Assessing the performance of the Cell Painting assay across different imaging systems.

Quantitative microscopy is a powerful method for performing phenotypic screens from which image-based profiling can extract a wealth of information, termed profiles. These profiles can be used to elucidate the changes in cellular phenotypes across cell populations from different patient samples or following genetic or chemical perturbations. One such image-based profiling method is the Cell Painting assay, which provides morphological insight through the imaging of eight cellular compartments.

Assessing the performance of the Cell Painting assay across different imaging systems.

Quantitative microscopy is a powerful method for performing phenotypic screens from which image-based profiling can extract a wealth of information, termed profiles. These profiles can be used to elucidate the changes in cellular phenotypes across cell populations from different patient samples or following genetic or chemical perturbations. One such image-based profiling method is the Cell Painting assay, which provides morphological insight through the imaging of eight cellular compartments.

A Homogeneous Cell-Based Halide-Sensitive Yellow Fluorescence Protein Assay to Identify Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator Ion Channel.

Cystic fibrosis (CF), an inherited genetic disease, is caused by mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which encodes an ion channel involved in hydration maintenance by anion homeostasis. Ninety percent of CF patients possess one or more copies of the F508del CFTR mutation. This mutation disrupts trafficking of the protein to the plasma membrane and diminishes function of mature CFTR.

Use of a High-Throughput Phenotypic Screening Strategy to Identify Amplifiers, a Novel Pharmacological Class of Small Molecules That Exhibit Functional Synergy with Potentiators and Correctors.

Cystic fibrosis (CF) is a lethal genetic disorder caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Despite recent groundbreaking approval of genotype-specific small-molecule drugs, a significant portion of CF patients still lack effective therapeutic options that address the underlying cause of the disease. Through a phenotypic high-throughput screen of approximately 54,000 small molecules, we identified a novel class of CFTR modulators called amplifiers.

Characterization and optimization of a novel protein-protein interaction biosensor high-content screening assay to identify disruptors of the interactions between p53 and hDM2.

We present here the characterization and optimization of a novel imaging-based positional biosensor high-content screening (HCS) assay to identify disruptors of p53-hDM2 protein-protein interactions (PPIs). The chimeric proteins of the biosensor incorporated the N-terminal PPI domains of p53 and hDM2, protein targeting sequences (nuclear localization and nuclear export sequence), and fluorescent reporters, which when expressed in cells could be used to monitor p53-hDM2 PPIs through changes in the subcellular localization of the hDM2 component of the biosensor. Coinfection with the recombinant adenovirus biosensors was used to express the NH-terminal domains of p53 and hDM2, fused to green fluorescent protein and red fluorescent protein, respectively, in U-2 OS cells.

Early safety assessment using cellular systems biology yields insights into mechanisms of action.

The integration of high-content screening (HCS) readers with organ-specific cell models, panels of functional biomarkers, and advanced informatics is a powerful approach to identifying the toxic liabilities of compounds early in the development process and forms the basis of "early safety assessment." This cellular systems biology (CSB) approach (CellCiphr profile) has been used to integrate rodent and human cellular hepatic models with panels of functional biomarkers measured at multiple time points to profile both the potency and specificity of the cellular toxicological response. These profiles also provide initial insights on the mechanism of the toxic response.

Implementation of a 220,000-compound HCS campaign to identify disruptors of the interaction between p53 and hDM2 and characterization of the confirmed hits.

In recent years, advances in structure-based drug design and the development of an impressive variety of high-throughput screening (HTS) assay formats have yielded an expanding list of protein-protein interaction inhibitors. Despite these advances, protein-protein interaction targets are still widely considered difficult to disrupt with small molecules. The authors present here the results from screening 220,017 compounds from the National Institute of Health's small-molecule library in a novel p53-hDM2 protein-protein interaction biosensor (PPIB) assay.

Cellular systems biology profiling applied to cellular models of disease.

Building cellular models of disease based on the approach of Cellular Systems Biology (CSB) has the potential to improve the process of creating drugs as part of the continuum from early drug discovery through drug development and clinical trials and diagnostics. This paper focuses on the application of CSB to early drug discovery. We discuss the integration of protein-protein interaction biosensors with other multiplexed, functional biomarkers as an example in using CSB to optimize the identification of quality lead series compounds.

Systems cell biology based on high-content screening.

