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：
8469525
负责人：
DAVID W CHRISTIANSON
金额：
$ 40.93万
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 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8469525
关键词：
A549 Active Sites Affinity Bicarbonate Ion Bicarbonate Ions Bicarbonates Binding Biochemical Biological Biological Assay Biological Markers Biological Models Biology Biosensor Calorimetry Cancer Detection Cancer Diagnostics Cancer Etiology Carbon Dioxide Carbonic Anhydrase I Carbonic Anhydrase II Carbonic Anhydrase Inhibitors Cell Line Cell Nucleus Cells Cessation of life Chemicals Chemistry Cocrystallography Collaborations Complex Computer Simulation Computing Methodologies Contrast Media Crystallography Data Detection Development Diagnostic Disease Dissociation Drug Targeting Early Diagnosis Encapsulated Environment Enzymes Epilepsy Evaluation Fluorescence Fluorescence Microscopy Funding Generations Glaucoma Grant Human Hydration status Hydroxide Ion Hydroxides Image Ions Isoenzymes Lung Magnetic Resonance Magnetic Resonance Imaging Malignant Epithelial Cell Malignant Neoplasms Malignant neoplasm of lung Measures Medicine Membrane Methods Milk Mitochondria Modeling Molecular Mutation NMR Spectroscopy Non-Small-Cell Lung Carcinoma Nuclear Magnetic Resonance Operative Surgical Procedures Organic Synthesis Patients Pennsylvania Positioning Attribute Process Property Protein Binding Proteins Protons Roentgen Rays Saliva Sampling Screening for cancer Signal Transduction Solutions Structure Structure of parenchyma of lung Testing Tissue Sample Tissues Titrations United States National Institutes of Health Universities Water X-Ray Crystallography Xenon Zinc base benzenesulfonamide cancer cell carbonate dehydratase carbonic anhydrase XII design dipole moment enantiomer extracellular folate-binding protein frontier human disease improved in vivo innovation lung cancer screening lung small cell carcinoma metalloenzyme molecular imaging outcome forecast overexpression professor programs research study

项目摘要

DESCRIPTION (provided by applicant): This application involves a collaboration between two PIs in the Department of Chemistry at the University of Pennsylvania, Professors Dmochowski and Christianson. We are combining our complementary expertise in organic synthesis, xenon-based and fluorescence-based molecular imaging, carbonic anhydrase inhibitor design, and protein X-ray crystallography to develop a new class of xenon magnetic resonance imaging (MRI) agents for early lung cancer detection. Unlike most atomic nuclei, xenon-129 can be hyperpolarized, which produces a ~100,000-fold signal enhancement in a MRI scanner. Furthermore, xenon is very polarizable, which allows it to bind water-soluble organic cages called cryptophanes with micromolar dissociation constants, and the 129Xe magnetic resonance chemical shift is very sensitive to the molecular environment of the cryptophane. These properties motivate the development of xenon biosensors for the detection of cancer biomarkers. Our collaborative studies indicate that hyperpolarized 129Xe nuclear magnetic resonance (NMR) biosensors show tremendous promise for the detection of specific isozymes of the zinc metalloenzyme carbonic anhydrase (CA), as Xe biosensors targeting CA I and II gave distinct resonances with very large chemical shifts and narrow linewidths. We also determined the first crystal structure of a CAII-Xe-biosensor complex, and this structure clearly shows the cryptophane encapsulating a single xenon atom and the benzenesulfonamide moiety coordinating to the active site zinc ion, as designed. We now propose an efficient synthesis for single-enantiomer Xe biosensors, which will greatly facilitate the interpretation of 129Xe NMR spectra, as well as the cocrystallization of these compounds with the CA isozymes. Carbonic anhydase is a validated drug target and cancer biomarker. For example, several CA isozymes, including CA IX and XII, are highly overexpressed in malignant tumors. We have chosen to focus on the development of xenon biosensors for small cell lung cancer (NSCLC) for four reasons: (1) lung cancer is the leading cause of cancer death worldwide, (2) early detection of NSCLC allows treatment with surgical procedures and dramatically improves patient prognosis; (3) CA IX and XII are highly overexpressed in most forms of NSCLC, and (4) hyperpolarized 129Xe is readily delivered to the lungs, where it provides useful spectroscopic signatures. In these studies, we propose to elucidate the full range of CA-cryptophane interactions that produce large 129Xe NMR chemical shifts by determining the structures of multiple CA-Xe biosensor complexes and measuring the hyperpolarized 129Xe NMR spectra for these complexes in solution. Together with Penn Chemistry colleague Jeffery Saven, we will analyze these biophysical data and elaborate computationally designed mutations in CA II that will alter the dipole moment while maintaining protein stability. Computational methods for predicting 129Xe NMR chemical shifts for protein-bound xenon biosensors will also be developed, using CA II as a model system. Using these models, we will then focus on the development of 129Xe NMR biosensors for the early detection of NSCLC, targeting CA IX and XII. Xe biosensors will be developed that give very distinct resonances for CA I, II, IX, and XII, and these will be tested in NSCLC cells via fluorescence microscopy and hyperpolarized 129Xe NMR spectroscopy and imaging. Multiplexing experiments will be performed in lung cancer cells and tissues, using xenon biosensors to identify multiple CA isozymes.

