Template Scaffold Research Articles

The Design and Characterization of Oligospecific Antibodies for Simultaneous Targeting of Multiple Disease Mediators

Monoclonal antibodies are traditionally used to block the function of a specific target in a given disease. However, some diseases are the consequence of multiple components or pathways and not the result of a single mediator; thus, blocking at a single point may not optimally control disease. Antibodies that simultaneously block the functions of two or more disease-associated targets are now being developed. Herein, we describe the design, expression, and characterization of several oligospecific antibody formats that are capable of binding simultaneously to two or three different antigens. These constructs were generated by genetically linking single-chain Fv fragments to the N-terminus of the antibody heavy and light chains and to the C-terminus of the antibody C H3 domain. The oligospecific antibodies were expressed in mammalian cells, purified to homogeneity, and characterized for binding to antigens, Fcγ receptors, FcRn, and C1q. In addition, the oligospecific antibodies were assayed for effector function, protease susceptibility, thermal stability, and size distribution. We demonstrate that these oligospecific antibody formats maintain high expression level, thermostability, and protease resistance. The in vivo half-life, antibody-dependent cellular cytotoxicity function, and binding ability to Fcγ receptors and C1q of the test oligospecific antibodies remain similar to the corresponding properties of their parental IgG antibodies. The excellent expression, biophysical stability, and potential manufacturing feasibility of these multispecific antibody formats suggest that they will provide a scaffold template for the construction of similar molecules to target multiple antigens in complex diseases.

Fabrication of microtemplates for the control of bacterial immobilization

The authors described a region-selective immobilization methods of bacteria by using superhydrophobic/superhydrophilic and superhydrophobic/poly(ethylene glycol) (PEG) micropatterns for culture scaffold templates. In the case of superhydrophobic/superhydrophilic micropatterns, the superhydrophobic surface was prepared first by microwave-plasma enhanced chemical vapor deposition (MPECVD) from trimethylmethoxysilane. Then the superhydrophilic regions were fabricated by irradiating the superhydrophobic surface with vuv light through a stencil mask. In the case of the superhydrophobic/PEG micropatterned surfaces, PEG surfaces were fabricated first by chemical reaction of ester groups of p-nitrophenyl PEG with NH2 group of NH2-terminated self assembled monolayer from n-6-hexyl-3-aminopropyltrimethoxysilane. The superhydrophobic regions were fabricated by MPECVD thorough a stencil mask. In this study four bacteria were selected from viewpoint of peptidoglycan cell wall (E. coli versus B. subtilis), extracellular polysaccharide (E.coli versus P. stutzeri, P. aeruginosa), and growth rate (P. stutzeri versus P. aeruginosa). The former micropattern brought discrete adhesions of E. coli and B. subtilis specifically on the hydrophobic regions, Furthermore, using the superhydrophobic/PEG micropattern, adhesion of bacteria expanded for E. coli, B. subtilis, P. stutzeri, and P. aeruginosa. They observed a high bacterial adhesion onto superhydrophobic surfaces and the inhibitive effect of bacterial adhesion on PEG surfaces.

Gradient collagen/nanohydroxyapatite composite scaffold: Development and characterization

This paper reports an in situ diffusion method for the fabrication of compositionally graded collagen/nanohydroxyapatite (HA) composite scaffold. The method is diffusion based and causes the precipitation of nano-HA crystallites in situ. A collagen matrix acts as a template through which calcium ions (Ca2+) and phosphate ions (PO43-) diffuse and precipitate a non-stoichiometric HA. It was observed that needle-like prismatic nano-HA crystallites (about 2×2×20nm) precipitated in the interior of the collagen template onto the collagen fibrils. Chemical and microstructural analysis revealed a gradient of the Ca to P ratio across the width of the scaffold template, resulting in the formation of a Ca-rich side and a Ca-depleted side of scaffold. The Ca-rich side featured low porosity and agglomerates of the nano-HA crystallites, while the Ca-depleted side featured higher porosity and nano-HA crystallites integrated with collagen fibrils to form a porous network structure.

