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

• 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
• 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

 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类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 Xe 生物传感器计划的基础上，该计划在过去 10 年中一直受到 DoD、NIH R21、R33 和 R01 拨款的持续资助（PI：Dmochowski）。现在需要 NIH R01 更新资金来继续这一充满活力且高效的计划。该研究项目的重点是开发 129Xe MRI 造影剂以改善肺癌的诊断。迄今为止，我们在 Xe 生物传感器的合成、氙亲和力、超极化 (hp) 129Xe NMR 光谱以及利用 Cryptophane 部分进行 Xe 封装的生物应用方面取得了关键进展。在 129Xe 超极化技术的最新改进的推动下，下一代 129Xe MRI 造影剂的开发正在迅速推进。 “开源”系统可产生人体肺部成像所需的约 1 L 数量的近乎一致的偏振。在 S10 资金（PI：Rizi）的支持下，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，该技术由伯克利的 Pines 实验室于 2006 年率先使用 Cryptophane 作为氙主体，并结合了由 Mugler 和 Ruppert 在弗吉尼亚州于 2000 年首次描述的氙偏振转移对比度 (XTC) 的概念。2012 年，我们的实验室表明 1 皮摩尔 Cryptophane 提供了有用的对比度 使用 Hyper-CEST NMR，其灵敏度比标准 MRI 造影剂增强 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，仍然大约是 100 倍 比法国和德国的研究人员对单点加密货币实体进行的 Hyper-CEST 测量更敏感。我们只能将 Hyper-CEST 效率中的部分差异归因于我们的三官能化水溶性加密烷具有更大的 Xe 亲和力和更快的 Xe 交换动力学。这就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？能否开发小分子和基因编码的氙结合 CEST 试剂以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，我们假设 Xe“气泡”围绕着 Cryptophane，其中许多弱关联的外部 Xe 原子与单个内部 Xe 原子在短程内进行快速磁化转移。这一假设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。虽然密码学能够探索氙生物传感，但其稀缺性限制了全球少数实验室的使用。因此，在目标 1.2 中，我们建议开发新的小分子 Hyper-CEST 制剂，可广泛用于生物医学研究。我们实验室最近发现，市售的葫芦脲 CB[6] 可以通过 Hyper-CEST NMR 检测到 1 皮摩尔浓度，类似于水溶性 Cryptophane。此外，我们确定可以通过 129Xe NMR 检测细胞和细胞裂解液中的 CB[6]。 CB[6] 的一个缺点是难以用单一靶向部分功能化该宿主分子。为了克服这个问题，我们将开发“打开”CB[6]氙生物传感器，利用CB[6]对许多有机小分子的亲和力。与 Cryptophane 一样，我们将通过计算和实验方法寻求阐明和改进 CB[6] Hyper-CEST 对比。我们的实验室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 波谱和成像研究。在目标 2 中，我们建议开发绿色荧光蛋白 (GFP) 和颜色变体的基因编码“MRI 类似物”，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，并且还避免了细胞递送、定位和降解的许多问题。同样，基于蛋白质的氙生物传感器将扩大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力。最近关于实现 Hyper-CEST 的气体囊泡 (GV) 蛋白的报告提供了有用的先例。然而，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，我们专注于开发更通用的单蛋白 Hyper-CEST 试剂。 Geissler 实验室发表的 MD 模拟使我们正确假设 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，这些变构位点在 Xe 可能短暂驻留的蛋白质内部提供了约 1 纳米的疏水袋。在目标 2.1 中，我们将与 Temple 合作者（Carnevale、Klein）合作，使用多种计算方法研究 Xe 与 β-内酰胺酶的相互作用，并开发可增加 CEST 对比度的 β-内酰胺酶变体，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 波谱和成像研究。