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: 8965163
• 负责人: Ivan Julian Dmochowski
• 金额: $ 38.46万
• 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/8965163
• 关键词: Address; Affinity; Allosteric Site; Amino Acid Motifs; Area; Bacteria; Binding; Binding Sites; Biological; Biological Assay; Biological Markers; 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; Human; Isoenzymes; Kinetics; Label; Laboratories; Lead; Life; Lung; Magnetic Resonance; Magnetic Resonance Imaging; Malignant Neoplasms; Malignant neoplasm of lung; Mammalian Cell; Measurement; Mediating; Mining; NMR Spectroscopy; Non-Small-Cell Lung Carcinoma; Patients; Penetration; Peptides; Prevalence; Process; Production; Protein Binding; Proteins; Publishing; Relative (related person); Reporting; Research; Research Personnel; Resolution; Roentgen Rays; Scheme; Signal Transduction; Site; Solutions; 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 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; new technology; next generation; open source; overexpression; programs; protein protein interaction; protein structure; public health relevance; 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（由申请人提供）：129 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。拟议的研究建立在一个生物传感器计划的基础上，该计划在过去10年中一直由DoD，NIH R21，R33和R 01赠款。NIH R 01更新资金现在被要求继续这个充满活力和高生产力的计划。该研究计划的重点是开发129 μ mol/L MRI造影剂，以改善肺癌的诊断。到目前为止，我们已经取得了关键的进展，在合成，氙亲和力，超极化（HP）129核磁共振光谱，和生物应用的生物传感器利用cryptophane部分的生物封装。下一代129 μ M MRI造影剂的开发正在迅速推进，目前受到129 μ M超极化技术的最新改进的推动。“开源”系统产生人类肺部成像所需的~1-L数量的接近统一的偏振。在S10基金的支持下，Dmochowski实验室将在未来两年内获得最先进的氙偏振器（PI：Rizi）。该提案集中于采用化学交换饱和转移（“Hyper-CEST”）的129 NMR技术，该技术是由伯克利的Pines实验室在2006年使用cryptophane作为氙主体而开创的，并结合了由弗吉尼亚的Mugler和Ruppert在2000年首次描述的氙偏振转移对比度（XTC）的概念。在2012年，我们的实验室表明，1皮摩尔的cryptophane使用Hyper-CEST NMR提供有用的对比度，比标准MRI造影剂灵敏度提高109倍。这改进了在Berkeley报道的原始5 nM隐石烷检测灵敏度，并且仍然是大约100倍 比法国和德国的研究人员对单点隐石实体进行的Hyper-CEST测量更敏感。我们已经能够将Hyper-CEST效率中的这些差异中的仅一些归因于我们的三官能化的水溶性隐吩的更大的亲合力和更快的亲合力交换动力学。这就提出了几个重要的问题：小分子介导的129 Ω磁化转移的运作机制是什么？这些过程是否可以优化以达到飞摩尔（或更高）的检测灵敏度？小分子和基因编码的氙结合CEST试剂能否开发用于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，我们假设一个“气泡”包围的cryptophane，与许多弱相关的，外部的natoms进行快速磁化转移在短距离与单个内部natoms。这一假设将在目标1.1中通过计算和实验方法进行严格测试，与宾夕法尼亚大学化学合作者Saven合作。虽然隐晶可以探索氙的生物传感，但它们的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试剂，可广泛用于生物医学研究。我们的实验室最近发现，市售的葫芦脲CB[6]可以通过Hyper-CEST NMR在1皮摩尔浓度下检测到，类似于水溶性的cryptophane。此外，我们确定CB[6]可以通过129 NMR在细胞和细胞裂解物中检测到。CB[6]的一个缺点是难以用单个靶向部分官能化该主体分子。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，我们将寻求通过计算和实验方法来阐明和改进CB[6] Hyper-CEST对比度。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光谱和成像研究。在目标2中，我们提出了绿色荧光蛋白（GFP）和颜色变体的遗传编码的“MRI类似物”的发展，这是目前通过荧光显微镜观察许多细胞过程的标准。GFP的细胞产生增加了由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。最近的一份报告的气体囊泡（GV）蛋白，实现超CEST提供了有用的先例。然而，GV由8-14种不同的蛋白质组成，这些蛋白质在细菌中自组装，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。由Geissler实验室发表的MD模拟使我们正确地假设β-内酰胺酶应该能够实现Hyper-CEST对比，这是基于其大量的隐蔽变构位点，这些位点在蛋白质内部提供了约1纳米的疏水口袋，其中β-内酰胺酶可以暂时驻留。在与Temple合作者（Kavalale，Klein）的合作中，在Aim 2.1中，我们将使用几种计算方法研究β-内酰胺酶与β-内酰胺酶的相互作用，并开发增加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光谱和成像研究。