Human iPS Cell-Derived Patient Tissues and 3D Cell Culture Part 2: Spheroids, Organoids, and Disease Modeling.

Human induced pluripotent stem cells (HiPSCs) provide several advantages for drug discovery, but principally they provide a source of clinically relevant tissue. Furthermore, the use of HiPSCs cultured in three-dimensional (3D) systems, as opposed to traditional two-dimensional (2D) culture approaches, better represents the complex tissue architecture in vivo. The use of HiPSCs in 3D spheroid and organoid culture is now growing, but particularly when using myocardial, intestinal enteric nervous system, and retinal cell lines. However, organoid cell culture is perhaps making the most notable impact in research and drug discovery, in which 3D neuronal cell cultures allow direct modeling of cortical cell layering and neuronal circuit activity. Given the specific degeneration seen in discrete neuronal circuitry in Alzheimer's disease (AD) and Parkinson's disease (PD), HiPSC culture systems are proving to be a major advance. In the present review, the second part of a two-part review, we discuss novel methods in which 3D cell culture systems (principally organoids) are now being used to provide insights into disease mechanisms. (The use of HiPSCs in target identification was reviewed in detail in Part 1.).

Human iPS Cell-Derived Patient Tissues and 3D Cell Culture Part 1: Target Identification and Lead Optimization.

Human-induced pluripotent stem cells (HiPSCs), and new technologies to culture them into functional cell types and tissues, are now aiding drug discovery. Patient-derived HiPSCs can provide disease models that are more clinically relevant and so more predictive than the currently available animal-derived or tumor cell-derived cells. These cells, consequently, exhibit disease phenotypes close to the human pathology, particularly when cultured under conditions that allow them to recapitulate the tissue architecture in three-dimensional (3D) systems. A key feature of HiPSCs is that they can be cultured under conditions that favor formation of multicellular spheroids or organoids. By culturing and differentiating in systems mimicking the human tissue in vivo, the HiPSC microenvironment further reflects patient in vivo physiology, pathophysiology, and ultimately pharmacological responsiveness. We assess the rationale for using HiPSCs in several phases of preclinical drug discovery, specifically in disease modeling, target identification, and lead optimization. We also discuss the growing use of HiPSCs in compound lead optimization, particularly in profiling compounds for their potential metabolic liability and off-target toxicities. Collectively, we contend that both approaches, HiPSCs and 3D cell culture, when used in concert, have exciting potential for the development of novel medicines.

Three-Dimensional Cell Cultures in Drug Discovery and Development.

The past decades have witnessed significant efforts toward the development of three-dimensional (3D) cell cultures as systems that better mimic in vivo physiology. Today, 3D cell cultures are emerging, not only as a new tool in early drug discovery but also as potential therapeutics to treat disease. In this review, we assess leading 3D cell culture technologies and their impact on drug discovery, including spheroids, organoids, scaffolds, hydrogels, organs-on-chips, and 3D bioprinting. We also discuss the implementation of these technologies in compound identification, screening, and development, ranging from disease modeling to assessment of efficacy and safety profiles.

Drug Discovery Goes Three-Dimensional: Goodbye to Flat High-Throughput Screening?

Immortalized cells, generated from two-dimensional cell culture techniques, are widely used in compound screening, lead optimization, and drug candidate selection. However, such cells lack many characteristics of cells in vivo. This could account for the high failure rates of lead candidates in clinical evaluation. New approaches from cell biology, materials science, and bioengineering are increasing the utility of three-dimensional (3D) culture. These approaches have become more compatible with automation and, thus, provide more physiologically relevant cells for high-throughput/high-content screening, notably in oncology drug discovery. Techniques range from simple 3D spheroids, comprising one or more cell types, to complex multitissue organoids cultured in extracellular matrix gels or microfabricated chips. Furthermore, each approach can be applied to stem cells, such as induced pluripotent stem cells, thereby providing additional phenotypic relevance and the exciting potential to enable screening in disease-specific cell types.

Overview of muscarinic receptor subtypes.

The physiological role of muscarinic receptors is highly complex and, although not completely understood, has become clearer over the last decade. Recent pharmacological evidence with novel compounds, together with data from transgenic mice, suggests that all five subtypes have defined functions in the nervous system as well as mediating the non neuronal, hormonal actions of acetylcholine. Numerous novel agonists, allosteric regulators, and antagonists have now been identified with authentic subtype specificity in vitro and in vivo. These compounds provide additional pharmacological opportunities for selective subtype modulation as well as a new generation of muscarinic receptor-based therapeutics.