描述（由申请人提供）：此申请涉及宾夕法尼亚大学化学系的两个PI之间的合作，Dmochowski教授和克里斯蒂安森教授。我们正在结合有机合成，基于XENON和荧光的分子成像，碳酸酐酶抑制剂设计和蛋白质X射线晶体学的互补专业知识，以开发一类新的XENON磁共振成像（MRI）剂，以进行早期肺癌检测。与大多数原子核不同，Xenon-129可以被超极化，在MRI扫描仪中产生约100,000倍的信号增强。此外，氙气非常极化，它使其可以用微摩尔解离常数结合水溶性有机笼，称为隐孢子虫，而129XE磁共振化学移位对隐构物的分子环境非常敏感。这些特性激发了氙气生物传感器的发展以检测癌症生物标志物。我们的协作研究表明，超极化的129XE核磁共振（NMR）生物传感器对检测发现特定的锌金属酶碳酸盐酶（CA）的特定同工酶具有巨大的希望，因为针对Ca I和II的XE生物传感器靶向Ca I和II具有很大的化学偏移和较大的化学偏移和狭窄的旋转范围。我们还确定了CAII-XE生物传感器复合物的第一个晶体结构，并且该结构清楚地显示了封装单氙原子和苯甲磺酰胺部分的隐态固定剂，该部分是苯甲烯酰胺部分协调到活性位点锌离子的设计。现在，我们提出了对单一构想体XE生物传感器的有效合成，这将极大地促进129XE NMR光谱的解释，以及与Ca同工酶对这些化合物的共晶化。碳酸碱酶是经过验证的药物靶标和癌症生物标志物。例如，包括CA IX和XII在内的几个CA同工酶在恶性肿瘤中高表达。由于四个原因，我们选择着重于针对小细胞肺癌（NSCLC）的氙气生物传感器的开发：（1）肺癌是全球癌症死亡的主要原因，（2）NSCLC的早期发现可以通过手术程序进行治疗，并大大改善患者的预后；（3）CA IX和XII在大多数NSCLC中高度过表达，（4）（4）超极化的129倍很容易地传递到肺部，在那里它提供了有用的光谱特征。在这些研究中，我们建议阐明通过确定多个CA-XE生物传感器复合物的结构并测量溶液中这些复合物的超极化的129xE NMR光谱，从而阐明各种CA-丙烷相互作用，从而产生了129xE NMR化学移位。与Penn Chemistrague Jeffery Saven一起，我们将分析这些生物物理数据，并在CA II中进行详细的计算设计突变，这些突变将改变偶极矩，同时保持蛋白质稳定性。还将使用Ca II作为模型系统来开发用于预测蛋白质结合XENON生物传感器的129倍NMR化学移位的计算方法。然后，使用这些模型，我们将重点介绍129xE NMR生物传感器的开发，以早日检测NSCLC，以CA IX和XII为目标。将开发XE生物传感器，从而为Ca I，II，IX和XII产生非常不同的共振，并通过荧光显微镜和超极化的129xE NMR光谱和成像在NSCLC细胞中测试这些共振。多路复用实验将在肺癌细胞和组织中，使用XENON生物传感器鉴定多个CA同工酶。