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: 8185940
• 负责人: DAVID W CHRISTIANSON
• 金额: $ 37.4万
• 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 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8185940
• 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 Delivery Systems; 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. PUBLIC HEALTH RELEVANCE: This application involves a collaboration between two PIs in the Department of Chemistry at the University of Pennsylvania, and we are combining our complementary expertise in organic synthesis, molecular imaging, and X-ray protein crystallography in the development of a new class of imaging agents for early cancer detection. Our recent studies indicate that xenon magnetic resonance imaging (MRI) contrast agents show tremendous promise for the detection of specific isozymes of the zinc metalloenzyme carbonic anhydrase (CA): CA is a validated drug target and cancer biomarker, and Xe biosensors are designed to coordinate to the active site zinc ion. For example, several CA isozymes, including CA IX and XII, are highly overexpressed in malignant tumors, including non-small cell lung cancer (NSCLC). We now propose an efficient synthesis for single-enantiomer Xe biosensors, which will promote the cocrystallization of these compounds with the CA isozymes to enable the optimization of biosensor designs and also facilitate the interpretation of isozyme- dependent differences in 129Xe chemical shifts. Using computational approaches together with Penn colleague Jeffery Saven, and mutational analysis of CA II as a model system, we propose to elucidate CA- cryptophane interactions that produce large changes in 129Xe NMR chemical shift, and in the process, develop a new class of xenon-based MRI contrast agents for the early detection of NSCLC.

描述(由申请人提供)：这项申请涉及宾夕法尼亚大学化学系的两名个人主管Dmochowski教授和Christian Senson教授之间的合作。我们正在结合我们在有机合成、基于氙气和基于荧光的分子成像、碳酸氢酶抑制剂设计和蛋白质X射线结晶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气磁共振成像(MRI)试剂。与大多数原子核不同，氙气-129可以超极化，这将在核磁共振扫描仪中产生约10万倍的信号增强。此外，氙气是极易极化的，这使得它可以将称为隐形分子的水溶性有机笼子与微摩尔解离常数结合在一起，而129Xe的磁共振化学位移对隐形分子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记物的氙气生物传感器的发展。我们的合作研究表明，超极化的129Xe核磁共振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生物传感器具有明显的共振，具有非常大的化学位移和很窄的线宽。我们还测定了CaII-Xe-生物传感器络合物的第一个晶体结构，该结构清楚地显示了包裹单个氙原子的隐形原子和与活性中心锌离子配位的苯磺酰胺部分。我们现在提出了一种高效的单对映体Xe生物传感器的合成方法，这将极大地促进129Xe核磁共振谱的解释，以及这些化合物与CA同工酶的共结晶。无水碳酸酶是一种有效的药物靶点和癌症生物标记物。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。我们选择关注用于小细胞肺癌(NSCLC)的氙气生物传感器的开发有四个原因：(1)肺癌是全球癌症死亡的主要原因，(2)早期发现非小细胞肺癌能够通过手术治疗，极大地改善患者预后；(3)CAIX和XII在大多数形式的非小细胞肺癌中高度表达，(4)超极化的129Xe很容易进入肺部，在那里它提供了有用的光谱信号。在这些研究中，我们建议通过确定多个CA-Xe生物传感器络合物的结构和测量这些络合物在溶液中的超极化129Xe核磁共振谱，来阐明产生大的129Xe核磁共振化学位移的CA-隐含物相互作用的全部范围。我们将与宾夕法尼亚大学化学同事杰弗里·塞文一起分析这些生物物理数据，并精心设计CA II中的计算设计突变，这些突变将在保持蛋白质稳定性的同时改变偶极矩。使用CA II作为模型系统，还将开发预测蛋白质结合氙气生物传感器的129Xe核磁共振化学位移的计算方法。利用这些模型，我们将重点开发针对CAIX和XII的129Xe核磁共振生物传感器，用于NSCLC的早期检测。Xe生物传感器将被开发出来，为CA I、II、IX和XII提供非常明显的共振，并将通过荧光显微镜和超极化129Xe核磁共振光谱和成像在NSCLC细胞中进行测试。多重实验将在肺癌细胞和组织中进行，使用氙气生物传感器来鉴定多个CA同工酶。 公共卫生相关性：这项申请涉及宾夕法尼亚大学化学系两名PI之间的合作，我们正在结合我们在有机合成、分子成像和X射线蛋白质结晶学方面的互补专业知识，开发用于早期癌症检测的新型显像剂。我们最近的研究表明，氙气磁共振成像(MRI)造影剂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：CA是一个有效的药物靶标和癌症生物标志物，而Xe生物传感器旨在与活性部位的锌离子配位。例如，几种CA同工酶，包括CA IX和XII，在包括非小细胞肺癌(NSCLC)在内的恶性肿瘤中高度过度表达。我们现在提出了一种高效的单对映体Xe生物传感器的合成方法，这将促进这些化合物与CA同工酶的共结晶，从而实现生物传感器的优化设计，并有助于解释129Xe化学位移中依赖于同工酶的差异。利用与宾夕法尼亚大学同事Jeffery Saven的计算方法，并以CA II的突变分析为模型系统，我们建议阐明CA与密码相互作用导致129Xe核磁共振化学位移的巨大变化，并在此过程中开发一类新的基于氙气的MRI造影剂用于NSCLC的早期检测。