Cellular Screening Research Articles

Using Genetic Methods To Define the Targets of Compounds with Antimalarial Activity

Although phenotypic cellular screening has been used to drive antimalarial drug discovery in recent years, in some cases target-based drug discovery remains more attractive. This is especially true when appropriate high-throughput cellular assays are lacking, as is the case for drug discovery efforts that aim to provide a replacement for primaquine (4-N-(6-methoxyquinolin-8-yl)pentane-1,4-diamine), the only drug that can block Plasmodium transmission to Anopheles mosquitoes and eliminate liver-stage hypnozoites. At present, however, there are no known chemically validated parasite protein targets that are important in all Plasmodium parasite developmental stages and that can be used in traditional biochemical compound screens. We propose that a plethora of novel, chemically validated, cross-stage antimalarial targets still remain to be discovered from the ~5,500 proteins encoded by the Plasmodium genomes. Here we discuss how in vitro evolution of drug-resistant strains of Plasmodium falciparum and subsequent whole-genome analysis can be used to find the targets of some of the many compounds discovered in whole-cell phenotypic screens.

New structure–activity relationships of chalcone inhibitors of breast cancer resistance protein: polyspecificity toward inhibition and critical substitutions against cytotoxicity

Adenosine triphosphate-binding cassette subfamily G member 2 (ABCG2) plays a major role in cancer cell multidrug resistance, which contributes to low eifficacy of chemotherapy. Chalcones were recently found to be potent and specific inhibitors, but unfortunately display a significant cytotoxicity. A cellular screening against ABCG2-mediated mitoxantrone efflux was performed here by flow cytometry on 54 chalcone derivatives from three different series with a wide panel of substituents. The identified leads, with submicromolar IC50 (half maximal inhibitory concentration) values, showed that the previously identified 2′-OH-4′,6′-dimethoxyphenyl, as A-ring, could be efficiently replaced by a 2′-naphthyl group, or a 3′,4′-methylenedioxyphenyl with lower affinity. Such a structural variability indicates 3polyspecificity of the multidrug transporter for inhibitors. At least two methoxyl groups were necessary on B-ring for optimal inhibition, but substitution at positions 3, 4, and 5 induced cytotoxicity. The presence of a large O-benzyl substituent at position 4 and a 2′-naphthyl as A-ring markedly decreased the cytotoxicity, giving a high therapeutic ratio, which constitutes a critical requirement for future in-vivo assays in animal models.

A small molecule modulates Jumonji histone demethylase activity and selectively inhibits cancer growth

The pharmacological inhibition of general transcriptional regulators has the potential to block growth through targeting multiple tumorigenic signaling pathways simultaneously. Here, using an innovative cell-based screen, we identify a structurally unique small molecule (named JIB-04) which specifically inhibits the activity of the Jumonji family of histone demethylases in vitro, in cancer cells, and in tumors in vivo. Unlike known inhibitors, JIB-04 is not a competitive inhibitor of α-ketoglutarate. In cancer but not in patient-matched normal cells, JIB-04 alters a subset of transcriptional pathways and blocks viability. In mice, JIB-04 reduces tumor burden and prolongs survival. Importantly, we find that patients with breast tumors that overexpress Jumonji demethylases have significantly lower survival. Thus JIB-04, a novel inhibitor of Jumonji demethylases in vitro and in vivo, constitutes a unique potential therapeutic and research tool against cancer, and validates the use of unbiased cellular screens to discover chemical modulators with disease relevance.

Cellular fluorescent high-throughput screening assays of the ATP-gated P2X7 receptor

The P2X7 receptor (P2X7R) is an important member of the P2X family of ligand-gated ion channels that respond to ATP as the endogenous agonist. Studies suggest that P2X7R plays a potentially pivotal role in a variety of physiological functions, including peripheral and central neuronal transmission, smooth muscle contraction, and inflammation. Thus, P2X7R may be a potential target for drug development. Here, we used a FlexStation to examine the function of recombinant P2X7R stably expressed in human embryonic kidney 293 cells and to compare three high-throughput screening assays: a membrane potential assay, an ethidium bromide uptake assay, and a calcium influx assay. We found that all three assays were suitable for the analysis of P2X7R, but the calcium influx assay was the most robust and is the best choice as a first high-throughput screening assay when embarking on a P2X7R drug discovery project.

