A Novel Epigenetic Drug-Eluting Balloon Angioplasty Device: Evaluation in a Large Animal Model of Neointimal Hyperplasia.

PURPOSE: Drug-eluting balloon catheters (DEBc) coated with paclitaxel (PTX) have been associated with potential safety concerns. An efficacious but less toxic balloon coating may reduce these outcomes. We evaluated a novel DEBc, Epi-Solve, coated with metacept-3 (MCT-3), a member of the histone deacetylase inhibitor (HDACi) class of epigenetic agents, in a large animal model of neointimal hyperplasia (NIH). METHODS: Plain balloon angioplasty (PABA) catheters were ultrasonically coated with MCT-3 to generate Epi-Solve DEBc. An ovine model of NIH formation was established utilising partial left common carotid artery (LCA) ligation. Twenty-eight days post neointima (NI) induction, PABA, Epi-Solve or PTX-coated DEBc were deployed at the site of induced NI formation. Twenty-eight days post-intervention, ligated vessels were evaluated for attenuation of NI formation, gene expression profiles and immunohistochemical analysis. RESULTS: Epi-Solve DEBc demonstrated attenuation of NIH over no intervention and a trend to inhibition of NIH over PABA. Gene expression analysis and immunohistochemical studies identified significant anti-proliferative and anti-inflammatory signatures and reduced vascular endothelial cell activation compared to PABA. CONCLUSIONS: Epi-Solve is a novel HDACi-coated DEBc which demonstrates significant anti-proliferative and anti-inflammatory signatures and reduced vascular endothelial cell activation compared to PABA in an ovine model and may afford endothelial protection.

ß3-tripeptides act as sticky ends to self-assemble into a bioscaffold.

Peptides comprised entirely of ß3-amino acids, commonly referred to as ß-foldamers, have been shown to self-assemble into a range of materials. Previously, ß-foldamers have been functionalised via various side chain chemistries to introduce function to these materials without perturbation of the self-assembly motif. Here, we show that insertion of both rigid and flexible molecules into the backbone structure of the ß-foldamer did not disturb the self-assembly, provided that the molecule is positioned between two ß3-tripeptides. These hybrid ß3-peptide flanked molecules self-assembled into a range of structures. α-Arginlyglycylaspartic acid (RGD), a commonly used cell attachment motif derived from fibronectin in the extracellular matrix, was incorporated into the peptide sequence in order to form a biomimetic scaffold that would support neuronal cell growth. The RGD-containing sequence formed the desired mesh-like scaffold but did not encourage neuronal growth, possibly due to over-stimulation with RGD. Mixing the RGD peptide with a ß-foldamer without the RGD sequence produced a well-defined scaffold that successfully encouraged the growth of neurons and enabled neuronal electrical functionality. These results indicate that ß3-tripeptides can form distinct self-assembly units separated by a linker and can form fibrous assemblies. The linkers within the peptide sequence can be composed of a bioactive α-peptide and tuned to provide a biocompatible scaffold.

Discovery, Development, and Cellular Delivery of Potent and Selective Bicyclic Peptide Inhibitors of Grb7 Cancer Target.

Grb7 is a signaling protein with critical roles in tumor cell proliferation and migration and an established cancer therapeutic target. Here we explore chemical space to develop a new bicyclic peptide inhibitor, incorporating thioether and lactam linkers that binds with affinity (KD = 1.1 µM) and specificity to the Grb7-SH2 domain. Structural analysis of the Grb7-SH2/peptide complex revealed an unexpected binding orientation underlying the binding selectivity by this new scaffold. We further incorporated carboxymethylphenylalanine and carboxyphenylalanine phosphotyrosine mimetics and arrived at an optimized inhibitor that potently binds Grb7-SH2 (KD = 0.13 µM) under physiological conditions. X-ray crystal structures of these Grb7-SH2/peptide complexes reveal the structural basis for the most potent and specific inhibitors of Grb7 developed to date. Finally, we demonstrate that cell permeable versions of these peptides successfully block Grb7 mediated interactions in a breast cancer cell line, establishing the potential of these peptides in the development of novel therapeutics targeted to Grb7.

Unexpected involvement of staple leads to redesign of selective bicyclic peptide inhibitor of Grb7.

