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

我首先重申澜湄合作核磁共振设施的功能：首先是作为LMC和NCI的整个化学家社区的资源，Frederick将其用作常规使用设施，用于收集为其他PI主持的项目准备的合成化合物的NMR数据。2)它用于解决与药物分子或其他生物聚合物（肽，糖肽类）的结构和二维构象相关的更具挑战性的问题。寡核苷酸和寡糖)在实验室的各个部分研究。在过去的一年里，我率先在NCI弗雷德里克进行了一些仪器的重组，以更有效地处理校园核磁共振的工作量。这个项目与第(2)项相关，因为我使用了部分时间来进行基于核磁共振的构象分析，这是我的工作和整个LMC的关键部分。因此，这个新项目的创建是为了定义致力于对LMC和NCI整体具有生物学意义的分子构象分析领域的原创研究的努力。在过去的财政年度，我们通过核磁共振x射线晶体学和分子模型完成了对三种二氟化核苷的全面研究。使用不同的程序和不同的力场和参数集，我们非常精确地定义了这些结构，并能够提出氟如何影响核苷构象参数的模型。我们对非常有趣的糖肽抗增殖因子（APF）进行了各种结构研究，这是一种唾液化的糖肽，是一种称为间质性膀胱炎的膀胱坏死性疾病的病原体。该分子在包括膀胱癌在内的多种癌症中也具有强大的抗肿瘤活性，将成为抗癌药物设计的有用工具。该肽在水溶液中几乎是无结构的，但在膜模拟环境（三氟乙醇）中具有一定的结构。核磁共振已经研究了其他几种类似物，试图解释结构发生微小变化时产生的不寻常的生物效应。这项工作取得了进展，合成了近50个糖肽的肽段类似物，并在J. Med. Chem上发表了一项SAR研究。今年。由于这个SAR项目的负责人Christopher Michejda不幸去世，我接替了他之前的博士后的工作，他将继续这项工作到2009年。Piotr Kaczmarek博士一直在我的小组工作，并准备了更多的类似物供研究。他现在正致力于对分子的糖部分进行修饰，几个类似物即将完成。2007年中期开始的另一个项目是探索免疫球蛋白与蛋清溶菌酶（HEL）的单链可变片段（scFV）的结合位点，该项目由桑德拉·史密斯-吉尔（Sandra Smith-Gill）和她的同事设计、克隆并全面分析。由于很难获得scFV-HEL复合物的晶体或核磁共振结构，他们进行了一系列含有5-氟色氨酸代替其他芳香氨基酸的突变体的表达，我们正在与该小组合作检查19F核磁共振光谱。我们收集了游离蛋白（6个突变体）的光谱，并根据每种蛋白的稳定性在不同温度下用HEL进行了多次滴定研究。我们已经证明，光谱只有在特定浓度的酶存在下才能完全分辨，并且我们已经开始通过观察19F信号的化学位移变化来绘制结合位点：6个信号中只有3个随着酶的加入而发生明显变化，这表明它们参与了结合。此外，通过收集Smith-Gill实验室制备的缺失突变体的光谱，我们完成了色氨酸残基的分配。在突变体上收集的弛豫数据使我们能够计算T1和T2弛豫时间，并将这些时间与蛋白质在氟化位点的运动联系起来。一份手稿草稿是可用的，是非常接近提交