Phenotypic Characterization of Retinoic Acid Differentiated SH-SY5Y Cells by Transcriptional Profiling

Multiple genetic and environmental factors play a role in the development and progression of Parkinson’s disease (PD). The main neuropathological hallmark of PD is the degeneration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta. To study genetic and molecular contributors to the disease process, there is a great need for readily accessible cells with prominent DAergic features that can be used for reproducible in vitro cellular screening. Here, we investigated the molecular phenotype of retinoic acid (RA) differentiated SH-SY5Y cells using genome wide transcriptional profiling combined with gene ontology, transcription factor and molecular pathway analysis. We demonstrated that RA induces a general neuronal differentiation program in SH-SY5Y cells and that these cells develop a predominantly mature DAergic-like neurotransmitter phenotype. This phenotype is characterized by increased dopamine levels together with a substantial suppression of other neurotransmitter phenotypes, such as those for noradrenaline, acetylcholine, glutamate, serotonin and histamine. In addition, we show that RA differentiated SH-SY5Y cells express the dopamine and noradrenalin neurotransmitter transporters that are responsible for uptake of MPP(+), a well known DAergic cell toxicant. MPP(+) treatment alters mitochondrial activity according to its proposed cytotoxic effect in DAergic neurons. Taken together, RA differentiated SH-SY5Y cells have a DAergic-like phenotype, and provide a good cellular screening tool to find novel genes or compounds that affect cytotoxic processes that are associated with PD.

Abstract 2212: Cellular genetics approaches to defining toxicity pathways.

Abstract Background: Significant, nonspecific cytotoxic adverse effects that complicate non-Hodgkin lymphoma treatment vary between patients and are likely due to complex genetic differences between individuals. Our ultimate goal is to identify genetic biomarkers that signal adverse clinical outcomes, maximizing patient safety and minimizing drug cost by identifying patients most likely to have an advantageous risk/benefit ratio for a particular therapy. We are particularly interested in the investigation of the novel anti-lymphoma agents BEZ235, a dual PI3K/mTOR inhibitor, and selumetinib, a MEK inhibitor. Our study is innovative in that we are identifying genetic biomarkers using a strategy that can be employed during preclinical development. Methods: Genome-wide association analysis requires diversity of both the genotype and phenotype among individuals in a mapping population. Thus, we are taking a model organism approach to evaluate pharmacotherapeutic response in a panel of isogenic mice based on priority strains from the Jackson Laboratory's Mouse Phenome Diversity Panel. Association mapping in this fixed and inbred population utilizes existing dense maps of SNP genotype information, providing precision (greater than 1 to 2 Mb) in localizing quantitative trait loci using SNPster and EMMA algorithms. We have developed a cellular genetics screening approach with robust, replicable, multiplexed assays to accurately describe toxicity response using flow cytometry. Our population of immune cells was derived from splenocytes isolated from 35 strains of mice using standard procedures. Of note, the splenocytes were not stimulated to avoid confounding effects, as identification of genes would be attributed to cellular stimulation rather than associated with toxicity. Cells at a density of 100,000 cells per well with 100 μl media were incubated with anti-lymphoma compounds on a 10-point logarithmic dosing scale ranging from 0 to 100 μM (37°C, 5% CO2). At 4 hours post-treatment, cells were labeled with antibodies and physiological indicator dyes and fixed with 4% paraformaldehyde. Cellular phenotypes (e.g., viability, mitochondrial membrane potential, and caspase activity) were collected with the BD Biosciences FACSCanto II flow cytometer and analyzed with Flow Jo version X. Dose-response curves with response normalized to the zero dose as a function of log concentration were subsequently generated using GraphPad Prism 5. Results: Phenotypes have been quantified using flow cytometry, yielding interstrain variation for measured endpoints for different immune function cells. The heritability for viability of T-cells at the 1 μM concentration of doxorubicin, idarubicin, BEZ235, and selumetinib is respectively 86, 83, 56, and 57%. Conclusion: Using this approach, we aim to identify genetic determinants of cellular response to drugs, including candidate genes and cellular toxicity pathways for future validation in human studies. Citation Format: Amber Frick, Rusty Thomas, Kristy Richards, Blossom Damania, Yuri Fedoriw, Bethany Parks, Emmanuel Chan, Tim Wiltshire. Cellular genetics approaches to defining toxicity pathways. [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 2212. doi:10.1158/1538-7445.AM2013-2212

