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: 8069436
• 负责人: STEVEN W GRAVES
• 金额: $ 3.77万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/8069436
• 关键词: Active Sites; Anthrax disease; Avidin; Bacillus anthracis; Binding; Binding Sites; Biological; Biological Assay; Bontoxilysin; Botulinum Toxin Type A; Botulinum Toxins; Categories; Cells; Chimeric Proteins; Collaborations; Detection; Development; Distal; Dose; Elements; Exocytosis; Family; Flow Cytometry; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Infection; Inhibitory Concentration 50; Kinetics; Lead; Length; Libraries; Light; Mammalian Cell; Measurement; Measures; Metalloproteases; Methods; Microspheres; Molecular; Molecular Probes; Monitor; Neurons; New Mexico; Organism; Pathway interactions; Pattern; Peptide Hydrolases; Peptides; Pharmacologic Substance; Poisoning; Process; Production; Protease Inhibitor; Proteins; Reader; S-nitro-N-acetylpenicillamine; SNAP receptor; Screening Result; Screening procedure; Series; Solutions; Specificity; Speed; Staging; Surface; Testing; Toxic effect; Toxin; Universities; VAMP-2; Zinc; anthrax lethal factor; base; biothreat; botulinum toxin type B; cost; cross reactivity; design; ebselen; follow-up; high throughput screening; inhibitor/antagonist; interest; novel; protein complex; response; small molecule

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）：来自肉毒杆菌神经毒素（BONT）的锌金属蛋白酶和炭疽芽孢杆菌的致命毒素（LT）对其宿主生物的毒性至关重要，并且是极好的药物靶标。两种毒素都令人感兴趣，因为它们是A类生物治疗剂的组成部分，但是肉毒杆菌毒素尤其重要，因为它们是常用的药物，具有悲惨的滥用。为了发现这些蛋白酶的活性化合物，我们开发了高通量流式细胞术的蛋白酶测定法，以执行Prestwick库的试验筛选，该筛选鉴定了3个用于肉毒杆菌神经毒素A型A型轻链蛋白酶（BONT/A LC）的铅抑制剂，并确定了2种对致死因子蛋白酶蛋白酶蛋白酶蛋白酶蛋白酶蛋白酶蛋白酶蛋白酶（LF）的铅抑制剂。我们已经证实，Ebselen是一种有前途的抑制剂，特异性是BONT/A LC，IC50为5 5M。 根据我们有希望的筛查结果，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进行广泛的抑制性化合物进行广泛的抑制性化合物。我们的筛选测定法使用高吞吐量流式细胞仪来测量附着在多重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，该测定值得注意，因为它可以在多个全长底物上同时测量几种蛋白酶的活性。这提供了几个优点，包括降低筛查成本，同质端点活动分析，靶向完全蛋白酶所需的已知远端结合元件的抑制剂的检测以及通过对不同蛋白酶的抑制模式对复合特异性进行立即估算。在我们的筛网中发现的铅化合物将通过随访微球测定中的活性测量和两个FRET蛋白酶测定方法确认，这些测定方法使用简单的FRET肽或荧光标记的Avidin和我们的生物素化融合蛋白含有全长蛋白酶底物和终端GFP标签。我们的第二个FRET分析支持全长蛋白酶底物的使用，是基于溶液的，并且可以在荧光板读取器上进行。最后，有希望的化合物将在细胞毒性测定中进行测试。 LF毒性测定将对用LT处理的细胞和通过ATP生产的发光读数测量的细胞进行。我们将通过将BONT/A＆F LC施用到哺乳动物细胞来测试靶向BONT/A＆F LC的化合物，该哺乳动物细胞内细胞内表达的融合蛋白轴承CFP和YFP由Snap-25或VAMP-2底物隔开。与未处理的细胞相比，将通过流式细胞仪监测化合物活性，以减少CFP为YFP FRET的损失。作为最终测定，通过剂量响应测定法比较了通过剂量响应测定法与其他商业上可用的BONT LCS（B，C，D和E）进行比较的BONT抑制剂，这些抑制剂预计将在我们所有的所有底物上都活跃。由于所有BONT的显着同源性，我们期望与这些其他蛋白酶的显着交叉反应。 我们的新型筛选方法与我们的确认测定法的结合将使您能够使用一组简单的筛选来快速地发现BONT/A＆F LC和LF的活性化合物，从而同时实现三种蛋白酶，从而大大降低成本和劳动。此外，我们的方法具有使用全长底物的新型能力，可以发现抑制剂化合物，以靶向蛋白酶与活性位点远端的底物结合位点相互作用。因此，该筛查提案提供了一种廉价的方法来快速发现新的抑制剂，包括新型抑制剂类型，这些抑制剂类型是其他方法是看不见的。这些分子将对药物开发工作，生物学项目，侧重于蛋白酶动力学的生物学项目很有价值，并且有可能作为神经元胞胞菌病途径中的SNARE蛋白质复合物形成的探针。 公共卫生相关性：该提案将发现新的重要分子，这些分子可以用作特定的活性探针和抑制剂，从而对肉毒杆菌神经毒素和炭疽芽孢杆菌的重要毒素蛋白酶进行抑制。这样，该提案将发现重要的新化合物，这些新化合物可能是治疗肉毒杆菌神经毒素中毒或后期炭疽感染的新药物的基础。