Synthetic Binders Research Articles

Design, construction, and characterization of a second‐generation DARPin library with reduced hydrophobicity

Designed ankyrin repeat proteins (DARPins) are well-established binding molecules based on a highly stable nonantibody scaffold. Building on 13 crystal structures of DARPin-target complexes and stability measurements of DARPin mutants, we have generated a new DARPin library containing an extended randomized surface. To counteract the enrichment of unspecific hydrophobic binders during selections against difficult targets containing hydrophobic surfaces such as membrane proteins, the frequency of apolar residues at diversified positions was drastically reduced and substituted by an increased number of tyrosines. Ribosome display selections against two human caspases and membrane transporter AcrB yielded highly enriched pools of unique and strong DARPin binders which were mainly monomeric. We noted a prominent enrichment of tryptophan residues during binder selections. A crystal structure of a representative of this library in complex with caspase-7 visualizes the key roles of both tryptophans and tyrosines in providing target contacts. These aromatic and polar side chains thus substitute the apolar residues valine, leucine, isoleucine, methionine, and phenylalanine of the original DARPins. Our work describes biophysical and structural analyses required to extend existing binder scaffolds and simplifies an existing protocol for the assembly of highly diverse synthetic binder libraries.

Biological and synthetic binders for immunoassay and sensor-based detection: generation and characterisation of an anti-AFB2 single-chain variable fragment (scFv)

Aflatoxins are highly carcinogenic in nature and occur in a variety of food and feed samples. To ensure food is free from aflatoxins and safe for human consumption, methods to detect these toxins are necessary. Both aflatoxin B1 and aflatoxin B2 (AFB2) co-occur in a variety of foods. The present study focuses on the generation of single-chain variable antibody fragments (scFvs) that are highly specific to AFB2. A panel of murine scFvs was isolated from a recombinant AFB2 library that was subjected to biopanning against AFB2-bovine serum albumin. The scFvs (E9 and H12) were found to be highly specific to AFB2, with no cross reactivity for other aflatoxins. The analytical potential of these two scFvs was tested by competitive inhibition ELISA and half maximal inhibitory concentrations (IC50) of 85 and 138 ng/ml, respectively, were shown. The affinity and sensitivity of scFv-E9 was determined by kinetic analysis and surface plasmon resonance (SPR)-based competitive inhibition assay using an AFB2-immobilised sensor chip. An SPR-based inhibition assay with IC50 of 8 ng/ml and LOD of 0.9 ng/ml was developed and subsequently successfully applied for AFB2 detection in spiked almond extracts.

Biological and synthetic binders for immunoassay and sensor-based detection: generation and characterisation of an anti-AFB<sub>2</sub> single-chain variable fragment (scFv)

Biological and synthetic binders for immunoassay and sensor-based detection: generation and characterisation of an anti-AFB<sub>2</sub> single-chain variable fragment (scFv)

A second-generation DNA-binding pyrrole-imidazole polyamide with antitumor activity.

e13588 Background: Pyrrole-imidazole polyamides are synthetic minor groove binders with programmable sequence recognition. A polyamide that targets the DNA sequence 5’-WGWWCW-3’ (W=A/T) found in the Androgen Receptor (AR) response element inhibits a subset of AR driven transcripts and is cytotoxic in a number of cancer cell lines including LNCaP (prostate cancer), without evidence of DNA damage. Pharmacokinetic studies show bioavailability after intravenous or subcutaneous injection. This polyamide suppressed growth of LNCaP tumor xenografts in mice. However, escalated dosing resulted in significant weight loss that could deter further development. We now report a second-generation polyamide with decreased systemic toxicity that retains efficacy towards LNCaP xenografts. Methods: We investigated the toxicity of our lead polyamide in normal (C57BL/6)mice by necropsy. We then synthesized a focused library of four polyamides that retain the DNA-recognition sequence of our lead molecule, but with chemical substitutions at non-recognition elements. We treated groups of normal male mice (n=4 each) with escalating doses up to 10 mg/kg and observed them for 10 days or until signs of significant distress, after which mice were necropsied. The least toxic polyamide was then tested for suppression of LNCaP xenograft growth (n=14 per treatment arm: 1 mg/kg polyamide versus saline vehicle alone). Tumors were allowed to grow to 200 mm3 prior to treatment by subcutaneous injection every three days for a cycle of six injections. Tumors were then resected and measured. Results: The primary target organ of toxicity for our lead polyamide was the liver, which demonstrated toxicity at 3 mg/kg and dose-limiting toxicity at less than 10 mg/kg. One polyamide in our library demonstrated no evidence of toxicity by necropsy and serum analysis up to 10 mg/kg. This polyamide also suppressed growth of established LNCaP xenografts (T/C: 51%, p=0.0073) at a dose of 1 mg/kg. The other polyamides demonstrated greater toxicity and were not tested further. Conclusions: We report a second-generation polyamide with minimal systemic toxicity in rodents that retains in vivo antitumor activity in prostate cancer xenografts.

