Genomics Analysis of the Effects of Boron Compounds on a Cell

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By - Miroslava Cuperlovic-Culf, PhD,      Adrian S. Culf, PhD,
      Mark Laflamme, PhD, Dr. Rodney J. Ouellette, PhD,
Stephen A. Westcott,PhD

Abstract
In recent years, several research groups have begun pioneering chemical genomics for drug discovery. Libraries of compounds are screened for a beneficial effect against a specific disease without any previous knowledge of the precise molecular target. This article will describe briefly the synthetic procedures used in the preparation of a library of boronic acid compounds, use of synthetic long oligonucleotide DNA microarrays and experimental and data analysis methods for the determination of drug targets.

Interest in chemical genomics for drug discovery — in which the starting point for a screen is a specific disease as defined by a particular genetic or genomic state1,2 — has begun to grow considerably.

In this approach, libraries of compounds are screened for a beneficial effect against the disease either in the cell culture or in animal models, without any prior knowledge of a precise molecular target. As part of our ongoing program of preparing novel amino boronic acid compounds,3 we are using chemical genomics in an effort to design efficient candidates for the treatment of breast cancer.

We have found that simple cyclic diamines containing boronic acids have potent antifungal and moderate antibacterial properties, and are currently investigating potential anticancer properties. Although some understanding of the general effects of boronic acid compounds on cells is available, a more in-depth understanding of the mechanism of action is still warranted.

Microarray Technology
Biological processes are complicated, with many molecules working together in concert. As such, biological scientists are eager to reveal the "whole picture" of this molecular interplay in order to understand disease states at the molecular level. Although most cells of the body contain a full set of chromosomes with identical genes, only a fraction of these genes are turned on (expressed), leading to the basis of differentiation of cellular function. Gene expression describes the transcription of information contained in nuclear DNA into messenger RNA (mRNA) molecules that are used to direct protein synthesis. Proteins perform most aspects of cellular function and disruption or changes in gene expression are responsible for many diseases.

DNA microarray technology allows scientists to quickly and efficiently analyze expression of all of an organism's genes (its transcriptome) in a single experiment. Thus, the microarray platform is an automated, enabling high-throughput technology that allows researchers to see the "whole picture" for the first time. A DNA microarray consists of an orderly arrangement of single-stranded DNA gene probes on a solid support (e.g. chemically modified glass, plastic or silicon surfaces).

These probes are synthesized as a linear polymer of four bases: adenine (A), thymine (T), guanine (G) and cytosine (C). Adenine will always pair with (complement) with thymine, and guanine is the complement of cytosine. When two complementary sequences find each other, such as surface-bound gene probes and fluorescently labelled complementary DNA (cDNA, synthesized in vitro from isolated mRNA) in solution, they lock together, or hybridize. DNA hybridization is the underlying principle of DNA microarrays and Northern blots. The microarray will capture and display the expressed genes of the biological sample under investigation. At the Atlantic Microarray Facility (Moncton, NB), we have capabilities for high-throughput DNA synthesis and silicon-pin contact microarray printing.

Pharmacogenomics Experimentation
Prior to microarray analyses, cancerous, as well as normal cells, are used to determine the activity and concentration of boron compounds that will be used for microarray experimentation. The cell viability in these experiments is measured from the ability of living cells to convert a redox dye (resazurin) into a fluorescent end product (resorufin) (CellTiter-Blue™ method).

Following these measurements, cells are treated with APO-1 reagent, which consists of a substrate that is cleaved by activated Caspase 3/7 enzymes to produce a fluorescent end product. The APO-1 end product is accumulated in a linear manner over time, and is a direct indication of the amount of Caspase 3/7 activity, which in turn is directly related to the level of cellular apoptosis. Compared to more traditional MTT analysis, CellTiter-Blue method has several advantages in this context:

a) it is based on fluorescence rather than absorbance, which provides greater sensitivity; b) both substrate and product are soluble, reducing the need for strong agitation, and the worry that precipitate will accumulate in an unmeasured area of the plate well; and c) it is easily multiplexed with other enzymatic tests, such as APO-1.

