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Proof of Principle for a Novel Class of Antihypertensives That Target the Oxidative Activation of PKG Iα (Protein Kinase G Iα).

ABSTRACT: Arterial hypertension continues to be a major health burden. Development of new antihypertensive drugs that engage vasodilatory mechanisms not harnessed by available therapies offer therapeutic potential. Oxidants induce an interprotein disulfide in PKG Iα (protein kinase G Iα) at C42, which is associated with its targeting and activation, resulting in vasodilation and blood pressure lowering. Consequently, we developed an assay and screened for electrophilic drugs that activate PKG Iα by selectively targeting C42, as such compounds have potential as novel antihypertensives with a mechanism of action that differs from current therapies. In this way, a drug that we termed G1 was identified, which targets C42 of PKG Iα to induce vasodilation of isolated resistance blood vessels and blood pressure lowering in a mouse model of angiotensin II-induced hypertension. In contrast, these antihypertensive effects were deficient in angiotensin II-induced hypertensive C42S PKG Iα knockin mice. These transgenic mice were engineered to have the reactive cysteinyl thiol replaced with a hydroxyl so that it cannot react with endogenous vasodilatory oxidants or electrophiles such as drug G1. These studies, therefore, provide validation of PKG Iα C42 as the target of G1, as well as proof-of-principle for a new class of antihypertensive drugs that have potential for further development for clinical use in humans.

SUBMITTER: Burgoyne JR

PROVIDER: S-EPMC5548503 | biostudies-other | 2017 Sep

REPOSITORIES: biostudies-other

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Project description:In this work we demonstrate a novel method of methylation detection that utilises immunoaffinity to detect the presence of methylated DNA hybridised to a cDNA microarray. We use a monoclonal antibody specific to 5 methylcytosine to detect the presence of 5 methylcytosine in genomic DNA from human fibroblasts bearing the karyotype 45 XO. We report that over 2900 genes show the presence of methylation in this condition. We also report that 165 genes are consistently methylated in all replicates of these experiments. The methylated genes show a uniform distribution over all the chromosomes. The gene ontology of these also indicates no functional correlation between the genes that are methylated. We detect the presence of methylation in IGF2, an imprinted gene and thus known to harbour DNA methylation. The method is extremely specific and offers a quick and efficient way to analyse the methylation landscape on a high throughput scale. This method uses existing technology to assess methylation and thus can integrate very efficiently into any platform used. A method that detects DNA methylation that is not restricted to specific sequences would provide a useful platform to analyse methylation distribution among the genes in any given phenotypic condition.Availability of additional approaches to examine DNA methylation would lead to increase in our understanding of the methylome and would help define the intrinsic and extrinsic factors which govern it. Microarray based high throughput methods have been extensively used for gene expression analysis. The method described uses a microarray to analyse DNA methylation levels in a gene/cDNA/oligo specific manner. Here, genomic DNA is fragmented and used for hybridization to microarray slides. The presence of DNA cytosine methylation in the hybridized DNA is detected by employing Cy3 labelled anti 5mC( 5-methyl cytidine) antibody (monoclonal). The resolution provided by this method is dependent upon the microarray platform used. This method can be adapted to any platform thus enabling seamless integration with existing technology of gene expression analysis

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Proof of Principle for a Novel Class of Antihypertensives That Target the Oxidative Activation of PKG Iα (Protein Kinase G Iα).

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