Abstract 1130: Antitumor activity of a pyrrole-imidazole polyamide in three tumor xenograft models.

Abstract Background: Pyrrole-imidazole polyamides are synthetic minor groove binders with modular sequence recognition. We developed a polyamide that targets the DNA sequence 5’-WGWWCW-3’ (W=A/T). This molecule is cytotoxic in a number of cancer cell lines including LNCaP (prostate cancer), DU145 (prostate cancer), and A549 (lung cancer). Data in cell culture suggests that this molecule interferes with RNA polymerase 2 activity resulting in proteasome-mediated degradation of the RNA Polymerase 2 large subunit, activation of p53 and induction of p53 target genes, and apoptosis. This polyamide demonstrates no detectable DNA damage by alkaline comet assay nor does it induce detectable phosphorylation of γH2A.X, ATM, DNA-PKcs, p53, or Chk2. This polyamide circulates in mice after intravenous and subcutaneous injection in saline vehicle. Methods: Male immune compromised mice were implanted with LNCaP, DU145, or A549 tumors. Tumors were allowed to grow until approximately 100 mm3 and then stratified into vehicle (5% DMSO/PBS) and treatment arms (1 mg/kg polyamide, in vehicle). Treatment was administered via subcutaneous injection every three days, for a total of three cycles (LNCaP), eight cycles (DU145), or six cycles (A549). At the experimental endpoint, tumors were resected and weighed immediately. For LNCaP xenografts, serum PSA pre- and post-treatment were also measured. In parallel, a group of immune compromised mice without xenografts were treated with an identical regimen for eight cycles and subsequently observed for three months. Results: Tumors resected from polyamide-treated mice were statistically smaller than those of vehicle-treated mice. The effect was greatest on LNCaP tumors (n=12, Treated (T)/Control (C): 32%, p=1.6E-5). DU145 tumors (n=6, T/C: 34%, p=0.013), and A549 tumors (n=8, T/C: 62%, p=0.047) were also affected. Post-treatment serum PSA in the polyamide-treated mice with LNCaP xenografts were lower than for the vehicle treated mice (p = 0.024). Statistical differences between treated and control groups were determined using two-tailed t-tests. Polyamide treatment resulted in no significant weight loss or observable distress in tumor-free mice and these mice appeared healthy for at least three months after treatment was complete. Conclusions: The polyamide demonstrated in vivo antitumor activity in this panel of tumor xenografts with limited host toxicity. Citation Format: Nicholas G. Nickols, Fei Yang, Benjamin C. Li, Georgi K. Marinov, Jonathan W. Said, Peter B. Dervan. Antitumor activity of a pyrrole-imidazole polyamide in three tumor xenograft models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1130. doi:10.1158/1538-7445.AM2013-1130

Characterization of date palm lignocellulosic by-products and self-bonded composite materials obtained thereof

In the present work, we investigated the possibility of valorizing four date palm tree by-products: leaflets, rachis, leaf sheath and fibrillum as self-bonded materials. First, their chemical composition and morphological properties were studied and showed high neutral detergent fiber values for all samples (65–91%) as normally required for self-bonding adhesion. Particleboard composites were then manufactured by thermopressing without any pretreatment or addition of synthetic binders. The properties of the resulting composites were assessed using bending test, internal bonding measurement, thickness swelling, water absorption and dynamical mechanical analysis. The fibrillum, leaflets and rachis based- composites have sufficient modulus of rupture to meet the Japanese Industrial Standard. However, only the fibrillum panels meet the French Standards. Furthermore, the results of dynamic mechanical analysis of leaflets composites reveal a relaxation peak which could be attributed to higher molecular weight carbohydrates. These preliminary results will allow to discriminate the various sources of fibrous particles on the same tree and to choose adequate preprocessing for further research.

