Mechanisms and Barriers in Cancer Nanomedicine: Addressing Challenges, Looking for Solutions.

Remarkable progress has recently been made in the synthesis and characterization of engineered nanoparticles for imaging and treatment of cancers, resulting in several promising candidates in clinical trials. Despite these advances, clinical applications of nanoparticle-based therapeutic/imaging agents remain limited by biological, immunological, and translational barriers. In order to overcome the existing status quo in drug delivery, there is a need for open and frank discussion in the nanomedicine community on what is needed to make qualitative leaps toward translation. In this Nano Focus, we present the main discussion topics and conclusions from a recent workshop: "Mechanisms and Barriers in Nanomedicine". The focus of this informal meeting was on biological, toxicological, immunological, and translational aspects of nanomedicine and approaches to move the field forward productively. We believe that these topics reflect the most important issues in cancer nanomedicine.

High content translocation assays for pathway profiling.

This chapter describes the design and development of cell-based assays, in which quantitation of the intracellular translocation of a target protein--rather than binding or catalytic activity--provides the primary assay readout. These are inherently high content assays, and they provide feedback on cellular response at the systems level, rather than data on activities of individual, purified molecules. Multiple protein translocation assays can be used to profile cellular signaling pathways and they can play a key role in determination of mechanism of action for novel classes of compounds with therapeutic potential. This assay technology has developed from laboratory curiosity into main stream industrial research over the past decade, and its promise is beginning to be realized as data acquisition and analysis technology evolve to take advantage of the rich window into systems biology provided by translocation assays.

Protein translocation assays: key tools for accessing new biological information with high-throughput microscopy.

Redistribution technology is a cell-based assay technology that uses protein translocation as the primary readout for the activity of cellular signaling pathways and other intracellular events. Protein targets are labeled with the green fluorescent protein, and stably transfected cell lines are generated. The assays are read using a high-throughput, optical microscope-based instrument, several of which have become available commercially. Protein translocation assays can be formatted as agonist assays, in which compounds are tested for their ability to promote protein translocation, or as antagonist assays, in which compounds are tested for their ability to inhibit protein translocation caused by a known agonist. Protein translocation assays are high-content, high-throughput assays primarily used for profiling of lead series, primary screening of compound libraries, and as readouts for gene-silencing studies using siRNAs. This chapter describes two novel high-content Redistribution assay technologies: (1) The p53:hdm2 GRIP interaction assay, in which one high-content image feature is used for detection of primary hits, whereas a different feature is used to deselect compounds with unwanted mode of action, and (2) application of siRNAs to Redistribution assays, exemplified by knockdown of Akt isoforms in a FKHR translocation assay reporting on the PI3 kinase signaling pathway.

High content screening for G protein-coupled receptors using cell-based protein translocation assays.

G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.

Emerging classes of protein-protein interaction inhibitors and new tools for their development.

Protein-protein interactions play a key role in the signal transduction pathways that regulate cellular function. Three years ago, few descriptions of small molecule protein-protein interaction inhibitors (SMPPIIs) existed in the literature. Today, the number of examples of both the biology and chemistry of such interaction inhibitors is growing rapidly. This growth occurs at the convergence of medicinal chemistry, signaling biology and novel assay technology for profiling emerging compound classes and modes of action. Protein translocation assays provide a unique new tool for identifying, profiling, and optimizing SMPPIIs. This review summarizes recent work in the field, and outlines future developments we can anticipate.

Nuclear export inhibitors and kinase inhibitors identified using a MAPK-activated protein kinase 2 redistribution screen.

Redistribution (BioImage) A/S, Søborg, Denmark) is a novel high-throughput screening technology that monitors translocation of specific protein components of intracellular signaling pathways within intact mammalian cells, using green fluorescent protein as a tag. A single Redistribution assay can be used to identify multiple classes of compounds that act at, or upstream of, the level of the protein target used in the primary screening assay. Such compounds may include both conventional and allosteric enzyme inhibitors, as well as protein-protein interaction modulators. We have developed a series of Redistribution assays to discover and characterize compounds that inhibit tumor necrosis factor-alpha biosynthesis via modulation of the p38 mitogen-activated protein kinase (MAPK) pathway. A primary assay was designed to identify low-molecular-weight compounds that inhibit the activation-dependent nuclear export of the p38 kinase substrate MAPK-activated protein kinase 2 (MK2). Hits from the primary screen were categorized, using secondary assays, either as direct inhibitors of MK2 nuclear export, or as inhibitors of the upstream p38 MAPK pathway. Activity profiles are presented for a nuclear export inhibitor, and a compound that structurally and functionally resembles a known p38 kinase inhibitor. These results demonstrate the utility of Redistribution technology as a pathway screening method for the identification of diverse and novel compounds that are active within therapeutically important signaling pathways.

A simple technique for reducing edge effect in cell-based assays.

Several factors are known to increase the noise and variability of cell-based assays used for high-throughput screening. In particular, edge effects can result in an unacceptably high plate rejection rate in screening runs. In an effort to minimize these variations, the authors analyzed a number of factors that could contribute to edge effects in cell-based assays. They found that pre-incubation of newly seeded plates in ambient conditions (air at room temperature) resulted in even distribution of the cells in each well. In contrast, when newly seeded plates were placed directly in the CO(2) incubator, an uneven distribution of cells occurred in wells around the plate periphery, resulting in increased edge effect. Here, the authors show that the simple, inexpensive approach of incubating newly seeded plates at room temperature before placing them in a 37 degrees C CO(2) incubator yields a significant reduction in edge effect.