Combinatorial Chemistry & High Throughput Screening - Volume 7, Issue 3, 2004

The introduction of microarrays in the early nineties by Fodor and co-workers, represented a major conceptual revolution in the field of parallel experimentation. At that time, the progress in microelectronics had achieved the level necessary to provide the technology upon which the first generation of microarrays was developed. It became possible to synthesize tens to hundreds of thousands of oligomers (peptide or nucleotide based) on a small, well-defined area of a glass slide, using standard photolithographic protocols. Thus, light-directed synthesis met the demands of molecular diversity generation as well as highthroughput screening in a simple and appealing fashion. The potential of this miniaturization technique was quickly realized by other researchers, and a few years later the gene-chip was introduced by Schena and co-workers as a method for tracking genetic diversity. Since microarrays provide massive quantities of data, this research tool became an important technology in genomic projects. In this issue of Combinatorial Chemistry & High Throughput Screening, we present a series of timely and insightful reviews dealing with several distinct applications of microarrays. One of the most important benefits of monitoring the transcriptome lies in its applications in disease management. In cancer, for example, the classification of a tumor type with precise prognostic implications could help determine the optimal therapeutic regimen. This subject is covered in an in-depth discussion by Kim, who describes some recent studies on the potential and limitations of microarrays to impact clinical management of selected tumor types through molecular classifications, and measurements of prognosis and responses to chemotherapy. The assessment of the toxicity of a given drug candidate is a critical step in the drug development process. Analysis of gene expression profiles of tissues undergoing known toxic events could ideally allow the identification of a limited number of toxicity related genes that could be subsequently used in low-density microarrays to assess drug candidates. Some recent applications and liabilities of this approach are carefully discussed by de Longueville in a review on toxicogenomics. The strategies employed for immobilization of macromolecules on glass slides have evolved dramatically over the last decade. Glass surfaces have been derivatized successfully with functional groups other than the amino group, offering new modes of attachment of macromolecules. The latest approaches for site-specific immobilization of proteins, peptides and carbohydrates are thoroughly reviewed by Yao. The advent of microarray technology also significantly impacted the field of immunology. Miniaturized immunoassays performed on a planar platform have proven very sensitive and accurate, opening opportunities for the investigation of protein expression of diseased tissue and cells. The principles and applications of a multiplexed sandwich immunoassay, for a recent innovation in immunoassays, is described in detail by Joos. A comparative analysis of microarray-based and bead-based assay systems is also discussed. Finally, a genome-based approach can help decipher key issues related to the modulation of the immune system. The use of transcriptional profiling data to define the molecular signature of a specific cell lineage is showcased by Falciani. The challenges and alternatives to performing such analysis in complex biological systems is also discussed. The reviews presented in this issue leave no doubt that, despite its incipient stage, microarrays have been firmly established as a research tool for the investigation of biological phenomena at the molecular level with high accuracy. This issue is intended to provide the reader an overview of some applications of microarrays in various fields of research.

Genomics has enabled the examination of the totality of disease at the transcriptome level. Dependent upon a myriad of genetic aberrations for its pathogenesis, cancer has been the focus of gene expression profiling studies that have highlighted the potential clinical applications of this technology. This type of molecular profiling has the potential to enhance the ability of pathologists and oncologists to correctly classify tumors, not just into existing subgroups which may or may not have clear prognostic implications, but into new groups which carry predictable correlations with outcomes. Ultimately, these outcome predictions can be tied to specific treatment regimens, allowing clinicians to predict at the time of diagnosis to which therapy a given patient may best respond. Although this ultimate goal of personalized therapy remains in the future, the numerous studies to date have clearly demonstrated the overall feasibility of this approach. This review will showcase a few of these studies in several key tumor types with the goal of demonstrating which type of studies have been conducted and what types of results are currently possible.

Toxicogenomics is an emerging technology that defines the use of novel genomic techniques to investigate the adverse effects of xenobiotic on gene expression. Toxicogenomics is based on the fact that most of relevant toxicological effects of a compound affect directly or indirectly the gene expression. The most common methods to profile gene expression at the transcript level are Northern Blotting and the real-time PCR. While commonly used and well accepted, these techniques are now superseded by new technologies allowing the analysis of the expression for multiple genes simultaneously. DNA microarrays are now developed for simultaneous gene analysis but inherent to such multiple assays, their quantitative aspect and their relevance for toxicogenomics have been questioned. We will review here recent studies on their use for toxicogenomics and examine the possible future of such technology in complementation with the other toxicology methods.

Recent advances in the generation of peptide and protein microarrays are reviewed, with special focuses on different strategies available for site-specific immobilization of proteins and peptides.

