic lactam‐bridged peptidomimetic MiniAp‐4 was the most permeable. This molecule is capable of translocating proteins and nanoparticles in a human‐cell‐based BBB model. Furthermore, MiniAp‐4 can efficiently deliver a cargo across the BBB into the brain parenchyma of mice.

MiniAp‐4: A Venom‐Inspired Peptidomimetic for Brain Delivery

AbstractDrug delivery across the blood–brain barrier (BBB) is a formidable challenge for therapies targeting the central nervous system. Although BBB shuttle peptides enhance transport into the brain non‐invasively, their application is partly limited by lability to proteases. The present study proposes the use of cyclic peptides derived from venoms as an affordable way to circumvent this drawback. Apamin, a neurotoxin from bee venom, was minimized by reducing its complexity, toxicity, and immunogenicity, while preserving brain targeting, active transport, and protease resistance. Among the analogues designed, the monocyclic lactam‐bridged peptidomimetic MiniAp‐4 was the most permeable. This molecule is capable of translocating proteins and nanoparticles in a human‐cell‐based BBB model. Furthermore, MiniAp‐4 can efficiently deliver a cargo across the BBB into the brain parenchyma of mice.

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.

In memoriam: A true statesman of science, Elkan R. Blout

On December 20, 2006, Elkan R. Blout, Edward S. Harkness Professor of Biological Chemistry and Molecular Pharmacology, Emeritus, at Harvard Medical School and a former dean for academic affairs at Harvard School of Public Health, a National Medal of Science winner, and a leading contributor to the development of instant film died at the age of 87 after a brief illness. Each of us who knew him sampled only one small component of a life that encompassed many careers. In those careers, Elkan's passion for science, his personal warmth, his creativity, his ability to work with people and to get them to work together, and his aptitude for finances and business all combined to leave a multifaceted and impressive legacy. In the field of peptide science, we have been touched by Elkan's second career: his work on poly-α-amino acids and their conformational propensities, which presaged the still ongoing goal of relating amino acid sequence to three-dimensional structures of proteins; his pioneering use of cyclic peptides to model structural features in proteins and natural products; and his fundamental contributions to spectroscopy, from electronic, to vibrational, to nuclear magnetic resonance. We also appreciate the extent to which Elkan built alliances across the globe, with strong ties to Russian (then Soviet Union) scientists, Israeli scientists, Italian scientists, and so on. Those of us who had the opportunity to work with him also know that he had superb ability as a mentor and advisor, encouraging and questioning without ever conveying anything but a positive attitude. But most of us did not realize how bold a move Elkan made from the practical chemistry of dye molecules in his first career at Polaroid, working with Edwin Land on instant color films. He seamlessly transitioned to conformational studies of protein models; he was a poster child for chemical biology before the term was “in vogue.” In his third, fourth, and fifth careers, Elkan went beyond the laboratory and made major contributions to society, using his unique combination of basic science knowledge, sound scientific judgment, and early industrial experience at Polaroid. He stewarded the finances of the National Academy of Sciences for 12 years in his role as Treasurer, single-handedly giving the Academy financial autonomy through good investments so that this important scientific body could freely pursue issues crucial to society. In his next career, Elkan served as Dean of Academic Affairs at the Harvard School of Public Health and led the school to a major renaissance in scientific research and education. And long after most of us would have slowed down, Elkan was chosen by David Kessler to serve as Senior Advisor to the Food and Drug Administration to bring more basic science grounding within the agency, and according to Kessler his impact was huge. This last career took place between 1991 and 1999, while Elkan was entering his eighties. In all of these positions, Elkan generated much affection and appreciation for his solid scientific judgment and his kindness in dealing with people. Elkan will be missed by more people and in more ways than any one of us can fathom. He was a true statesman of science, and our field was so much the better for his presence. Elkan Blout was a Founding Editor of Biopolymers. A special issue of Biopolymers dedicated to his memory is being prepared, with guest editors Barbara Brodsky, Charles Deber, and myself.

Selection of active ScFv to G-protein-coupled receptor CCR5 using surface antigen-mimicking peptides.

