Combinatorial Chemistry & High Throughput Screening - Volume 7, Issue 6, 2004

The post-genomic era heralds a multitude of challenges for chemists and biologists alike, with the study of protein functions at the heart of much research. The elucidation of protein structure, localization, stability, post-translational modifications and protein interactions will steadily unveil the role of each protein and its associated biological function in the cell. The push to develop new technologies has necessitated the integration of various disciplines in science. This special issue of CCHTS will cover some of the emerging technologies developed in recent years in the areas of combinatorial chemistry and high-throughput screenings, with special focuses on microarray-based and other promising technologies. One of most promising methods used in current proteomics research is the microarray-based techniques, which enable tens of thousands of biological assays to be carried out simultaneously on a microscope-size glass slide (2.5 cm × 7.5 cm) in a highly parallel and extremely high-throughput fashion. DNA- and Protein-based microarrays have received a great deal of interests from scientists working in life sciences-related fields. Other types of microarrays, namely those based on synthetic chemical entities (i.e. small molecules, peptides, carbohydrates and lipids, etc), are equally important as useful tools for potential highthroughput screenings and drug discovery. Sridar Chittur discussed the currently available DNA microarrays that have various applications in gene expression and genotyping studies. Also discussed are the novel formats of microarrays being used that push the limits of this technology to achieve higher throughput. Predlki et al. from Protometrix highlighted some of the major challenges faced when using protein microarray technologies for potential high-throughput drug discovery and development. Protometrix, by the way, is the biotech company which successfully commercialized the “yeast proteome array”. While fabricating a functional protein microarray is difficult, the generation of a peptide-based microarray is considerably much more straightforward, mainly because peptides are a lot more stable than proteins - they don't get denatured and subsequently lose their biological properties easily. Consequently, peptide arrays have increasingly become an important tool for highthroughput screening of potential protein substrates, binders and even inhibitors. Some of the applications of peptide array are reviewed by Yao et al. Unlike proteins and peptides, which are typically considered poor drug candidates due to their limited bioavailability, small molecules are the focus of the pharmaceutical industry and drug discovery. No wonder small molecule micraorrays, as Chang et al. discussed in their timely review, hold a great potential for direct and high-throughput discovery of “drugable” drugs against a variety of protein target. Carbohydrate-protein interaction plays an important role for biological recognitions, in their review; Shin et al. convincingly illustrated how microarray technologies based on carbohydrates could be used to study carbohydrate-binding proteins and carbohydrate-processing enzymes, the diagnosis of diseases and even drug discovery. Hewitt et al. present a well-investigated and thorough summary of issues including Tissue Microarray (TMA) design, construction, sectioning, staining, scoring and statistical analysis, as well as mention of developing techniques including image analysis and their own novel immunodetection technique. John Cowell presents a detailed review of the use of BAC arrays in human genetics and chronicles the development of this technology over the last 50 years. The benefit of a physical map approach to the human genome rather than the expression map approach of many investigators using array CGH on expression cDNA platforms is also discussed in this review. Lastly, by looking beyond the microarray-based technologies, Yeo et al. discussed an emerging area in modern chemical biology - bioimaging, which sheds lights on how precisely proteins behave inside a living, cellular environment. Various chemical approaches for in vivo labeling of proteins such that they become “visible” were highlighted in the review.

Profiling of gene expression patterns with microarray technology is widely used in both basic and applied research. DNA microarrays have also shown great promise in clinical medicine and are paving the way toward effective pharmaceutical drug discovery and individualized drug regimens. With growing utilization of this high-throughput technology, new applications are making headlines on a regular basis. This review aims to outline the pros and cons of this methodology and direct the reader towards available useful resources. Various major array formats such as high-density oligonucleotide arrays and spotted cDNA arrays are examined, and advantages and options for using each format are presented. Factors important for the design and analysis of microarray experiments are also discussed.

Functional protein microarrays promise new approaches to address longstanding challenges in drug discovery and development, with applications ranging from target identification to clinical trial design. However, their widespread adoption will be contingent upon a robust ability to develop and manufacture arrays in support of these applications. This review will address the major areas of relevance to the development of functional protein microarrays; protein content, surface chemistry, manufacture and assay development. Successful development will empower multiple drug research applications, help fill future HTS pipelines and guide next generation combinatorial chemistry efforts.

Peptide array is a rapidly growing tool that provides both large-scale and high-throughput capabilities for protein detection and activity studies. Materials presented in this review will examine the recent advances in the field of peptide microarray with special emphasis on the generation and applications of high-density arrays of peptides on glass slides.