Screening for compounds that modulate epigenetic regulation of the transcriptome: an overview.

Epigenetic control of the transciptome is a complex and highly coordinated cellular process. One critical mechanism involves DNA methylation, mediated by distinct but related DNA methyltransferases (DNMTs). Although several DNMT inhibitors are available, most are nonselective; selective DNMT inhibitors, therefore, could be optimal as therapeutics, as well acting as chemical probes to elucidate the fundamental biology of individual DNMTs. DNA methylation is a stable chemical modification, yet posttranslational modification of histones is transitory, with reversible effects on gene expression. Histone posttranslational modifications influence access of transcription factors to DNA target sites to affect gene activity. Histones are regulated by several enzymes, including acetylases (HATs), deacetylases (HDACs), methyltransferases (HMTs), and demethylases (HDMTs). Generally, HATs activate, whereas HDACs suppress gene activity. Specifically, HMTs and HDMTs can either activate or inhibit gene expression, depending on the site and extent of the methylation pattern. There is growing interest in drugs that target enzymes involved in epigenetic control. Currently, a range of high-throughput screening (HTS) technologies are used to identify selective compounds against these enzymes. This review focuses on the rationale for drug development of these enzymes, as well the utility of HTS methods used in identifying and optimizing novel selective compounds that modulate epigenetic control of the human transcriptome.

Human kinome drug discovery and the emerging importance of atypical allosteric inhibitors.

IMPORTANCE OF THE FIELD: Protein kinases are important targets for drug discovery because they possess critical roles in many human diseases. Several protein kinase inhibitors have entered clinical development with others having already been approved for treating a host of diseases. However, many kinase inhibitors suffer from non-selectivity because they interact with the ATP binding region which has similar structures amongst the protein kinases and this non-selectivity sometimes can cause side effects. As a consequence, there is much interest in developing drugs that inhibit kinases through non-classical mechanisms with the hope of avoiding the side effects of previous kinase drugs. AREAS COVERED IN THIS REVIEW: This review covers emerging information on kinase biology and discusses new approaches to design selective inhibitors that do not compete with ATP. WHAT THE READER WILL GAIN: The reader will gain a better understanding of the importance of the field of allosteric inhibitor drug discovery and how this has required the adoption of a new generation of high-throughput screening techniques. TAKE HOME MESSAGE: Discovery and development of allosteric modulators will result in a family of novel kinase therapies with greater selectivity and more varied ways to control activity of disease causing kinase targets.

New insights into GPCR function: implications for HTS.

G protein-coupled receptors (GPCRs) are a large family of proteins that represent targets for approximately 40% of all approved drugs. They possess unique structural motifs that allow them to interact with a diverse series of extracellular ligands, as well as intracellular signaling proteins, such as G proteins, RAMPs, arrestins, and indeed other receptors. Extensive efforts are under way to discover new generations of drugs against GPCRs with unique targeted therapeutic uses, including "designer" drugs such as allosteric regulators, inverse agonists, and drugs targeting hetero-oligomeric complexes. This has been facilitated by the development of new screening technologies to identify novel drugs against both known and orphan GPCRs.

The current status of drug discovery against the human kinome.

Protein kinases are important targets in drug discovery programs aimed at treating many devastating diseases, including cancer, autoimmune disorders, diabetes, and neurological disorders. Most "classical" drug discovery efforts employ rational drug design methods based upon structural information to identify compounds targeting the enzyme catalytic domain. Novel information on kinase biology is opening up other approaches in the design of selective inhibitors that may provide more subtle modulation of these drug discovery targets. The identification of such modulators requires adoption of a new generation of high-throughput screening techniques. These approaches will allow measurement of conformational changes in kinases, as well as protein-protein interactions via assessment of functional responses such as cellular translocation. Therefore a range of novel techniques, together with the understanding that numerous "orphan" kinases will provide targets for therapeutics, suggests that a new era of kinase therapies is rapidly emerging.

Photoproteins: important new tools in drug discovery.