This study describes the use of cyclic peptides for use in the selection of single-chain (ScFv) antibodies specific for the HIV-1 coreceptor CCR5, a representative G-protein-coupled receptor (GPCR). A tandem ligation strategy was developed for preparing biotinylated cyclic peptides, first through an orthogonal end-to-end ligation and then a chemoselective ligation with functionalized biotin. Cyclic peptides mimicking the extracellular loops of CCR5 and their unconstrained counterparts were then used for solution-phase selection of ScFv antibodies from a phage display antibody library. Antibodies reactive with CCR5 on cells were detected using a homogeneous high throughput assay. Of 19 isolated ScFv antibodies that bound to CCR5+ cells, three inhibited CCR5-mediated but not CXCR4-mediated HIV infection. Only ScFvs selected by binding to cyclic constrained peptides exhibited inhibitory activity. Our results demonstrate that surface-antigen mimetics of a GPCR are effective tools for selecting active, site-specific ScFv antibodies that hold promise as immunological reagents and therapeutics.

Hepatic fibrosis: from bench to bedside.

Antifibrotic therapies are preferentially targeted to the activated mesenchymal cells in the liver that synthesize an excess of matrix proteins and resemble the myofibroblasts of healing wounds. These cells derive from normally quiescent hepatic stellate cells and (myo-) fibroblasts. Their activation is triggered and maintained by several fibrogenic modulators and cytokines, but also by mechanical stress. Whereas many agents inhibit stellate cell/myofibroblast proliferation and collagen synthesis in vitro, only few of them are tolerable or effective in suitable animal models in vivo. An antifibrotic effect was demonstrated for silymarin, a defined mixture of flavonoids, sho-saiko-to which contains the related compound baicalein, for halofuginone, another plant-derived agent, for the phosphodiesterase inhibitor pentoxifylline and for LU135252, an oral inhibitor of the endothelin-A-receptor. The retrospective finding that interferon-alpha therapy for hepatitis C may halt or even reverse fibrosis, has to be confirmed in prospective randomized trials. Strategies to inhibit the profibrogenic cytokines transforming growth factor (TGF)-beta or connective tissue growth factor (e.g. by soluble decoy receptors) are evolving, but have not been convincing yet. Drug targeting to the fibrogenic liver cells is now possible by use of cyclic peptides that bind to receptors which are specifically up-regulated on activated stellate cells, for example those for platelet-derived growth factors or collagen type VI. In addition, blockade of such activation receptors can induce stress-relaxation which reverts the fibrogenic cells to a fibrolytic, collagen degrading phenotype. Combined with the evolving validation of serological markers of fibrogenesis and fibrolysis an effective and individualized treatment of liver fibrosis can be anticipated.

Use of cyclic peptides to mimic the active sites of metalloproteins: 13C and 1H NMR, ESR and visible spectra of copper(II) complexes with the cyclo(GlyHisGlyHisGlyHisGly) peptide in aqueous solution

Cyclic peptides have been used to model various aspects of protein conformation and active sites [1]. In such models the usual flexibility of peptide chains is substantially reduced through cyclization. Furthermore, it is generally assumed that specific amino acid residues such as Glu, Asp, His, Lys⋯ have side chains that can bind to transition-metal ions. We chose to synthesize cyclo-(GlyHisGlyHisGlyHisGly) (hereinafter denoted (G 4H 3)) because the histidine residue plays an important role in metal ion coordination of metalloproteins and frequently encountered in vivo. Complexation of (G 4H 3) with the transition metal ion Cu(II) in aqueous solution over a wide pH range and with different peptide/ metal ratios has been studied using carbon-13 and proton NMR, ESR and visible spectroscopy. From analyses of the spectral data, it is concluded that Cu(II) binds at two metal-binding sites depending on the pH. At physiological pH, the binding of Cu(II) at the first site involves the three HisN(3) imidazole groups to give a 1:1 species in a tetracoordinated structure; whereas, at higher pH, the binding of Cu(II) at the second site uses four deprotonated peptide nitrogens. The present results further confirm the concept of molecular design and the feasibility of designing peptide molecules mimicking the complicated metal-binding sites of biological macromolecules.