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(申请人提供):肉毒杆菌神经毒素(BONT)和炭疽杆菌致死毒素(LT)中的锌金属蛋白酶对其宿主生物的毒性至关重要,是极好的药物靶标。这两种毒素都是令人感兴趣的,因为它们是A类生物制剂的组成部分,但肉毒杆菌毒素特别重要,因为它们是经常使用的药物,有可能发生悲惨的误用。为了发现这些酶的活性化合物,我们建立了一种基于高通量流式细胞术的蛋白酶分析方法来对Prestwick文库进行中试筛选,其中鉴定了3种肉毒神经毒素A型轻链蛋白酶(BONT/A LC)的先导抑制剂和2种炭疽杆菌致死因子蛋白酶(LF)的先导抑制剂。我们已经证实ebselen是一种很有前途的BONT/A LC的特异性抑制剂,其IC50为5.5M。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,对针对BoNT A&F和LF型的抑制性化合物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复合微球上的荧光融合蛋白的切割。除了筛选化合物的速度之外,这种分析值得注意的是它同时测量了多个全长底物上的几种蛋白酶的活性。这提供了几个优点,包括降低筛选成本,均一终点活性分析,针对完整的蛋白酶活性所需的已知末端结合元件的抑制剂的检测,以及通过对不同的蛋白酶的抑制模式立即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分析和两个FRET蛋白酶分析中的活性测量来确认,这两个分析使用的是简单的FRET肽或荧光标记的亲和素,以及我们的包含全长蛋白酶底物和末端GFP标记的生物素化融合蛋白。我们的第二个FRET分析支持使用全长的蛋白酶底物,是基于溶液的,可以在荧光平板阅读器上进行。最后,有希望的化合物将在细胞毒性测试中进行测试。LF毒性分析将在LT处理的细胞上进行,并通过发光读数测量ATP产生的活性。我们将测试针对BONT/A&F LC的化合物,方法是将BONT/A&F LC注射到哺乳动物细胞中,这些细胞内表达的融合蛋白含有由SNAP-25或VAMP-2底物分离的CFP和YFP。与未经处理的细胞相比,化合物的活性将通过流式细胞术进行监测,以减少CFP对YFP FRET的损失。作为最终的测试,通过这一过程的BONT抑制剂将在剂量反应分析中与其他商业上可获得的BONT LC(B、C、D和E)进行比较,这些药物预计在我们的所有底物上都有效。由于所有BoNTs的同源性很高,我们预计会与这些其他蛋白酶发生显著的交叉反应。我们新的筛选方法与我们的确认分析相结合,将使我们能够使用一套同时实施三种蛋白酶的简单筛选来快速多路发现BONT/A&F LC和LF的活性化合物,从而显著降低成本和劳动力。此外,我们的方法具有使用全长底物的新颖能力,这将使发现以蛋白酶与活性部位远端底物结合部位为靶点的抑制剂化合物为目标。因此,这种筛选方案提供了一种廉价的方法来快速发现新的抑制剂,包括其他方法看不到的新型抑制剂类型。这些分子将对药物开发工作、专注于表面蛋白酶动力学的生物项目有价值,并可能作为神经元胞吐途径中SNARE蛋白复合体形成的探针。 与公共卫生相关:这项提案将发现新的重要分子,这些分子可以作为肉毒杆菌神经毒素和炭疽芽孢杆菌的重要毒素蛋白酶的特异性活性探针和抑制剂。通过这种方式,这项提议将发现重要的新化合物,这些化合物可以作为治疗肉毒神经毒素中毒或晚期炭疽感染的新药物的基础。

项目成果

期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)

数据更新时间:{{ journalArticles.updateTime }}

{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

数据更新时间:{{ journalArticles.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ monograph.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ sciAawards.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ conferencePapers.updateTime }}

{{ item.title }}
  • 作者:
    {{ item.author }}

数据更新时间:{{ patent.updateTime }}

STEVEN W GRAVES其他文献

STEVEN W GRAVES的其他文献

{{ item.title }}
{{ item.translation_title }}
  • DOI:
    {{ item.doi }}
  • 发表时间:
    {{ item.publish_year }}
  • 期刊:
  • 影响因子:
    {{ item.factor }}
  • 作者:
    {{ item.authors }}
  • 通讯作者:
    {{ item.author }}

{{ 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:

相似海外基金

The Importance and Function of Heme Degrading Enzymes during Anthrax Disease
炭疽病期间血红素降解酶的重要性和功能
  • 批准号:
    9323699
  • 财政年份:
    2017
  • 资助金额:
    $ 3.77万
  • 项目类别:
Nutrient Uptake during Anthrax Disease
炭疽病期间的营养吸收
  • 批准号:
    10053299
  • 财政年份:
    2012
  • 资助金额:
    $ 3.77万
  • 项目类别:
Nutrient Uptake during Anthrax Disease
炭疽病期间的营养吸收
  • 批准号:
    10296654
  • 财政年份:
    2012
  • 资助金额:
    $ 3.77万
  • 项目类别:
Edema Toxin Suppression of Immune Responses During Anthrax Disease
炭疽病期间水肿毒素抑制免疫反应
  • 批准号:
    7695606
  • 财政年份:
    2009
  • 资助金额:
    $ 3.77万
  • 项目类别:
Edema Toxin Suppression of Immune Responses During Anthrax Disease
炭疽病期间水肿毒素抑制免疫反应
  • 批准号:
    8716418
  • 财政年份:
  • 资助金额:
    $ 3.77万
  • 项目类别:
Edema Toxin Suppression of Immune Responses During Anthrax Disease
炭疽病期间水肿毒素抑制免疫反应
  • 批准号:
    8379006
  • 财政年份:
  • 资助金额:
    $ 3.77万
  • 项目类别:
Edema Toxin Suppression of Immune Responses During Anthrax Disease
炭疽病期间水肿毒素抑制免疫反应
  • 批准号:
    8137849
  • 财政年份:
  • 资助金额:
    $ 3.77万
  • 项目类别:
Edema Toxin Suppression of Immune Responses During Anthrax Disease
炭疽病期间水肿毒素抑制免疫反应
  • 批准号:
    8320309
  • 财政年份:
  • 资助金额:
    $ 3.77万
  • 项目类别:
{{ showInfoDetail.title }}

作者:{{ showInfoDetail.author }}

知道了