Flexible cellulose/LiFePO4 paper-cathodes: toward eco-friendly all-paper Li-ion batteries

Today most of commercial Li-ion batteries (LIBs) are manufactured using toxic solvents and synthetic polymer binders. In order to lower the cost and the environmental impact of LIBs an effort must be made to identify low-cost and environmentally friendly materials and processes. In this work, flexible, self-standing and easily recyclable LiFePO4 cathodes are obtained using cellulose fibers as biosourced binder and a quick, aqueous filtration process, easily upscalable capitalizing the well-established papermaking know-how. The obtained paper-cathodes show very good mechanical properties, with Young’s modulus as high as 100 MPa, discharge capacity values up to 110 mAh g−1 and very good cycling performances, comparable with conventional polymer-bonded LiFePO4 cathodes. Moreover, a complete paper-cell, constituted by a paper-cathode, a paper-separator and a paper-anode is presented, showing good cycling performances in terms of specific capacity, efficiency and stability.

Characterisation of sago pith waste and its composites

Sago pith waste (SPW), a fibrous residue resulted from the sago starch extraction process, was characterised in terms of moisture content (82%) and starch content (62%, dry weight basis). The dried and ground SPW was irregular in shape and has a CE diameter of 29.41μm, similar to that of pure sago starch (28.43μm). SEM micrograph showed that SPW is a mixture which consists of sago starch and fibre, verified by FTIR spectrum and XRD diffractogram observed from peaks which were attributed to sago starch and SPW fibre. With glycerol and water as plasticisers, SPW was then plasticised successfully to form a natural fibre filled thermoplastic starch composite using a twin screw extruder in the presence of various quantities of glycerol. No synthetic polymer binder was added. Plasticisation led to the disruption of the original C-type crystallinity of sago starch. However, VH and B type crystallinities were developed after processing as a result of the reorganisation of amylose, amylose–lipid complex (very fast) and amylopectin chains (slow).

Development of Next-Generation Peptide Binders Using In vitro Display Technologies and Their Potential Applications

During the last decade, a variety of monoclonal antibodies have been developed and used as molecular targeting drugs in medical therapies. Although antibody drugs tend to have intense pharmacological activities and negligible side effects, several issues in their development and prescription remain to be resolved. Synthetic peptides with affinities and specificities for a desired target have received significant attention as alternatives to antibodies. In vitro display technologies are powerful methods for the selection of such peptides from combinatorial peptide libraries. Various types of peptide binders are being selected with such technologies for use in a wide range of fields from bioscience to medicine. This mini review article focuses on the current state of in vitro display selection of synthetic peptide binders and compares the selected peptides with natural peptides/proteins to provide a better understanding of the target affinities and inhibitory activities derived from their amino acid sequences and structural frameworks. The potential of synthetic peptide binders as alternatives to antibody drugs in therapeutic applications is also reviewed.

A simple new competition assay for heparin binding in serum applied to multivalent PAMAM dendrimers

We report a competition assay using our recently reported dye Mallard Blue, which allows us to identify synthetic heparin binders in competitive media, including human serum - using this we gain insight into the ability of PAMAM dendrimers to bind heparin, with the interesting result that low-generation G2-PAMAM is the preferred heparin binder.

A synthetic polypeptide conjugate from a 42‐residue polypeptide and salicylhydroxamic acid binds human myeloperoxidase with high affinity

Myeloperoxidase (MPO) is a 150 kD tetrameric heme protein consisting of two heavy chains and two light chains, which is present in neutrophils, white blood cells, at concentrations between 2% and 5% and plays an important role in the innate immune system. The MPO concentration in serum or plasma has been shown to be linked to the risk for cardiovascular diseases, and MPO is considered to be a high potential diagnostic biomarker. To develop a molecule that binds MPO, salicylhydroxamic acid (SHA), a substrate analog inhibitor of MPO with a KD=2 μM, was conjugated to a designed set of 42-residue polypeptide scaffolds via 9- and 11-carbon atom aliphatic spacers to form 20 different protein binder candidates, and their interactions with MPO were evaluated by surface plasmon resonance analysis. The polypeptide conjugate 4C37L34C11SHA was found to bind to MPO with an affinity that could be estimated to have a dissociation constant of around 400 pM, nearly four orders of magnitude higher than that of SHA. Inhibition of binding to MPO by free SHA was observed in competition experiments demonstrating that the binding of the polypeptide conjugate is dominated by the interactions of SHA with the heme cavity. Although still in the future, the discovery of these new synthetic binders for MPO suggests a route to clinical diagnostic tests in vivo or in vitro, independent of antibodies.

