Structural Studies of Drug Agents by Nuclear Magnetic Resonance Spectroscopy

我首先重申LMC的NMR设施的功能：首先是作为一个 LMC和NCI Frederick的整个化学家社区的资源，作为常规使用 设施步行和收集NMR数据的合成化合物准备的项目下 其他PI的支持，2）用于解决与以下方面相关的更具挑战性的问题 药物分子或其它生物聚合物（肽， 糖肽、寡核苷酸和低聚糖）在《生物学杂志》的各个部分进行了研究 实验室在过去的一年里，我带头在这里重组了一些仪器， NCI fRederick更有效地处理校园内NMR的工作量。这个项目 我用部分时间做基于NMR的构象分析，这与（2）有关 这是我的工作和整个LMC的关键部分。因此，这一新的创造 该项目旨在确定致力于构象领域原创性研究的努力， 分析对LMC和NCI整体具有生物学意义的分子。过去 2009财年，我们完成了三种二氟核苷的核磁共振综合研究 X射线晶体学和分子模拟。使用不同的程序与不同的力量 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。我们有 对这种高度有趣的糖肽进行了各种结构研究 抗增殖因子（APF），一种唾液酸化糖肽，是肿瘤的病原体， 膀胱坏死性疾病，称为间质性膀胱炎。这种分子还具有 在包括膀胱癌在内的多种癌症中具有抗肿瘤活性，并且它将成为 在设计抗癌疗法中的有用工具。该肽实际上是非结构化的 在水溶液中，但在膜模拟环境中呈现一些结构 （三氟乙醇）。其他几个类似物已经研究了核磁共振，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 已经合成了近50种糖肽的肽段类似物 一项SAR研究发表在今年的《医学化学杂志》上。由于英年早逝的 克里斯托弗·米切达，这个搜救项目的负责人，我已经接管了 监督他的前博士后谁将继续这项工作到2009年。皮奥特·卡兹莫科博士 一直在我的小组工作，并准备了几个类似物的研究。他现在工作 对分子的糖部分进行修饰， 建成2007年年中开始的另一个项目是探索 鸡卵清溶菌酶（HEL）的免疫球蛋白的单链可变片段（scFV） 由桑德拉·史密斯-吉尔和她的 同事由于难以获得scFV-HEL的晶体或NMR结构， 复杂，他们进行了一系列突变体的表达，含有5-氟 色氨酸代替其他芳香族氨基酸，我们正在检查19 F NMR光谱， 与这个团体合作。我们已经收集了游离蛋白质（六种突变体）的光谱， 并已进行了几个滴定研究与HEL在不同的温度下，根据 每种蛋白质的稳定性概况。我们已经证明，只有在 在特定浓度的酶存在下，我们已经开始绘制 通过观察19 F信号的化学位移变化来确定结合位点：6个中只有3个显示 加入酶后有明显的变化，表明它们参与了结合。此外，本发明还提供了一种方法， 我们通过收集缺失的光谱， 由史密斯-吉尔实验室制备的突变体从变种人身上收集到的弛豫数据 我们来计算T1和T2弛豫时间，并将其与蛋白质中的运动联系起来。 氟化位点。一份手稿草稿已经准备好，并且非常接近提交

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