The G protein-coupled receptor (GPCR) family is a major target for drug discovery, and most, if not all, GPCRs can couple to Ca2+ signaling. Consequently, there are a number of cellbased, primary, high-throughput screening (HTS) assays used for drug discovery that assess changes in intracellular Ca2+ as a functional readout of GPCR activation. Historically, changes in intracellular Ca2+ levels have been readily detected using fluorescent dyes that emit light in proportion to changes in intracellular Ca2+ concentration. An alternative approach to indirectly measure changes in Ca2+ concentrations involves the use of recombinantly expressed biosensor photoproteins, of which aequorin is a prototypic example. These biosensors have the advantage that they provide an intense luminescent signal in response to elevations in intracellular Ca2+. This exquisite sensitivity, the high signal-to-noise ratios, and the ability to target expression to discrete subcellular sites (in order to detect Ca2+ microdomains) have made photoproteins a principal choice in a wide range of GPCR drug discovery programs. Photoproteins are also finding increasing use in detecting activation of other molecular target classes such as ligand-gated ion channels and transporters. This review focuses upon the use of calcium photoproteins principally for use in GPCR drug discovery and HTS.

The use of immortalized cell lines in GPCR screening: the good, bad and ugly.

For most membrane-bound molecular targets, including G protein linked receptors (GPCRs), the optimal approach in drug discovery involves the use of cell based high throughput screening (HTS) technologies to identify compounds that modulate target activity. Most GPCRs have been cloned and can therefore be routinely expressed in immortalized cell lines. These cells can be easily and rapidly grown in unlimited quantities making them ideal for use in current HTS technologies. A significant advantage of this approach is that immortalized recombinant cells provide a homogenous background for expression of the target which greatly facilitates consistency in screening, thus allowing for a better understanding of the mechanism of action of the interacting compound or drug. Nonetheless, it is now evident that numerous disparities exist between the physiological environment of screening systems using recombinant cells and natural tissues. This has lead to a problem in the validity of the pharmacological data obtained using immortalized cells in as much as such cells do not always reflect the desired clinical efficacy and safety of the compounds under examination. This brief review discusses these issues and describes how they influence the discovery of drugs using modern HTS.

An overview of drug screening using primary and embryonic stem cells.

Cellular technologies are widely used in drug discovery to treat human diseases. Most studies involve the expression of recombinant targets in immortalized cells and measure drug interactions using simple, quantifiable responses. Such cells are also amenable to high throughput screening (HTS) methods. However, the cell phenotype employed in HTS is often determined by the assay technology available, rather than the physiological relevance of the cell background. They are, therefore, suboptimal surrogates for cells that accurately reflect human diseases. Consequently, there is growing interest in adopting primary and embryonic stem cells in drug discovery. Primary cells are already used in secondary screening assays in conjunction with confocal imaging techniques, as well as in target validation studies employing, for example, gene silencing approaches. Stem cells can be grown in unlimited quantities and can be derived from transgenic animals engineered to express disease causing proteins better coupling the molecular target with function in vivo. Human stem cells also offer unique opportunities for drug discovery in that they can be directed to specific phenotypes thus providing a framework to identify tissue-selective agents. Organizing stem cells into networks resembling those in native tissues, potentially returns drug discovery back to the highly successful pharmacological methods of the past, in which organ and tissue based systems were used, but with the advantage that they can be utilized using modern HTS technologies. This emerging area will lead to discovery of compounds whose effect in vivo is more predictable thereby increasing the efficiency of drugs that ameliorate human disease.

The use of AlphaScreen technology in HTS: current status.

AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) is versatile assay technology developed to measuring analytes using a homogenous protocol. This technology is an example of a bead-based proximity assay and was developed from a diagnostic assay technology known as LOCI (Luminescent Oxygen Channeling Assay). Here, singlet oxygen molecules, generated by high energy irradiation of Donor beads, travel over a constrained distance (approx. 200 nm) to Acceptor beads. This results in excitation of a cascading series of chemical reactions, ultimately causing generation of a chemiluminescent signal.In the past decade, a wide variety of applications has been reported, ranging from detection of analytes involved in cell signaling, including protein:protein, protein:peptide, protein:small molecule or peptide:peptide interactions. Numerous homogeneous HTS-optimized assays have been reported using the approach, including generation of second messengers (such as accumulation of cyclic AMP, cyclic GMP, inositol [1, 4, 5] trisphosphate or phosphorylated ERK) from liganded GPCRs or tyrosine kinase receptors, post-translational modification of proteins (such as proteolytic cleavage, phosphorylation, ubiquination and sumoylation) as well as protein-protein and protein-nucleic acid interactions.Recently, the basic AlphaScreen technology was extended in that the chemistry of the Acceptor bead was modified such that emitted light is more intense and spectrally defined, thereby markedly reducing interference from biological fluid matrices (such as trace hemolysis in serum and plasma). In this format, referred to as AlphaLISA, it provides an alternative technology to classical ELISA assays and is suitable for high throughput automated fluid dispensing and detection systems.Collectively, AlphaScreen and AlphaLISA technologies provide a facile assay platform with which one can quantitate complex cellular processes using simple no-wash microtiter plate based assays. They provide the means by which large compound libraries can be screened in a high throughput fashion at a diverse range of therapeutically important targets, often not readily undertaken using other homogeneous assay technologies. This review assesses the current status of the technology in drug discovery, in general, and high throughput screening (HTS), in particular.

