Cellular Screening Research Articles

Synthetic Biomaterials to Rival Nature's Complexity-a Path Forward with Combinatorics, High-Throughput Discovery, and High-Content Analysis.

Cells in tissue receive a host of soluble and insoluble signals in a context-dependent fashion, where integration of these cues through a complex network of signal transduction cascades will define a particular outcome. Biomaterials scientists and engineers are tasked with designing materials that can at least partially recreate this complex signaling milieu towards new materials for biomedical applications. In this progress report, recent advances in high throughput techniques and high content imaging approaches that are facilitating the discovery of efficacious biomaterials are described. From microarrays of synthetic polymers, peptides and full-length proteins, to designer cell culture systems that present multiple biophysical and biochemical cues in tandem, it is discussed how the integration of combinatorics with high content imaging and analysis is essential to extracting biologically meaningful information from large scale cellular screens to inform the design of next generation biomaterials.

Flow Cytometry Multiplexing and its Use in Detailed Characterization of Cell Health

Abstract High throughput screening (HTS) is extremely effective method for allowing researchers to identify putative compounds of interest which play a role in their area of cellular research. Recently, flow cytometry has emerged as a powerful HTS tool with the added benefit of cell-by-cell analysis. Flow cytometry not only allows a researcher to study drug effectiveness towards different cell types but also can be used to analyze protein-protein interactions, metabolic activities, as well as DNA/mRNA content in a single or multiplexed assay format. In this study, we screened the MicroSource Discovery System Killer Plate compound library and compared HTS using a plate reader with flow cytometer multiplexing. Jurkat and Ramos cells, T and B lymphocyte cell types respectively, were used as cell models and cultured under standard conditions and screened either at hypoxic (1%) or hyperoxic (19%) oxygen levels at varied lengths of time (24, 48, or 72 hrs). To assess compound veracity, post-screening analysis was implemented to establish EC50s of “hits” from the compound library. Membrane integrity and metabolic activity were measured as an initial screening output for evaluating cellular viability. Using the multiplexed capabilities of flow cytometry, we performed secondary and tertiary assays to further characterize the possible mechanism of action by analysis of cell cycle, specific RNA expression levels, oxidative stress, and caspase activation across differing concentrations of the compounds. This method and analysis highlights the complicated nature of assessing toxicity in cellular screening assays and the advantage of Flow Cytometry in particular for characterizing cell health.

Next‐generation nutritional screening

The biochemical identification of essential vitamins and minerals during the early 20th century led to major public health advances through treatment and prevention of deficiency diseases. Today, we face an epidemic of overnutrition. Chronic metabolic conditions such as obesity and diabetes are responsible for millions of deaths each year and constitute an enormous global health burden. Despite the major influence of diet on this epidemic, nutrition knowledge has not been applied as successfully as for vitamin and mineral deficiency diseases. In addition to vitamins and minerals, the foods we eat contain thousands of biologically active nutritional compounds that are managed at the cellular level by molecular networks which sense the cellular nutritional state and respond to maintain homeostasis. A key hub in these networks, and the focus of this project, is the endoplasmic reticulum (ER), a cellular organelle that is a broad sensor of nutritional and metabolic status. Impaired ER function is implicated in the development of obesity and diabetes, and improvement of ER function is a common therapeutic target that can potentially improve health in many tissues and disease states. Among its many functions, the ER is a major site for metabolism of lipids, a class of nutritional compounds that includes fatty acids and bile acids. Enzymes located on the ER membrane metabolize lipids from the diet, and also synthesize lipids de novo. While lipid synthesis at the ER has been widely studied, little is known about how the ER handles the many lipids from the diet, and how the response of the ER to dietary lipids impacts health. Without new tools to detect how cells sense and respond to nutritional compounds, we are unable to make continued progress in nutrition to reduce chronic metabolic conditions.The aim of this project is to create a novel cellular screening system to detect the effects of nutritional compounds. This project will focus on lipids, as they are rich in diversity and biology, and our lab is well‐equipped to study them. I will begin by curating a small collection of the predominant lipids in the human diet. These lipids are included in an existing collection of 688 lipids that our lab has established in collaboration with the Broad Institute for screening. In order to detect the cellular effects of lipids, I will focus on indicators of ER function. We have developed luminescent cellular reporters for real‐time monitoring of ER function in live cells. I will introduce these reporters into adipocytes, cells that manage systemic nutrient levels through storage and release of lipids. These reporter cells will be integrated into a microfluidic chip the size of a microscope slide. It will contain chambers, where we will seed cells expressing the ER reporters, and channels to flow culture medium and nutritional compounds to the cells. This system will enable high‐throughput screening of dietary lipids, continuous sampling of the culture medium for evaluation of the dynamic response of the ER, and co‐culture of related cell types, such as adipocytes and macrophages, to model cellular interactions.Support or Funding InformationWe acknowledge the generous support of the Sabri Ülker Food Research Foundation, Sabri Ülker Center, and Harvard Training Program in Nutritional Metabolism T32 training grant (4T32HD052961‐10).

