Novel Approaches to Drug Discovery in Signal Transduction

Cover illustration: This issue of BTJ contains three special articles featuring novel approaches to drug discovery in signal transduction. On the one hand side, there is a focus on cell-based assays for GPCRs – one of the main drug targets and technologies for label-free cell-based assays. On the other hand, it is shown how computational approaches are important to predict binding activity of chemical structures and thus support drug discovery. Image@Photodisc/Getty images.     

Engineered Co-culture Strategies Using Stem Cells for Facilitated Chondrogenic Differentiation and Cartilage Repair

Osteoarthritis (OA) is a chronic disease in elders and athletes due to limited regenerative capacities of cartilage tissues and subsequently insufficient recovery of damaged sites. Recent clinical treatments for OA have utilized progenitor cell-based therapies for cartilage tissue regeneration. Administration of a single type of cell population such as stem cells or chondrocytes does not guarantee a full recovery of cartilage defects. Therefore, current tissue engineering approaches using co-culture techniques have been developed to mimic complex and dynamic cellular interactions in native cartilage tissues and facilitate changes in cellular phenotypes into chondrogenesis. Therefore, this paper introduces recently developed co-culture systems using two major cell populations, mesenchymal stem cells (MSCs) and chondrocytes. Specifically, a series of examples to describe (1) synergistic in vitro activations of MSCs by paracrine signaling molecules from adult chondrocytes in co-culture systems and (2) functional in vivo tissue regeneration via co-administration of both cell types were reviewed. Based on these findings, it could be speculated that engineered co-culture systems using MSC/ chondrocyte is a promising and feasible cell-based OA therapy in clinical aspects.     

Micro- and Nanofluidics – Applications in Biotechnology

Cover illustration: Micro- and Nanofluidics – Applications in Biotechnology. This issue of BTJ edited by Hikmet Geckil and Utkan Demirci covers recent advances on BioMEMs (microelectromechanical systems used in biology) and lab-on-a-chip devices for cell and fluid manipulation, e.g. picoliter sequencing and PCR, as well as cytometry and imaging technologies. The cover image provided by Gurkan Yilmaz shows fluorescent particles moving inside a spiral channel in an dielectrophoretic particle separation experiment displayed by a fluorescent microscope. DOI: http://dx.doi.org/10.1002/biot.201000204     

Systems biology in drug discovery

The hope of the rapid translation of 'genes to drugs' has foundered on the reality that disease biology is complex, and that drug development must be driven by insights into biological responses. Systems biology aims to describe and to understand the operation of complex biological systems and ultimately to develop predictive models of human disease. Although meaningful molecular level models of human cell and tissue function are a distant goal, systems biology efforts are already influencing drug discovery. Large-scale gene, protein and metabolite measurements ('omics') dramatically accelerate hypothesis generation and testing in disease models. Computer simulations integrating knowledge of organ and system-level responses help prioritize targets and design clinical trials. Automation of complex primary human cell-based assay systems designed to capture emergent properties can now integrate a broad range of disease-relevant human biology into the drug discovery process, informing target and compound validation, lead optimization, and clinical indication selection. These systems biology approaches promise to improve decision making in pharmaceutical development.     

Biotech Reviews

Cover illustration: Biotech Reviews. This special issue, edited by Sophia Hober (Stockholm, Sweden) covers review articles on key areas of biotechnology, synthetic biology, metabolic engineering as well as protein and biofuel engineering, nanobiotechnology, siRNA therapeutics and food biotechnology. Cover image: SPDM/localization microscopy of nanostructures in human cell nuclei, H2A proteins (red) and Snf2H proteins (green) in a human osteosarcoma cell nucleus (U2OS cells), provided by Manuel Gunkel, Heidelberg, Germany, taken from the Review on superresolution imaging by Cremer et al. http://dx.doi.org/10.1002/biot.201100031 .     

The use of 3-D cultures for high-throughput screening: the multicellular spheroid model

Over the past few years, establishment and adaptation of cell-based assays for drug development and testing has become an important topic in high-throughput screening (HTS). Most new assays are designed to rapidly detect specific cellular effects reflecting action at various targets. However, although more complex than cell-free biochemical test systems, HTS assays using monolayer or suspension cultures still reflect a highly artificial cellular environment and may thus have limited predictive value for the clinical efficacy of a compound. Today's strategies for drug discovery and development, be they hypothesis free or mechanism based, require facile, HTS-amenable test systems that mimic the human tissue environment with increasing accuracy in order to optimize preclinical and preanimal selection of the most active molecules from a large pool of potential effectors, for example, against solid tumors. Indeed, it is recognized that 3-dimensional cell culture systems better reflect the in vivo behavior of most cell types. However, these 3-D test systems have not yet been incorporated into mainstream drug development operations. This article addresses the relevance and potential of 3-D in vitro systems for drug development, with a focus on screening for novel antitumor drugs. Examples of 3-D cell models used in cancer research are given, and the advantages and limitations of these systems of intermediate complexity are discussed in comparison with both 2-D culture and in vivo models. The most commonly used 3-D cell culture systems, multicellular spheroids, are emphasized due to their advantages and potential for rapid development as HTS systems. Thus, multicellular tumor spheroids are an ideal basis for the next step in creating HTS assays, which are predictive of in vivo antitumor efficacy.     