Using this combination of tests permits us to assess cell viability and apoptosis on the exact same cells, which greatly increases confidence in the obtained results. The results of these studies are used to establish the concentrations of boron compounds, which will be used in microarray studies. In each case the smallest concentration of compound that shows significant inhibition of cell proliferation due to apoptosis over a five-day period will be used for these studies. Time course set up of these experiments provides us with sufficient amount of information for the determination of both affected genes as well as pathway changes.

Microarray Data Analysis for
Pharmacogenomics
Pharmacogenomics is the determination and analysis of the genome (DNA) and its products (RNA and proteins) as they relate to drug response. These types of experiments yield enormous amounts of data that need to be organized, stored, analyzed and disseminated. From the point of view of pharmacogenomics, it is very important to determine potential gene networks that will respond to boron compounds tested in these experiments.
Gene networks can ideally have the potential to predict a chemical compound's mode of action, discover more effective drug targets and predict side-effects.

In addition, it is extremely useful to determine genes with significantly altered expression and also significant clusters of gene ontology terms and their overall changes. Results are compared in these experiments to the gene expression measurements of the cells treated with solvent (control samples). As in all microarray experiments, it is crucial to ensure high RNA quality prior to microarray experimentation for which a microfluidic gel electrophoresis system is used with only minor sample losses.

Once the data is prepared for analysis, different methods—of which there are many developed for the determination of the most significantly differentially expressed genes in standard experiments4—are applied in order to extract all required information. These methods, as well as many methods developed specifically for pharmacogenomics experiments, are successfully utilized to determine gene expression changes between stimulated cells and controls, as well as gene networks. Information obtained in this way can be used to determine effects as well as side-effects of boron compounds. In addition, collaborations between chemists, bioinformaticians and biologists will result in the development of more specific and potent compounds.

Summary
Overall, the application of transcriptional profiling to the drug-discovery workbench provides all the necessary elements to identify, characterize and discriminate between compounds with novel activity. As the technologies continue to be applied, the true benefits of the genomics revolution will be realized in the discovery and development of new medicines.

References
1.     Bernardo, D., M.J. Thompson, T.S. Gardner, S.E. Chobot, E.L. Eastwood, A.P. Wojtovich,
et al. "Chemogenomics Profiling on a Genome-Wide Scale Using Reverse-Engineered Gene Networks." Nature Biotech 23 (2005): 377-383.
2.     Ohlstein, E.H., A.G. Johnson, J.D. Elliott and A.M. Romanic. "New Strategies in Drug Discovery." Methods Mol Biol 316 (2006): 1-11.
3.     Blacquiere, J.M., O. Sicora, C.M. Vogels, M. Cuperlovic-Culf, A. Decken, R.J. Ouellette, and S.A. Westcott. "Dihydropyrimidinones Containing Boronic Acids." Can. J. Chem 83 (2005): 2052-2059.
4.     Cuperlovic-Culf, M., N. Belacel, R.J. Ouellette. "Determination of Tumour Marker Genes from Gene Expression Data." Drug Discovery Today 10 (2005): 429-437.

Stephen A. Westcott, PhD is an associate professor in the department of chemistry at Mount Allison University (MTA)(Sackville, NB). Miroslava
Cuperlovic-Culf, PhD, Adrian S. Culf, PhD, Mark Laflamme, PhD and Dr. Rodney J. Ouellette, PhD are all affiliated with the Atlantic Cancer Research Institute (Moncton, NB). Westcott would like to thank the Canada Research Chairs program, the Canada Foundation for Innovation (Ottawa, ON), the Atlantic Innovation Fund, the Natural Science and Engineering Research Council of Canada (Ottawa, ON) and MTA for financial support. Cuperlovic-Culf, Culf, Laflamme and Ouellette are grateful to the Atlantic Innovation Fund and the Georges L.-Dumont Hospital Foundation for their generous support.