Accurate nanoelectrode recording of human pluripotent stem cell-derived cardiomyocytes for assaying drugs and modeling disease

The measurement of the electrophysiology of human pluripotent stem cell-derived cardiomyocytes is critical for their biomedical applications, from disease modeling to drug screening. Yet, a method that enables the high-throughput intracellular electrophysiology measurement of single cardiomyocytes in adherent culture is not available. To address this area, we have fabricated vertical nanopillar electrodes that can record intracellular action potentials from up to 60 single beating cardiomyocytes. Intracellular access is achieved by highly localized electroporation, which allows for low impedance electrical access to the intracellular voltage. Herein, we demonstrate that this method provides the accurate measurement of the shape and duration of intracellular action potentials, validated by patch clamp, and can facilitate cellular drug screening and disease modeling using human pluripotent stem cells. This study validates the use of nanopillar electrodes for myriad further applications of human pluripotent stem cell-derived cardiomyocytes such as cardiomyocyte maturation monitoring and electrophysiology-contractile force correlation.

Poloxamer-Based Thermoreversible Gel for Topical Delivery of Emodin: Influence of P407 and P188 on Solubility of Emodin and Its Application in Cellular Activity Screening.

Emodin is a component in a Chinese herb, Rheum officinale Baill, traditionally used for diabetes and anticancer. Its poor solubility is one of the major challenges to pharmaceutical scientists. We previously reported on thermoreversible gel formulations based on poloxamer for the topical delivery of emodin. The present study was to understand the effect of poloxamer type on emodin solubility and its application in cellular activity screening. Various gel formulations composed of poloxamer 407 (P407), poloxamer 188 (P188) and PEG400 were prepared and evaluated. Major evaluation parameters were the gelation temperature (Tgel) and solubility of emodin. The emodin solubility increased with increasing poloxamer concentration and the Tgel was modulated by the proper combination of P407. In particular, this study showed that the amount of P407 in thermoreversible poloxamer gel (PG) was the dominant factor in enhancing solubility and P188 was effective at fixing gelation temperature in the desired range. A thermoreversible emodin PG was selected as the proper composition with the liquid state at room temperature and gel state at body temperature. The gel showed the solubility enhancement of emodin at least 100-fold compared to 10% ethanol or water. The thermoreversible formulation was applied for in vitro cellular activity screening in the human dermal fibroblast cell line and DLD-1 colon cancer cell line after dilution with cell culture media. The thermoreversible gel formulation remained as a clear solution in the microplate, which allowed reliable cellular activity screening. In contrast, emodin solution in ethanol or DMSO showed precipitation at the corresponding emodin concentration, complicating data interpretation. In conclusion, the gel formulation is proposed as a useful prototype topical formulation for testing emodin in vivo as well as in vitro.