Abstract 2243: Clonogenic 3D high throughput screening in mutant KRAS dependent cancer cells - a chemogenomic approach.

Abstract Activating KRAS mutations are oncogenic and prevalent in multiple tumor types, being found in as high as 90% of pancreatic carcinomas and 50% of colorectal carcinomas. In spite of its attractiveness for therapeutic intervention to date no KRAS-targeted drug therapies have been approved. Most of the reported mutant KRAS directed cellular screening efforts utilize 2-dimensional assays. Because mutant KRAS driven tumor cell lines are much more strongly dependent on activated K-Ras signaling for anchorage independent growth relative to growth when anchored to plastic, we report herein a screening strategy that leverages a 3-dimensional clonogenic growth assay in soft agar. We have performed a multi-cell line parallel phenotypic high throughput screen with our proprietary compound collection to identify pathways, targets and chemical matter with selective anti-tumor activity in mutant KRAS dependent cell lines - a Synthetic Lethal approach. Our strategy has led to the identification of several chemical classes that inhibit the growth of multiple mutant KRAS cell lines of pancreatic and colorectal carcinoma origin, while sparing multiple wild-type KRAS cancer cell lines. Studies to elucidate their molecular mechanisms of action are underway. Citation Format: Malvika Koundinya, Judith Sudhalter, Albane Courjaud, Bruno Lionne, Gaetan Touyer, Luc Bonnet, Isabelle Menguy, Isabelle Schreiber, Christelle Perrault, Stephanie Vougier, Brigitte Benhamou, Daniel Simard, Maria Paola Castaldi, Ronald Tomlinson, Stephan Reiling, Hui Cao, David Harper, Monsif Bouaboula, Jack Pollard, Claudine Grepin, Carlos Garcia-Echeverria, Francisco Adrian, Ivan Cornella-Taracido, Rosalia Arrebola, Aaron J. Morris. Clonogenic 3D high throughput screening in mutant KRAS dependent cancer cells - a chemogenomic approach. [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 2243. doi:10.1158/1538-7445.AM2013-2243

Surface Charge and Cellular Processing of Covalently Functionalized Multiwall Carbon Nanotubes Determine Pulmonary Toxicity

Functionalized carbon nanotubes (f-CNTs) are being produced in increased volume because of the ease of dispersion and maintenance of the pristine material physicochemical properties when used in composite materials as well as for other commercial applications. However, the potential adverse effects of f-CNTs have not been quantitatively or systematically explored. In this study, we used a library of covalently functionalized multiwall carbon nanotubes (f-MWCNTs), established from the same starting material, to assess the impact of surface charge in a predictive toxicological model that relates the tubes' pro-inflammatory and pro-fibrogenic effects at cellular level to the development of pulmonary fibrosis. Carboxylate (COOH), polyethylene glycol (PEG), amine (NH2), sidewall amine (sw-NH2), and polyetherimide (PEI)-modified MWCNTs were successfully established from raw or as-prepared (AP-) MWCNTs and comprehensively characterized by TEM, XPS, FTIR, and DLS to obtain information about morphology, length, degree of functionalization, hydrodynamic size, and surface charge. Cellular screening in BEAS-2B and THP-1 cells showed that, compared to AP-MWCNTs, anionic functionalization (COOH and PEG) decreased the production of pro-fibrogenic cytokines and growth factors (including IL-1β, TGF-β1, and PDGF-AA), while neutral and weak cationic functionalization (NH2 and sw-NH2) showed intermediary effects. In contrast, the strongly cationic PEI-functionalized tubes induced robust biological effects. These differences could be attributed to differences in cellular uptake and NLRP3 inflammasome activation, which depends on the propensity toward lysosomal damage and cathepsin B release in macrophages. Moreover, the in vitro hazard ranking was validated by the pro-fibrogenic potential of the tubes in vivo. Compared to pristine MWCNTs, strong cationic PEI-MWCNTs induced significant lung fibrosis, while carboxylation significantly decreased the extent of pulmonary fibrosis. These results demonstrate that surface charge plays an important role in the structure-activity relationships that determine the pro-fibrogenic potential of f-CNTs in the lung.