Morphology-controlled fabrication of polygonal ZnO nanobowls templated from spherical polymeric nanowell arrays

We report a facile and effective strategy for synthesizing morphology-controlled patterned ZnO nanostructures. Polymeric nanowell arrays were employed as scaffold templates, followed by solution dipping and calcination process, polygonal ZnO nanobowl structures were fabricated on silicon substrate. The ordered polymeric nanowell arrays not only provided confined areas for depositing desired materials, but also induced shape transition of ZnO nanobowls from circular to polygonal. The morphology of the patterned ZnO nanostructures can be easily controlled by tuning parameters of polymeric nanowell arrays and other treatment conditions. The patterned structures were characterized by field emission scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray diffraction (XRD) and thermogravimetric analysis (TGA).

Full-sequence Computational Design and Solution Structure of a Thermostable Protein Variant

Computational protein design procedures were applied to the redesign of the entire sequence of a 51 amino acid residue protein, Drosophila melanogaster engrailed homeodomain. Various sequence optimization algorithms were compared and two resulting designed sequences were experimentally evaluated. The two sequences differ by 11 mutations and share 22% and 24% sequence identity with the wild-type protein. Both computationally designed proteins were considerably more stable than the naturally occurring protein, with midpoints of thermal denaturation greater than 99 °C. The solution structure was determined for one of the two sequences using multidimensional heteronuclear NMR spectroscopy, and the structure was found to closely match the original design template scaffold.

Rapid aqueous photo‐polymerization route to polymer and polymer‐composite hydrogel 3D inverted colloidal crystal scaffolds

Successful regeneration of biological tissues in vitro requires the utilization of three-dimensional (3D) scaffolds that provide a near natural microenvironment for progenitor cells to grow, interact, replicate, and differentiate to form target tissues. In this work, a rapid aqueous photo-polymerization route was developed toward the fabrication of a variety of polymer hydrogel 3D inverted colloidal crystal (ICC) scaffolds having different physical and chemical properties. To demonstrate the versatility of this technique, a variety of polymer hydrogel ICC scaffolds were prepared, including (1) polyacrylamide (pAAM) scaffolds, (2) poly(2-hydroxyethyl methacrylate) (pHEMA) scaffolds, (3) poly(2-hydroxyethyl acrylate) (pHEA) scaffolds, and composite scaffolds including (4) pAAM-pHEMA scaffolds, (5) pHEMA-pMAETAC [poly(2-methacryloyloxy) trimethyl ammonium] scaffolds, and (6) pHEA-pMEATAC scaffolds. Templates for scaffolds incorporated both uniform sized (104 microm diameter) and nonuniform sized (100 +/- 20 microm diameter) closely packed noncrosslinked poly(methyl methacrylate) beads. Human bone marrow stromal HS-5 cells were cultured on the six different types of scaffolds to demonstrate biocompatibility. Experimental results show that cells can remain viable in these scaffolds for at least 5 weeks. Of the six scaffolds, maximal cell adhesion and proliferation are obtained on the positively charged composite hydrogel pHEMA-pMEATAC and pHEA-pMAETAC scaffolds.

Bone modeling adaptation as a method for promoting development of bone tissue engineered construct in vitro

It was widely accepted that bone adjusts its mass and structure to altered external mechanical environment. In the modeling process, in vivo bone increases bone formation in order to adapt to overload strains. Therefore the bone modeling adaptation as a method may be applied to promote the development of bone tissue engineered construct in vitro. External mechanical force subjected on cell-seeded scaffolds during culture produces overload zone strains in construct, which activates bone cells differentiation and/or extracellular matrix deposition, and promotes bone formation on the interior surface of scaffolds. The mechanical environment enhances the development of bone tissue engineered construct, which recreates the process of bone modeling. Though the overload strains will be restore to the adaptation zone strains due to modeling adaptation, the overload strains made by increasing the amount of external force step by step always remain the bone-forming state of construct. The development of construct will be in progress to accommodate to the new mechanical environment until the bone-like construct is obtained. The method may not only accomplish the bone formation on the base of template (scaffold), but also optimize the mechanical properties of new formation, and finally may produce the off-the-shelf, bone-like substitutes.

