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Biomedical subjects

Kenneth A Giuliano

Publications and source records attributed to Kenneth A Giuliano.

12 recordsLinked to original sources
PubMed2007

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. The use measurement and manipulation reagents in multiplexed high content screening assays promises to enable a systems cell biology approach to drug discovery and biomedical research.

Animals↗
PubMed2007

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. Here, an example multiplexed fixed end point HCS assay is used to guide potential assay developers on how to optimize complex, yet cellular information rich, multiplexed HCS assays although avoiding some common pitfalls.

Antibodies↗
PubMed2006

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. Therefore, an SCB profile is a cellular systemic response as measured by a panel of reagents that quantify a specific set of biomarkers.

Animals↗
PubMed2005

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. Pharmacol. 65, 75-82). We used synthetic (-)-dictyostatin to study its biochemical and cytological activities in greater detail. The antiproliferative activity of dictyostatin did not differ greatly from that of paclitaxel or discodermolide. Like discodermolide, dictyostatin retained antiproliferative activity against human ovarian carcinoma cells resistant to paclitaxel due to beta-tubulin mutations and caused conversion of cellular soluble tubulin pools to microtubules. Detailed comparison of the abilities of dictyostatin and discodermolide to induce tubulin assembly demonstrated that the compounds had similar potencies. Dictyostatin inhibited the binding of radiolabeled discodermolide to microtubules more potently than any other compound examined, and dictyostatin and discodermolide had equivalent activity as inhibitors of the binding of both radiolabeled epothilone B and paclitaxel to microtubules. These results are consistent with the idea that the macrocyclic structure of dictyostatin represents the template for the bioactive conformation of discodermolide.

Alkanes↗
PubMed2005

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 microM 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 microM, 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.

Alkaloids↗
PubMed2005

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. We show how this new systems cell biology knowledge can be used to design a lead compound optimization strategy for at least two members of the panel, (-)-laulimalide and (+)-discodermolide, that exploits cell killing activity while minimally perturbing the regulation of the cell cycle and the stability of microtubules. Furthermore, this methodology can also be applied to basic biomedical research on cells.

Antineoplastic Agents↗
PubMed2004

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.

Antineoplastic Agents, Phytogenic↗
PubMed2003

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.

Alkanes↗
PubMed2003

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.

Drug Design↗
PubMed2003

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. The heterogeneity of the response, addressed through population analysis and multiparameter comparisons within single cells, was consistent with vinblastine and curacin A having similar effects on nuclear morphology and 90 kDa ribosomal s6 kinase (RSK90) phosphorylation despite having distinct effects on the microtubule cytoskeleton. Ketoconazole, originally developed as an antifungal agent, exhibited concentration-dependent inhibitory and potentiating effects on both drugs in HeLa and PC-3 cells at concentration ranges near the plasma levels of ketoconazole attained in human subjects. Thus, high-content profiling was used to dissect the cellular and molecular responses to interacting drugs and is therefore a potentially important tool in the selection, characterization, and optimization of lead therapeutic compounds.

Animals↗
PubMed2002

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. The goal of this study was to prepare simplified analogues of (+)-discodermolide and to analyze their biological activities to expand structure-activity relationships. A small library of analogues was prepared wherein the (+)-discodermolide methyl groups at C-14 and C-16 and the C-7 hydroxyl were removed, and the lactone was replaced by simple esters. The library components were analyzed for microtubule-stabilizing actions in vitro, antiproliferative activity against a small panel of human carcinoma cells, and cell signaling, microtubule architecture and mitotic spindle alterations by a multiparameter fluorescence cell-based screening technique. The results show that even drastic structural simplification can lead to analogues with actions related to microtubule targeting and signal transduction, but that these subtle effects were illuminated only through the high information content cell-based screen.

Alkanes↗