Structural Studies of Drug Agents by Nuclear Magnetic Resonance Spectroscopy

通过核磁共振波谱法研究药物的结构

• 批准号: 7733184
• 负责人: Joseph John Barchi
• 金额: $ 22.96万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733184
• 关键词: Affect; Area; Aromatic Amino Acids; Binding; Binding Sites; Biological; Biopolymers; Bladder Diseases; CCR; Cessation of life; Chemicals; Collaborations; Communities; Complex; Data; Environment; Enzymes; Fluorine; Gills; Glycopeptides; Goals; Immunoglobulins; Interstitial Cystitis; Malignant Neoplasms; Malignant neoplasm of urinary bladder; Manuscripts; Maps; Membrane; Minor; Mission; Modeling; Modification; Molecular Conformation; Motion; Muramidase; NMR Spectroscopy; Necrosis; Nucleosides; Numbers; Oligonucleotides; Oligosaccharides; Peptides; Pharmaceutical Preparations; Postdoctoral Fellow; Proteins; Publishing; Relaxation; Research; Resources; Scientist; Series; Signal Transduction; Site; Structure; Temperature; Therapeutic; Time; Titrations; Trifluoroethanol; Tryptophan; Walking; Work; Workload; X-Ray Crystallography; analog; anti-cancer therapeutic; base; design; instrumentation; interest; molecular modeling; mutant; programs; success; sugar; tool; tumor; two-dimensional; water solution

I will first reiterate the functions of the NMR facility of the LMC: The first is as a resource for the entire chemist community of the LMC and NCI Frederick to use as a routine use facility to walk up and collect NMR data on synthetic compounds prepared for projects under the auspices of other PI's and 2) It is used to tackle more challenging problems related to the structure and 2-dimensional conformations of drug molecules or other biopolymers (peptide, glycopeptides, oligonucleotides and oligosaccharides) studied in the various sections of the lab. In the past year I have spearheaded the reorganization of some instrumentation here at NCI fRederick to more efficiently handle the workload for NMR here on campus. This project relates to number (2) in that I use part of my time to do NMR-based conformational analysis that is a critical part of my work and of the entire LMC. Thus the creation of this new project is to define the effort dedicated to original research in the area of conformational analysis of molecules of biological significance to the LMC and NCI as a whole. In the past fiscal year, we have completed a comprehensive study of three difluorinated nucleosides by NMR X-ray crystallography and molecular modeling. Using various programs with different force fields and parameter sets, we defined these structures very precisely and were able to propose a model for how fluorine affects the conformational parameters of nucleosides. We have performed a variety of structural studies on the highly interesting glycopeptide Antiproliferative Factor (APF), a sialylated glycopeptide that is the causative agent of a necrotic disease of the bladder called interstitial cystitis. This molecule also has potent anti tumor activity in a variety of cancers including bladder cancer, and it will become a useful tool in the design of anti cancer therapeutics. The peptide is virtually unstructured in water solution but assumes some structure in membrane mimicking environments (trifluoroethanol). Several other analogues have been studied by NMR in an attempt to explain the unusual biological effects that occur with very minor changes to the structure. This work has progressed where nearly 50 peptide-segment analogues of the glycopeptide were synthesized and an SAR study was published in J. Med. Chem. this year. Due to the untimely death of Christopher Michejda, the leader of this SAR project, I have taken over the responsibility of supervising his former post doc who will continue this work into 2009. Dr. Piotr Kaczmarek has been working in my group and has prepared several more analogues for study. He is now working on modifications to the sugar portion of the molecule and several analogues are near completion. A separate project that was started in mid 2007 is to explore the binding site of a Single Chain Variable Fragment (scFV) of an immunoglobulin to hen eggwhite lysozyme (HEL) that was designed, cloned and comprehensively analyzed by Sandra Smith-Gill and her colleagues. Due to difficulties in obtaining crystal or NMR structures of the scFV-HEL complex, they undertook the expression of a series of mutants that contained 5-fluoro tryptophan in place of other aromatic amino acids and we are examining the 19F NMR spectra in collaboration with this group. We have collected spectra of the free proteins (six mutants), and have performed several titration studies with HEL at various temperatures according to the stability profile of each protein. We have shown that the spectra are completely resolved only in the presence of the enzyme at specific concentrations, and we have begun to mapped the binding site by observing changes in chemical shift of the 19F signals: Only 3 of 6 show marked changes with addition of enzyme, suggesting they are involved in binding. In addition, we have completed the assignments of the tryptophan residues by collecting spectra of deletion mutants prepared by the Smith-Gill lab. Relaxation data collected on the mutants has allowed us to calculate the T1 and T2 relaxation times and relate these to motion in the protein at he fluorinated sites. A manuscript draft is available and is very close to submission