Preparation, characterization, and in vitro dosimetry of dispersed, engineered nanomaterials.

Evidence continues to grow of the importance of in vitro and in vivo dosimetry in the hazard assessment and ranking of engineered nanomaterials (ENMs). Accurate dose metrics are particularly important for in vitro cellular screening to assess the potential health risks or bioactivity of ENMs. To ensure meaningful and reproducible quantification of in vitro dose, with consistent measurement and reporting between laboratories, it is necessary to adopt standardized and integrated methodologies for (i) generation of stable ENM suspensions in cell culture media; (ii) colloidal characterization of suspended ENMs, particularly of properties that determine particle kinetics in an in vitro system (size distribution and formed agglomerate effective density); and (iii) robust numerical fate and transport modeling for accurate determination of the ENM dose delivered to cells over the course of the in vitro exposure. Here we present an integrated comprehensive protocol based on such a methodology for in vitro dosimetry, including detailed standardized procedures for each of these three critical aims. The entire protocol requires ∼6-12 h to complete.

Cellular and molecular screening of connective tissue dysplasia in adolescent athletes (pilot study)

The purpose of the study is to evaluate the cellular and molecular parameters of bone remodeling in the blood as potential markers of undifferentiated forms of connective tissue dysplasia Materials and methods. The structural and functional status of cellular elements of in vitro culturing of mononuclear leukocytes of peripheral blood in adolescent athletes connected with phenotypic manifestations of undifferentiated connective tissue dysplasia (UCTD) were investigated. 25 pupils of sport schools from 10–14 years old (main disciplines: figure skating, gymnastics, athletics) were examined with the help of express analysis. The average age of the examined adolescents was (12,0 ± 1,7) years. Clinical examination of adolescents allowed ranking the UCTD signs on a scale of 4–11,5 points. Results. A comparison of questionnaire survey results and an evaluation of bone remodeling distant markers allowed the revelation of 2 groups in the distribution of adolescent athletes: those with minimal signs of UCTD (scores lesser than 7 points – 10 pupils), and those with expressed UCTD phenotype (scores are equal or more than 7 points –15 pupils). Significant statistical decrease in the content of collagen type I degradation products (CrossLaps) (by 80%) and ionized calcium (by 5%) has been determined in the peripheral blood of adolescent athletes with expressed UCTD phenotype. In conditions of short-term 72-h cultivation of mononuclear leukocytes in the presence of a 3D matrix imitating the properties of the mineral substance of the regenerating bone tissue, morphofunctional features of cellular reaction in adolescent athletes with clinical manifestations of UCTD, as well the heterogeneity of the cell population associated with the appearance of cells with an osteoblast-like phenotype in the blood have been revealed. The results of investigation propose the use of distant cellular and molecular parameters of bone remodeling to screen the mechanisms and dynamics of dysplastic disorders in adolescent athletes.

Mask-free fabrication of a versatile microwell chip for multidimensional cellular analysis and drug screening.

Cells are frequently studied because they are basic structural, functional, and biological units of living organisms. Extracting features from cellular behaviors can facilitate decision making in medical diagnoses and represents an important aspect in the development of biomedical engineering. Previous studies have just focused on either the individual cell or cell clusters separately, which leads to a great lack of information. Microwell technologies could address the challenges of in vitro cellular studies, from individual cell studies to 3D functional assays, by providing more information from smaller sample volumes and enabling the incorporation of low-cost high-throughput assays in the drug discovery process. To this end, the present study describes an easy-to-use method for fabricating a versatile microwell chip that utilizes a digital micro-mirror device printing system, and the chip can be employed in multidimensional cellular analysis, ranging from the single cell to the 3D spheroid level. The microwell manufacturing process, using a digital mask in place of a conventional physical mask, is based on shadowed light and is full of flexibility. Three different dimensions (single cell (1D), cell monolayer (2D) and cell spheroid (3D)) of one cell type can be formed using a microwell array and the analyses of biological characteristics are achieved separately. Single cells and cell clusters can be controlled via customized geometries of microfabricated selectively adhesive poly(ethylene glycol) diacrylate (PEGDA) wells. The effects of shape on cellular growth and hybrid tissue layers were investigated by peeling off the microwells. Furthermore, 3D multicellular spheroids were successfully established in a controllable and high-throughput format. Preclinical drug screening was investigated and distinct differences were observed in the tolerance response to drug testing between the 2D and 3D conditions. The study results further demonstrate that the high-density microwell chip is an easy-to-use multidimensional cellular analysis and rapid drug screening technique, and it could be easily adapted for a wide range of biological research applications.

