Van Eyk, Dunn - Proteomic and Genomic Analysis of Cardiovascular Disease - 2003 (522919), страница 95
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Transgenic animal models are commonly used todetermine the impact of organ-specific over-expression of the candidate gene. Inversely, knock out and gene-targeting experiments are used to determine the impact of absence or reduced expression levels of the candidate genes in the pathogenesis of the disease of the interest. Gene-targeting experiments are sometimesconfounded by the functional redundancy of the candidate genes and embryoniclethality.
Therefore, several methods of conditional gene expression have been developed whereby expression of the gene of interest could be induced and regulated in a specific organ as desired. In addition, gene transfer experiments andantisense oligonucleotides are used to validate the function of the candidate genein the pathogenesis of the disease of interest.Molecular genetics and biology studies also have led to identification of severalattractive drug targets for treatment of dyslipidemia including targets involved in“reverse-cholesterol transport”.
A variety of experiments have explored the utilityof ATP binding cassette 1 (ABCA1) protein, scavenger receptor class B, type 1(SR-B1), Lecithin:cholesterol acyltransferase (LCAT), and cholesteryl ester transferprotein (CETP) as targets for lipid lowering drugs.
The process usually includesidentification of the causal gene for a familial phenotype characterized by dyslipidemia. With regard to ABCA1, molecular genetic studies of Tangier disease, aphenotype characterized by very low plasma levels of HDL-C and ApoA1 and an34734820 Genomics Perspective for Drug Discoveryincreased risk of premature coronary atherosclerosis, led to identification of mutations in the ABCA1 gene [34, 35].
In addition, SNPs in ABCA1 have been associated with the severity of coronary atherosclerosis in population studies [36, 37].Therefore, ABCA1 protein has emerged as an attractive drug target for manipulating plasma levels of HDL and treatment of atherosclerosis. Subsequent generation of transgenic and knock out mouse models has provided additional credenceto ABCA1 as a potential drug target for regulating plasma levels of lipids and reducing the risk atherosclerosis [38–43]. Accordingly, over-expression of ABCA1 results in increased plasma levels of HDL-C and apoA1 and confers protectionagainst atherosclerosis [39–41]. In contrast, knock out deletion of ABCA1 leads tovirtual absence of HDL-C [28, 43]. Collectively, the results of these experimentsalong with the result of many others validate ABCA1 as a potential drug target fortreatment of dyslipidemia in humans.Genetic and molecular biology studies have provided credence to the utility ofCETP, LCAT and Acyl coenzyme A-cholesterol acyltransferase (ACAT) as potentialdrug targets for lipid lowering effects.
With regard to CETP, the first direct clueto the potential involvement of CETP in reverse cholesterol transport emergedfrom the identification of 2 Japanese families who had CETP deficiency and extremely high levels of HDL-C [44, 45]. Subsequent cloning of the CETP cDNA[46], SNPs association studies [47], and transgenesis [48], provided further evidence as for the validity of CETP as a potential drug target to raise plasma levelsof HDL-C.
An example of a lead molecule to inhibit CETP is JTT-705, designedbased on amino acid sequence of CETP. It forms a disulfide bond with CETP, inhibits its activity, increases HDL cholesterol, decreases non-HDL cholesterol andslows the progression of atherosclerosis in animal models [49]. Phase 1 clinicaltrials are ongoing to determine safety and efficacy of the inhibition of CETP inhumans. Another attractive target for drug inhibition is ACAT, since partial inhibition of ACAT improves dyslipidemia and induces regression of atherosclerosis inexperimental models [50].
An alternative approach to inhibition is to promote expression of the target gene. This approach has been tested for two targets in dyslipidemia. One example is upregulation of expression of LCAT, which provide salutary effects on lipid profile [51]. Similarly, promotion of expression of SCAP,which cleaves and activates SREBP, is an attractive modality for treatment of highplasma levels of LDL-C, as discussed earlier [21]. Collectively, these examples illustrate the role of genomics in target identification and validation and the potentialutility of such targets in treatment of dyslipidemia in the general population.Genomics also have been used to validate the role of renin-angiotensin-aldosterone system (RAAS) and the kallikrein-kinin system as effective targets in the treatment of systemic cardiovascular diseases such as hypertension, heart failure, andrenal failure.
Overexpression of human tissue kallikrein in transgenic mice lowersthe blood pressure [52]. Similarly, gene transfer and expression of human tissuekallikrein lowers blood pressure that persists for longer than 6 weeks, attenuatescardiac hypertrophy, and improves renal function [53, 54]. Several sets of experiments also have validated components of the renin-angiotensin-aldosterone system (RAAS) as potential drug targets, despite the well-established role of inhibi-20.3 Genomics and the Process of Drug Discoverytion of the RAAS in treatment of hypertension and heart failure. Transgenic miceexpressing angiotensinogen exhibit a dose-dependent rise in blood pressure [55].In contrast, anti-sense oligonucleotides targeted against angiotensinogen mRNAlowers the blood pressure and attenuate cardiac hypertrophy in spontaneously hypertensive rats [56–58]. Similar experiments using antisense technology and genetargeting experiments [59, 60] also validate other components of the RAAS asdrug targets for chronic long-term treatment of hypertension.The adrenergic system plays a major role in a variety of cardiovascular pathologies, in particular heart failure.
Experimental studies implicate activation ofb-adrenergic kinase 1 (bARK1) in phosphorylation and desensitization of theb-adrenergic receptors in heart failure [61]. Over-expression of bARK1 in the heartattenuates myocardial contractile response to adrenergic stimulation [62]. In contrast, cardiac-restricted expression of bARK1 inhibitor leads to an enhanced cardiac contractile performance [62]. In addition, blockade of b-receptors is associatedwith reduction of bARK1 activity [63]. Collectively, these data suggest bARK1 is asuitable target for drug targeting in heart failure.
To validate the potential significance of inhibition of bARK1 in heart failure, transgenic mice have been generated that over-express an inhibitor of bARK1 in the heart and crossed to mousemodels of heart failure. Over-expression of inhibitor of bARK1 rescued the phenotype of heart failure in transgenic mice that overexpress the sarcoplasmic reticulum Ca2+-binding protein, calsequestrin [63]. The results of target validation studies have paved the way toward development and refinement of lead compounds toinhibit function of bARK1 or down-regulate its expression in the heart.20.3.3Genomics and Lead Identification and RefinementIdentification of new drug targets and their subsequent validation shifts the emphasis toward search for a lead compound that interacts with the target proteinand affects its expression or function. The lead compound is subsequently refinedto design and develop a specific drug with the desirable therapeutic index (median toxic dose/median effective dose).
The conventional approach is to performhigh-throughput screening of combinatorial libraries, comprised of large collections of all possible combinations of a set of smaller chemical structures, againstthe potential targets. Then the large pool of combinatorial libraries is fractionatedinto smaller pools optimized for an activity of interest until compounds with thehighest levels of activity are identified (deconvlution). Advances in combinatorialchemistry and high-throughput screening techniques have made it possible to testthe effect of thousands of compounds at several concentrations on a target molecule in a short period of time.
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