Structural Studies of Drug Agents by Nuclear Magnetic Resonance Spectroscopy

我将首先重申LMC的核磁共振设施的功能：第一是作为LMC和NCI Frederick整个化学家社区的资源，用作常规使用设施，收集为其他PI主持的项目准备的合成化合物的核磁共振数据；2)它用于解决与实验室不同部分研究的药物分子或其他生物聚合物(肽、糖肽、寡核苷酸和寡糖)的结构和二维构象有关的更具挑战性的问题。在过去的一年里，我带头对NCI Frederick的一些仪器进行了重组，以便更有效地处理校园内的核磁共振工作。这个项目与第(2)项有关，因为我使用部分时间进行基于核磁共振的构象分析，这是我工作的关键部分，也是整个LMC的关键部分。因此，这个新项目的创建是为了确定致力于对LMC和NCI作为一个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。在过去的一个财政年度里，我们完成了三个二氟核苷的核磁共振、X射线结晶学和分子模拟的全面研究。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，并能够提出一个关于氟如何影响核苷构象参数的模型。我们对高度感兴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。这种分子在包括膀胱癌在内的多种癌症中也具有很强的抗肿瘤活性，它将成为设计抗癌治疗药物的有用工具。该多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。核磁共振还研究了其他几种类似物，试图解释结构发生微小变化就会产生的不寻常的生物效应。这项工作已经取得了进展，合成了近50个糖肽的肽段类似物，并在《医学杂志》上发表了一项SAR研究。化学。今年。由于这个特别行政区项目负责人Christopher Michejda的英年早逝，我已经接管了监督他以前的博士后工作的责任，他将继续这项工作到2009年。Piotr Kaczmarek博士一直在我的团队中工作，并准备了更多的类似物用于研究。他现在正在对分子中的糖部分进行修饰，几个类似物即将完成。2007年年中启动的另一个项目是探索免疫球蛋白的单链可变片段(ScFv)与鸡蛋白溶菌酶(HEL)的结合部位，这是由Sandra Smith-Gill和她的同事设计、克隆和综合分析的。由于难以获得scFv-HEL复合体的晶体或核磁共振结构，他们承担了一系列含有5-氟色氨酸的突变体的表达，我们正在与该小组合作研究19F核磁共振谱。我们收集了游离蛋白质(6个突变体)的光谱，并根据每个蛋白质的稳定性曲线在不同温度下用HEL进行了几次滴定研究。我们已经证明，只有在特定浓度的酶存在下，光谱才能完全分解，我们已经开始通过观察19F信号的化学位移的变化来绘制结合位置：只有6个信号中的3个随着酶的加入而发生显著变化，表明它们参与了结合。此外，我们还收集了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。在突变体上收集的松弛数据使我们能够计算T1和T2松弛时间，并将这些时间与蛋白质在氟化位置的运动联系起来。手稿草稿已经准备好，即将提交

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