Protein microarray technology allows the simultaneous determination of a large variety of parameters from a minute amount of sample within a single experiment. Assay systems based on this technology are currently applied for the identification, quantitation and functional analysis of proteins. Protein microarray technology is of major interest for proteomic research in basic and applied biology as well as for diagnostic applications. Miniaturized and parallelized assay systems have reached adequate sensitivity and hence have the potential to replace singleplex analysis systems. However, robustness and automation needs to be demonstrated before this technology will finally prove suitable for high-throughput applications. Miniaturized and parallelized sandwich immunoassays are the most advanced assays formats among the different protein microarray applications. Multiplexed sandwich immunoassays can be used for the identification of biomarkers and the validation of potential target molecules. In this review an overview will be given on the current stage of protein microarray technology with a special focus on miniaturized multiplexed sandwich immunoassays.

The development of Functional Genomics technologies has opened new avenues to investigate the complexity of the immune system. Microarray technology has been particularly successful because of its relatively low cost and high genome coverage. Consequently to our ability to monitor the expression of a significant proportion of an organism genome, our understanding of the molecular dynamics behind cell differentiation and cell response has greatly improved. Molecular signatures associated to immune cells have provided important tools to investigate the molecular basis of diseases and have been often associated to diagnostic and prognostic markers. The availability of such large collection of data has stimulated the application of complex machine learning techniques in the attempt to link molecular signatures and cell physiology. Here we review the most recent developments in the analysis of molecular signatures in the immune system.

Lipopolysaccharides (LPS), otherwise termed ‘endotoxins’, are outer-membrane constituents of Gram-negative bacteria. Lipopolysaccharides play a key role in the pathogenesis of ‘Septic Shock’, a major cause of mortality in the critically ill patient. Therapeutic options aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide do not exist at the present time. We have defined the pharmacophore necessary for small molecules to specifically bind and neutralize LPS, and have shown using animal models of sepsis that the sequestration of circulatory LPS by small molecules is a therapeutically viable strategy. Assays reported previously in the literature do not lend themselves well to the rapid screening of large numbers of structurally diverse compounds. In this report, we describe a highly sensitive and robust fluorescent displacement assay using BODIPY TR cadaverine (BC), which binds specifically to the toxic center of LPS, lipid A, and is competitively displaced by compounds displaying an affinity for lipid A. The assay clearly discriminates subtle differences in the binding of polymyxin B, and its nonapeptide derivative, with LPS. The spectral properties of the BODIPY fluorophore are ideally suited for screening diverse structural classes of compounds, including those with conjugated aromatic groups, or with chromophores in the 260-500 nm range. The fluorescent probe: LPS complex is stable under physiologically relevant salt concentrations, resulting in the rapid rejection of spurious binders interacting via non-specific electrostatic interactions, and, therefore, in greatly improved dispersion of ED50 values.

Liquid phasel synthesis of biheterocyclic benzimidazoles by controlled microwave irradiation was investigated. Polymer immobilized o-phenylenediamines was synthesized under microwave irradiation. The resulting PEG bound diamines was N-acylated with 4-fluoro-3-nitrobenzoic acid selectively in primary aromatic amino moiety. Nucleophilic aromatic substitution of amide was performed with various amines then cyclized to form the first benzimidazole scaffold in acidic condition. Successive reduction, cyclization with isothiocyanates yielded 5-(benzimidazol-2-yl)benzimidazoles. The desired products were released from the polymer support to afford the tri-substituted bis-benzimidazoles in good yields and purity.

Edson Luiz da Silva Lima received his B.S. degree from the Federal University of Rio de Janeiro in 1987 and the Ph.D.degree in Organic Synthesis in the same University in 1992. After a postdoctoral fellowship at Harvard University in thelaboratories of Prof. David A. Evans, he moved back to Brazil to join the Oswaldo Cruz Foundation as a research associate in1995. After two years he joined the Institute of Chemistry of the Federal University of Rio de Janeiro where he currently holdsthe position of Professor of Chemstry. His research interest lie in the development of synthetic methodologies, and thesynthesis of compounds active against tropical diseases. Selected Publications Simon J. Garden, Jose C. Torres, Simone C. de Souza Melo, Alexandre S. Lima, Angelo C. Pinto, Edson L. S. Lima;Aromatic Iodination in aqueous solution. A new lease of life for aqueous potassium dichloroiodate. Tetrahedron Lett., 2001,42, 2089. Emerson T. Silva, Andrea S. Cunha and Edson L. S. Lima; An efficient protocol for solution and solid-phase end-groupdifferentiation of spermidine. Bioorg. Med. Chem. Lett., 2002, 12, 3207. Emerson T. da Silva, Edson L. S. Lima; Reaction of 1,3-dimethyl-5-acetyl-barbituric acid (DAB) with primary amines. Acessto intermediates for selectively protected spermidines. Tetrahedron Lett., 2003, 44, 3621.