Muscarinic acetylcholine receptors: mutant mice provide new insights for drug development.

Muscarinic acetylcholine receptors (mAChRs), M(1)-M(5), regulate the activity of numerous fundamental central and peripheral functions. The lack of small-molecule ligands that can block or activate specific mAChR subtypes with high selectivity has remained a major obstacle in defining the roles of the individual receptor subtypes and in the development of novel muscarinic drugs. Recently, phenotypic analysis of mutant mouse strains deficient in each of the five mAChR subtypes has led to a wealth of new information regarding the physiological roles of the individual receptor subtypes. Importantly, these studies have identified specific mAChR-regulated pathways as potentially novel targets for the treatment of various important disorders including Alzheimer's disease, schizophrenia, pain, obesity and diabetes.

Emerging concepts of guanine nucleotide-binding protein-coupled receptor (GPCR) function and implications for high throughput screening.

Guanine nucleotide binding protein (G protein) coupled receptors (GPCRs) comprise one of the largest families of proteins in the human genome and are a target for 40% of all approved drugs. GPCRs have unique structural motifs that allow them to interact with a wide and diverse series of extracellular ligands, as well as intracellular proteins, G proteins, receptor activity-modifying proteins, arrestins, and indeed other receptors. This distinctive structure has led to numerous efforts to discover drugs against GPCRs with targeted therapeutic uses. Such "designer" drugs currently include allosteric regulators, inverse agonists, and drugs targeting hetero-oligomeric complexes. Moreover, the large family of orphan GPCRs provides a rich and novel field of targets to discover drugs with unique therapeutic properties. The numerous technologies to discover GPCR drugs have also greatly advanced over the years, facilitating compound screening against known and orphan GPCRs, as well as in the identification of unique designer GPCR drugs. Indeed, high throughput screening (HTS) technologies employing functional cell-based approaches are now widely used. These include measurement of second messenger accumulation such as cyclic AMP, calcium ions, and inositol phosphates, as well as mitogen-activated protein kinase activation, protein-protein interactions, and GPCR oligomerization. This review focuses on how the improved understanding of the molecular pharmacology of GPCRs, coupled with a plethora of novel HTS technologies, is leading to the discovery and development of an entirely new generation of GPCR-based therapeutics.

Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery.

Many cell-based assays interrogating cell pathway activation employ protocols that require microscopic imaging techniques. However, such assays are not in general widely adopted for primary screening. Protein complementation, particularly of enzymes, provides an alternative approach for cell pathway analysis, with a principal advantage that is amenable to high throughput screening using microtiter plate protocols. Notably, alpha complementation of the enzyme beta-galactosidase has been exploited as a technology in this regard, using substrates that generates luminescent signals. This review describes the various uses of this flexible technology to cell-based assay development.

Using ligand-induced conformational change to screen for compounds targeting G-protein-coupled receptors.

The authors describe a novel drug strategy designed as a primary screen to discover either antagonist or agonist compounds targeting G-protein-coupled receptors (GPCRs). The incorporation of a nuclear localization sequence (NLS, a 5 amino acid substitution), in a location in helix 8 of the GPCR structure, resulted in ligand-independent receptor translocation from the cell surface to the nucleus. Blockade of the GPCR-NLS translocation from the cell surface was achieved by either antagonist or agonist treatments, each achieving their result in a sensitive concentration-dependent manner. GPCR-NLS translocation and blockade occurred regardless of the identity of the G-protein-coupling, and thus this assay is also ideally suited for identification of compounds targeting orphan GPCRs. The GPCR-NLS trafficking was visualized by fusion to fluorescent detectable proteins. Quantification of this effect was measured by determining the density of cell surface receptors, using enzyme fragment complementation in a manner suitable for high-throughput screening. Thus, the authors have developed a cellular assay for GPCRs suitable for compound screening without requiring prior identification of an agonist or knowledge of G-protein-coupling.