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: 8901574
• 负责人: DAVID W CHRISTIANSON
• 金额: $ 5.59万
• 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 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8901574
• 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

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）：该申请涉及宾夕法尼亚大学化学系的两位 PI（Dmochowski 教授和 Christianson 教授）之间的合作。我们正在结合我们在有机合成、基于氙和基于荧光的分子成像、碳酸酐酶抑制剂设计和蛋白质 X 射线晶体学方面的互补专业知识，开发一种用于早期肺癌检测的新型氙磁共振成像 (MRI) 试剂。与大多数原子核不同，氙 129 可以超极化，从而在 MRI 扫描仪中产生约 100,000 倍的信号增强。此外，氙具有很强的极化性，这使得它可以与具有微摩尔解离常数的水溶性有机笼（称为cryptphanes）结合，并且129Xe磁共振化学位移对cryptphane的分子环境非常敏感。这些特性推动了用于检测癌症生物标志物的氙生物传感器的开发。我们的合作研究表明，超极化 129Xe 核磁共振 (NMR) 生物传感器在检测锌金属酶碳酸酐酶 (CA) 的特定同工酶方面显示出巨大的前景，因为针对 CA I 和 II 的 Xe 生物传感器给出了具有非常大的化学位移和窄线宽的独特共振。我们还确定了 CAII-Xe-生物传感器复合物的第一个晶体结构，该结构清楚地显示了封装单个氙原子的 Cryptophane 和与活性位点锌离子配位的苯磺酰胺部分，正如设计的那样。我们现在提出了一种单对映异构体 Xe 生物传感器的有效合成方法，这将极大地促进 129Xe NMR 谱的解释，以及这些化合物与 CA 同工酶的共结晶。碳酸酐酶是经过验证的药物靶点和癌症生物标志物。例如，几种CA同工酶，包括CA IX和XII，在恶性肿瘤中高度过度表达。我们选择重点开发用于小细胞肺癌（NSCLC）的氙生物传感器，原因有四个：（1）肺癌是全世界癌症死亡的主要原因，（2）早期发现 NSCLC 可以进行外科手术治疗，并显着改善患者预后； (3) CA IX 和 XII 在大多数形式的 NSCLC 中高度过表达，并且 (4) 超极化 129Xe 很容易传递到肺部，在肺部提供有用的光谱特征。在这些研究中，我们建议通过确定多个 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 129Xe NMR 谱来阐明产生大 129Xe NMR 化学位移的全方位 CA-cryptophane 相互作用。我们将与宾夕法尼亚大学化学系的同事 Jeffery Saven 一起分析这些生物物理数据，并详细阐述 CA II 中计算设计的突变，这些突变将改变偶极矩，同时保持蛋白质稳定性。还将开发使用 CA II 作为模型系统来预测蛋白质结合氙生物传感器的 129Xe NMR 化学位移的计算方法。使用这些模型，我们将重点开发 129Xe NMR 生物传感器，用于早期检测 NSCLC，针对 CA IX 和 XII。将开发 Xe 生物传感器，为 CA I、II、IX 和 XII 提供非常独特的共振，并将通过荧光显微镜和超极化 129Xe NMR 光谱和成像在 NSCLC 细胞中测试这些传感器。将在肺癌细胞和组织中进行多重实验，使用氙生物传感器来识别多种 CA 同工酶。