Discovery of host-targeted covalent inhibitors of dengue virus

We report here on an approach targeting the host reactive cysteinome to identify inhibitors of host factors required for the infectious cycle of Flaviviruses and other viruses. We used two parallel cellular phenotypic screens to identify a series of covalent inhibitors, exemplified by QL-XII-47, that are active against dengue virus. We show that the compounds effectively block viral protein expression and that this inhibition is associated with repression of downstream processes of the infectious cycle, and thus significantly contributes to the potent antiviral activity of these compounds. We demonstrate that QL-XII-47's antiviral activity requires selective, covalent modification of a host target by showing that the compound's antiviral activity is recapitulated when cells are preincubated with QL-XII-47 and then washed prior to viral infection and by showing that QL-XII-47R, a non-reactive analog, lacks antiviral activity at concentrations more than 20-fold higher than QL-XII-47's IC90. QL-XII-47's inhibition of Zika virus, West Nile virus, hepatitis C virus, and poliovirus further suggests that it acts via a target mediating inhibition of these other medically relevant viruses. These results demonstrate the utility of screens targeting the host reactive cysteinome for rapid identification of compounds with potent antiviral activity.

The Novel ADCC-Optimized Human CD54 (ICAM-1) Antibody MSH-TP15e Has Potent Anti-Myeloma Activity

Background: Monoclonal antibodies directed against various target antigens have proven efficacy in cancer therapy including hematological malignancies. Recently, daratumumab and elotuzumab became the first antibodies approved for the treatment of multiple myeloma (MM). Due to the substantial number of patients still in need for long-term disease control, the investigation of additional target structures and potent molecule formats for antibody-based therapy of MM remains important.Methods: Human synthetic single-chain fragment variable (scFv) phage display libraries were subjected to a cellular screening approach. The MSH-TP15 clone was selected based on its binding to various MM cell lines and patient-derived CD138-positive malignant plasma cells. Intercellular adhesion molecule-1 (ICAM-1/CD54), known to be important for the interaction of malignant plasma cells with the bone marrow microenvironment, was identified as target antigen. Based on the scFv sequence, fully human IgG1 antibody variants with selected mutations in the Fc domain were constructed to generate wild-type, antibody-dependent cell-mediated cytotoxicity (ADCC)-optimized and Fc-knockout variants. First, these variants were analyzed for their capacity to kill MM cells in vitro. Apoptosis induction and growth inhibition were tested by flow cytometry and in MTS proliferation assays. ADCC and complement-dependent cytotoxicity were investigated in chromium-release assays using human serum, peripheral blood mononuclear cells or purified NK cells of healthy donors. In vivoefficacy of an ADCC-optimized variant was analyzed in the INA-6 myeloma xenograft model.Results: The MSH-TP15 antibodies target an epitope on the N-terminal part of the extracellular domain of human ICAM-1/CD54. The antibody variants showed no anti-proliferative effects on patient-derived bone marrow stromal cells, nor directly induced apoptosis or inhibited proliferation of myeloma cells. While complement-dependent cytotoxic activity was generally absent, the Fc-engineered antibody variant MSH-TP15e significantly triggered ADCC against various MM cell lines and freshly isolated patient myeloma cells. The recruitment of human NK cells was crucial for the cytotoxic effects observed in vitro. Importantly, an ADCC-optimized variant completely prevented tumor engraftment in the INA-6 xenograft model.Conclusions: The novel ADCC-optimized fully human antibody MSH-TP15e directed against ICAM-1/CD54 exerts potent anti-myeloma activity in vitro and in vivo. Therefore, it has promising characteristics and will be further evaluated for MM immunotherapy. DisclosuresGuenther:Novartis: Consultancy, Honoraria; Celgene: Honoraria; Takeda: Consultancy, Honoraria.