A new discipline of biology has emerged since 2004, which we call "systems cell biology" (SCB). Systems cell biology is the study of the living cell, the basic unit of life, an integrated and interacting network of genes, proteins, and myriad metabolic reactions that give rise to function. SCB takes advantage of high-content screening platforms, but delivers more detailed profiles of cellular systemic function, including the application of advanced reagents and informatics tools to sophisticated cellular models.

Optimizing the integration of immunoreagents and fluorescent probes for multiplexed high content screening assays.

Immunoreagents formed the basis of early fixed end point high content screening (HCS) assays and their use in HCS applications in drug discovery will continue to increase. One important application of immunoreagents is their incorporation into multiplexed HCS assays in which multiple physiological features are simultaneously measured and related in the same cells. However, creating multiplexed HCS assays that incorporate multiple immunoreagents presents issues such as reagent compatibility, spectral signal overlap, and reproducibility that must be addressed.

Reagents to measure and manipulate cell functions.

Reagents that are used as part of a discovery platform for the measurement and manipulation of cell functions are at the heart of single and multiplexed high content screening assays. Measurement reagents include physiological indicators, immunoreagents, fluorescent analogs of macromolecules, positional biosensors, and fluorescent protein biosensors. Recent developments in reagents that manipulate specific cell functions including small inhibitory RNAs, caged peptides, proteins, and RNAs, and gene switches complement measurement reagents, especially when both classes of reagents are used in the same living cells.

Systems cell biology knowledge created from high content screening.

High content screening (HCS), the large-scale automated analysis of the temporal and spatial changes in cells and cell constituents in arrays of cells, has the potential to create enormous systems cell biology knowledge bases. HCS is being employed along with the continuum of the early drug discovery process, including lead optimization where new knowledge is being used to facilitate the decision-making process. We demonstrate methodology to build new systems cell biology knowledge using a multiplexed HCS assay, designed with the aid of knowledge-mining tools, to measure the phenotypic response of a panel of human tumor cell types to a panel of natural product-derived microtubule-targeted anticancer agents and their synthetic analogs.

Tubulin assembly, taxoid site binding, and cellular effects of the microtubule-stabilizing agent dictyostatin.

(-)-Dictyostatin is a sponge-derived, 22-member macrolactone natural product shown to cause cells to accumulate in the G2/M phase of the cell cycle, with changes in intracellular microtubules analogous to those observed with paclitaxel treatment. Dictyostatin also induces assembly of purified tubulin more rapidly than does paclitaxel, and nearly as vigorously as does dictyostatin's close structural congener, (+)-discodermolide (Isbrucker et al. (2003), Biochem.

Multiplexed high content screening assays create a systems cell biology approach to drug discovery.

High content screening (HCS) has emerged as an important platform technology for early drug discovery from target identification through in vitro ADME/Tox. The focus is now on implementing multiplexed assays, developing and using advanced reagents and developing and harnessing more sophisticated informatics tools. Multiplexed HCS assays have the potential to dramatically improve the early drug discovery process by creating systems cell biology profiles on the activities of compounds.

Multiplexed high content screening assays create a systems cell biology approach to drug discovery.

High content screening (HCS) has emerged as an important platform technology for early drug discovery from target identification through in vitro ADME/Tox. The focus is now on implementing multiplexed assays, developing and using advanced reagents and developing and harnessing more sophisticated informatics tools. Multiplexed HCS assays have the potential to dramatically improve the early drug discovery process by creating systems cell biology profiles on the activities of compounds.

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.

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.

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.

High-content profiling of drug-drug interactions: cellular targets involved in the modulation of microtubule drug action by the antifungal ketoconazole.

Drug-drug interactions play an important role in the discovery and development of therapeutic agents. High-content profiling was developed to unravel the complexity of these interactions by providing multiparameter measurements of target activity at the cellular and subcellular levels. Two microtubule drugs, vinblastine and curacin A, were shown to modulate multiple cellular processes, including nuclear condensation, the activation of the extracellular signal-regulated kinase pathway as measured by RSK90 phosphorylation, and the regulation of the microtubule cytoskeleton as measured in detergent-extracted cells.

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.

Synthesis and high content cell-based profiling of simplified analogues of the microtubule stabilizer (+)-discodermolide.

(+)-Discodermolide, a C24:4, trihydroxylated, octamethyl, carbamate-bearing fatty acid lactone originally isolated from a Caribbean sponge, has proven to be the most potent of the microtubule-stabilizing agents. Recent studies suggest that it or its analogues may have advantages over other classes of microtubule-stabilizing agents. (+)-Discodermolide's complex molecular architecture has made structure-activity relationship analysis in this class of compounds a formidable task.