Intra- and Intermolecular Dynamics of Dihydrofolate Reductase

二氢叶酸还原酶的分子内和分子间动力学

• 批准号: 7749030
• 负责人: Andrew L Lee
• 金额: $ 27.17万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-07 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7749030
• 关键词: Address; Affect; Affinity; Anti-Bacterial Agents; Antineoplastic Agents; Arts; Attention; Behavior; Binding; Biochemical; Biophysics; Cell Proliferation; Chemicals; Communicable Diseases; Communication; Complex; Coupling; DNA biosynthesis; Detection; Dihydrofolate Reductase; Disease; Dissociation; Drug Delivery Systems; Drug Design; Drug Interactions; Drug usage; Enzyme Kinetics; Enzymes; Epitopes; Equilibrium; Escherichia coli; Event; Fluorescence Spectrometry; Folate; Folic Acid Antagonists; Foundations; Goals; Holoenzymes; Human; Indium; Kinetics; Knowledge; Life; Ligand Binding; Ligands; Light; Link; Location; Malignant Neoplasms; Maps; Metabolism; Methods; Methotrexate; Modeling; Molecular Conformation; Motion; NADP; Names; One-Step dentin bonding system; Organism; Pathway interactions; Pharmaceutical Preparations; Principal Investigator; Process; Property; Protein Dynamics; Protein NMR Spectroscopy; Proteins; Relaxation; Research; Role; Science; Series; Side; Site; Solutions; Specificity; Structure; System; Tetrahydrofolates; Trimethoprim; Urea; Vertebral column; base; cofactor; comparative; conformational conversion; design; dihydrofolate; drug mechanism; enzyme mechanism; fighting; flexibility; inhibitor/antagonist; insight; millisecond; nanosecond; programs; protein structure; research study; response; small molecule

DESCRIPTION (provided by applicant): Dihydrofolate reductase (DHFR) is a small, ~20 kD enzyme that catalyzes the reduction of 7,8- dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF), a key metabolite required for DNA biosynthesis. Because of DHFR's central location in metabolism and required activity for cell proliferation, it has become an attractive drug target for the treatment of human cancers and infectious diseases. Furthermore, differences in sequence and structural properties in DHFRs from different organisms allow many of these drugs to act with high specificity. Under a separate light, extensive biochemical and structural studies on E. coli DHFR have led to DHFR becoming a paradigm for how dynamic fluctuations in protein structure facilitate enzyme function. DHFR is now known to undergo switching between distinct conformational states on a microsecond to millisecond timescale, in a manner that connects one step in the functional cycle to adjacent steps. Here, E. coli DHFR is studied in light of its importance as a model for both protein-drug interactions and enzyme dynamics. An interdisciplinary experimental approach combining protein NMR relaxation with transient and pre-steady-state kinetics will be employed to study the response of DHFR behavior to chemical denaturants and antifolate inhibitors with affinities spanning five orders of magnitude. Since off-rates are a key determinant of binding affinity, attention will be paid to the role of internal dynamics and conformational changes to ligand dissociation, in the cases of both natural substrates and antifolates. The role of picosecond-nanosecond fluctuations in stabilizing bound ternary states and promoting concerted conformational changes will be assessed, with particular focus on side-chain mobility. The DHFR system presents an excellent opportunity to study the influence of internal dynamics on folate/antifolate ejection from different conformational states; conversely, these studies will address how conformational context defines the dynamics of ligand occupancy and release. Given that conformational changes are collective motions, the inherent connectivity between residues will be mapped using an NMR perturbation-response approach. Throughout this research, emphasis will be placed on DHFR complexes containing reduced nicotinamide adenine dinucleotide phosphate cofactor (NADPH). In summary, this application seeks to gain mechanistic insights into the role of internal dynamics in protein-drug interactions, slow conformational changes, ligand ejection, and intramolecular communication in DHFR. Project Narrative Dihydrofolate reductase is the target for drugs used to treat cancer and infectious diseases, and it serves as a model for understanding protein-drug interactions. By using protein NMR spectroscopy and enzyme kinetics to identify mechanisms of protein flexibility that either stabilize or destabilize drug occupancy, a greater understanding of the determinants of drug binding affinity will be obtained. This new knowledge will increase the efficiency of the design of small molecule inhibitors to dihydrofolate reductase and other proteins.

描述（由申请人提供）：二氢叶酸还原酶 (DHFR) 是一种约 20 kD 的小型酶，可催化 7,8-二氢叶酸 (DHF) 还原为 5,6,7,8-四氢叶酸 (THF)，这是一种关键代谢物DNA生物合成所需。由于 DHFR 在代谢中的中心位置以及细胞增殖所需的活性，它已成为治疗人类癌症和传染病的有吸引力的药物靶点。此外，来自不同生物体的 DHFR 的序列和结构特性的差异使得许多此类药物具有高度特异性。另一方面，对大肠杆菌 DHFR 的广泛生化和结构研究已使 DHFR 成为蛋白质结构动态波动如何促进酶功能的范例。现在已知 DHFR 在微秒到毫秒的时间尺度上经历不同构象状态之间的切换，其方式是将功能循环中的一个步骤与相邻步骤连接起来。在此，研究了大肠杆菌 DHFR 作为蛋白质-药物相互作用和酶动力学模型的重要性。将采用将蛋白质 NMR 弛豫与瞬态和前稳态动力学相结合的跨学科实验方法来研究 DHFR 行为对化学变性剂和抗叶酸抑制剂的响应，其亲和力跨越五个数量级。由于解离速率是结合亲和力的关键决定因素，因此在天然底物和抗叶酸剂的情况下，将关注内部动力学和构象变化对配体解离的作用。将评估皮秒-纳秒波动在稳定束缚三元态和促进协同构象变化中的作用，特别关注侧链流动性。 DHFR 系统提供了一个极好的机会来研究内部动力学对不同构象状态的叶酸/抗叶酸排出的影响；相反，这些研究将解决构象背景如何定义配体占据和释放的动力学。鉴于构象变化是集体运动，残基之间的固有连接性将使用 NMR 扰动响应方法进行映射。在整个研究中，重点将放在含有还原烟酰胺腺嘌呤二核苷酸磷酸辅因子 (NADPH) 的 DHFR 复合物上。总之，本申请旨在获得内部动力学在 DHFR 中蛋白质-药物相互作用、缓慢构象变化、配体喷射和分子内通讯中的作用的机制见解。 项目叙述 二氢叶酸还原酶是用于治疗癌症和传染病的药物的靶标，它可以作为理解蛋白质-药物相互作用的模型。通过使用蛋白质核磁共振波谱和酶动力学来确定稳定或不稳定药物占据的蛋白质灵活性机制，将更好地了解药物结合亲和力的决定因素。这一新知识将提高二氢叶酸还原酶和其他蛋白质小分子抑制剂的设计效率。