Design, synthesis, and biological evaluation of (E)-3-(4-methanesulfonylphenyl)-2-(aryl)acrylic acids as dual inhibitors of cyclooxygenases and lipoxygenases

A group of (E)-3-(4-methanesulfonylphenyl)acrylic acids possessing a substituted-phenyl ring (4-H, 4-Br, 3-Br, 4-F, 4-OH, 4-OMe, 4-OAc, and 4-NHAc) attached to the acrylic acid C-2 position were prepared using a stereospecific Perkin condensation reaction. A related group of compounds having 4- and 3-(4-isopropyloxyphenyl)phenyl, 4- and 3-(2,4-difluorophenyl)phenyl and 4- and 3-(4-methanesulfonylphenyl)phenyl substituents attached to the acrylic acid C-2 position were also synthesized, using a palladium-catalyzed Suzuki cross-coupling reaction, for evaluation as dual cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) inhibitors. (E)-2-(3-Bromophenyl)-3-(4-methanesulfonylphenyl)acrylic acid (9h), and compounds having 4-(4-isopropyloxyphenyl-, 2,4-difluorophenyl-, or 4-methylsulfonylphenyl)phenyl moieties at the acrylic acid C-2 position (11a,b,d), were particularly potent COX-2 inhibitors with a high COX-2 selectivity index (COX-2 IC50≈0.32μM, SI>316) similar to the reference drug rofecoxib (COX-2 IC50=0.5μM, SI>200). Acrylic acid analogs with a C-2 4-hydoxyphenyl (9d, IC50=0.56μM), or 4-acetamidophenyl (9g, IC50=0.11μM), substituent were particularly potent 5-LOX inhibitors that may participate in an additional specific hydrogen-bonding interaction. A number of compounds possessing a C-2 substituted-phenyl moiety (4-Br, 4-F, and 4-OH), or a 4- or 3-(2,4-difluorophenyl)phenyl moiety, showed potent 15-LOX inhibitory activity (IC50 values in the 0.31–0.49μM range) relative to the reference drug luteolin (IC50=3.2μM). Compounds having a C-2 4-acetylaminophenyl, or 4-(2,4-difluorophenyl)phenyl, moiety exhibited anti-inflammatory activities that were equipotent to aspirin, but less than that of celecoxib. The structure–activity data acquired indicate the acrylic acid moiety constitutes a suitable scaffold (template) to design novel acyclic dual inhibitors of the COX and LOX isozymes.

Noninvasive Optical Imaging of Ovarian Metastases Using Cy5-labeled RAFT-c(-RGDfK-)4

Our group has developed a new molecular tool based on the use of a regioselectively addressable, functionalized template (RAFT) scaffold, where four cyclic (Arg-Gly-Asp) (cRGD) peptide motifs were grafted. The aim of this study was to determine whether RAFT-c(-RGDfK-)4 combined with optical imaging could allow noninvasive detection of deep ovarian metastases. Human ovarian adenocarcinoma IGROV1 cells expressing low levels of integrin alphaVbeta3 (the main receptor for the cRGD peptide) were used for in vitro and in vivo assays in combination with Cy5-labeled RAFT-c(-RGDfK-)4, cRGD, or RAFT-c(-RbetaADfK-)4. In vivo fluorescence imaging was performed on subcutaneous (SC) tumors and intraperitoneal IGROV1 metastases in nude mice. The accumulation of RGD-Cy5 conjugates in cultured cells or in tumor tissues was examined using confocal laser scanning microscopy. RAFT-c(-RGDfK-)4 exhibited stronger staining in vitro, enhanced tumor-to-background ratio for sc tumors, and allowed early detection of 1- to 5-mm large intraabdominal nodules using noninvasive optical imaging. Histological study revealed that RAFT-c(-RGDfK-)4 accumulated into tumor neovasculature but also into tumor cells. Our data demonstrate that a Cy5-labeled RAFT-c(-RGDfK-)4 is an efficient optical probe for early and noninvasive tumor detection.