The design of potent and specific peptide inhibitors to therapeutic targets is of enormous utility for both proof-of-concept studies and for the development of potential new therapeutics. Grb7 is a key signaling molecule in the progression of HER2 positive and triple negative breast cancers. Here we report the crystal structure of a stapled bicyclic peptide inhibitor G7-B1 in complex with the Grb7-SH2 domain. This revealed an unexpected binding mode of the peptide, in which the staple forms an alternative contact with the surface of the target protein. Based on this structural information, we designed a new series of bicyclic G7 peptides that progressively constrain the starting peptide, to arrive at the G7-B4 peptide that binds with an approximately 2-fold enhanced affinity to the Grb7-SH2 domain (KD = 0.83 µM) compared to G7-B1 and shows low affinity binding to Grb2-, Grb10- and Grb14-SH2 domains (KD > 100 µM). Furthermore, we determined the structure of the G7-B4 bicyclic peptide in complex with the Grb7-SH2 domain, both before and after ring closing metathesis to show that the closed staple is essential to the target interaction. The G7-B4 peptide represents an advance in the development of Grb7 inhibitors and is a classical example of structure aided inhibitor development.

Orthogonal strategy for the synthesis of dual-functionalised ß(3)-peptide based hydrogels.

We have described a new class of hydrogelator based on helical ß(3)-peptides carrying a bioactive payload. The ß(3)-peptides self-assemble in aqueous solution to form a nanofibrous mesh resulting in a stable hydrogel. The simple design provides the versatility for attaching different functional payloads to the ß(3)-peptide scaffold to develop new materials.

Self-assembled nanomaterials based on beta (ß(3)) tetrapeptides.

ß(3)-amino acid based polypeptides offer a unique starting material for the design of self-assembled nanostructures such as fibres and hierarchical dendritic assemblies, due to their well-defined helical geometry in which the peptide side chains align at 120° due to the 3.0-3.1 residue pitch of the helix. In a previous work we have described the head-to-tail self-assembly of N-terminal acetylated ß(3)-peptides into infinite helical nanorods that was achieved by designing a bioinspired supramolecular self-assembly motif. Here we describe the effect of consecutively more polar side chains on the self-assembly characteristics of ß(3)-tetrapeptides Ac-ß (3)Ala-ß(3)Leu-ß(3)Ile-ß(3)Ala (Ac-ß(3)[ALIA]), Ac-ß(3)Ser-ß(3)Leu-ß(3)Ile-ß(3)Ala (Ac-ß(3)[SLIA]) and Ac-ß (3)Lys-ß (3)Leu-ß(3)Ile-ß (3)Glu (Ac-ß(3)[KLIE]). ß(3)-tetrapeptides complete 1 1/3 turns of the helix: thus in the oligomeric form the side chain positions shift 120° with each added monomer, forming a regular periodic pattern along the nanorod. Dynamic light scattering (DLS) measurements confirmed that these peptides self-assemble even in highly polar solvents such as water and DMSO, while diffusion-ordered NMR spectroscopy revealed the presence of a substantial monomeric population. Temperature dependence of the size distribution in DLS measurements suggests a dynamic equilibrium between monomers and oligomers. Solution casting produced distinct fibrillar deposits after evaporating the solvent. In the case of the apolar Ac-ß(3)[ALIA] the longitudinal helix morphology gives rise to geometrically defined (∼70°) junctions between fibres, forming a mesh that opens up possibilities for applications e.g. in tissue scaffolding. The deposits of polar Ac-ß(3)[SLIA] and Ac-ß(3)[KLIE] exhibit fibres in regular parallel alignment over surface areas in the order of 10 µm.

Geometrically Precise Building Blocks: the Self-Assembly of ß-Peptides.

Peptides comprised entirely of ß-amino acids, or ß-peptides, have attracted substantial interest over the past 25 years due to their unique structural and chemical characteristics. ß-Peptides form well-defined secondary structures that exhibit different geometries compared with their α-peptide counterparts, giving rise to their foldamer classification. ß-Peptide foldamers can be functionalized easily and are metabolically stable and, together with the predictable side-chain topography, have led to the design of a growing number of bioactive ß-peptides with a range of biological targets. The strategic engineering of chemical and topographic properties has also led to the design of ß-peptide mimics of higher-order oligomers. More recently, the ability of these peptides to self-assemble into complex structures of controlled geometries has been exploited in materials applications. The focus of this mini-review is on how the unique structural features of ß-peptide assemblies have been exploited in the design of self-assembled proteomimetic bundles and nanomaterials.

Synthesis and evaluation of antibacterial activity of (R)-2-methylheptyl isonicotinate, a putative naturally occurring bioactive agent.

A putative antibacterial and antifungal compound, (R)-2-methylheptyl isonicotinate, was synthesized via reductive lactone alkylation of (R)-4-methyldihydrofuran-2(3H)-one. Structural characterisation data as well as bioassay results (with Bacillus subtilis or Escherichia coli) contradict those previously reported.