A “Clickable” MTX Reagent as a Practical Tool for Profiling Small‐Molecule–Intracellular Target Interactions via MASPIT

We present a scalable synthesis of a versatile MTX reagent with an azide ligation handle that allows rapid γ-selective conjugation to yield MTX fusion compounds (MFCs) appropriate for MASPIT, a three-hybrid system that enables the identification of mammalian cytosolic proteins that interact with a small molecule of interest. We selected three structurally diverse pharmacologically active compounds (tamoxifen, reversine, and FK506) as model baits. After acetylene functionalization of these baits, MFCs were synthesized via a CuAAC reaction, demonstrating the general applicability of the MTX reagent. In analytical mode, MASPIT was able to give concentration-dependent reporter signals for the established target proteins. Furthermore, we demonstrate that the sensitivity obtained with the new MTX reagent was significantly stronger than that of a previously used non-regiomeric conjugate mixture. Finally, the FK506 MFC was explored in a cellular array screen for targets of FK506. Out of a pilot collection of nearly 2000 full-length human ORF preys, FKBP12, the established target of FK506, emerged as the prey protein that gave the highest increase in luciferase activity. This indicates that our newly developed synthetic strategy for the straightforward generation of MFCs is a promising asset to uncover new intracellular targets using MASPIT cellular array screening.

A Computational model for compressed sensing RNAi cellular screening

BackgroundRNA interference (RNAi) becomes an increasingly important and effective genetic tool to study the function of target genes by suppressing specific genes of interest. This system approach helps identify signaling pathways and cellular phase types by tracking intensity and/or morphological changes of cells. The traditional RNAi screening scheme, in which one siRNA is designed to knockdown one specific mRNA target, needs a large library of siRNAs and turns out to be time-consuming and expensive.ResultsIn this paper, we propose a conceptual model, called compressed sensing RNAi (csRNAi), which employs a unique combination of group of small interfering RNAs (siRNAs) to knockdown a much larger size of genes. This strategy is based on the fact that one gene can be partially bound with several small interfering RNAs (siRNAs) and conversely, one siRNA can bind to a few genes with distinct binding affinity. This model constructs a multi-to-multi correspondence between siRNAs and their targets, with siRNAs much fewer than mRNA targets, compared with the conventional scheme. Mathematically this problem involves an underdetermined system of equations (linear or nonlinear), which is ill-posed in general. However, the recently developed compressed sensing (CS) theory can solve this problem. We present a mathematical model to describe the csRNAi system based on both CS theory and biological concerns. To build this model, we first search nucleotide motifs in a target gene set. Then we propose a machine learning based method to find the effective siRNAs with novel features, such as image features and speech features to describe an siRNA sequence. Numerical simulations show that we can reduce the siRNA library to one third of that in the conventional scheme. In addition, the features to describe siRNAs outperform the existing ones substantially.ConclusionsThis csRNAi system is very promising in saving both time and cost for large-scale RNAi screening experiments which may benefit the biological research with respect to cellular processes and pathways.

