Structure-Based Design of Xe-129 NMR Biosensors for Multiplexed Cancer Detection

用于多重癌症检测的 Xe-129 NMR 生物传感器的基于结构的设计

基本信息

批准号：
9315851
负责人：
Ivan Julian Dmochowski
金额：
$ 36.2万
依托单位：
UNIVERSITY OF PENNSYLVANIA
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：
2011-09-15 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9315851
关键词：
Address Affinity Allosteric Site Amino Acid Motifs Area Bacteria Binding Binding Proteins Binding Sites Biological Biological Assay Biomedical Research Biophysics Biosensing Techniques Biosensor Cancer Detection Cancer Diagnostics Cell physiology Cell surface Cells Chemicals Chemistry Collaborations Color Contrast Media Crystallization Databases Detection Development Diagnosis Diagnostic Procedure Disease Enzymes Escherichia coli Eukaryotic Cell Exhibits Floods Fluorescence Microscopy France Funding Gases Gene Expression Germany Goals Grant Green Fluorescent Proteins Human Hydrophobicity Isoenzymes Kinetics Label Laboratories Lung Magnetic Resonance Magnetic Resonance Imaging Malignant neoplasm of lung Mammalian Cell Measurement Mediating Mining NMR Spectroscopy Non-Small-Cell Lung Carcinoma Patients Penetration Peptides Prevalence Process Production Proteins Publishing Reporting Research Research Personnel Resolution Roentgen Rays Scheme Signal Transduction Site Structure System Techniques Technology Testing Thermodynamics Tissues United States National Institutes of Health Variant Vesicle Virginia Water X-Ray Crystallography Xenon analog base beta-Lactamase cancer biomarkers cancer cell cancer diagnosis cancer therapy carbonate dehydratase cellular imaging cold temperature computational chemistry computer studies design experimental study fluorophore gas vesicle protein imaging study improved in vivo interest light microscopy lung imaging molecular dynamics molecular imaging multiplex detection mutant nanometer nanomolar new technology next generation open source overexpression pressure programs protein E protein biomarkers protein protein interaction protein structure public health relevance simulation small molecule