Rheological characteristics of synthetic wax-modified asphalt binders

This paper presents the test results of modification of bitumen 35/50 with synthetic aliphatic wax (SW) obtained during the Fisher-Tropsha synthesis. The aim of the modification is an improvement of rheological properties of the bitumen as well as possibilities to lay the asphalt pavement at decreased compaction temperature by up to 30 °C. Experiments have confirmed that the modification of asphalt with synthetic wax resulted in beneficial changes in complex modulus (G*) and phase angle (d) of modified bitumen 35/50. In consequence, the modified bitumen increased resistance of the asphalt pavement to permanent deformation and decreased the compaction temperature to 100 °C. On the basis of the MSCR test it was revealed that the existence of aliphatic wax in bitumen compound affected the compliance (Jnr) and elastic recover (R) of bitumen. It resulted in an increase in the resilient modulus of elasticity of asphalt pavement and also it will contribute to its higher durability.

Designed protein binders in combination with nanocrystalline diamond for use in high-sensitivity biosensors

A platform for diagnostic applications showing signal-to-noise ratios that by far surpass those of traditional bioanalytical test formats has been developed. It combines the properties of modified nanocrystalline diamond (NCD) surfaces and those of polyethylene oxide and polypropylene oxide based block copolymers for surface passivation and binder conjugation with a new class of synthetic binders for proteins. The NCD surfaces were fluorine-, hydrogen-, or oxygen-terminated prior to further biofunctionalization and the surface composition was characterized by X-ray photoelectron spectroscopy. In a proof of principle demonstration targeting the C-reactive protein, an ELISA carried out using an F-terminated diamond surface showed a signal-to-noise ratio of 3,900 which compares well to the signal-to-noise of 89 obtained in an antibody-based ELISA on a polystyrene microtiter plate, a standard test format used in most life science laboratories today. The increase in signal-to-noise ratio is to a large extent the result of extremely efficient passivation of the diamond surface. The results suggest that significant improvements can be obtained in standardized test formats using new materials in combination with new types of chemical coatings and receptor molecules.

Improvement Fastnesses and Color Strength of Pigment Printed Textile Fabric

This paper focused on the improvement of fastnesses and color strength of pigment printed textile fabrics. To improve fastness and color strength of the polyester fabrics, atmospheric plasma treatment was used. Polyester fabric was treated with argon or air plasma at atmospheric pressure, and plasma pre-treatment effect on the pigment printing was evaluated. A printing paste comprising synthetic thickener, binder, and pigment was applied using a flat screen printing technique. The fabric was then dried and exposed to heat. The cured prints were evaluated for color property, color fastness to washing, and dry/wet rubbing. The effects of atmospheric plasma treatments were evaluated by XPS, SEM and AFM. When the properties of atmospheric plasma treated samples were compared to those of untreated sample, higher K/S values were obtained. Also, the fastness of treated samples increased between ½ and 1 point.

Abstract SY06-01: Intracellular antibody therapy: Specific delivery of synthetic antibodies to cytoplasmic targets in cancer cells

Abstract Synthetic Antigen Binders (sABs) are a new class of antibody-based reagents engineered using novel phage display libraries and selection strategies. These attributes provide for the ability to generate sABs that are customized to: 1) target specific regions on the surface of the protein, 2) recognize specific conformational or oligomeric states, 3) induce conformational changes, and 4) capture and stabilize multi-protein complexes. As such, they have the potential to outperform traditional monoclonal antibodies as therapeutic modalities for biomedical applications. However, a critical limitation is that antibodies can only be directed at extracellular targets because they cannot penetrate the cell membrane. This eliminates a rich set of potential cytoplasmic targets that function as crucial components of signaling pathways or structural networks. To overcome this limitation we have developed receptor-mediated delivery systems that have proven effective in internalizing a novel class of synthetic antigen binders in their fully functional form. Further, this delivery system inherently seeks out certain types of cancer cells, while being inert towards cells on normal tissue. As a demonstration of the power of the approach, we have generated a set of sABs that have been tailored to induce conformational changes in F-actin filaments that substantially alter actin cytoskeletal structure by mechanisms involving depolymerizing, severing, bundling and capping of actin filaments. We introduce these sABs into cancer cells using receptor-mediated delivery and show the properties selected for in the in vitro phage display sorting are exactly recapitulated in the live cells. The effects of actin filament bundling are shown to be particularly detrimental to the cancer cells suggesting that sABs performing this function may be useful therapeutic agents. This approach is being expanded to make agents that target nuclear components to alter cell function at the DNA level. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY06-01. doi:1538-7445.AM2012-SY06-01

