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
• 负责人: STEVEN W GRAVES
• 金额: $ 3.78万
• 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/8206465
• 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; public health relevance; 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）和炭疽芽孢杆菌致死毒素（LT）的锌金属蛋白酶对其宿主生物体的毒性至关重要，并且是极好的药物靶点。这两种毒素都令人感兴趣，因为它们是 A 类生物威胁剂的成分，但肉毒杆菌毒素尤其重要，因为它们是常用药物，有可能发生悲剧性的滥用。为了发现这些蛋白酶的活性化合物，我们开发了一种基于高通量流式细胞术的蛋白酶测定法，以对 Prestwick 库进行试点筛选，该测定法确定了 A 型肉毒杆菌神经毒素轻链蛋白酶 (BoNT/A LC) 的 3 种主要抑制剂和 2 种肉毒杆菌神经毒素 A 型轻链蛋白酶 (BoNT/A LC) 的主要抑制剂炭疽芽孢杆菌 LT 的致死因子蛋白酶 (LF)。我们已经证实 Ebselen 是一种很有前途的 BoNT/A LC 特异性抑制剂，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 A 型、F 型和 LF 型 BoNT 抑制化合物进行广泛的高通量筛选。我们的筛选测定使用高通量流式细胞术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，该测定还值得注意，因为它可以同时测量多个全长底物上多种蛋白酶的活性。这提供了几个优点，包括降低筛选成本、同质终点活性测定、检测针对完整蛋白酶活性所需的已知远端结合元件的抑制剂，以及通过针对不同蛋白酶的抑制模式立即估计化合物特异性。我们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进行确认，其中使用简单的 FRET 肽或荧光标记的亲和素以及含有全长蛋白酶底物和末端 GFP 的生物素化融合蛋白标签。我们的第二个 FRET 测定支持使用全长蛋白酶底物，基于溶液，并且可以在荧光板读数器上进行。最后，有前途的化合物将在细胞毒性测定中进行测试。 LF 毒性测定将在用 LT 处理的细胞上进行，并通过 ATP 产生的发光读数来测量活力。我们将通过向哺乳动物细胞施用 BoNT/A 和 F LC 来测试靶向 BoNT/A 和 F LC 的化合物，这些哺乳动物细胞在细胞内表达带有由 SNAP-25 或 VAMP-2 底物分隔的 CFP 和 YFP 的融合蛋白。将通过流式细胞术监测化合物活性，作为与未处理的细胞相比，CFP 至 YFP FRET 的损失的减少。作为最终测定，通过该过程的 BoNT 抑制剂将在剂量反应测定中与其他市售 BoNT LC（B、C、D 和 E）进行比较，预计这些 BoNT LC 对我们的所有底物都有活性。由于所有 BoNT 具有显着的同源性，我们预计与这些其他蛋白酶具有显着的交叉反应性。 我们的新颖筛选方法与确认测定相结合，将能够使用一组简单的筛选同时实现三种蛋白酶，从而快速多重发现 BoNT/A & F LC 和 LF 的活性化合物，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，这将使得能够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。因此，该筛选方案提供了一种廉价的方法来快速发现新的抑制剂，包括其他方法看不见的新抑制剂类型。这些分子对于药物开发工作、专注于表面蛋白酶动力学的生物项目以及潜在地作为神经元胞吐途径中 SNARE 蛋白复合物形成的探针具有重要价值。 公共健康相关性：该提案将发现新的重要分子，这些分子可以作为来自肉毒杆菌神经毒素和炭疽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，该提案将发现重要的新化合物，这些化合物可以成为治疗肉毒杆菌神经毒素中毒或晚期炭疽感染的新药物的基础。