项目摘要

DESCRIPTION (provided by applicant): 129Xe NMR biosensors represent a fundamentally new class of biophysical probes with tremendous potential as cancer diagnostic agents. The proposed studies build on a Xe biosensor program that has been continuously funded (PI: Dmochowski) for the past 10 years by DoD, NIH R21, R33, and R01 grants. NIH R01 renewal funding is now requested to continue this dynamic and highly productive program. A focus of this research program is the development of 129Xe MRI contrast agents for improved diagnosis of lung cancer. To date, we have made key advances in the synthesis, xenon affinity, hyperpolarized (hp) 129Xe NMR spectroscopy, and biological application of Xe biosensors utilizing a cryptophane moiety for Xe encapsulation. The development of next-generation 129Xe MRI contrast agents is rapidly advancing, now propelled by recent improvements in 129Xe hyperpolarization technology. An 'open source' system produces near-unity polarization in ~1-L quantities required for human lung imaging. The Dmochowski laboratory will gain access to a state-of the-art xenon polarizer within the next two years, with support from S10 funding (PI: Rizi). This proposal focuses on a 129Xe NMR technique employing chemical exchange saturation transfer ('Hyper-CEST'), which was pioneered using cryptophane as the xenon host by the Pines lab at Berkeley in 2006, and incorporates concepts of xenon polarization transfer contrast (XTC) first described by Mugler and Ruppert at Virginia in 2000. In 2012, our laboratory showed that 1 picomolar cryptophane provides useful contrast using Hyper-CEST NMR, a 109-fold sensitivity enhancement over standard MRI contrast agents. This improved upon the original 5 nM cryptophane detection sensitivity reported at Berkeley, and is still roughly 100-fold more sensitive than Hyper-CEST measurements performed for single-site cryptophane entities by researchers in France and Germany. We have been able to attribute only some of these differences in Hyper-CEST efficiency to the greater Xe affinity and faster Xe exchange kinetics of our trifunctionalized, water-soluble cryptophanes. This raises several important questions: What is the operative mechanism for small molecule-mediated 129Xe magnetization transfer? Can these processes be optimized to achieve femtomolar (or better) detection sensitivity? Can small molecule and genetically encoded xenon-binding CEST agents be developed for wide distribution to labs interested in molecular imaging? To address the first question, we hypothesize that a Xe "bubble" surrounds the cryptophane, with many weakly-associated, exterior Xe atoms undergoing rapid magnetization transfer at short-range with the single interior Xe atom. This hypothesis will be rigorously tested by computational and experimental approaches in Aim 1.1, working with UPenn Chemistry collaborator Saven. While cryptophanes enable explorations of xenon biosensing, their scarcity limits use to a handful of labs worldwide. Thus, in Aim 1.2 we propose to develop new small-molecule Hyper-CEST agents that can be widely distributed for biomedical research. Our lab made the recent discovery that commercially available cucurbituril CB[6] can be detected at 1 picomolar concentration via Hyper-CEST NMR, similar to water-soluble cryptophane. Moreover, we determined that CB[6] can be detected by 129Xe NMR in cells and cell lysate. One shortcoming of CB[6] is the difficulty of functionalizing this host molecule with single targeting moieties. To overcome this problem, we will develop "turn on" CB[6] xenon biosensors that exploit the affinity of CB[6] for many organic small molecules. As with cryptophane, we will seek to elucidate and improve upon CB[6] Hyper-CEST contrast by computational and experimental approaches. Our lab will develop water-soluble cryptophane and CB[6] solutions for targeting lung cancer cells, and perform Hyper-CEST NMR spectroscopy and imaging studies. In Aim 2, we propose the development of genetically encoded "MRI analogs" of green fluorescent protein (GFP) and color variants, which are the current standard for visualizing many cellular processes by fluorescence microscopy. Cellular production of GFP increases the spatial and temporal information encoded by this fluorophore, and also circumvents many problems of cell delivery, localization, and degradation. Similarly, protein-based xenon biosensors will expand the repertoire of cellular and in vivo studies, while taking advantage of the much greater tissue penetration of MRI relative to light microscopy. A recent report of gas vesicle (GV) proteins that achieve Hyper-CEST provides useful precedent. GVs, however, are composed of 8-14 different proteins that self-assemble in bacteria but cannot be expressed in eukaryotic cells. Thus, we are focused on developing more versatile single-protein Hyper-CEST agents. MD simulations published by the Geissler laboratory led us to hypothesize correctly that beta-lactamase should enable Hyper-CEST contrast, based on its large number of cryptic allosteric sites that provide ~1-nanometer hydrophobic pockets in the protein interior where Xe may transiently reside. In collaboration with Temple collaborators (Carnevale, Klein), in Aim 2.1, we will study Xe interactions with beta-lactamase using several computational approaches, and develop variants of beta-lactamase that increase CEST contrast, while also enabling multiplexing experiments (similar to CFP, GFP, YFP, RFP for fluorescence microscopy). In Aim 2.2, we will perform Hyper-CEST NMR spectroscopy and imaging studies using beta-lactamase variants.