Design, synthesis, and biological evaluation of linear 1-(4-, 3- or 2-methylsulfonylphenyl)-2-phenylacetylenes: A novel class of cyclooxygenase-2 inhibitors

A group of regioisomeric 1-(methylsulfonylphenyl)-2-phenylacetylenes possessing a COX-2 SO 2Me pharmacophore at the para-, meta- or ortho-position of the C-1 phenyl ring, in conjunction with a C-2 phenyl or substituted-phenyl ring substituent (3-F, 3-OMe, 3-OH, 3-OAc, 4-Me), were designed for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. These target linear 1,2-diarylacetylenes were synthesized via a palladium-catalyzed Sonogashira cross-coupling reaction followed by oxidation of the respective 1-(methylthiophenyl)-2-phenylacetylene intermediate. In vitro COX-1/COX-2 isozyme inhibition structure–activity studies identified 1-(3-methylsulfonylphenyl)-2-(4-methylphenyl)acetylene ( 12d) as a potent COX-2 inhibitor (IC 50 = 0.32 μM) with a high COX-2 selectivity index (SI > 320) comparable to the reference compound rofecoxib (COX-2 IC 50 = 0.50 μM; COX-2 SI > 200). A molecular modeling study where ( 12d) was docked in the binding site of COX-2 showed that the MeSO 2 COX-2 pharmacophore was positioned in the vicinity of the secondary COX-2 binding site near Val 523. The 1-(4-methylsulfonylphenyl)-2-(3-acetoxyphenyl)acetylene ( 11f, COX-1 IC 50 = 1.00 μM; COX-2 IC 50 = 0.06 μM; COX-2 SI = 16.7) and 1-(3-methylsulfonylphenyl)-2-(3-acetoxyphenyl)acetylene ( 12f, COX-1 IC 50 = 6.5 μM; COX-2 IC 50 = 0.05 μM; COX-2 SI = 130) regioisomers exhibited comparable COX-2 inhibition, and moderately lower selective COX-2 selectivity, relative to the reference drug celecoxib (COX-1 IC 50 = 33.1 μM; COX-2 IC 50 = 0.07 μM; COX-2 SI = 472). The most potent anti-inflammatory agent 1-(3-methylsulfonylphenyl)-2-(4-methylphenyl)acetylene ( 12d) exhibited moderate oral anti-inflammatory activity (ED 50 = 129 mg/kg) at 3 h postdrug administration relative to the reference drug celecoxib (ED 50 = 10.8 mg/kg) in a carrageenan-induced rat paw edema assay. The structure–activity data acquired indicate that the acetylene moiety constitutes a suitable scaffold (template) to design novel acyclic 1,2-diarylacetylenes with selective COX-2, or dual COX-1/COX-2, inhibitory activities.

Virtual Screening and Scaffold Hopping Based on GRID Molecular Interaction Fields

In this study, a set of strategies for structure-based design using GRID molecular interaction fields (MIFs) to derive a pharmacophoric representation of a protein is reported. Thrombin, one of the key enzymes involved in the blood coagulation cascade, was chosen as the model system since abundant published experimental data are available related to both crystal structures and structurally diverse sets of inhibitors. First, a virtual screening methodology was developed either using a pharmacophore representation of the protein based on GRID MIFs or using GRID MIFs from the 3D structure of a set of chosen thrombin inhibitors. The search was done in a 3D multiconformation version of the Available Chemical Directory (ACD) database, which had been spiked with 262 known thrombin inhibitors (multiple conformers available per compound). The model managed to find 80% of the known thrombin inhibitors among the 74,291 conformers in the ACD by only searching 5% of the database; hence, a 15-fold enrichment of the library was achieved. Second, a scaffold hopping methodology was developed using GRID MIFs, giving the scaffold interaction pattern and the shape of the scaffold, together with the distance between the anchor points. The scaffolds reported by Dolle in the Journal of Combinatorial Chemistry summaries (2000 and 2001) and scaffolds built or derived from ligands cocomplexed with the thrombin enzyme were parameterized using a new set of descriptors and saved into a searchable database. The scaffold representation from the database was then compared to a template scaffold (from a thrombin crystal structure), and the thrombin-derived scaffolds included in the database were found among the top solutions. To validate the usefulness of the methodology to replace the template scaffold, the entire molecule was built (scaffold and side chains) and the resulting compounds were docked into the active site of thrombin. The docking solutions showed the same binding pattern as the cocomplexed compound, hence, showing that this method can be a valuable tool for medicinal chemists to select interchangeable core structures (scaffolds) in an easy manner and retaining the binding properties from the original ligand.

