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

我首先要重申 LMC NMR 设施的功能：第一个是作为 LMC 和 NCI Frederick 整个化学家社区的资源，用作常规使用设施，以获取和收集为其他 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的结构和二维构象相关的更具挑战性的问题 或在实验室各个部分研究的其他生物聚合物（肽、糖肽、寡核苷酸和寡糖）。去年，我带头重组了 NCI fRederick 的一些仪器，以更有效地处理校园 NMR 的工作量。该项目与第 (2) 项相关，因为我利用部分时间进行基于 NMR 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 具有生物学意义的分子构象分析领域的原创研究的努力。在上一财年，我们通过核磁共振X射线晶体学和分子建模完成了对三种二氟化核苷的全面研究。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，并能够提出一个关于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，这是一种唾液酸化糖肽，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有效的抗肿瘤活性，它将成为设计抗癌疗法的有用工具。该肽在水溶液中实际上是非结构化的，但在膜模拟环境（三氟乙醇）中呈现出某种结构。通过核磁共振研究了其他几种类似物，试图解释结构发生微小变化时发生的不寻常的生物效应。这项工作已取得进展，合成了近 50 种糖肽的肽段类似物，并且 SAR 研究发表在 J. Med 上。化学。今年。由于这个SAR项目的负责人Christopher Michejda的英年早逝，我接手了监督他的前博士后的责任，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。他现在正致力于对该分子的糖部分进行修饰，一些类似物已接近完成。 2007 年中期启动的一个单独项目是探索免疫球蛋白单链可变片段 (scFV) 与鸡蛋清溶菌酶 (HEL) 的结合位点，该项目由 Sandra Smith-Gill 及其同事设计、克隆和全面分析。由于难以获得 scFV-HEL 复合物的晶体或 NMR 结构，他们表达了一系列含有 5-氟色氨酸代替其他芳香族氨基酸的突变体，我们正在与该小组合作检查 19F NMR 谱。我们收集了游离蛋白质（六个突变体）的光谱，并根据每种蛋白质的稳定性概况，在不同温度下用 HEL 进行了多项滴定研究。我们已经证明，只有在特定浓度的酶存在下，光谱才能完全解析，并且我们已经开始通过观察 19F 信号化学位移的变化来绘制结合位点：6 个信号中只有 3 个显示出添加酶后的显着变化，表明它们参与了结合。此外，我们还通过收集Smith-Gill实验室制备的缺失突变体的光谱，完成了色氨酸残基的归属。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，并将这些时间与氟化位点的蛋白质运动联系起来。手稿草稿已经可用并且即将提交