Automated high-content live animal drug screening using C. elegans expressing the aggregation prone serpin α1-antitrypsin Z

The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.     

Cellular Responses and Tissue Depots for Nanoformulated Antiretroviral Therapy

Long-acting nanoformulated antiretroviral therapy (nanoART) induces a range of innate immune migratory, phagocytic and secretory cell functions that perpetuate drug depots. While recycling endosomes serve as the macrophage subcellular depots, little is known of the dynamics of nanoART-cell interactions. To this end, we assessed temporal leukocyte responses, drug uptake and distribution following both intraperitoneal and intramuscular injection of nanoformulated atazanavir (nanoATV). Local inflammatory responses heralded drug distribution to peritoneal cell populations, regional lymph nodes, spleen and liver. This proceeded for three days in male Balb/c mice. NanoATV-induced changes in myeloid populations were assessed by fluorescence-activated cell sorting (FACS) with CD45, CD3, CD11b, F4/80, and GR-1 antibodies. The localization of nanoATV within leukocyte cell subsets was determined by confocal microscopy. Combined FACS and ultra-performance liquid chromatography tandem mass-spectrometry assays determined nanoATV carriages by cell-based vehicles. A robust granulocyte, but not peritoneal macrophage nanoATV response paralleled zymosan A treatment. ATV levels were highest at sites of injection in peritoneal or muscle macrophages, dependent on the injection site. The spleen and liver served as nanoATV tissue depots while drug levels in lymph nodes were higher than those recorded in plasma. Dual polymer and cell labeling demonstrated a nearly exclusive drug reservoir in macrophages within the liver and spleen. Overall, nanoART induces innate immune responses coincident with rapid tissue macrophage distribution. Taken together, these works provide avenues for therapeutic development designed towards chemical eradication of human immunodeficiency viral infection.     

Cover Picture: Biotechnology Journal 6/2010

Cover illustration: Biotech Methods and Advances. This issue of Biotechnology Journal covers methods for systems metabolic engineering as well as downstream processing. The cover image shows an artist's impression of a magnetic fusion protein consisting of antibody like particles (pale blue) and ferritin subunits (pale green) encapsulating an iron core (red). These “Magnetizable antibody-like proteins” are described by Dehal et al. pp. 596–605 (http://dx.doi.org/10.1002/biot.200900273). Art by Mark Bradshaw, Pixelweave 3D.     

Living in three dimensions

Research focused on deciphering the biochemical mechanisms that regulate cell proliferation and function has largely depended on the use of tissue culture methods in which cells are grown on two-dimensional (2D) plastic or glass surfaces. However, the flat surface of the tissue culture plate represents a poor topological approximation of the more complex three-dimensional (3D) architecture of the extracellular matrix (ECM) and the basement membrane (BM), a structurally compact form of the ECM. Recent work has provided strong evidence that the highly porous nanotopography that results from the 3D associations of ECM and BM nanofibrils is essential for the reproduction of physiological patterns of cell adherence, cytoskeletal organization, migration, signal transduction, morphogenesis, and differentiation in cell culture. In vitro approximations of these nanostructured surfaces are therefore desirable for more physiologically mimetic model systems to study both normal and abnormal functions of cells, tissues, and organs. In addition, the development of 3D culture environments is imperative to achieve more accurate cell-based assays of drug sensitivity, high-throughput drug discovery assays, and in vivo and ex vivo growth of tissues for applications in regenerative medicine.     

Development and validation of algorithms for measuring G-protein coupled receptor activation in cells using the LSC-based imaging cytometer platform.

