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: 8332753
• 负责人: DAVID W CHRISTIANSON
• 金额: $ 41.47万
• 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/8332753
• 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.

描述（由申请人提供）：本申请涉及宾夕法尼亚大学化学系两名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、氙基和荧光基分子成像、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，开发一类新的氙磁共振成像（MRI）试剂，用于早期肺癌检测。与大多数原子核不同，氙-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，氙是非常可极化的，这使得它能够结合称为隐石的水溶性有机笼，具有微摩尔解离常数，并且129磁共振化学位移对隐石的分子环境非常敏感。这些特性激发了氙生物传感器用于检测癌症生物标志物的发展。我们的合作研究表明，超极化129核磁共振（NMR）生物传感器显示出巨大的前景，用于检测特定的同工酶的锌金属酶碳酸酐酶（CA），作为靶向CA的生物传感器I和II给出了不同的共振非常大的化学位移和窄线宽。我们还确定了第一个晶体结构的CAII-β-生物传感器复合物，这个结构清楚地显示了cryptophane封装一个单一的氙原子和苯磺酰胺部分协调的活性位点锌离子，如设计。现在，我们提出了一个有效的合成单对映异构体的生物传感器，这将极大地促进129 NMR谱的解释，以及这些化合物与CA同工酶的共结晶。碳酸酐酶是一种经过验证的药物靶标和癌症生物标志物。例如，几种CA同工酶，包括CA IX和XII，在恶性肿瘤中高度过表达。我们选择专注于开发用于小细胞肺癌（NSCLC）的氙生物传感器有四个原因：（1）肺癌是全球癌症死亡的主要原因，（2）NSCLC的早期检测允许外科手术治疗并显著改善患者预后;（3）CA IX和XII在大多数形式的NSCLC中高度过表达，和（4）超极化129 β容易递送至肺，在那里它提供有用的光谱特征。在这些研究中，我们建议阐明全方位的CA-cryptophane的相互作用，产生大的129 NMR化学位移，通过确定多个CA-129生物传感器复合物的结构和测量这些复合物在溶液中的超极化129 NMR光谱。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，并详细说明CA II中的计算设计突变，这些突变将改变偶极矩，同时保持蛋白质稳定性。还将开发用于预测蛋白质结合氙生物传感器的129 NMR化学位移的计算方法，使用CA II作为模型系统。使用这些模型，我们将专注于开发129 NMR生物传感器，用于早期检测NSCLC，靶向CA IX和XII。将开发Xe生物传感器，为CA I、II、IX和XII提供非常不同的共振，并将通过荧光显微镜和超极化129 Xe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，使用氙生物传感器来识别多种CA同工酶。