DNA Microarrays Technique: Application In Pharmacy

Abstract

Natural products are gaining increased applications in drug discovery and development. Being chemically diverse they are able to modulate several targets simultaneously in a complex system. Analysis of gene expression becomes necessary for better understanding of molecular mechanisms. Conventional strategies for expression profiling are optimized for single gene analysis. DNA microarrays serve as suitable high throughput tool for simultaneous analysis of multiple genes. Major practical applicability of DNA microarrays remains in DNA mutation and polymorphism analysis. This review highlights applications of DNA microarrays in pharmacodynamics, pharmacogenomics, toxicogenomics and quality control of herbal drugs and extracts.

Keywords: Drug discovery, evidence-based medicine, gene expression, genotyping, pharmacodynamics, transcription profiling

Introduction

Understanding the functions of genes is a major post-genomics challenge. Strategies like proteomics, transcriptomics, metabolomics are implemented to assign the role of genes in molecular networks. The gene expression profile of a cell determines its phenotype, function and response to the environment. The complement of genes expressed by a cell is very dynamic and responds rapidly to external stimuli. Therefore, analysis of gene expression becomes necessary for providing clues about regulatory mechanisms, biochemical pathways and broader cellular function. Conventional strategies for expression profiling such as northern blot, reverse northern blot, reverse transcriptase-polymerase chain reaction (RT–PCR), nuclease protection, enzyme-linked immunosorbent assay (ELISA), western blot, in situ hybridization and immunohistochemistry are optimized for single gene analysis. Although, it is possible to modify at least some of these techniques for multiplexing, the procedure becomes increasingly technically cumbersome. For genome wide expression analysis it is necessary to develop technologies having high degree of automation, since in any living organism thousands of genes and their products function in a complicated and orchestrated way. DNA microarrays were developed in response to the need for a high-throughput, efficient and comprehensive strategy that can simultaneously measure all the genes, or a large defined subset, encoded by a genome ^(1,2). Several different methodologies including differential display PCR, northern blots ⁽³⁾, quantitative PCR, serial analysis of gene expression (SAGE) ^(4,5) and TIGR Orthologous Gene Alignments (TOGA) ^(6,7) are used alongside microarrays as research tools.

With an initial focus in the post-genomic era on tracking gene expression changes for target identification, microarray applications soon widened to span the entire drug discovery pipeline ^{(8, 9)}. DNA microarrays are being used to study the transcriptional profile in various physiological and pathological conditions, leading to the mining of novel genes and molecular markers for diagnosis, prediction or prognosis of those specific states ^(10,11).

Success in the DNA microarray field has led to protein array development ^(12–13). Protein microarrays are mainly applied to protein function studies, screening the production of antibodies ⁽¹⁴⁾ and recombinant proteins ⁽¹⁵⁾, discovery of proteins implicated in disease or those that are potential drug targets, rapid detection or diagnosis of disease ^(16,17) screening for protein–protein, DNA–protein and enzyme–substrate interactions ^(18,19). High-density human protein arrays are undermined by the incomplete knowledge of full-length human gene clones and the vast idiosyncrasies of proteins with respect to stability and structure ⁽²⁰⁾. Although protein microarrays may not have reached the stage of maturity of DNA microarrays, recent developments have shown that many of the barriers holding back the technology can be overcome ^{(21 )}.

Recently cell-based arrays employing matrices of living cells engineered to express select proteins have rapidly emerged as versatile tools for the high-throughput analysis of gene function. Such arrays can facilitate genome-scale studies on different aspects of protein function, including biochemical activities, gene disruption phenotypes and protein–protein interactions ^(22). Another version of living arrays called transfection microarray has been demonstrated as an alternative to protein microarrays for the identification of drug targets, and as an expression cloning system for the discovery of gene products that alter cellular physiology ^(23). A siRNA transfected cell microarray has been developed to facilitate large-scale, high-throughput functional genomics studies using RNAi ^(24).

Chemical microarrays, which are arrays of small organic compounds, represent a novel approach towards analysis of chemical libraries. They are widely used to analyze the interaction of proteins with organic compounds in a miniaturized and high-throughput fashion ^(25,26). Several modified technologies spanning wide range of macromolecular microarrays to cell arrays have opened new horizons in molecular and physiological systems.