BACKGROUND: A cell-based assay system (Transfluor) has been developed for measurement of G-protein coupled receptor (GPCR) activity by using cells transfected to express a fusion protein of arrestin plus green fluorescent protein (GFP) and the target GPCR. Upon agonist stimulation, the arrestin-GFP translocates to and binds the activated GPCR at the plasma membrane. The receptor/arrestin-GFP complexes then localize in clathrin-coated pits and/or intracellular vesicles. This redistribution of arrestin-GFP into condensed fluorescent spots is useful for visually monitoring the active status of GPCRs and its quantitation is possible with certain types of digital image analysis systems. METHODS: We designed two lines of image processing algorithms to carry out quantitative measurement of the arrestin-GFP movement on an inverted version of laser scanning cytometry (iCyte) as an imaging platform. We used a cell line expressing arrestin-GFP and the wild-type beta2-adrenergic receptor or a modified version of this receptor with enhanced affinity for arrestin. Each cell line was challenged with various concentrations of agonist. RESULTS: A dose-dependent signal was measured and half-maximal effective concentration values were obtained that agreed well with results determined by other methods previously reported. CONCLUSIONS: The results indicate that the combination of Transfluor, iCyte, and our algorithms is suitable for robust and pharmacologically relevant GPCR ligand exploration.     

Functional characterization of UCP1 in mammalian HEK293 cells excludes mitochondrial uncoupling artefacts and reveals no contribution to basal proton leak

Mechanistic studies on uncoupling proteins (UCPs) not only are important to identify their cellular function but also are pivotal to identify potential drug targets to manipulate mitochondrial energy transduction. So far, functional and comparative studies of uncoupling proteins in their native environment are hampered by different mitochondrial, cellular and genetic backgrounds. Artificial systems such as yeast ectopically expressing UCPs or liposomes with reconstituted UCPs were employed to address crucial mechanistic questions but these systems also produced inconsistencies with results from native mitochondria. We here introduce a novel mammalian cell culture system (Human Embryonic Kidney 293 - HEK293) to study UCP1 function. Stably transfected HEK293 cell lines were derived that contain mouse UCP1 at concentrations comparable to tissue mitochondria. In this cell-based test system UCP1 displays native functional behaviour as it can be activated with fatty acids (palmitate) and inhibited with purine nucleotides guanosine-diphosphate (GDP). The catalytic centre activity of the UCP1 homodimer in HEK293 is comparable to activities in brown adipose tissue supporting functionality of UCP1. Importantly, at higher protein levels than in yeast mitochondria, UCP1 in HEK293 cell mitochondria is fully inhibitable and does not contribute to basal proton conductance, thereby emphasizing the requirement of UCP1 activation for therapeutic purposes. These findings and resulting analysis on UCP1 characteristics demonstrate that the mammalian HEK293 cell system is suitable for mechanistic and comparative functional studies on UCPs and provides a non-confounding mitochondrial, cellular and genetic background.     

Unveiling the molecular crosstalk in a human induced pluripotent stem cell-derived cardiac model

In vitro cell-based models that better mimic the human heart tissue are of utmost importance for drug development and cardiotoxicity testing but also as tools to understand mechanisms related with heart disease at cellular and molecular level. Besides, the implementation of analytical tools that allow the depiction and comprehensive understanding of the molecular mechanisms of the crosstalk between the different cell types is also relevant. In this work, we implemented a human cardiac tissue-like in vitro model, derived from human-induced pluripotent stem cell (hiPSC), and evaluated the relevance of the cell–cell communication between the two of the most representative cell populations of the human heart: cardiomyocytes (hiPSC-CM) and endothelial cells (hiPSC-EC). We observed that heterotypic cell communication promotes: (a) structural maturation of hiPSC-CM and (b) deposition of several extracellular matrix components (such as collagens and fibronectin). Overall, the toolbox of analytical techniques used in our study not only enabled us to validate previous reports from the literature on the importance of the presence of hiPSC-EC on hiPSC-CM maturation, but also bring new insights on the molecular mechanisms involved in the communication between these two cell types when cocultured in vitro.     

Polymer and Textile Biotech

Cover illustration: Polymer and Textile Biotech. Polymer biotechnology has led to highly functional materials with wideranging applications from textiles to medicine. This includes protective clothing, which could for example be used for future space trips, as well as for tissue engineering. This special issue of BTJ, edited by Georg Guebitz, Graz University of Technology, Austria (co-edited by Giuliano Freddi and Artur Cavaco-Paulo) provides a broad overview of novel biotechnological approaches for processing of materials and textiles. Cover image: Astronaut © Lasse Kristensen – Fotolia.com     

Engineering stem cell niches in bioreactors

Stem cells, including embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells and amniotic fluid stem cells have the potential to be expanded and differentiated into various cell types in the body. Efficient differentiation of stem cells with the desired tissue-specific function is critical for stem cell-based cell therapy, tissue engineering, drug discovery and disease modeling. Bioreactors provide a great platform to regulate the stem cell microenvironment, known as “niches”, to impact stem cell fate decision. The niche factors include the regulatory factors such as oxygen, extracellular matrix (synthetic and decellularized), paracrine/autocrine signaling and physical forces (i.e., mechanical force, electrical force and flow shear). The use of novel bioreactors with precise control and recapitulation of niche factors through modulating reactor operation parameters can enable efficient stem cell expansion and differentiation. Recently, the development of microfluidic devices and microbioreactors also provides powerful tools to manipulate the stem cell microenvironment by adjusting flow rate and cytokine gradients. In general, bioreactor engineering can be used to better modulate stem cell niches critical for stem cell expansion, differentiation and applications as novel cell-based biomedicines. This paper reviews important factors that can be more precisely controlled in bioreactors and their effects on stem cell engineering.     