Cyclic Peptides Incorporating Phosphotyrosine Mimetics as Potent and Specific Inhibitors of the Grb7 Breast Cancer Target.

The Grb7 adaptor protein is a therapeutic target for both TNBC and HER2+ breast cancer. A nonphosphorylated cyclic peptide 1 (known as G7-18NATE) inhibits Grb7 via targeting the Grb7-SH2 domain, but requires the presence of phosphate ions for both affinity and specificity. Here we report the discovery of malonate bound in the phosphotyrosine binding pocket of the apo-Grb7-SH2 structure. Based on this, we carried out the rational design and synthesis of two analogues of peptide 1 that incorporate carboxymethylphenylalanine (cmF) and carboxyphenylalanine (cF) as mimics of phosphotyrosine (pY). Binding studies using SPR confirmed that affinity for Grb7-SH2 domain is improved up to 9-fold over peptide 1 under physiological phosphate conditions (KD = 2.1-5.7 µM) and that binding is specific for Grb7-SH2 over closely related domains (low or no detectable binding to Grb2-SH2 and Grb10-SH2). X-ray crystallographic structural analysis of the analogue bearing a cmF moiety in complex with Grb7-SH2 has identified the precise contacts conferred by the pY mimic that underpin this improved molecular interaction. Together this study identifies and characterizes the tightest specific inhibitor of Grb7 to date, representing a significant development toward a new Grb7-targeted therapeutic.

ß-Pro7Ang III is a novel highly selective angiotensin II type 2 receptor (AT2R) agonist, which acts as a vasodepressor agent via the AT2R in conscious spontaneously hypertensive rats.

We have previously shown that individual ß-amino acid substitution in angiotensin (Ang) II reduced Ang II type 1 receptor (AT1R) but not Ang II type 2 receptor (AT2R)-binding and that the heptapeptide Ang III exhibited greater AT2R:AT1R selectivity than Ang II. Therefore, we hypothesized that ß-amino-acid-substituted Ang III peptide analogues would yield highly selective AT2R ligands, which we have tested in binding and functional vascular assays. In competition binding experiments using either AT1R- or AT2R-transfected human embryonic kidney (HEK)-293 cells, novel ß-substituted Ang III analogues lacked appreciable AT1R affinity, whereas most compounds could fully displace (125)I-Sar(1)Ile(8) Ang II from AT2R. The rank order of affinity at AT2R was CGP42112 > Ang III > ß-Pro(7) Ang III=Ang II > ß-Tyr(4) Ang III ≥ PD123319 >> ß-Phe(8) Ang III >> ß Arg(2) Ang III=ß-Val(3) Ang III >> ß-Ile(5) Ang III. The novel analogue ß-Pro(7) Ang III was the most selective AT2R ligand tested, which was >20,000-fold more selective for AT2R than AT1R. IC50 values at AT2R from binding studies correlated with maximum vasorelaxation in mouse aortic rings. Given that ß-Pro(7) Ang III was an AT2R agonist, we compared ß-Pro(7) Ang III and native Ang III for their ability to reduce blood pressure in separate groups of conscious spontaneously hypertensive rats. Whereas Ang III alone increased mean arterial pressure (MAP), ß-Pro(7) Ang III had no effect. During low-level AT1R blockade, both Ang III and ß-Pro(7) Ang III, but not Ang II, lowered MAP (by ∼30 mmHg) at equimolar infusions (150 pmol/kg/min for 4 h) and these depressor effects were abolished by the co-administration of the AT2R antagonist PD123319. Thus, ß-Pro(7) Ang III has remarkable AT2R selectivity determined in binding and functional studies and will be a valuable research tool for insight into AT2R function and for future drug development.

Structural determinants for binding to angiotensin converting enzyme 2 (ACE2) and angiotensin receptors 1 and 2.