The DNA Microarray

DNA microarray is an orderly arrangement of thousands of oligonucleotides or identified sequenced genes printed on an impermeable solid support, usually glass, silicon chips or nylon membrane. A number of terms such as DNA arrays, gene chips and biochips are often used to describe these devices. The DNA microarray field is a combination of several technologies, including automated DNA sequencing, DNA amplification by PCR, oligonucleotide synthesis, nucleic acid labeling chemistries and bioinformatics. Two most common types of DNA microarrays include one in which the DNA (in the form of a single stranded short oligonucleotide) is synthesized in situ using photolithographic or other techniques and another where the DNA (usually in the form of a cDNA or full-length ORF) is post-synthetically attached to a solid support ^(27).

Depending on the kind of probe (immobilized DNA) used to generate the array and, ultimately, the information that is derived from it, DNA microarrays are used for different applications ^(28). As a definition in the context of microarrays, a ‘probe’ is the (partial) genomic sequence of a gene deposited and fixed on the Microarray, whereas the ‘target’ is the biological sample material.

Although there are many protocols and types of microarray experiments, the basic steps involved in any microarray experiment are isolating RNA or mRNA from appropriate biological samples; applying a fluorescent tag to the RNA or cDNA copy of it; hybridizing the labeled RNA or cDNA (target) to a microarray (probe) for a period of time after which the excess is washed off; scanning the microarray under laser light and data analysis using appropriate software. A brief description of three basic types of DNA micro array experiments and their applications is provided in Table 1 .

Applications Of DNA Microarrays In Pharma Field And Its Development

Natural product research is often based on ethnobotanical information and many of the drugs used today were employed in indigenous societies ^(29). One of the aims of ethnopharmaceutical research is better understanding of the pharmacological effects of different medicinal plants traditionally used in healthcare ^(30). Plants are regarded as a promising source of novel therapeutic agents due to their higher structural diversity as compared to standard synthetic chemistry. Plants have applications in the development of therapeutic agents: as a source of bioactive compounds for possible use as drugs. There are three approaches to natural product-based drug discovery: screening of crude extracts; screening of pre-fractionated extracts; screening of pure compounds ^(31).

There are three main applications of DNA microarrays: first, in pharmacodynamics for discovery of new diagnostic and prognostic indicators and biomarkers of therapeutic response; elucidation of molecular mechanism of action of a herb, its formulations or its phytochemical components and identification and validation of new molecular targets for herbal drug development. Second, in pharmacogenomics for prediction of potential side-effects of the herbal drug during preclinical activity and safety studies; identification of genes involved in conferring drug sensitivity or resistance and prediction of patients most likely to benefit from the drug and use in general pharmacogenomic studies. Third, in pharmacognosy for correct botanical identification and authentication of crude plant materials as part of standardization and quality control. These applications are described here with some examples ^(32,33) by figure 1.

DNA Microarrays In Pharmacodynamics

Herbal products are usually whole herbs, their formulations or extracts consisting of several bioactive compounds. With the increased demand for scientifically validated and standardized herbal product there is a need for better understanding of the molecular mechanisms underlying their biological activity. Although, the physiological actions of many herbal drugs are being studied at the molecular level, it remains unclear what are the targets of individual phytochemical components of herbs and how these molecules contribute to biological activities.

Purified Compounds /Specific Phytochemical Groups

The molecular pathways underlying diverse biological activity of the triterpenoid compounds isolated from the tropical medicinal plant Centella asiatica when studied with gene microarrays showed that Centella triterpenes evoke a gene-expression response consistent with their prevailing medical uses in the treatment of connective tissue disorders such as wound healing and microangiopathy. The identification of genes modulated by these compounds provides the basis for a molecular understanding of Centella's bioactivity, and opportunities for the quantitative correlation of this activity with clinical effectiveness at a molecular level ^(34). Similarly, the antiproliferative activity of Coptidis rhizoma, a medicinal herb, and its major component berberine was investigated in human pancreatic cancer cell lines. Gene expression patterns associated with sensitivities to each agent were analyzed with oligonucleotide arrays that comprised approximately 11,000 genes. It was possible to identify common and distinct genes related to anti-proliferative activities of purified berberine and C. rhizoma ^(35).

Involvement of genes in apoptosis inducing activity of alkaloids from Chinese medicinal herb Tripterygium hypoglaucum (levl.) Hutch (Celastraceae) root was examined using cDNA microarrays containing 3000 human genes derived from a leukocyte cDNA library. Induction of apoptosis by Tripterygium hypoglaucum alkaloids was found to be through c-myc and NF-kappa B signaling pathways ^(36).

