Terry Reisine

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.

Drug discovery and the human kinome: Recent trends

A major new trend in drugs targeted at protein kinases is the discovery of allosteric modulators. These compounds differ from ATP-centric drugs in that they do not compete with ATP for binding to the catalytic domain, generally acting by inducing conformational changes to modulate activity. They could provide a number of advantages over more classical protein kinase drugs. For example, they are likely to be more selective, since they bind to unique regions of the kinase and may be useful in overcoming resistance that has developed to drugs that compete with ATP. They offer the ability of activating the kinases either by removing factors that inhibit kinase activity or by simply producing changes to the enzyme to foster catalytic activity. Furthermore, they provide more subtle modulation of kinase activity than simply blocking ATP access to inhibit activity. One hurdle to overcome in discovering these compounds is that allosteric modulators may need to inhibit protein-protein interactions; generally difficult to accomplish with small molecules. Despite the technical problems of identifying allosteric modulators, major gains have been made in identifying allosteric inhibitors and activators of the growth factor receptors as well as soluble tyrosine and serine/threonine kinases and some of these drugs are now in various stages of clinical trials. This review will focus on the discovery of novel allosteric modulators of protein kinases and drug discovery approaches that have been employed to identify such compounds.

Primary Cells and Stem Cells in Drug Discovery: Emerging Tools for High-Throughput Screening

Many drug discovery screening programs employ immortalized cells, recombinantly engineered to express a defined molecular target. Several technologies are now emerging that render it feasible to employ more physiologically, and clinically relevant, cell phenotypes. Consequently, numerous approaches use primary cells, which retain many functions seen in vivo, as well as endogenously expressing the target of interest. Furthermore, stem cells, of either embryonic or adult origin, as well as those derived from differentiated cells, are now finding a place in drug discovery. Collectively, these cells are expanding the utility of authentic human cells, either as screening tools or as therapeutics, as well as providing cells derived directly from patients. Nonetheless, the growing use of phenotypically relevant cells (including primary cells or stem cells) is not without technical difficulties, particularly when their envisioned use lies in high-throughput screening (HTS) protocols. In particular, the limited availability of homogeneous primary or stem cell populations for HTS mandates that novel technologies be developed to accelerate their adoption. These technologies include detection of responses with very few cells as well as protocols to generate cell lines in abundant, homogeneous populations. In parallel, the growing use of changes in cell phenotype as the assay readout is driving greater use of high-throughput imaging techniques in screening. Taken together, the greater availability of novel primary and stem cell phenotypes as well as new detection technologies is heralding a new era of cellular screening. This convergence offers unique opportunities to identify drug candidates for disorders at which few therapeutics are presently available.

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.

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.

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.

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.

GPCRs Revisited: New Insights Lead to Novel Drugs

GPCRs play a critical role in human physiology and are a prime target for drug discovery globally. Novel insights into the functions of GPCRs are providing unique approaches to modulate these proteins to generate unique drug candidates. Next generation ligands include those with novel pharmacologies such as allosteric regulators as well pepducins, that affect the interaction of GPCRs with G proteins, to either block selective receptor signaling pathways or mimic the actions of intracellular domains of receptors, thereby activating GPCRs to signal selectively to intracellular pathways. We will review these new concepts and then discuss how they may be exploited using modern discovery technologies to provide novel drug candidates for the future.

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.