High-throughput multiplex microsphere screening for toxin protease inhibitors

毒素蛋白酶抑制剂的高通量多重微球筛选

基本信息

  • 批准号:
    8069436
  • 负责人:
  • 金额:
    $ 3.77万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2011
  • 资助国家:
    美国
  • 起止时间:
    2011-01-01 至 2012-12-31
  • 项目状态:
    已结题

项目摘要

DESCRIPTION (provided by applicant): The zinc metalloproteases from Botulinum neurotoxin (BoNT) and the Lethal Toxin (LT) of B. anthracis are critical for the toxicity of their host organisms and are excellent pharmaceutical targets. Both toxins are of interest as they are components of category A biothreat agents, but the botulinum toxins are particularly significant as they are commonly used pharmaceuticals with the potential for tragic misuse. To discover active compounds for these proteases we developed a high throughput flow cytometry based protease assay to perform pilot screens of the Prestwick Library, which identified 3 lead inhibitors for Botulinum neurotoxin type A light chain protease (BoNT/A LC) and 2 lead inhibitors for the Lethal Factor protease (LF) of B. anthracis LT. We have confirmed that Ebselen is a promising inhibitor specific for BoNT/A LC with an IC50 of 5 5M. Based on our promising screening results we propose to perform extensive high throughput screening for inhibitory compounds against BoNT types A & F and LF in collaboration with the University of New Mexico Center for Molecular Discovery. Our screening assay uses high throughput flow cytometry to measure the cleavage of fluorescent fusion proteins that are attached to multiplexed microspheres. Beyond the speed at which compounds are screened, this assay is notable because it measures the activity of several proteases simultaneously across multiple full-length substrates. This offers several advantages that include reduced screening costs, homogenous endpoint activity assays, detection of inhibitors targeting known distal binding elements required for full protease activity, and immediate estimation of compound specificity via the inhibition pattern against different proteases. Lead compounds discovered in our screens will be confirmed via activity measurements in follow-up microsphere assays and two FRET protease assays, which use a simple FRET peptide or a fluorescently tagged avidin and our biotinylated fusion proteins containing full-length protease substrate and a terminal GFP tag. Our second FRET assay supports the use of full-length protease substrates, is solution based, and can be performed on fluorescence plate readers. Finally, promising compounds will be tested in cellular toxicity assays. LF toxicity assays will be performed on cells treated with LT and viability measured through luminescent readout of ATP production. We will test compounds targeting BoNT/A & F LC by administering BoNT/A & F LC to mammalian cells that intracellularly express fusion protein bearing CFP and YFP separated by a SNAP-25 or VAMP-2 substrate. Compound activity will be monitored via flow cytometry as a reduction of the loss of CFP to YFP FRET as compared to untreated cells. As a final assay, BoNT inhibitors that pass through this process will be compared in dose response assays against other commercially available BoNT LCs (B, C, D, & E) that are predicted to be active on all of our substrates. Due to the significant homology of all the BoNTs, we expect significant cross reactivity to these other proteases. The combination of our novel screening approach with our confirmation assays will enable the rapid mulitplexed discovery of active compounds for BoNT/A & F LC and LF using a simple set of screens that implements three proteases simultaneously to dramatically reduce cost and labor. Furthermore, our approach has the novel ability to use full-length substrates that will enable the discovery of inhibitor compounds that target protease interactions with substrate binding sites distal to the active site. Thus, this screening proposal offers an inexpensive approach to rapidly discover new inhibitors including novel inhibitor types that would be invisible by other methods. These molecules will be valuable to pharmaceutical development efforts, biological projects focused on protease kinetics at surfaces, and potentially as probes for the SNARE protein complex formation in the neuronal exocytosis pathway. PUBLIC HEALTH RELEVANCE: This proposal will discover new and important molecules that can serve as specific activity probes and inhibitors for the important toxin proteases from Botulinum neurotoxin and Bacillus anthracis. In this way, this proposal will discover important new compounds that can be the basis of new pharmaceuticals to treat Botulinum neurotoxin poisoning or late stage Anthrax infections.
描述(由申请人提供):来自炭疽杆菌的肉毒杆菌神经毒素(BoNT)和致死毒素(LT)的锌金属蛋白酶对其宿主生物的毒性至关重要,是极好的药物靶点。这两种毒素都令人感兴趣,因为它们是A类生物威胁剂的组成部分,但肉毒杆菌毒素尤其重要,因为它们是常用的药物,有可能造成悲剧性的滥用。为了发现这些蛋白酶的活性化合物,我们开发了一种基于流式细胞术的高通量蛋白酶测定方法,对Prestwick文库进行中试筛选,发现了3种肉毒杆菌神经毒素a型轻链蛋白酶(BoNT/ a LC)的先导抑制剂和2种炭疽杆菌lt的致死因子蛋白酶(LF)的先导抑制剂。我们已经证实Ebselen是一种有希望的BoNT/ a LC特异性抑制剂,IC50为55 m。基于我们有希望的筛选结果,我们建议与新墨西哥大学分子发现中心合作,对BoNT类型A、F和LF进行广泛的高通量筛选。我们的筛选试验使用高通量流式细胞术来测量附着在多路微球上的荧光融合蛋白的裂解。除了筛选化合物的速度之外,该试验还值得注意,因为它同时测量了多个全长底物上几种蛋白酶的活性。该方法具有以下优点:降低筛选成本、均质终点活性测定、检测针对已知远端结合元件的抑制剂以获得充分的蛋白酶活性,以及通过对不同蛋白酶的抑制模式直接估计化合物的特异性。在我们的筛选中发现的先导化合物将通过后续微球测定和两次FRET蛋白酶测定中的活性测量来确认,其中使用简单的FRET肽或荧光标记的亲和素和我们的生物素化融合蛋白包含全长蛋白酶底物和末端GFP标签。我们的第二个FRET试验支持使用全长蛋白酶底物,是溶液为基础的,可以在荧光板阅读器上进行。最后,有希望的化合物将在细胞毒性试验中进行测试。将对经LT处理的细胞进行LF毒性试验,并通过发光读出ATP生成来测量活力。我们将测试靶向BoNT/A & F LC的化合物,方法是将BoNT/A & F LC施用于细胞内表达融合蛋白的哺乳动物细胞,这些融合蛋白含有CFP和YFP,由SNAP-25或VAMP-2底物分离。与未经处理的细胞相比,将通过流式细胞术监测化合物活性,以减少CFP对YFP FRET的损失。作为最后的试验,通过这一过程的BoNT抑制剂将在剂量反应试验中与其他市售的BoNT lc (B、C、D和E)进行比较,这些lc预计在我们的所有底物上都有活性。由于所有bont具有显著的同源性,我们预计与这些其他蛋白酶具有显著的交叉反应性。我们的新筛选方法与我们的确认分析相结合,将使BoNT/A & F LC和LF的活性化合物的快速多路发现成为可能,使用一组简单的筛选,同时实现三种蛋白酶,从而显着降低成本和劳动力。此外,我们的方法具有使用全长底物的新能力,这将使发现靶向蛋白酶与活性位点远端的底物结合位点相互作用的抑制剂化合物成为可能。因此,这种筛选建议提供了一种廉价的方法来快速发现新的抑制剂,包括其他方法无法发现的新型抑制剂。这些分子将对药物开发工作、关注表面蛋白酶动力学的生物学项目,以及可能作为神经元胞吐途径中SNARE蛋白复合物形成的探针有价值。