Novel composite materials based on cavitated wood

A topical trend in the industrial and ecological development of Russia is elaboration of the methods for integrated use of all components of natural raw materials, in particular, woodbased biomass. Note that the integrated processing of the woodbased bio� mass is possible only in close cooperation of various woodbased industries. These include production of traditional materials, energy sources, chemical prod� ucts, and biologically active compounds approaching the geochemically and energetically closed cycle tech� nology. Currently, all woodbased industries related to either mechanical or chemical processing of wood produce specific wastes although in different amounts. The difficulty of targeted standardized processing of these wastes is related to the necessity of strict indus� trial classification of the wastes and their averaging taking into account the physicochemical properties of the substances that form the material and parts of the trees. It is important that bulky wastes of woodbased biomass are formed upon extraction from wood of bio� logically active compounds such as terpenes, polypre� nols, and flavonoids, which are usually present in small amounts in the initial raw material (1). For example, the larch wood remainders after the removal of arabinogalactan and dihydroquercetin are large� scale wastes, their weight being about 85% of the wood weight (2, 3). A promising method for waste wood recycling is the production of plate materials without or with mini� mized use of synthetic binders. This trend is promising as regards the environmental safety of constructional materials. It was proposed to manufacture plate mate� rials from the compression molding material resulting from the extensive physicomechanical modification of wood by explosive autohydrolysis occurring at elevated

Erratum to: “Silicon nanopowder as active material for hybrid electrodes of lithium-ion batteries”

Cycling parameters (reversible specific capacity, first-cycle coulombic efficiency, accumulated irreversible capacity, and reversible capacity retention) of hybrid electrodes based on mechanical mixtures of a silicon nanopowder with KS6 and MAG-20 synthetic graphites and binders of varied nature were subjected to an integrated analysis in comparison with graphite electrodes.

Advances in binder identification and characterisation: the case of oligonucleotide aptamers

Aptamers represent an important class of synthetic protein binders useful for proteome-wide applications. The identification and characterisation of such molecules have been greatly facilitated by the development of Systematic Evolution of Ligands by Exponential Amplification (SELEX). Since then numerous advances and alternatives to improve efficient aptamer discovery have been reported. In the present manuscript we discuss the recent advances performed around the SELEX approach that may help to expand the availability of new aptamers and the subsequent applications that may be developed.

Formulation of new synthetic binders: Thermomechanical properties of resin/recycled polymer blends

AbstractSynthetic binders are special paving materials manufactured by mixing polymers, resins, and oils. These materials may have improved mechanical properties as compared with the traditional modified bitumen. This work is part of a comprehensive study on the design of synthetic binders with selected mechanical properties. In this sense, upgraded mechanical properties of the final synthetic binder can be attained by understanding and correlating the mechanical properties of its individual constituents as a function of composition and temperature. With this aim, this work deals with the thermomechanical properties of recycled polymer/resin blends, over a wide range of temperature and composition. Recycled polymer/resin blends are thermorheologically complex materials, due to the development of multiphase domains depending on polymer concentration. Thus, these blends show a predominantly gel‐like behavior at high polymer concentrations and a predominantly viscous behavior, with high thermal susceptibility, for low polymer concentrations. The dynamic viscosities of the blends, as a function of polymer concentration and temperature, can be predicted using a logarithmic mixing rule. POLYM. ENG. SCI., 2012. © 2011 Society of Plastics Engineers

키토산과 천연색소를 이용한 Eco-Printing(제1보)

The aim of this study is to develope eco-printing method using natural pigments and chitosan as a natural binder. Three chitosans with different molecular weights were employed to find appropriate conditions including chitosan concentration and pigment/binder ratio. Dye uptake, color and fastnesses of the printed fabrics were evaluated to find optimum conditions within the range of experiments carried out in this study. The effectiveness of chitosan as a printing binder was examined in comparison with color, dye uptake, and fastnesses of conventional synthetic binder and guar gum. It was found that chitosans with low or medium molecular weight were appropriate. Using low molecular weight chitosan, optimum concentrations were 1.7% for charcoal, madder and chlorophyll, whereas 2.2% for ocher, yellow soil, indigo and cochineal. Regardless of molecular weight and concentration of chitosan, the color fastnesess of fabrics printed with mineral pigments were superior to those of the fabrics printed with plant and animal pigments. As pigment/chitosan ratio became higher, rubbing fastness was decreased by 1-3 grade. The colorfastness of printed fabric with chitosan binder was similar to that with synthetic binder, which was higher than that with guar gum.