Scaling up a chemically-defined aggregate-based suspension culture system for neural commitment of human pluripotent stem cells.

The demand of high cell numbers for applications in cellular therapies and drug screening requires the development of scalable platforms capable to generating highly pure populations of tissue-specific cells from human pluripotent stem cells. In this work, we describe the scaling-up of an aggregate-based culture system for neural induction of human induced pluripotent stem cells (hiPSCs) under chemically-defined conditions. A combination of non-enzymatic dissociation and rotary agitation was successfully used to produce homogeneous populations of hiPSC aggregates with an optimal (140 μm) and narrow distribution of diameters (coefficient of variation of 21.6%). Scalable neural commitment of hiPSCs as 3D aggregates was performed in 50 mL spinner flasks, and the process was optimized using a factorial design approach, involving parameters such as agitation rate and seeding density. We were able to produce neural progenitor cell cultures, that at the end of a 6-day neural induction process contained less than 3% of Oct4-positive cells and that, after replating, retained more than 60% of Pax6-positive neural cells. The results here presented should set the stage for the future generation of a clinically relevant number of human neural progenitors for transplantation and other biomedical applications using controlled, automated and reproducible large-scale bioreactor culture systems.

Trehalose upregulates progranulin expression in human and mouse models of GRN haploinsufficiency: a novel therapeutic lead to treat frontotemporal dementia.

BackgroundProgranulin (PGRN) is a secreted growth factor important for neuronal survival and may do so, in part, by regulating lysosome homeostasis. Mutations in the PGRN gene (GRN) are a common cause of frontotemporal lobar degeneration (FTLD) and lead to disease through PGRN haploinsufficiency. Additionally, complete loss of PGRN in humans leads to neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Importantly, Grn−/− mouse models recapitulate pathogenic lysosomal features of NCL. Further, GRN variants that decrease PGRN expression increase the risk of developing Alzheimer’s disease (AD) and Parkinson’s disease (PD). Together these findings demonstrate that insufficient PGRN predisposes neurons to degeneration. Therefore, compounds that increase PGRN levels are potential therapeutics for multiple neurodegenerative diseases.ResultsHere, we performed a cell-based screen of a library of known autophagy-lysosome modulators and identified multiple novel activators of a human GRN promoter reporter including several common mTOR inhibitors and an mTOR-independent activator of autophagy, trehalose. Secondary cellular screens identified trehalose, a natural disaccharide, as the most promising lead compound because it increased endogenous PGRN in all cell lines tested and has multiple reported neuroprotective properties. Trehalose dose-dependently increased GRN mRNA as well as intracellular and secreted PGRN in both mouse and human cell lines and this effect was independent of the transcription factor EB (TFEB). Moreover, trehalose rescued PGRN deficiency in human fibroblasts and neurons derived from induced pluripotent stem cells (iPSCs) generated from GRN mutation carriers. Finally, oral administration of trehalose to Grn haploinsufficient mice significantly increased PGRN expression in the brain.ConclusionsThis work reports several novel autophagy-lysosome modulators that enhance PGRN expression and identifies trehalose as a promising therapeutic for raising PGRN levels to treat multiple neurodegenerative diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0114-3) contains supplementary material, which is available to authorized users.

High throughput toxicity screening and intracellular detection of nanomaterials

With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety—preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read‐across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter‐experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM‐cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose‐ and time‐dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label‐free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance‐based monitoring, Multiplex analysis of secreted products, and genotoxicity methods—namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413For further resources related to this article, please visit the WIREs website.