项目成果

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STEVEN W GRAVES其他文献

STEVEN W GRAVES的其他文献

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{{ truncateString('STEVEN W GRAVES', 18)}}的其他基金

Demonstration of repeated Positionally Assisted Negative particle Rejection for High-Speed Sorting
用于高速分选的重复位置辅助负粒子剔除演示
  • 批准号:
    10081332
  • 财政年份:
    2021
  • 资助金额:
    $ 3.77万
  • 项目类别:
Amplified detection of viral RNA using catalytic DNA logic circuits
使用催化 DNA 逻辑电路放大检测病毒 RNA
  • 批准号:
    8970675
  • 财政年份:
    2014
  • 资助金额:
    $ 3.77万
  • 项目类别:
Amplified detection of viral RNA using catalytic DNA logic circuits
使用催化 DNA 逻辑电路放大检测病毒 RNA
  • 批准号:
    8806318
  • 财政年份:
    2014
  • 资助金额:
    $ 3.77万
  • 项目类别:
A biomimetic nanoparticle protease assay platform
仿生纳米颗粒蛋白酶检测平台
  • 批准号:
    8582398
  • 财政年份:
    2013
  • 资助金额:
    $ 3.77万
  • 项目类别:
High volume high throughput affordable parallel acoustic flow cytometry
高容量、高通量、经济实惠的并行声学流式细胞仪
  • 批准号:
    8575382
  • 财政年份:
    2013
  • 资助金额:
    $ 3.77万
  • 项目类别:
High volume high throughput affordable parallel acoustic flow cytometry
高容量、高通量、经济实惠的并行声学流式细胞仪
  • 批准号:
    8721985
  • 财政年份:
    2013
  • 资助金额:
    $ 3.77万
  • 项目类别:
KINETIC ANALYSIS OF TOXIN-RECEPTOR INTERACTIONS
毒素-受体相互作用的动力学分析
  • 批准号:
    8361745
  • 财政年份:
    2011
  • 资助金额:
    $ 3.77万
  • 项目类别:
High-throughput multiplex microsphere screening for toxin protease inhibitors
毒素蛋白酶抑制剂的高通量多重微球筛选
  • 批准号:
    8206465
  • 财政年份:
    2011
  • 资助金额:
    $ 3.77万
  • 项目类别:
MICROFABRICATION FOR SORTING LARGE PARTICLES
用于分选大颗粒的微加工
  • 批准号:
    8361777
  • 财政年份:
    2011
  • 资助金额:
    $ 3.77万
  • 项目类别:
DEVELOPMENT OF A HAND-HELD FLOW CYTOMETER
手持式流式细胞仪的开发
  • 批准号:
    8361759
  • 财政年份:
    2011
  • 资助金额:
    $ 3.77万
  • 项目类别:

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