Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 94
Текст из файла (страница 94)
Potential drug targets are identified through genome-wide chromosomal mapping,profiling of mRNA expression (also referred to as transcriptome), profiling of protein expression (proteomics), genotype-phenotype correlation studies and largescale SNP/haplotype association studies. Consequently, a genomic approach couldprovide for identification of novel molecular pathways as targets for drug therapy.In addition, it could lead to identification of known pathways that were not previously implicated in the pathogenesis of disease of interest and thus, were notconsidered targets for drug therapy.
Given the number of genes in the human genome, estimated at approximately 35,000, and the number of encoded proteinsand their isoforms, it is quite conceivable that the potential targets for drug development may be amplified at least by an order of magnitude or even greater.Genomics has had the greatest impact on identification of the causal genes forsingle-gene disorders. As of 02/09/2002 over 30,000 mutations in more than 1,000human genes have been identified (http://archive.uwcm.ac.uk/uwcm/mg/docs/hahaha.html).
During the past 15 years, the causal genes for more than 100 differentdiseases involving the cardiovascular system have been identified. The direct consequence of identification of the causal gene is to design and develop drugs thatcould specifically target the mutant protein. For a recessive disorder, whereby thedisease results from the lack of a specific protein, identification of the causal genecould lead to replacement of the deficient protein. Enzyme replacement therapyhas been tested in human patients with Fabry disease; an X-linked recessive lysosomal storage disorder caused by a deficiency of a-galactosidase A [17]. The phenotype manifests as painful neuropathy, progressive renal, cardiovascular, and cerebrovascular dysfunction and premature death [17].
Preliminary studies showsafety and efficacy of single infusions of a-galactosidase A prepared from transfected human fibroblasts [18]. For an autosomal dominant disorder, specific drugscould be designed to manipulate expression or function of the mutant protein.This is illustrated in the case of familial hypercholesterolemia, caused by muta-20.3 Genomics and the Process of Drug Discoverytions in the low-density lipoprotein receptor (LDLR) gene [19].
The phenotype ischaracterized by the deficiency of LDL receptors in hepatocytes, severe hyperlipidemia and premature atherosclerosis. While the primary therapeutic target protein for reduction of LDL-cholesterol is HMG CoA reductase, insight into the regulation of the LDLR gene has provided additional targets.
It is shown that the expression of LDL receptors is regulated by the sterol regulatory binding protein(SREBP) transcription factors and SREBP cleavage protein (SCAP) [20]. New compounds have been developed that bind to SCAP and activate cleavage of SREBPs[21]. Increased expression level of the active SREBPs in the cell nucleus activatestranscription of LDL receptors in the hepatocytes [21], resulting in increased LDLreceptor density, increased hepatic uptake of LDL and decreased levels of plasmaLDL and triglycerides [21]. Thus, SCAP ligands have emerged as potential newdrugs for treatment of patients with dyslipidemia that could be used in conjunction with HMG CoA reductase inhibitors to maximize the benefits.Genetic and molecular biology studies also could provide significant insightinto the pathogenesis of the disease of interest and thus identification of potentialtargets.
One example is the case of hypertrophic cardiomyopathy, an autosomaldominant disease for which currently no specific therapy is available. During thepast 10 years the molecular genetic basis of HCM has been elucidated and significant insight has been gained into the pathogenesis of its phenotype [22] .
In addition, expression profiling has led to identification of genes that are differentiallyexpressed in the heart of patients with HCM [23]. The collective results of theseexperiments have led to the notion that hypertrophy and fibrosis are secondaryphenotypes due to activation of stress-responsive signaling molecules and kinases,and thus, potentially reversible [24]. Insight into the pathogenesis of HCM – derived from genetic studies, has led to the new application of existing drugs to reverse and attenuate cardiac phenotype [25, 26]. We have shown blockade of signaling kinases implicated in cardiac hypertrophy with simvastatin could reverse evolving cardiac hypertrophy and fibrosis and improve cardiac function in a transgenic rabbit model of HCM [25].
Similarly, we have shown that losartan, an angiotensin II receptor 1 blocker, could reverse and normalize interstitial fibrosis in atransgenic mouse model of HCM [26]. Thus, genomics, by providing insight intothe pathogenesis of disease, not only could provide for the opportunity to developnew drugs, but also could provide for new applications of existing drugs.While thus far, genomics has had the greatest impact on identification of thecausal genes for monogenic disorders, construction of SNP and haplotype mapsof the human genome is now shifting the paradigm toward mapping and identification of the susceptibility genes for common complex disorders. Similar tomonogenic disorders, identification of genes involved in susceptibility to complexphenotypes, such as atherosclerosis, hypertension, and dyslipidemia, will identifya variety of new targets for drug development. This point is illustrated for identification and cloning of human angiotensin-1 converting enzyme 2 (ACE2) [27],which was identified through 5' sequencing of a human heart failure ventricularcDNA library.