Angiotensin converting enzyme 2 (ACE2) is a zinc carboxypeptidase involved in the renin-angiotensin system (RAS) and inactivates the potent vasopressive peptide angiotensin II (Ang II) by removing the C-terminal phenylalanine residue to yield Ang1-7. This conversion inactivates the vasoconstrictive action of Ang II and yields a peptide that acts as a vasodilatory molecule at the Mas receptor and potentially other receptors. Given the growing complexity of RAS and level of cross-talk between ligands and their corresponding enzymes and receptors, the design of molecules with selectivity for the major RAS binding partners to control cardiovascular tone is an on-going challenge. In previous studies we used single ß-amino acid substitutions to modulate the structure of Ang II and its selectivity for ACE2, AT1R, and angiotensin type 2 (AT2R) receptor. We showed that modification at the C-terminus of Ang II generally resulted in more pronounced changes to secondary structure and ligand binding, and here, we further explore this region for the potential to modulate ligand specificity. In this study, (1) a library of 47 peptides derived from the C-terminal tetrapeptide sequence (-IHPF) of Ang II was synthesized and assessed for ACE2 binding, (2) the terminal group requirements for high affinity ACE2 binding were explored by and N- and C-terminal modification, (3) high affinity ACE2 binding chimeric AngII analogs were then synthesized and assessed, (4) the structure of the full-length Ang II analogs were assessed by circular dichroism, and (5) the Ang II analogs were assessed for AT1R/AT2R selectivity by cell-based assays. Studies on the C-terminus of Ang II demonstrated varied specificity at different residue positions for ACE2 binding and four Ang II chimeric peptides were identified as selective ligands for the AT2 receptor. Overall, these results provide insight into the residue and structural requirements for ACE2 binding and angiotensin receptor selectivity.

Evaluation of comprehensive two-dimensional gas chromatography with accurate mass time-of-flight mass spectrometry for the metabolic profiling of plant-fungus interaction in Aquilaria malaccensis.

To explore the possible obligate interactions between the phytopathogenic fungus and Aquilaria malaccensis which result in generation of a complex array of secondary metabolites, we describe a comprehensive two-dimensional gas chromatography (GC × GC) method, coupled to accurate mass time-of-flight mass spectrometry (TOFMS) for the untargeted and comprehensive metabolic profiling of essential oils from naturally infected A. malaccensis trees. A polar/non-polar column configuration was employed, offering an improved separation pattern of components when compared to other column sets. Four different grades of the oils displayed quite different metabolic patterns, suggesting the evolution of a signalling relationship between the host tree (emergence of various phytoalexins) and fungi (activation of biotransformation). In total, ca. 550 peaks/metabolites were detected, of which tentative identification of 155 of these compounds was reported, representing between 20.1% and 53.0% of the total ion count. These are distributed over the chemical families of monoterpenic and sesquiterpenic hydrocarbons, oxygenated monoterpenes and sesquiterpenes (comprised of ketone, aldehyde, oxide, alcohol, lactone, keto-alcohol and diol), norterpenoids, diterpenoids, short chain glycols, carboxylic acids and others. The large number of metabolites detected, combined with the ease with which they are located in the 2D separation space, emphasises the importance of a comprehensive analytical approach for the phytochemical analysis of plant metabolomes. Furthermore, the potential of this methodology in grading agarwood oils by comparing the obtained metabolic profiles (pattern recognition for unique metabolite chemical families) is discussed. The phytocomplexity of the agarwood oils signified the production of a multitude of plant-fungus mediated secondary metabolites as chemical signals for natural ecological communication. To the best of our knowledge, this is the most complete information available so far about essential oils of A. malaccensis, which represents a valuable extension to available data for advanced studies on microbial-mediated biotransformation of terpenes, and offers promise for potential discovery of unanticipated phytochemicals, and biotechnological exploitation.

Light-triggered release of ciprofloxacin from an in situ forming click hydrogel for antibacterial wound dressings.

Light triggered release of an antibiotic from a click crosslinked hydrogel was developed by conjugating ciprofloxacin through a photo-cleavable linker to the hydrogel network structure. Upon irradiation of the hydrogel material with UV light (365 nm) at low intensity, native ciprofloxacin was released into the surrounding environment and could be detected by HPLC. The antimicrobial activity of the released compound on Staphylococcus aureus was demonstrated.

Single ß³-amino acid substitutions to MOG peptides suppress the development of experimental autoimmune encephalomyelitis.

CD4(+) T-cells play a key role in the pathogenesis of multiple sclerosis (MS). Altered peptide ligands capable of modulating T-cell autoreactivity are considered a promising strategy for development of antigen-specific therapies for MS. Since peptides are inherently unstable, the current study explored single ß-amino acid substitution as a means of stabilizing an epitope of myelin oligodendrocyte glycoprotein. ß-Amino acid substitution at position 44, the major T-cell receptor contact residue, increased the half-life of active metabolites. Vaccination with one altered peptide, MOG44ßF, conferred protection from EAE, decreased T-cell autoreactivity and pro-inflammatory cytokine production. Additional studies using MOG44ßF in an oral treatment regimen, administered after EAE induction, also attenuated disease severity. Thus, altered peptides such as those reported here may lead to the development of novel and more specific treatments for MS.

Preparation of crystals for characterizing the Grb7 SH2 domain before and after complex formation with a bicyclic peptide antagonist.