At-Line Cellular Screening Methodology for Bioactives in Mixtures Targeting the α7-Nicotinic Acetylcholine Receptor

The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel expressed in different regions of the central nervous system (CNS). The α7-nAChR has been associated with Alzheimer’s disease, epilepsy, and schizophrenia, and therefore is extensively studied as a drug target for the treatment of these diseases. Important sources for new compounds in drug discovery are natural extracts. Since natural extracts are complex mixtures, identification of the bioactives demands the use of analytical techniques to separate a bioactive from inactive compounds. This study describes screening methodology for identifying bioactive compounds in mixtures acting on the α7-nAChR. The methodology developed combines liquid chromatography (LC) coupled via a split with both an at-line calcium (Ca2+)-flux assay and high-resolution mass spectrometry (MS). This allows evaluation of α7-nAChR responses after LC separation, while parallel MS enables compound identification. The methodology was optimized for analysis of agonists and positive allosteric modulators, and was successfully applied to screening of the hallucinogen mushroom PsilocybeMckennaii. The crude mushroom extract was analyzed using both reversed-phase and hydrophilic interaction liquid chromatography. Matching retention times and peak shapes of bioactives found with data from the parallel MS measurements allowed rapid pinpointing of accurate masses corresponding to the bioactives.

Living Cell Microarrays: An Overview of Concepts.

Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays.

A cellular high-throughput screening approach for therapeutic trans-cleaving ribozymes and RNAi against arbitrary mRNA disease targets

Major bottlenecks in development of therapeutic post transcriptional gene silencing (PTGS) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease. Methods for rapid and reliable cellular testing of PTGS agents are needed to identify potent lead candidates for optimization. Our goal was to develop a means of rapid assessment of many RNA agents to identify a lead candidate for a given mRNA associated with a disease state. We developed a rapid human cell-based screening platform to test efficacy of hammerhead ribozyme (hhRz) or RNA interference (RNAi) constructs, using a model retinal degeneration target, human rod opsin (RHO) mRNA. The focus is on RNA Drug Discovery for diverse retinal degeneration targets.To validate the approach, candidate hhRzs were tested against NUH↓ cleavage sites (N = G,C,A,U; H = C,A,U) within the target mRNA of secreted alkaline phosphatase (SEAP), a model gene expression reporter, based upon in silico predictions of mRNA accessibility. HhRzs were embedded in a larger stable adenoviral VAI RNA scaffold for high cellular expression, cytoplasmic trafficking, and stability. Most hhRz expression plasmids exerted statistically significant knockdown of extracellular SEAP enzyme activity when readily assayed by a fluorescence enzyme assay intended for high throughput screening (HTS). Kinetics of PTGS knockdown of cellular targets is measureable in live cells with the SEAP reporter. The validated SEAP HTS platform was transposed to identify lead PTGS agents against a model hereditary retinal degeneration target, RHO mRNA. Two approaches were used to physically fuse the model retinal gene target mRNA to the SEAP reporter mRNA. The most expedient way to evaluate a large set of potential VAI-hhRz expression plasmids against diverse NUH↓ cleavage sites uses cultured human HEK293S cells stably expressing a dicistronic Target-IRES-SEAP target fusion mRNA. Broad utility of this rational RNA drug discovery approach is feasible for any ophthalmological disease-relevant mRNA targets and any disease mRNA targets in general. The approach will permit rank ordering of PTGS agents based on potency to identify a lead therapeutic compound for further optimization.

