Kenneth A. Giuliano

High-Content Screening: A New Approach to Easing Key Bottlenecks in the Drug Discovery Process

Recent improvements in target discovery and high throughput screening (HTS) have increased the pressure at key points along the drug discovery pipeline. High-content screening (HCS) was developed to ease bottlenecks that have formed at target validation and lead optimization points in the pipeline. HCS defines the role of targets in cell functions by combining fluorescence-based reagents with the ArrayScan™ System to automatically extract temporal and spatial information about target activities within cells. The ArrayScan System is a tabletop instrument that includes optics for subcellular resolution of fluorescence signals from many cells in a field within a well of a microtiter plate. One demonstrated application is a high-content screen designed to measure the drug-induced transport of a green fluorescent protein-human glucocorticoid receptor chimeric protein from the cytoplasm to the nucleus of human tumor cells. A high-content screen is also described for the multiparametric measurement of apoptosis. This single screen provides measurements of nuclear size and shape changes, nuclear DNA content, mitochondrial potential, and actin-cytoskeletal rearrangements during drug-induced programmed cell death. The next generation HCS system is a miniaturized screening platform, the CellChip™ System, that will increase the throughput of HCS, while integrating HCS with HTS on the same platform.

Advances in high content screening for drug discovery.

Cell-based target validation, secondary screening, lead optimization, and structure-activity relationships have been recast with the advent of HCS. Prior to HCS, a computational approach to the characterization of the functions of specific target proteins and other cellular constituents, along with whole-cell functions employing fluorescence cell-based assays and microscopy, required extensive interaction among the researcher, instrumentation, and software tools. Early HCS platforms were instrument-centric and addressed the need to interface fully automated fluorescence microscopy, plate-handling automation, and seamless image analysis. HCS has since evolved into an integrated solution for accelerated drug discovery by encompassing the workflow components of assay and reagent design, robust instrumentation for automated fixed-end-point and live cell kinetic analysis, generalized and specific BioApplication software (Cellomics, Pittsburgh, PA) modules that produce information on drug responses from cell image data, and informatics/bioinformatics solutions that build knowledge from this information while providing a means to globalize HCS throughout an entire organization. This review communicates how these recent advances are incorporated into the drug discovery workflow by presenting a real-world use case.

The Benzo[c]phenanthridine Alkaloid, Sanguinarine, Is a Selective, Cell-active Inhibitor of Mitogen-activated Protein Kinase Phosphatase-1

Mitogen-activated protein kinase phosphatase-1 (MKP-1) is a dual specificity phosphatase that is overexpressed in many human tumors and can protect cells from apoptosis caused by DNA-damaging agents or cellular stress. Small molecule inhibitors of MKP-1 have not been reported, in part because of the lack of structural guidance for inhibitor design and definitive assays for MKP-1 inhibition in intact cells. Herein we have exploited a high content chemical complementation assay to analyze a diverse collection of pure natural products for cellular MKP-1 inhibition. Using two-dimensional Kolmogorov-Smirnov statistics, we identified sanguinarine, a plant alkaloid with known antibiotic and antitumor activity but no primary cellular target, as a potent and selective inhibitor of MKP-1. Sanguinarine inhibited cellular MKP-1 with an IC50 of 10 μm and showed selectivity for MKP-1 over MKP-3. Sanguinarine also inhibited MKP-1 and the MKP-1 like phosphatase, MKP-L, in vitro with IC50 values of 17.3 and 12.5 μm, respectively, and showed 5–10-fold selectivity for MKP-3 and MKP-1 over VH-1-related phosphatase, Cdc25B2, or protein-tyrosine phosphatase 1B. In a human tumor cell line with high MKP-1 levels, sanguinarine caused enhanced ERK and JNK/SAPK phosphorylation. A close congener of sanguinarine, chelerythrine, also inhibited MKP-1 in vitro and in whole cells, and activated ERK and JNK/SAPK. In contrast, sanguinarine analogs lacking the benzophenanthridine scaffold did not inhibit MKP-1 in vitro or in cells nor did they cause ERK or JNK/SAPK phosphorylation. These data illustrate the utility of a chemical complementation assay linked with multiparameter high content cellular screening.

Fluorescent-protein biosensors: New tools for drug discovery

Recent improvements in target discovery and high-throughput screening have increased the pressure at key points along the drug-discovery pipeline. High-content screening was developed to ease the bottlenecks formed at the target-validation and lead-optimization points, and a new generation of reagents that report on specific molecular processes in living cells (fluorescent-protein biosensors) have been important in its development. Creative designs of fluorescent-protein biosensors have emerged and been used to measure the molecular dynamics of macromolecules, metabolites and ions. Recent applications of fluorescent-protein biosensors to biological problems have provided a foundation for their use in biotechnology.