Cellular & Molecular Bioengineering

Cover illustration: This issue includes articles presented at the International Conference on Cellular and Molecular Bioengineering (ICCMB) held in Singapore, August 2–4, 2010 and other articles on this topic, featuring stem cell stimulation, nanoparticle delivery and primary cell culture. The cover image shows a stereoscopic image of a Xenopus laevis tadpole 72 h after fertilization. This embryo was injected with quantum dots by near infrared laser injection as reported by Umanzor-Alvarez et al., in this issue. http://dx.doi.org/10.1002/biot.201000205     

Journal of Cellular Biochemistry: Volume 114, Number 8, August, 2013

Cover: Fibroblasts within whole mouse connective tissue actively respond to static stretching of the tissue by expanding and remodeling their cytoskeleton. The tissue was stretched for two hours ex vivo, then fixed and immunohistochemically stained for β‐tubulin (green) and countersigned with DAPI (blue). Cover designed by Priscilla Vazquez.     

Can human embryonic stem cells contribute to the discovery of safer and more effective drugs?

Few scientific achievements have received such irresistible attention from scientists, clinicians, and the general public as the ability of human embryonic stem (hES) cells to differentiate into functional cell types for regenerative medicine. The most immediate benefit of neurons, cardiomyocytes, and insulin-secreting cells derived from hES cells, however, may reside in their application in drug discovery and toxicology. The availability of renewable human cells with functional similarities to their in vivo counterparts is the first landmark for a new generation of cell-based assays. The development of cell-based assays using human cells that are physiological targets of drug activity will increase the robustness of target validation and efficacy, high-throughput screening (HTS), structure-activity relationship (SAR), and should introduce safer drugs into clinical trials and the marketplace. The pluripotency of embryonic stem cells, that is, the capacity to generate multiple cell types, is a novel path for the discovery of 'regenerative drugs', the pursuit of small molecules that promote tissue repair (neurogenesis, cardiogenesis) or proliferation of resident stem cells in different organs, thus creating drugs that work by a novel mechanism.     

Bioprocess Scale-up/down

Cover illustration: Scale-up and scale-down strategies are a key for successful implementation of bioprocesses in industry. This issue of Biotechnology Journal, edited by Peter Neubauer (TU Berlin, Germany), features articles on methods for improved scale-up and -down strategies, e.g. measuring CO₂ gradients, drop breakage analysis and study of metabolic stress. One review article by Alvin Nienow presents strategies for improved beer production (http://dx.doi.org/10.1002/biot.201000414 ). Cover image: Outdoor fermentation vessels at a Spanish brewery. ©Olaf Hendel (Versuchs- und Lehranstalt für Brauerei in Berlin (VLB) e.V.).     

Evaluation of protein kinase activities of cell lysates using peptide microarrays based on surface plasmon resonance imaging

We developed a peptide microarray based on surface plasmon resonance (SPR) imaging for monitoring protein kinase activities in cell lysates. The substrate peptides of kinases were tethered to the microarray surface modified with a self-assembled monolayer of an alkanethiol with triethylene glycol terminus to create a low nonspecific binding surface. The phosphorylation of the substrate peptides immobilized on the surface was detected with the following phosphate specific binders by amplifying SPR signals: anti-phosphotyrosine antibody for tyrosine kinases and Phos-tag biotin (a phosphate-specific ligand with biotin tag) for serine/threonine kinases. Using the microarray, 9 kinds of protein kinases were evaluated as a pattern of phosphorylation of 26 kinds of substrate peptides. The pattern was unique for each protein kinase. The microarray could be used to evaluate the inhibitory activities of kinase inhibitors. The microarray was applied successfully for kinase activity monitoring of cell lysates. The chemical stimuli responsive activity changes of protein kinases in cell lysates could also be monitored by the peptide microarray. Thus, the peptide microarray based on SPR imaging would be applicable to cell-based drug discovery, diagnosis using tissue lysates, and biochemical studies to reveal signal transduction pathways.