A novel cell-based duplex high-throughput screening assay combining fluorescent Ca2+ measurement with homogeneous time-resolved fluorescence technology

Cell-based assays for G-protein-coupled receptor (GPCR) activation applied in high-throughput screening (HTS) monitor various readouts for second messengers or intracellular effectors. Recently, our understanding of diverging signaling pathways downstream of receptor activation and the capability of small molecules to selectively modulate signaling routes has increased substantially, underlining the importance of selecting appropriate readouts in cellular functional screens. To minimize the rate of false negatives in large-scale screening campaigns, it is crucial to maximize the chance of a ligand being detected, and generally applicable methods for detecting multiple analytes from a single well might serve this purpose. The few assays developed so far based on multiplexed GPCR readouts are limited to only certain applications and usually rely on genetic manipulations hindering screening in native or native-like cellular systems. Here we describe a more generally applicable and HTS-compatible homogeneous assay based on the combination of fluorometric detection of [Ca2+] with subsequent homogeneous time-resolved fluorescence (HTRF) cAMP readout in the same well. Besides describing development and validation of the assay, using a cell line recombinantly expressing the human PTH1 receptor screening of a small library is also presented, demonstrating the robustness and HTS compatibility of the novel paradigm.

Discovery of Novel Tricyclic Thiazepine Derivatives as Anti-Drug-Resistant Cancer Agents by Combining Diversity-Oriented Synthesis and Converging Screening Approach.

An efficient discovery strategy by combining diversity-oriented synthesis and converging cellular screening is described. By a three-round screening process, we identified novel tricyclic pyrido[2,3-b][1,4]benzothiazepines showing potent inhibitory activity against paclitaxel-resistant cell line H460TaxR (EC50 < 1.0 μM), which exhibits much less toxicity toward normal cells (EC50 > 100 μM against normal human fibroblasts). The most active hits also exhibited drug-like properties suitable for further preclinical research. This redeployment of antidepressing compounds for anticancer applications provides promising future prospects for treating drug-resistant tumors with fewer side effects.

Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL), a Resistance Mechanism for Two Distinct Compound Classes.

MMV007564 is a novel antimalarial benzimidazolyl piperidine chemotype identified in cellular screens. To identify the genetic determinant of MMV007564 resistance, parasites were cultured in the presence of the compound to generate resistant lines. Whole genome sequencing revealed distinct mutations in the gene named Plasmodium falciparum cyclic amine resistance locus (pfcarl), encoding a conserved protein of unknown function. Mutations in pfcarl are strongly associated with resistance to a structurally unrelated class of compounds, the imidazolopiperazines, including KAF156, currently in clinical trials. Our data demonstrate that pfcarl mutations confer resistance to two distinct compound classes, benzimidazolyl piperidines and imidazolopiperazines. However, MMV007564 and the imidazolopiperazines, KAF156 and GNF179, have different timings of action in the asexual blood stage and different potencies against the liver and sexual blood stages. These data suggest that pfcarl is a multidrug-resistance gene rather than a common target for benzimidazolyl piperidines and imidazolopiperazines.

High‐Throughput Topographic, Mechanical, and Biological Screening of Multilayer Films Containing Mussel‐Inspired Biopolymers

A high‐content screening method to characterize multifunctional multilayer films that combine mechanical adhesion and favorable biological response is reported. Distinct combinations of nanostructured films are produced using layer‐by‐layer methodology and their morphological, physicochemical, and biological properties are analyzed in a single microarray chip. Inspired by the composition of the adhesive proteins in mussels, thin films containing dopamine‐modified hyaluronic acid are studied. Flat biomimetic superhydrophobic patterned chips produced by a bench‐top methodology are used for the build‐up of arrays of multilayer films. The wettability contrasts imprinted onto the chips are allowed to produce individual, position controlled, multilayer films in the wettable regions. The flat configuration of the chip permits to perform a series of nondestructive measurements directly on the individual spots. In situ adhesion properties are directly measured in each spot, showing that nanostructured films richer in dopamine promote the adhesion. In vitro tests show an enhanced cell adhesion for the films with more catechol groups. The advantages presented by this platform include ability to control the uniformity and size of the multilayers films, its suitability to be used as a new low cost toolbox and for high‐content cellular screening, and capability for monitoring in situ a variety of distinct material properties.