The field of Small Molecule Microarray's (SMM's) is an ever-expanding part of the larger microarray field. SMM's are array based detection systems that use small molecules as probes immobilized on a variety of microarray surfaces that are screened against a number of targets for purposes including, but not limited to, protein-small molecule ligand recognition and protein function profiling. This review covers the recent advances in the field with particular emphasis on the successful applications of SMM's, as well as technical advances in platform optimization and novel small molecule immobilization strategies.

Carbohydrates, as components of glycoproteins, glycolipids and proteoglycans, play an important biological role as recognition markers through carbohydrate-protein interactions. For the most part, biophysical and biochemical methods have been used to analyze these biomolecular interactions. In contrast, less attention has been given to the development of high-throughput procedures to elucidate carbohydrateprotein recognition events. Recently, carbohydrate arrays were developed and employed as a novel highthroughput analytic tool for monitoring carbohydrate-protein interactions. This technique has been used to profile protein binding and enzymatic activity. The results have shown that carbohydrate binding to the corresponding lectins is highly selective and that the relative binding affinities are well correlated with those obtained from solution-based assays. In addition, this effort demonstrated that carbohydrate arrays could be also utilized to identify and characterize novel carbohydrate-binding proteins or carbohydrate-processing enzymes. Finally, the results of this investigation showed that lectin-carbohydrate binding affinities could be quantitatively assessed by determining IC50 values for soluble carbohydrates with the carbohydrate arrays. The results of these studies suggest that carbohydrate arrays have the potential of playing an important role in basic researches, the diagnoses of diseases and drug discovery.

Tissue microarrays (TMAs) are means of combining tens to hundreds of specimens of tissue onto a single slide for analysis at one time. TMAs are most frequently constructed from paraffin embedded tissue; however, they can be constructed from frozen tissue. The construction of TMAs is flexible, meeting the focused needs of the investigator. A TMA slide can be processed like an ordinary tissue section, and used for histochemical, immunohistochemical staining or in situ hybridization. Combined with automated new image analysis systems, TMAs are a powerful molecular profiling tool. By confirming the findings of microarray experiments or protein arrays, TMAs can be applied systematically to global cellular network analysis within tissue cell. TMAs are commonly used to confirm the results of expression microarrays as well as in the development of diagnostic and prognostics markers for clinical applications. This review will cover recent advancements in technology for the construction and use of TMAs. Because TMAs can be constructed from archival paraffin embedded tissue, they open up the vast archive of patient samples and make them accessible for medical research. TMAs play an ever increasing role in translational medicine, bridging the chasm of discovery at the research bench to the demonstration of clinical utility prior to implementation in patient care.

Chromosome analysis has been a cornerstone both for the identification of genetic defects that predispose to a variety of genetic syndromes as well as for the analysis of cancer progression. The relatively low resolution of metaphase chromosomes, however, only allows characterization of major genetic events which are defined at the megabase level. The development of the human genome-wide bacterial artificial chromosome (BACs) libraries which were used as templates for the human genome project made it possible to design microarrays containing these BACs which can theoretically span the genome uninterrupted. Comparative genomic hybridization to these arrays using test and reference DNA samples reveals numerical chromosome abnormalities (deletions, gains and amplifications) which can be accurately defined with a resolution depending on the density of the arrays. Analysis of test DNA samples using these arrays reveals low level deletions and amplifications that cannot be detected by chromosome analysis and provides a global view of these genetic changes in a single overnight hybridization using a high throughput approach. The extent of the genetic changes can then be determined precisely and the gene content of the affected regions established. These BAC arrays have widespread application to the analysis of constitutional genetic abnormalities associated with human diseases as well as cancer patients and their tumors. The development of similar BAC arrays for the mouse genome means that it is now possible to extend the CGHa approach to the study of genetic disorders and cancer models in mice.

We review various advancements in small molecule probes, intein-based labeling methods, and the incorporation of synthetic amino acids into proteins for live cell imaging experiments. Finally, recent developments in quantum dots-based labeling are briefly reviewed.

Shao Q. Yao graduated with a bachelor's degree in Chemistry from Ohio State University in Columbus, Ohio (USA) in 1993. He obtained his PhD in organic & bioorganic chemistry from Purdue University, USA in 1998 with Professor Jean Chmielewski. He conducted his post-doctoral research in the fields of bioorganic chemistry, functional genomics & molecular biology first at the University of California at Berkeley, USA, then at the Scripps Research Institute, USA, both under the supervision of Professor Peter G. Schultz. He joined National University of Singapore in 2001 and currently holds a joint appointment between the Department of Chemistry and Department of Biological Sciences. His research interests include combinatorial chemistry, chemical proteomics, protein engineering and Bioimaging.