Streamlining the Drug Discovery Process by Integrating Miniaturization, High Throughput Screening, High Content Screening, and Automation on the CellChip™ System

A major bottleneck to the early stages of drug discovery is the absence of integration of high throughput screening (HTS) with smarter assays that screen “hits” from HTS to identify leads (High content screening, HCS). We propose a solution using novel fluorescent engineered protein biosensors integrated into a miniaturized live-cell-based screening platform (CellChip™ System) that markedly shortens the early drug discovery process. Microarrays of selectively localized living cells, containing engineered fluorescent biosensors, serve to integrate HTS and HCS onto a single platform. HTS “hits” are identified using one biosensor while reading the whole chip array of cells. The high-biological content information is then obtained from probing target activity at inter-cellular, sub-cellular and molecular levels in the “hit” wells. HCS assays yield temporal-spatial dynamic maps of the drug-target interaction within each living cell. We predict that a new platform incorporating HTS and HCS assays that are automated, miniaturized, and information-rich will dramatically improve the decision making process in the pharmaceutical industry and optimize lead compounds during the early part of the drug discovery process. There is an opportunity to establish a new paradigm for drug discovery based on integration of fluorescence technology, micropatterning of living cells, automated optical detection and data analysis, and a new generation of knowledge building bioinformatics approaches. The technology will have an expansive impact spanning the fields of drug discovery, biomedical research, environmental monitoring, life sciences, and clinical diagnostics. The integrated CellChip™ Platform with miniaturized tissue-specific microarrayed cells capable of providing inter-cellular and sub-cellular spatio-temporal information in response to drug-cell, toxin-cell, or pathogen-cell interactions will serve to enhance the decision making process in drug discovery, toxicology, and clinical diagnostics.

High-Content Screening with siRNA Optimizes a Cell Biological Approach to Drug Discovery: Defining the Role of P53 Activation in the Cellular Response to Anticancer Drugs

Deciphering the effects of compounds on molecular events within living cells is becoming an increasingly important component of drug discovery. In a model application of the industrial drug discovery process, the authors profiled a panel of 22 compounds using hierarchical cluster analysis of multiparameter high-content screening measurements from nearly 500,000 cells per microplate. RNAi protein knockdown methodology was used with high-content screening to dissect the effects of 2 anticancer drugs on multiple target activities. Camptothecin activated p53 in A549 lung carcinoma cells pretreated with scrambled siRNA, exhibited concentration-dependent cell cycle blocks, and induced moderate microtubule stabilization. Knockdown of camptothecin-induced p53 protein expression with p53 siRNA inhibited the G1/S blocking activity of the drug and diminished its microtubule-stabilizing activity. Paclitaxel activated p53 protein at low concentrations but exhibited G2/M cell cycle blocking activity at higher concentrations where microtubules were stabilized. In cells treated with p53 siRNA, paclitaxel failed to activate p53 protein, but the knockdown did not have a significant effect on the ability of paclitaxel to stabilize microtubules or induce a G2/M cell cycle block. Thus, this model application of the use of RNAi technology within the context of high-content screening shows the potential to provide massive amounts of combinatorial cell biological information on the temporal and spatial responses that cells mount to treatment by promising therapeutic candidates.

[1] Light-optical-based reagents for the measurement and manipulation of ions, metabolites, and macromolecules in living cells

Publisher Summary The chemical processes that comprise living organisms are the result of the precise orchestration of ions, metabolites, macromolecules, macromolecular assemblies, and organelles in time and space. The regulation of free ion concentration is crucial to processes that include the maintenance of membrane potential, the control of biochemical energetics, and the mediation of signal transduction. This chapter describes the progress made in the design and use of fluorescent indicators of the major intracellular free ion concentrations. The development of fluorescent analogs of macromolecules, fluorogenic substrates, and protein-based optical biosensors is discussed in the chapter. The chapter also discusses the progress and the prospects of what they believe to be an exciting application of optical-based reagents––the manipulation of specific molecular events using photomodulated ions, metabolites, and macromolecules.

Immunofluorescence signal amplification by the enzyme-catalyzed deposition of a fluorescent reporter substrate (CARD).

Progress has been made in improving the immunohistochemical detection of antigens for imaging and flow cytometry. We report the synthesis of a novel fluorescent horseradish peroxidase substrate, Cy3.29-tyramide, and its application in an enzyme-based signal amplification system, catalyzed reporter deposition (CARD). The catalyzed deposition of Cy3.29-tyramide was used to detect cell surface markers such as CD8 and CD25 on tonsil tissue and human lymphocytes. We compared the fluorescence CARD method to standard indirect immunofluorescence detection methods and found that an amplification of up to 15-fold was possible with CARD. The detection of the intracellular protein myosin II in fibroblastic cells and rabbit serum proteins blotted onto nitrocellulose was also improved. Thus, fluorescent CARD is a simple modification that can be made to standard immunofluorescence staining protocols to enhance significantly the detection of antigens.

Measurement and manipulation of cytoskeletal dynamics in living cells

A new ear in cell biology is at hand with the development of tools for imaging molecular functions in living cells and tissues. Specific chemical and molecular events can now be measured and manipulated in cells in order to explore the mechanisms of cell functions. In particular, cytoskeletal processes are being dissected temporally and spatially in single cells from lower eukaryotes, plants, and animals using light-based reagents and electronic light microscopy.

Synthesis and biological assessment of simplified analogues of the potent microtubule stabilizer (+)-discodermolide

An efficient, convergent and stereocontrolled synthesis of simplified analogues of the potent antimitotic agent (+)-discodermolide has been achieved and several small libraries have been prepared. In all the libraries, the discodermolide methyl groups at C14 and C16 and the C7 hydroxy group were removed and the lactone was replaced by simple esters. Other modifications introduced in each series of analogues were related to C11, C17 and C19 of the natural product. Key elements of the synthetic strategy included (a) elaboration of the main subunits from a common intermediate and (b) fragment couplings using Wittig reactions to install the (Z)-olefins. Library components were analyzed for microtubule-stabilizing actions in vitro, for displacement of [3H]paclitaxel from its binding site on tubulin, for antiproliferative activity against human carcinoma cells, and for cell signaling and mitotic spindle alterations by a multiparameter fluorescence cell-based screening technique. The results show that even significant structural simplification can lead to analogues with actions related to microtubule targeting.