Reporter gene expression from LTR-circles as tool to identify HIV-1 integrase inhibitors

Early HIV-1 integrase inhibitors, such as compounds containing a β-diketo acid moiety, were identified by extensive high-throughput screening campaigns. Traditionally, in vitro biochemical assays, measuring the catalytic activities of integrase, have been used for this purpose. However, these assays are confounded by the absence of cellular processes or cofactors that play a role in the integration of HIV-1 DNA in the cellular genome. In contrast to regular cell-based virus inhibition assays, which targets all steps of the viral replication cycle, a novel cellular screening assays was developed to enable the specific identification of integrase inhibitors, employing a readout that is linked with the inhibition of integrase activity. Therefore, a HIV-1 lentiviral vector equipped with the enhanced green fluorescent protein (eGFP) reporter gene was used to detect expression from extrachromosomal viral DNA (1- or 2-long terminal repeat circles), formed when integration of vector DNA into the cellular genome is prevented by an integrase inhibitor. In this assay, eGFP expression from the low residual level of transcriptional activity of extrachromosomal DNA was measured via high-throughput flow cytometry. An algorithm for analysis of eGFP expression histograms enabled the specific identification of integrase inhibitors. This assay is amenable for high throughput screening to identify inhibitors of HIV-1 integrase.

Direct Molecular Evolution of Detergent-Stable G Protein-Coupled Receptors Using Polymer Encapsulated Cells

G protein-coupled receptors (GPCRs) are the largest class of pharmaceutical protein targets, yet drug development is encumbered by a lack of information about their molecular structure and conformational dynamics. Most mechanistic and structural studies as well as in vitro drug screening with purified receptors require detergent solubilization of the GPCR, but typically, these proteins exhibit only low stability in detergent micelles. We have developed the first directed evolution method that allows the direct selection of GPCRs stable in a chosen detergent from libraries containing over 100 million individual variants. The crucial concept was to encapsulate single Escherichia coli cells of a library, each expressing a different GPCR variant, to form detergent-resistant, semipermeable nano-containers. Unlike naked cells, these containers are not dissolved by detergents, allowing us to solubilize the GPCR proteins in situ while maintaining an association with the protein's genetic information, a prerequisite for directed evolution. The pore size was controlled to permit GPCR ligands to permeate but the solubilized receptor to remain within the nanocapsules. Fluorescently labeled ligands were used to bind to those GPCR variants inside the nano-containers that remained active in the detergent tested. With the use of fluorescence-activated cell sorting, detergent-stable mutants derived from two different family A GPCRs could be identified, some with the highest stability reported in short-chain detergents. In principle, this method (named cellular high-throughput encapsulation, solubilization and screening) is not limited to engineering stabilized GPCRs but could be used to stabilize other proteins for biochemical and structural studies.

A high-throughput screening assay using Krabbe disease patient cells

Globoid cell leukodystrophy (GLD) or Krabbe disease is a lysosomal disease caused by β-galactocerebrosidase (GALC) deficiency resulting in a rapidly progressive neurodegenerative disorder. Unfortunately, the only available treatment is hematopoietic bone marrow transplantation, which prevents its fulminant manifestation but without treating further neurological manifestations. Here, we describe the development of a cellular high-throughput screening (HTS) assay using GLD patient fibroblasts to screen for small molecules that enhance the residual mutant GALC enzymatic activity. Small molecules have substantial therapeutic potential in GLD because they are more prone to cross the blood–brain barrier, reaching the neuronal affected cells. The transformation of primary skin fibroblasts with SV40 large T antigen has been shown to maintain the biochemical characteristics of the GLD cells and generates sufficient cells for the HTS. Using a specific fluorescent substrate, residual GALC activity from an SV40-transformed GLD patient fibroblast was measurable in high-density microplates. The pilot quantitative HTS against a small compound collection showed robust statistics. The small molecules that showed active concentration–response curves were further studied in primary GLD fibroblasts. This cell-based HTS assay demonstrates the feasibility of employing live GLD patient cells to identify therapeutic agents that can potentially be used for the treatment of this progressive neurodegenerative disease.

