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）和B的致死毒素（LT）的锌金属蛋白酶。炭疽菌对其宿主生物体的毒性至关重要，并且是极好的药物靶标。这两种毒素都是令人感兴趣的，因为它们是A类生物威胁剂的成分，但肉毒杆菌毒素特别重要，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，我们开发了基于高通量流式细胞术的蛋白酶测定以进行Prestwick文库的中试筛选，其鉴定了肉毒杆菌神经毒素A型轻链蛋白酶（BoNT/A LC）的3种先导抑制剂和B的致死因子蛋白酶（LF）的2种先导抑制剂。Ebselen对BoNT/A LC有较好的特异性抑制作用，其IC_（50）为5.5M。 基于我们有希望的筛选结果，我们建议与新墨西哥州大学分子发现中心合作，对A型和F型BoNT和LF型BoNT的抑制性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于多重微球的荧光融合蛋白的切割。除了筛选化合物的速度之外，该测定法是值得注意的，因为它同时测量多种蛋白酶在多种全长底物中的活性。这提供了几个优点，包括降低的筛选成本、同质终点活性测定、检测靶向完全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。在我们的屏幕中发现的先导化合物将通过后续微球测定和两个FRET蛋白酶测定中的活性测量来确认，所述测定使用简单的FRET肽或荧光标记的抗生物素蛋白和我们的生物素化融合蛋白，其含有全长蛋白酶底物和末端GFP标签。我们的第二个FRET检测支持使用全长蛋白酶底物，是基于溶液的，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试验中进行测试。将对用LT处理的细胞进行LF毒性测定，并通过ATP产生的发光读数测量活力。我们将通过将BoNT/A & F LC施用于哺乳动物细胞来测试靶向BoNT/A & F LC的化合物，所述哺乳动物细胞在细胞内表达携带由SNAP-25或VAMP-2底物分离的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，将在剂量响应测定中将通过该过程的BoNT抑制剂与预测对我们的所有底物具有活性的其他市售BoNT LC（B、C、D和E）进行比较。由于所有BoNT的显著同源性，我们预期与这些其他蛋白酶的显著交叉反应性。 我们的新型筛选方法与我们的确认测定的组合将使得能够使用同时实施三种蛋白酶的一组简单筛选来快速多重发现BoNT/A & F LC和LF的活性化合物，以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，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，包括通过其他方法不可见的新型抑制剂类型。这些分子将是有价值的药物开发工作，生物学项目集中在蛋白酶动力学在表面上，并可能作为探针的SNARE蛋白复合物的形成在神经元胞吐途径。 公共卫生关系：该方案将发现新的和重要的分子，可以作为特异性活性探针和抑制剂的重要毒素蛋白酶从肉毒神经毒素和炭疽杆菌。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的基础。