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Riboswitch Based Methyltransferase HTS Assay for Epigenetic Drug Discovery

基于核糖开关的甲基转移酶 HTS 测定用于表观遗传药物发现

基本信息

批准号：
9140743
负责人：
Robert G Lowery
金额：
$ 47.05万
依托单位：
BELLBROOK LABS, LLC
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-15 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9140743
关键词：
Affinity Antineoplastic Agents Area Binding Biochemical Biological Biological Assay Chemicals Chemistry Clinical Complex Detection Development Diabetes Mellitus Disease Drug Targeting Elements Energy Transfer Enzyme-Linked Immunosorbent Assay Enzymes Epigenetic Process Event Fluorescence Polarization Fluorescence Resonance Energy Transfer Freezing Gene Expression Gene Expression Regulation Health Inflammation Lead Ligation Malignant Neoplasms Methods Methylation Methyltransferase Modification Performance Phase Production Quantum Dots RNA Reaction Reagent Research Personnel Resort S-Adenosylhomocysteine S-Adenosylmethionine Sampling Signal Transduction Site Solid Technology Therapeutic Time aptamer assay development base biomarker development catalyst commercialization companion diagnostics diagnostic assay drug discovery epigenetic drug epigenetic regulation high throughput screening histone methyltransferase improved inhibitor/antagonist innovation methyl group microbial molecular recognition nanomolar nanoparticle novel prevent scale up screening sensor small molecule stability testing targeted treatment therapeutic target

项目摘要

DESCRIPTION (provided by applicant): Epigenetic regulation of gene expression via methylation has been implicated in diverse diseases including cancer, diabetes and inflammation, and high throughput screening for histone methyltransferase (HMT) inhibitors is an area of intense drug discovery effort. However, there are significant shortcomings with existing HMT enzyme assay methods, and these are slowing exploration of the therapeutic potential of these emerging targets. Detection of specific methylation events can be quite complicated, and detection of S-adenosylhomocysteine (SAH), the invariant product of all HMT reactions, would be preferred in most cases. However, HMTs are very poor catalysts and many have very low SAM requirements - a combination of factors that creates very stringent sensitivity requirements for SAH-based assay methods. Moreover, direct detection of SAH is a very challenging molecular recognition problem as it requires a reagent capable of discriminating between SAH and S-adenosylmethionine (SAM), which differ by a single methyl group. The available SAH assays rely largely on enzymatic conversion of SAH to a detectable product, and are inherently prone to interference from screening compounds and lack the sensitivity needed for detection of some methyltransferases. The lack of suitable assay reagents is delaying and in some cases preventing the screening of potential therapeutic targets. To overcome this technical gap, we are using microbial SAH-sensing RNA aptamers, or "riboswitches", that bind SAH with nanomolar affinity and exquisite selectivity. In Phase I, we established the critical technical feasibility for this approach by showing that SAH binding to a riboswitch can be transduced into fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals without disrupting affinity or selectivity. To achieve this, we split the riboswitch into two halves, such that SAH binding induces assembly of a trimeric complex; this modification vastly improved the sensitivity, selectivity and stability of the signaling. We used the split aptamer assays, called AptaFluor SAH, to detect SAH produced by several HMTs at levels several-fold below the sensitivity limit for current assays. In Phase II we will leverage recent advances in aptamer and nanoparticle technologies to make the novel FP- and TR-FRET based assays suitable for industrial HTS, validate them extensively for inhibitor screening and profiling with HMTs, and establish stability and manufacturing aspects required for commercialization. In addition, we will develop an ultrasensitive ELISA-like assay for detecting HMT activity in biological samples using an innovative split aptamer proximity ligation method. By enabling direct, highly sensitive detection of SAH in homogenous the FP and TR-FRET AptaFluor SAH assay will provide a universal HMT assay platform for inhibitor discovery and lead optimization and allow pursuit of otherwise intractable targets. The solid phase AptaFluor SAH assay will enable discovery of biomarkers and development of companion diagnostic assays for clinical development of HMT targeted therapies. Taken together these developments will accelerate screening of new HMT targets and development of small molecule drugs for cancer, diabetes and other diseases with an epigenetic basis.