Novel Tetrahydropyrido[1,2-a]isoindolone Derivatives (Valmerins): Potent Cyclin-Dependent Kinase/Glycogen Synthase Kinase 3 Inhibitors with Antiproliferative Activities and Antitumor Effects in Human Tumor Xenografts

The development of CDK and GSK3 inhibitors has been regarded as a potential therapeutic approach, and a substantial number of diverse structures have been reported to inhibit CDKs and GSK-3β in recent years. Only a few molecules have gone through or are currently undergoing clinical trials as CDK and GSK inhibitors. In this paper, we prepared valmerins, a new family containing the tetrahydropyrido[1,2-a]isoindone core. The fused heterocycle was prepared with a straightforward synthesis that was functionalized by a (het)arylurea. Twelve valmerins inhibited the CDK5 and GSK3 with an IC(50) < 100 nM. A semiquantitative kinase scoring was realized, and a cellular screening was done. At the end of our study, we investigated the in vivo potency of one valmerin. Mice exhibited good tolerance to our lead, which proved its efficacy and clearly blocked tumor growth. Valmerins appear also as good candidates for further development as anticancer agents.

Glioma-Propagating Cells as an In Vitro Screening Platform: PLK1 as a Case Study

Gliomas are the most devastating of primary adult malignant brain tumors. These tumors are highly infiltrative and can arise from cells with extensive self-renewal capability and chemoresistance, frequently termed glioma-propagating cells (GPCs). GPCs are thus the plausible culprits of tumor recurrence. Treatment strategies that eradicate GPCs will greatly improve disease outcome. Such findings support the use of GPCs as in vitro cellular systems for small-molecule screening. However, the nuances in using GPCs as a cellular screening platform are not trivial. These slow-growing cells are typically cultured as suspension, spheroid structures in serum-free condition supplemented with growth factors. Consequently, replenishment of growth factors throughout the screening period must occur to maintain cells in their undifferentiated state, as the more lineage-committed, differentiated cells are less tumorigenic. We present a case study of a small-molecule screen conducted with GPCs and explain how unique sphere activity assays were implemented to distinguish drug efficacies against the long-term, self-renewing fraction, as opposed to transient-amplifying progenitors, the latter of which are detected in conventional viability assays. We identified Polo-like kinase 1 as a regulator of GPC survival. Finally, we leveraged on public glioma databases to illustrate GPC contribution to disease progression and patient survival outcome.

Fabrication of Two-Dimensional Gradient Layer-by-Layer Films for Combinatorial Biosurface Studies

We have developed a novel gradient fabrication method for combinatorial surface studies that provides the equivalent of 5000 individual polyelectrolyte multilayer (PEM) film physicochemical conditions in a single 7 cm square film. A simple, inexpensive, and versatile automated layering instrument was built, which can generate a gradient of physical properties on a film in 1 dimension laterally by simultaneously changing both the location of polyelectrolyte adsorption and the layering conditions, such as pH or salt concentration of the polyelectrolyte dipping solutions. By rotating the substrate 90° after each deposition cycle, full 2-dimensional gradient combinatorial films were fabricated over many layers, spanning virtually all previous combinations of stable deposition pH and salt conditions for both poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA), a process which previously required more than 10 000 separate film samples. Surface spatial profiles of film thickness, surface energy (wettability), density (refractive index), and stiffness (modulus) were generated and correlated to assembly conditions. Additionally, step gradient films were generated first by varying the number of bilayers along one axis and pH along the other, which enabled us to measure their combined effect on thickness. To test for biocompatibility, we incubated HEK 293 cells on step gradient films and 2D combinatorial films for 48 h and determined that film assembly conditions played a major role, especially in controlling the stiffness and the density, which could be tailored with deposition pH over a wide range. Optimal growth conditions were discovered not at the extremes of fabrication pH, but instead near PAH pHs of 4–6 and PAA pH around 4, demonstrating that these PEM biosurfaces and this technique are suitable for optimizing high-throughput cellular screening.