我将首先重申LMC的NMR设施的功能：首先是作为LMC和NCI Frederick的整个化学家社区的资源，以便将常规使用设施用作常规使用设施，以便在其他PI和2）中使用的合成化合物进行NMR数据，以解决与其他挑战性的杂音相关的综合构成，以解决其他问题，并与2）相关的问题和2）与2）相关的问题和2）或在实验室的各个部分中研究的其他生物聚合物（肽，糖肽，寡核苷酸和寡糖）。在过去的一年中，我率先在NCI Frederick的某些乐器重组，以更有效地处理校园NMR的工作量。该项目与数字（2）有关，因为我将部分时间用于进行基于NMR的构象分析，这是我工作和整个LMC的关键部分。因此，这个新项目的创建是定义针对对LMC和NCI的生物学意义分子构象分析领域的原始研究的努力。在过去的财政年度中，我们通过NMR X射线晶体学和分子建模完成了三种扩散核苷的全面研究。使用具有不同力场和参数集的各种程序，我们非常精确地定义了这些结构，并能够为氟如何影响核苷的构象参数提出模型。我们已经对非常有趣的糖肽抗增生因子（APF）进行了多种结构研究，这是一种溶解的糖肽，是膀胱坏死性疾病的病因，称为间质囊肿。该分子在包括膀胱癌在内的各种癌症中还具有有效的抗肿瘤活性，它将成为抗癌治疗设计的有用工具。该肽在水溶液中几乎是非结构化的，但假设在模仿环境（三氟乙醇）的膜中存在一些结构。 NMR已经研究了其他几种类似物，以解释结构发生很小的变化而发生的异常生物学效应。在合成糖肽的近50个肽段类似的情况下，这项工作已经进行了，并在J. Med中发表了SAR研究。化学今年。由于该SAR项目的负责人克里斯托弗·米奇达（Christopher Michejda）过早去世，我接管了监督他以前的邮政文档的责任，他将继续这项工作，直到2009年。他现在正在研究分子的糖分部分，几个类似物即将完成。 2007年中期开始的一个单独的项目是探索免疫球蛋白的单个链可变片段（SCFV）的结合位点，与桑德拉·史密斯·吉尔（Sandra Smith-Gill）和她的同事对母鸡蛋白溶菌酶（HEL）（HEL）进行了设计，克隆并全面分析。由于难以获得SCFV络合物的晶体或NMR结构，他们承担了一系列突变体的表达，这些突变体含有5-氟色氨酸，代替其他芳族氨基酸，我们正在研究与该组合作的19F NMR光谱。我们已经收集了游离蛋白（六个突变体）的光谱，并根据每种蛋白质的稳定性曲线在各种温度下对HEL进行了几项滴定研究。我们已经表明，仅在特定浓度的酶的情况下完全解析了光谱，并且我们开始通过观察19F信号的化学位移变化来映射结合位点：只有6个中的3个显示出酶的添加明显变化，这表明它们与结合有关。此外，我们通过收集史密斯 - 吉尔实验室制备的缺失突变体的光谱来完成色氨酸残基的分配。在突变体上收集的松弛数据使我们能够计算T1和T2松弛时间，并将其与He氟化位点的蛋白质相关联。手稿草稿可用，非常接近提交