描述（由申请人提供）： 129Xe NMR 生物传感器代表了一种全新的生物物理探针，具有作为癌症诊断剂的巨大潜力。拟议的研究建立在过去 10 年持续资助的 Xe 生物传感器计划的基础上（PI：Dmochowski）。现在请求国防部、NIH R21、R33 和 R01 续订资金来继续这一充满活力且高效的计划。该研究项目是开发 129Xe MRI 造影剂，以改善肺癌的诊断。迄今为止，我们在利用密码烷的 Xe 生物传感器的合成、氙亲和力、超极化 (hp) 129Xe NMR 光谱和生物应用方面取得了重大进展。下一代 129Xe MRI 造影剂的开发正在迅速推进，目前受到 129Xe 最新改进的推动。超极化技术。在支持下，Dmochowski 实验室将在未来两年内获得最先进的氙偏振器，该系统可产生人体肺部成像所需的接近统一的偏振。该提案重点关注采用化学交换饱和转移（“Hyper-CEST”）的 129Xe NMR 技术，该技术率先使用 Cryptophane 作为氙。 2006 年由伯克利的 Pines 实验室主持，并结合了 2000 年弗吉尼亚州的 Mugler 和 Ruppert 首次描述的氙偏振转移衬度 (XTC) 的概念。2012 年，我们的实验室使用 Hyper-CEST NMR 表明 1 皮摩尔的隐光子可提供有用的衬度，比标准 MRI 造影剂的灵敏度提高了 109 倍，这比伯克利报告的原始 5 nM 密码检测灵敏度有所提高，但仍然大致如此。 100倍比法国和德国的研究人员对单点加密烷实体进行的 Hyper-CEST 测量更敏感，我们只能将 Hyper-CEST 效率中的一些差异归因于我们的三官能化的更大的 Xe 亲和力和更快的 Xe 交换动力学。这提出了几个重要的问题：小分子介导的 129Xe 磁化转移的操作机制是什么？能否优化这些过程以实现飞摩尔（或更好）的检测灵敏度？分子和基因编码的氙结合 CEST 试剂是否可以开发并广泛分发给对分子成像感兴趣的实验室？为了解决第一个问题，我们捕获了一个 Xe“气泡”围绕着 cryptophane，其中许多弱关联的外部 Xe 原子经历了快速的反应。单个内部 Xe 原子的短程磁化转移将与宾夕法尼亚大学化学合作者 Saven 合作，通过 Aim 1.1 中的计算和实验方法进行严格测试。因此，在目标 1.2 中，我们建议开发可广泛用于生物医学研究的新型小分子 Hyper-CEST 试剂。市售的葫芦脲 CB[6] 可以通过 Hyper-CEST NMR 检测到 1 皮摩尔浓度，与水溶性 Cryptophane 类似，此外，我们确定可以通过以下方法检测 CB[6]。细胞和细胞裂解物中的 129Xe NMR 的一个缺点是难以用单一靶向部分功能化该宿主分子。为了克服这一问题，我们将开发利用亲和力的“打开”CB[6] 氙生物传感器。与 Cryptophane 一样，我们将通过计算和实验方法来阐明和改进 CB[6] Hyper-CEST 对比。将开发用于靶向肺癌细胞的水溶性 Cryptophane 和 CB[6] 解决方案，并进行 Hyper-CEST NMR 光谱和成像研究。在目标 2 中，我们建议开发绿色荧光蛋白 (GFP) 的基因编码“MRI 类似物”。）和颜色变体，这是通过荧光显微镜可视化许多细胞过程的当前标准，GFP 的细胞产生增加了该荧光团编码的空间和时间信息，并且还避免了细胞传递的许多问题，基于蛋白质的氙生物传感器将同样扩展细胞和体内研究的范围，同时利用 MRI 相对于光学显微镜更大的组织穿透性。然而，Hyper-CEST 提供了有用的先例，GV 由 8-14 种不同的蛋白质组成，它们在细菌中自组装，但不能在真核细胞中表达，因此，我们致力于开发更多的蛋白质。 Geissler 实验室发表的多功能单蛋白 Hyper-CEST 试剂使我们正确地重新认识到，β-内酰胺酶应该能够实现 Hyper-CEST 对比，这是基于其大量的神秘变构位点，这些位点在在目标 2.1 中，我们将与 Temple 合作者（Carnevale、Klein）合作，利用多种计算方法研究 Xe 与 β-内酰胺酶的相互作用，并开发变体。增加 CEST 对比度的 β-内酰胺酶，同时还可以进行多重实验（类似于荧光显微镜的 CFP、GFP、YFP、RFP）。在目标 2.2 中，我们将使用 β-内酰胺酶变体进行 Hyper-CEST NMR 光谱和成像研究。