Detection of vimentin serine phosphorylation by multicolor Quenchbodies

Protein phosphorylation is a key event in intracellular signal transduction, and fluorescent biosensor for the specific phosphorylation event in a target protein is considered highly useful as a tool of cellular biology and drug screening. Vimentin, the most abundant intermediate filament protein, is phosphorylated at its specific serine (Ser) residues in a cell cycle dependent manner. Its structural and functional characteristics are modified by the phosphorylation, which affects biological properties of the cell. Here we present the detection of the vimentin Ser71 phosphorylation (PS71) and the vimentin Ser82 phosphorylation (PS82) using a novel fluorescent biosensor Quenchbody, which works on the principle of antigen-dependent removal of a quenching effect by intrinsic tryptophan residues on a carboxytetramethylrhodamine (TAMRA) dye incorporated at the N-terminal region of single chain antibody variable region. First, we found that rhodamine 6G (R6G)-labeled Quenchbody shows superior response than TAMRA-labeled one. Next, we made several Quenchbodies to detect PS71 and PS82. After optimization of reaction conditions, the fluorescence intensity of VH-VL type PS71 Quenchbody labeled with R6G at two positions was increased to 4.0-fold in an antigen dependent manner. Furthermore, the fluorescence intensity of doubly R6G-labeled VL-VH type PS82 Quenchbody was increased to 6.7-fold immediately after adding antigen peptide, also suggesting deeper quenching due to H-dimer formation between the dyes. Due to its simplicity, the Quenchbody-based phosphorylation biosensors will be widely applicable to in vitro diagnostics, drug screening and imaging in a rapid, simple and high-sensitive manner.

High-throughput cellular screening of engineered ECM based on combinatorial polyelectrolyte multilayer films

The capacity to engineer the extracellular matrix is critical to better understand cell function and to design optimal cellular environments to support tissue engineering, transplantation and repair. Stacks of adsorbed polymers can be engineered as soft wet three dimensional matrices, with properties tailored to support cell survival and growth. Here, we have developed a combinatorial method to generate coatings that self assemble from solutions of polyelectrolytes in water, layer by layer, to produce a polyelectrolyte multilayer (PEM) coating that has enabled high-throughput screening for cellular biocompatibility. Two dimensional combinatorial PEMs were used to rapidly identify assembly conditions that promote optimal cell survival and viability. Conditions were first piloted using a cell line, human embryonic kidney 293 cells (HEK 293), and subsequently tested using primary cultures of embryonic rat spinal commissural neurons. Cell viability was correlated with surface energy (wettability), modulus (matrix stiffness), and surface charge of the coatings.Our findings indicate that the modulus is a crucial determinant of the capacity of a surface to inhibit or support cell survival.

Protein–Ligand Crystal Structures Can Guide the Design of Selective Inhibitors of the FGFR Tyrosine Kinase

The design of compounds that selectively inhibit a single kinase is a significant challenge, particularly for compounds that bind to the ATP site. We describe here how protein-ligand crystal structure information was able both to rationalize observed selectivity and to guide the design of more selective compounds. Inhibition data from enzyme and cellular screens and the crystal structures of a range of ligands tested during the process of identifying selective inhibitors of FGFR provide a step-by-step illustration of the process. Steric effects were exploited by increasing the size of ligands in specific regions in such a way as to be tolerated in the primary target and not in other related kinases. Kinases are an excellent target class to exploit such approaches because of the conserved fold and small side chain mobility of the active form.

Fabrication of Arrays of Polymer Gradients Using Inkjet Printing

Arrays of 84 polymer gradients, fabricated on a single glass microscope slide, were generated by inkjet printing, allowing a combination of high-throughput and true combinatorial methods. The gradual change of composition within the polymer gradients, consisting of two different monomers and a cross-linker, was validated by XPS and fluorescence analysis. Cellular screening of the gradients allowed the rapid identification of optimal polymer compositions for binding of the suspension cell line K562 and the adherent cell line HeLa. The polymers identified were identical to those identified by previous microarray data, providing proof of concept for the successful application of the polymer gradient arrays as a screening tool. In addition, the polymer gradients could be readily modified by conjugation enabling the generation of bio-molecule gradients.