Lignin-Hydroxyapatite/Tricalcium Phosphate Biocomposites: SEM/EDX and FTIR Characterization

In the present work, a novel biocomposite has been developed based on lignin (Lig) and HA/b-TCP bioceramic materials. The Lig/HA and Lig/b-TCP systems were investigated by Scanning Electron Microscopy coupled to EDX microprobe (SEM/EDX). Lignin is a lignosulfonate natural polymer with complex random coupling and the exact chemical structure is yet unknown. FTIR spectroscopy is an important tool for lignin characterization based on several major vibration bands associated with generally found chemical groups. Consequently, FTIR spectroscopy was used to evaluate the chemical functionalities and major components present in the material. SEM photomicrographs of biocomposites Lig/HA and Lig/b-TCP have shown uniform dispersion of the natural polymer binder in the bioceramic matrix. Also, chemical composition obtained by EDX spectra have indicated lignin evenly distributed in the HA and b-TCP compounds. Novel biocomposites based on lignin/HA and lignin/b-TCP were developed with porosity ranging from 50 to 100 µm. These results have confirmed the high potential application of lignin as a non-toxic natural polymer binder to be used as 3D scaffold template for biomedical applications.

Vascular tissue engineering: microtextured scaffold templates to control organization of vascular smooth muscle cells and extracellular matrix

The in vitro construction of tissue-engineered small diameter (<6mm) blood vessels with sufficient strength and mechanical compliance has evaded researchers. We hypothesize that the high spatial organization of the medial layer of vascular smooth muscle cells (VSMCs) and their surrounding matrix provides high burst strength, compliance, and stability. We investigated the effect of microfabricated polydimethylsiloxane (PDMS) scaffolds with various groove widths on VSMC organization. We found that the presence of these grooved topographical cues significantly enhanced VSMC aspect ratio, alignment, and oriented remodeling of the underlying extracellular matrix. This study suggests that topographical patterning of tissue scaffolds can influence cellular and matrix spatial organization and could provide a framework for achieving the required organization and physical properties for blood vessels.

Templated scaffolds of cis- and trans-tetrahydrofuran γ-amino acids: γ-azido-β-hydroxy-tetrahydrofuran-2-carboxylates from pentono-δ-lactones

Short syntheses of enantiomeric templated scaffolds of cis- and trans-tetrahydrofuran γ-amino acids from pentono-δ-lactones derived from arabinose and ribose are reported; an unexpectedly efficient synthesis of a templated tetrahydrofuran β-amino acid by azide displacement of a triflate β to an ester function proceeds with remarkably little elimination. These materials should allow evaluation of such peptidomimetics to induce predisposition towards secondary structures.

Accessory proteins bind a primed template and mediate rapid cycling of DNA polymerase III holoenzyme from Escherichia coli.

DNA polymerase III holoenzyme was assembled from pure proteins onto a primer template scaffold. The assembly process could be divided into two stages. In the time-consuming first stage, beta subunit and gamma.delta subunit complex were required in forming a tightly bound ATP-activated "preinitiation complex" with a single-stranded DNA bacteriophage circle uniquely primed with a synthetic pentadecadeoxyribonucleotide. This finding substantiates an earlier study using crude protein preparations in a homopolymer system lacking Escherichia coli single-stranded DNA binding protein (Wickner, S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3511-3515). In the second stage, the polymerase III core and the tau subunit rapidly seek out and bind the preinitiation complex to form DNA polymerase III holoenzyme capable of rapid and entirely processive replication of the circular DNA. ATP is not required beyond formation of the preinitiation complex. It is remarkable that the fully assembled DNA polymerase III holoenzyme is so stably bound to the primed DNA circle (4-min half-time of dissociation), yet upon completing a round of synthesis the polymerase cycles within 10 s to a new preinitiation complex on a challenge primed DNA circle. Efficient polymerase cycling only occurred when challenge primed DNA was endowed with a preinitiation complex implying that cycling is mediated by a polymerase subassembly which dissociates from its accessory proteins and associates with a new preinitiation complex. These subunit dynamics suggest mechanisms for polymerase cycling on the lagging strand of replication forks in a growing chromosome.