Human growth factor receptor-bound protein 7 (Grb7) is an adapter protein involved in cell growth, migration and proliferation. It is now recognized that Grb7 is an emerging therapeutic target in specific cancer subtypes. Recently, the discovery of a bicyclic peptide inhibitor that targets the Grb7 SH2 domain, named G7-B1, was reported. In an attempt to probe the foundation of its interaction with Grb7, the crystallization and preliminary data collection of both the apo and G7-B1-bound forms of the Grb7 SH2 domain are reported here. Diffraction-quality crystals were obtained using the hanging-drop vapour-diffusion method. After several rounds of microseeding, crystals of the apo Grb7 SH2 domain were obtained that diffracted to 1.8 Šresolution, while those of the G7-B1-Grb7 SH2 domain complex diffracted to 2.2 Šresolution. The apo Grb7 SH2 domain crystallized in the trigonal space group P63, whereas the G7-B1-Grb7 SH2 domain complex crystallized in the monoclinic space group P21. The experimental aspects of crystallization, crystal optimization and data collection and the preliminary data are reported.

An emerging reactor technology for chemical synthesis: surface acoustic wave-assisted closed-vessel Suzuki coupling reactions.

In this paper we demonstrate the use of an energy-efficient surface acoustic wave (SAW) device for driving closed-vessel SAW-assisted (CVSAW), ligand-free Suzuki couplings in aqueous media. The reactions were carried out on a mmolar scale with low to ultra-low catalyst loadings. The reactions were driven by heating resulting from the penetration of acoustic energy derived from RF Raleigh waves generated by a piezoelectric chip via a renewable fluid coupling layer. The yields were uniformly high and the reactions could be executed without added ligand and in water. In terms of energy density this new technology was determined to be roughly as efficient as microwaves and superior to ultrasound.

Studies on the nuances of the electrochemically induced room temperature isomerization of cis-stilbene in acetonitrile and ionic liquids.

Electrochemical reduction of cis-stilbene occurs by two well-resolved one-electron reduction steps in acetonitrile with (n-Bu)4NPF6 as the supporting electrolyte and in N-butyl-N-methylpyrrolidinium (Pyrr1,4(+)) and (trimethylamine)(dimethylethylamine)-dihydroborate bis(trifluoromethylsulfonyl)amide (NTf2(-)) ionic liquids (ILs). Mechanistic details of the electroreduction have been probed by dc and Fourier transformed ac voltammetry, simulation of the voltammetry, bulk electrolysis, and EPR spectroscopy. The first one-electron reduction induces fast cis to trans isomerization in CH3CN and ILs, most likely occurring via disproportionation of cis-stilbene radical anions and fast transformation of the cis-dianion to the trans-configuration. The second reduction process is chemically irreversible in CH3CN due to protonation of the dianion but chemically reversible in highly aprotic ILs under high cis-stilbene concentration conditions. Increase of the (n-Bu)4NPF6 supporting electrolyte concentration (0.01-1.0 M) in CH3CN induces substantial positive shifts in the potentials for reduction of cis-stilbene, consistent with strong ion pairing of the anion radical and dianion with (n-Bu)4N(+). However, protection by ion pairing against protonation of the stilbene dianions or electrochemically induced cis-trans-stilbene isomerization is not achieved. Differences in electrode kinetics and reversible potentials for cis-stilbene(0/•-) and trans-stilbene(0/•-) processes are less pronounced in the Pyrr1,4-NTf2 ionic liquid than in the molecular solvent acetonitrile.

2-(10',10'-Dimethyl-3'-sulfanyl-idene-4'-aza-tri-cyclo-[5.2.1.0(1,5)]decan-2'--yl)-10,10-dimethyl-4-aza-tri-cyclo-[5.2.1.0(1,5)]decane-3-thione.

The title compound, C28H40N2O2S2, was obtained as a minor product from an anti-aldol reaction between the corresponding N-propionyl-thiol-actam and benzaldehyde. The asymmetric unit contains one half-molecule, which is completed by inversion symmetry. The molecule displays a nearly eclipsed conformation along the central C-C bond with a C-C-C-C- torsion angle of 20.4 (3)°.

"One-pot" reductive lactone alkylation provides a concise asymmetric synthesis of chiral isoprenoid targets.

An efficient method, based on nucleophilic addition to lactones followed by modified in situ Clemmensen reduction, provides a short synthetic route to chiral isoprenoid targets. The efficacy of this method has been exemplified through the synthesis of several targets including the commercial fragrance Rosaphen, the side chain of Zaragozic acid C, the cotton leaf sex pheromone, and the side chains of vitamin E.