Unlike ACE1, ACE2 is expressed only in the heart, kidney, and testis and is localized predominantly to the endothelium of coronary and intra-renal34534620 Genomics Perspective for Drug Discoveryvessels and to renal tubular epithelium [27]. ACE2 hydrolyzes angiotensin 1 to angiotensin 1–9, but does not cleave bradykinin [27]. Therefore, given the organand cell-specific expression of ACE2 and its unique cleavage of key vasoactive peptides, it is an attractive drug target for blocking expression of the local renin-angiotensin system in the heart and kidney in patients with heart failure and hypertension.Genomic Tools for Drug Target Identification. A cadre of genomic tools is currently available for identification of potential drug targets and the list is expanding very rapidly. It encompasses tools used to profile gene expression – at mRNAor protein level – in a pathological state, bio-informatics and structural biology,and many others. The utility of these techniques is not restricted to identificationof potential drug targets but also extends to target validation as well.
The majorityof the currently used techniques, such as expression profiling using micro-arrayDNA chips and proteomics, have been discussed in separate chapters. Therefore,our discussion will be limited to their utility on drug discovery.Expression profiling is performed to identify genes that are differentially expressed, at the mRNA or protein level, in a disease state. Identification of the differentially expressed genes not only could provide insight into the pathogenesis ofthe disease under investigation but also could provide for new targets for drugs.Currently, several techniques are available for detection of differentially expressedgenes at the mRNA level, including subtraction hybridization, DNA micro-arraychips, and serial analysis of gene expression (SAGE).
High-throughput sequencing of Expressed Sequence Tags (ESTs) has led to development of a compendiumof differentially expressed genes in the cardiovascular system [28]. Similarly, subtraction hybridization has led to identification of differentially expressed genes –in part – in human hypertrophic cardiomyopathy [23]. The advantage of techniques, such as subtraction hybridization, EST sequencing and SAGE, is theirability to detect expression of the known and novel genes without a priori knowledge of their involvement in the pathogenesis of the disease.
However, these techniques are somewhat cumbersome and may require several sets of experimentsfor comprehensive analysis. In contrast, micro-array DNA chips, while restrictedto known sequences, provide for screening of a large number of genes in a singlehybridization assay. Until recently, microarray chips provided for screening of arelatively limited number of known genes as potential drug targets and thus precluded discovery of unknown genes. The completion of The Human Genome Project and identification of expressed sequences have diminished this problem significantly.
The recent microarray DNA chips comprise approximately 39,000 human transcripts from more than 33,000 well-characterized human genes (http://www.affymetrix.com). Micro-array technology has been utilized to detect changes ingene expression in a variety of cardiovascular pathobiologies including myocardialischemia, heart failure and reperfusion injury [29–31]. It also provides for the opportunity to monitor gene expression following pharmacological interventions [32,33]. DNA micro-array chips have been used to detect gene expression followingmyocardial infarction before and after treatment with an ACE-1 inhibitor [33]. Pro-20.3 Genomics and the Process of Drug Discoveryfiling gene expression before and after pharmacological intervention could identify additional previously unidentified targets.Proteomics provide global analysis of gene expression at the protein level andcould be used to analyze protein content of a given organ or cells at a given time.Several methods also have been developed to identify the differentially expressedproteins, the ultimate drug targets, in disease states.
The conventional approachof using 2D gel electrophoresis has been conjoined with the power technique ofmass spectrometry to identify the proteins based on their mass. Recently, a highthroughput protein analyzer based on Matrix Assisted Laser Desorption Ionization(MALDI) source and Time of Flight optics has been developed, which could accelerate discovery and identification of the differentially expressed proteins by a magnitude of order. Data derived from expression profiling along with advances incomputational techniques and bioinformatics have provided a wealth of information that could be used as potential drug targets.20.3.2Genomics and Target ValidationGenomic based techniques commonly lead to identification of a large number ofpotential drug targets.
To identify the most relevant candidates, it is necessary tovalidate the role of each specific candidate in the pathogenesis of the disease of interest. The initial approach is largely based on selection of targets that are biologically plausible candidates as suggested by the existing biological data. Subsequentexperiments, to validate individual genes and proteins as the potential candidatesfor drug development, may involve in vitro and in vivo experiments using techniques of functional genomics.
![](https://s.studizba.com/z.php?f=/templates/si/i/noavatar.png&w=100&h=100&t=1"){kind=avatar}