Gene expression pattern of inferior colliculus from DBA/2J mice with audiogenic seizure and those treated with Qingyangshenylycosides, a traditional Chinese medicine, were examined. Gene expression analysis using Agilent oligo microarray showed that total of 134 genes were either up- or down-regulated during audiogenic seizure. Qingyangshenylycosides prevented many of the audiogenic seizure induced gene expression changes. Nevertheless, some of the audiogenic seizure induced genes were further enhanced or reversed by Qingyangshenylycosides treatment. The data provided important information regarding the molecular mechanisms of audiogenic seizure and the mechanism of action of Qingyangshenylycosides ^{( 37).}

Mechanism of herbal glycoside recipes retrieving deficient ability of spatial learning memory in mice suffering from cerebral ischemia/repurfusion was studied using DNA micoarray system. Gene expression pattern was analyzed in the groups that showed increased ability of spatial learning. A 1.8-fold increase in expression was observed for many genes including genes in cell cycle regulation, signal transduction, nerve system transcription factors, DNA-binding protein, etc. Nine genes related to retrieving deficient ability of spatial learning memory when treated with glycoside recipes were found in this study ^(38).

DNA Microarrays In Pharmacogenomics

Pharmacogenomics is the study of genes and the gene products (proteins) essential for pharmacological or toxicological responses to pharmaceutical agents. Oligonucleotide-based DNA chip technology or cDNA microarray can be used to analyze gene expression profiles that are induced or repressed by xenobiotics ^{( 39 ).}

An attempt has been made to develop microarray genotyping system for multiplex analysis of a panel of single nucleotide polymorphisms (SNPs) in genes encoding proteins involved in blood pressure regulation, and to apply this system in a pilot study demonstrating its feasibility in the pharmacogenetics of anti-hypertensive drug response ^{( 40-42 ).} DNA microarray techniques might prove to be a reliable basis for predicting response (or lack of response) of individuals to herbal drugs.

Technologies designed to characterize genes and their products on a discovery scale are now having an impact on many areas of biology, including toxicology. Toxicogenomics is the sub-discipline that merges genomics with toxicology. In toxicology research, gene expression profiling facilitates mechanism-based research on toxicant action by comparing results for an experimental compound with a database. Recently several studies have demonstrated the utility of microarray analysis for studying genome-wide effects of xenobiotics and the rapid identification of toxic hazards for novel drug candidates ^(43,44).

An example of such a platform is ToxBlot II, a custom microarray containing cDNAs representing 12564 human genes chosen on the basis of their potential relevance to a broad range of toxicities. ToxBlot II allows the simultaneous expression profiling of genes representing entire cellular pathways, facilitating a very detailed investigation of potential mechanisms of toxicity ^(45). cDNA microarray analysis was used to study the expression level of genes in oral fibroblast cell lines in response to exposure to ripe areca nut extract. The results showed up-regulation of IL-6 expression and down-regulation of PDGFR, APP-1 and KGF-1 expressions in multiple cell lines assayed. The down-regulation of KGF-1 expression in oral fibroblast cell lines potentially impairs the proliferation of overlying keratinocytes, which could partially explain the frequent epithelial atrophy observed in chronic areca chewers in vivo. This study established a novel toxicogenomic database for areca nut extract ^(46). cDNA microarray analysis was also used to analyze the mRNA expression patterns of 1177 genes in ten oral cancer patients with betel quid chewing history. This study provides pilot data for understanding the pathogenesis of oral cancer in countries like Taiwan where betel quid chewing is prevalent ^(47).

DNA Microarray In Pharmacognosy

Use of authentic herbal materials is the first step to ensure quality, safety and efficacy of herbal medicines. DNA polymorphism-based assays have been developed for the identification of herbal medicines ^(48,49). In this approach, small amounts of DNA are amplified by the polymerase chain reaction and the reaction products are analyzed by gel electrophoresis, sequencing, or hybridization with species-specific probes. Recently, microarrays have been applied for the DNA sequence-based identification of medicinal plants ^(50,51).

To utilize DNA microarrays for identification and authentication of herbal material, it is necessary to identify a distinct DNA sequence that is unique to each species of medicinal plant. The DNA sequence information is then used to synthesize a corresponding probe on a silicon-based gene chip. These probes are capable of detecting complementary target DNA sequences if present in the test sample being analyzed.

