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: 9118178
• 负责人: Ivan Julian Dmochowski
• 金额: $ 35.01万
• 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/9118178
• 关键词: Address; Affinity; Allosteric Site; Amino Acid Motifs; Area; Bacteria; Binding; Binding Proteins; Binding Sites; Biological; Biological Assay; Biomedical Research; Biosensing Techniques; Biosensor; Cancer Detection; Cancer Diagnostics; Cell physiology; Cell surface; Cells; Chemicals; Chemistry; Collaborations; Color; Contrast Media; Databases; Detection; Development; Diagnosis; Diagnostic Procedure; Disease; Enzymes; Escherichia coli; Eukaryotic Cell; Exhibits; Floods; Fluorescence Microscopy; France; Funding; Gene Expression; Germany; Goals; Grant; Green Fluorescent Proteins; Health; Human; Isoenzymes; Kinetics; Label; Laboratories; Lead; Life; 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; Virginia; Water; Work; 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; fluorophore; gas vesicle protein; improved; in vivo; interest; light microscopy; lung imaging; molecular dynamics; molecular imaging; mutant; nanometer; nanomolar; new technology; next generation; open source; overexpression; programs; protein biomarkers; protein protein interaction; protein structure; research study; simulation; small molecule; Address; Affinity; Allosteric Site; Amino Acid Motifs; Area; Bacteria; Binding; Binding Proteins; Binding Sites; Biological; Biological Assay; Biomedical Research; Biosensing Techniques; Biosensor; Cancer Detection; Cancer Diagnostics; Cell physiology; Cell surface; Cells; Chemicals; Chemistry; Collaborations; Color; Contrast Media; Databases; Detection; Development; Diagnosis; Diagnostic Procedure; Disease; Enzymes; Escherichia coli; Eukaryotic Cell; Exhibits; Floods; Fluorescence Microscopy; France; Funding; Gene Expression; Germany; Goals; Grant; Green Fluorescent Proteins; Health; Human; Isoenzymes; Kinetics; Label; Laboratories; Lead; Life; 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; Virginia; Water; Work; 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; fluorophore; gas vesicle protein; improved; in vivo; interest; light microscopy; lung imaging; molecular dynamics; molecular imaging; mutant; nanometer; nanomolar; new technology; next generation; open source; overexpression; programs; protein biomarkers; protein protein interaction; protein structure; research study; 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（由适用提供）：129XE NMR生物传感器代表了一种从根本上新的生物物理问题，具有巨大潜力作为癌症诊断剂。拟议的研究以XE生物传感器计划为基础，该计划在过去10年中一直由DOD，NIH R21，R33和R01赠款持续资助（PI：DMochowski）。现在要求NIH R01更新资金继续这一动态且高产的计划。该研究计划的重点是开发129XE MRI对比剂，以改善肺癌的诊断。迄今为止，我们在合成，氙相亲和，超极化（HP）129XE NMR光谱方面取得了关键的进步，以及利用隐pophane部分用于XE封装的XE生物传感器的生物学应用。下一代129XE MRI对比剂的发展正在迅速发展，现在由129 Xe超极性技术的最新改善所推动。 “开源”系统在人肺成像所需的〜1-l量中产生几乎不合同的极化。 DMochowski实验室将在未来两年内获得最先进的Xenon偏光层，并得到S10资金（PI：RIZI）的支持。该提议着重于采用化学交换满意度转移（“超钟”）的129xE NMR技术，该技术是在2006年在伯克利的Pines Lab在伯克利的Xenon主机进行的，并在2012年使用Cryportoy在2000年的Cryporty profors profortos in Cryport of vircon。 Hyper-CEST NMR，比标准MRI对比剂的109倍灵敏度增强。这在伯克利报道的原始5 nm隐次检测灵敏度上有所改善，但仍大约100倍 比法国和德国的研究人员对单位隐pophane实体进行的超级测量更敏感。我们只能将这些效率上的某些差异归因于我们三官能化的水溶性隐液的更大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129倍磁化转移的工作机制是什么？这些过程是否可以优化以实现femtoloral（或更好）检测灵敏度？可以开发出小分子和一般编码的氙结合剂CEST剂，以扩大分布到分子成像中有趣的实验室分布吗？为了解决第一个问题，我们假设XE“气泡”周围环境，有许多弱相关的外部Xe原子在短距离时与单个内部Xe原子进行短程快速磁化转移。假设将通过AIM 1.1中的计算和实验方法进行严格的检验，并与Upenn Chemistry合作者Saven合作。虽然隐脚剂可探索氙气生物传感，但它们的稀缺限制用于全球少数实验室。在AIM 1.2中，我们建议开发新的小分子超晶体代理，可以广泛分布在生物医学研究中。我们的实验室最近发现，可以通过超晶体NMR在1层浓度下检测到市售的黄瓜CB [6]，类似于水溶性隐孢烷。此外，我们确定在细胞和细胞裂解物中，129xE NMR可以检测到CB [6]。 CB [6]的一个缺点是用单个靶向部分功能使该宿主分子官能化的困难。为了克服这个问题，我们将开发“打开” CB [6] XENON生物传感器，以利用CB [6]对许多有机小分子的亲和力。与隐二烷一样，我们将寻求通过计算和实验方法来阐明和改善CB [6]超级对比度。我们的实验室将开发出水溶性隐者和CB [6]溶液，用于靶向肺癌细胞，并进行超中的NMR光谱和成像研究。在AIM 2中，我们提出了绿色荧光蛋白（GFP）和颜色变体的一般编码的“ MRI类似物”的发展，这是当前通过荧光显微镜可视化许多细胞过程的标准。 GFP的细胞产生增加了该荧光团编码的空间和临时信息，并规定了许多细胞输送，定位和降解的问题。同样，基于蛋白质的氙气生物传感器将扩大细胞和体内研究的曲目，同时利用MRI相对于光学显微镜的组织渗透更大。最新的气囊泡（GV）蛋白的报告提供了有用的先例。但是，GV由8-14种不同蛋白质组成，这些蛋白质在细菌中自组装，但在真核细胞中无法表达。这是我们专注于开发更通用的单蛋白超局部剂。 Geissler实验室发布的MD模拟使我们正确地假设β-内酰胺酶应基于其大量加密变构位点，使其在蛋白质内部提供〜1纳米疏水口袋的大量加密变构位点，XE内部XE可以暂时居住。在AIM 2.1中，我们将与Temple合作者（Carnevale，Klein）合作，使用几种计算方法研究XE与β-内酰胺酶的互动，并开发β-内酰胺酶的变体，从而增加CEST对比度，同时启用多重实验（类似于CFP，GFP，GFP，YFP，yfp，rfp，rfp for Fluorealsmical Microsisme）。在AIM 2.2中，我们将使用β-内酰胺酶变体执行超级NMR光谱和成像研究。