A silicon-based DNA microarray using species-specific oligonucleotide probes is designed and fabricated to identify multiple toxic traditional Chinese medicinal plant species by parallel genotyping ^{( 52 ).} Chip-based authentication of medicinal plants may be useful as economical, precise tool for quality control and safety monitoring of herbal pharmaceuticals and neutraceuticals.

Identification of herbal materials, which commonly consist of dried or processed parts, is difficult. This is particularly true for similar looking herbal materials that can often vary greatly in their medicinal properties and market value. DNA microarray based technology can provide an efficient, accurate and cheaper means of testing the authenticity of hundreds of samples simultaneously while conventional chemical methodologies usually take several days for verification. Chip-based authentication of medicinal plants can be useful as a tool for quality control and safety monitoring of herbal pharmaceuticals and neutraceuticals and will significantly add to the medical potential and commercial profitability of herbal products.

This application of DNA microarrays will not only benefit the herbal drug industry but can also facilitate the identification of herbal products by regulatory authorities. Microarray analysis of gene expression can be useful for elucidating the molecular mechanisms and networks underlying the complex pharmacological function of herbal extracts and mixtures. Studying the patterns of gene expression at many stages during the treatment process can reveal mechanism from a modern genetic perspective and will help to identify biomarkers of adverse or favorable response. A positive correlation between the transcriptional response induced by an herbal drug and a database profile of an existing therapeutic agent can provide insight into target specificity, mechanism of action, as well as facilitate analysis of pathways downstream of the target. It will also help in identification of novel therapeutic applications of a herbal drug. DNA microarrays can also be used for activity-guided fractionation of herbal extracts thereby helping in narrowing down upon the active principle producing the desired effect. Microarrays have applications in the entire drug discovery pipeline to improve selection of biological targets and lead compounds. Correlation of gene expression data of herbal drug candidates with clinical outcome or biomarkers of response in biological system will facilitate selection of best candidate for drug development.

In the area of pharmacogenomics, DNA microarrays will facilitate development of individually optimized drugs based on differential gene expression patterns. Genetic polymorphism studies can be done to classify individuals according to their drug metabolizing capacities or response to disease ^(53).Microarray based approach can facilitate high throughput, fast track evidence-based herbal drug development following Reverse Pharmacology path. Phytomics, a technology platform for characterization of herbal compositions wherein, Herbal Bio Response Arrays (HBR Arrays) are used to determine bioactive constituents and biological activities of an herbal composition has recently been developed and patented ^(54).

DNA microarrays have the potential for application in different phases of herbal drug discovery and development. This includes quality control and standardization of the herbal drugs, identification and validation of new targets, the profiling of on-target and off-target effects during the optimization of new therapeutic agents, understanding molecular mechanisms of action, structure–activity relationships ⁽⁵⁵⁾ and the prediction of side-effects, and the discovery of diagnostic, prognostic, and pharmacodynamic biomarkers.

Future prospects

At present the major practical applicability of DNA microarrays remains in SNP mapping, genotyping and pharmacogenetics. In recent years, array-based sequencing (minisequencing / resequencing arrays) that combines target hybridization with enzymatic primer extension reactions has emerged as a powerful means to scan for all possible DNA sequence variations. Although DNA microarrays have huge potential for pharmacodynamics and toxigenomics applications, these are still in exploratory stage and need validation by other biological experiments. With the development of new, uniform and more sophisticated experimental designs, data management systems statistical tools and algorithms for data analysis DNA microarrays can be optimally used in herbal drug research. In spite of the huge potential offered by microarray technology, the importance of in vitro biological assays, cell-line studies and in vivo animal studies cannot be ignored. A comprehensive strategy integrating information from diverse scientific experiments and technologies will lead to molecular evidence-based herbal medicine.

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Figure 1: Applications of DNA microarray at various stages of typical drug discovery pipeline.

Table 1: Types of DNA micro array techniques and their applications:

About Authors:

Basant Kaushik , Pramod Dewangan, Laxmi Verma, Roshan Patel, Ravindra Gendle, and Shekhar Verma

Basant Kaushik
Institute of Pharmacy, RITEE, Raipur-492101, Chhattisgarh, India

Roshan Patel
Institute of Pharmacy, RITEE, Raipur, CG- 492101

Ravindra Gendle
Institute of Pharmacy, RITEE, Raipur, CG- 492101