Biomolecular Structure and Mechanism, Structure-Based Drug Design

生物分子结构与机制、基于结构的药物设计

• 批准号: 7592663
• 负责人: XINHUA JI
• 金额: $ 133.18万
• 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/7592663
• 关键词: 6-hydroxymethyl-7,8-dihydropterin; Alkylating Agents; Antibiotics; Antibodies; Antineoplastic Agents; Biological; Biological Models; Catalytic Domain; Cells; Chemistry; Class; Complex; DNA-Directed RNA Polymerase; Development; Dihydroneopterin aldolase; Dimerization; Diphosphotransferases; Double-Stranded RNA; Drug Design; Elements; Enzymes; Family; Family member; Folate; Glutathione; Glutathione S-Transferase; Hydrolysis; Ions; Lead; Mammals; Mediating; Modeling; Modification; Molecular Target; Nitric Oxide; Nucleotides; Object Attachment; Pathway interactions; Pharmaceutical Preparations; Process; Prodrugs; Proteins; RNA; RNA Interference; RNA Processing; Reaction; Resistance; Ribonuclease III; Shikimate kinase; Small Interfering RNA; Specificity; Structure; Sulfonamides; System; Trimethoprim; Virus; antimicrobial drug; base; cancer cell; design; endonuclease; enzyme mechanism; enzyme pathway; human DICER1 protein; member; microorganism; neoplastic cell; novel; phosphodiester; shikimate; shikimate dehydrogenase; transcription factor

RNA interference is mediated by small interfering RNAs produced by members of the ribonuclease III (RNase III) family, including Dicer. For mechanistic studies, bacterial RNase III has been a valuable model system for the entire family. Previously, we have shown how the dimerization of the endonuclease domain of the enzyme creates a catalytic valley where two catalytic sites are located, how the catalytic valley accommodates a dsRNA in a manner such that each of the two RNA strands is aligned with one of the two catalytic sites, how the hydrolysis of each strand involves both subunits (residues from one subunit are involved in the selection of the scissile bond, while those from the partner subunit are involved in the cleavage chemistry), and how RNase III uses the two catalytic sites to create the 2-nucleotide 3' overhangs in its products. Recently, we have demonstrated how Mg2+ is essential for the formation of a catalytically competent protein-RNA complex, how the use of two Mg2+ ions can drive the hydrolysis of each phosphodiester bond, and how conformational changes in both the substrate and the protein are critical elements for assembling the catalytic complex. Moreover, we have modeled a protein-substrate complex and a protein-reaction intermediate (transition state) complex in a meaningful way. Together, the models and crystal structures suggest a stepwise mechanism for the enzyme to execute the phosphoryl transfer reaction. The structural information of protein-dsRNA interactions and the mechanism of dsRNA processing by bacterial RNase III can be extrapolated to other family members, including eukaryotic Rnt1p, Drosha and Dicer. The folate and shikimate pathways are essential for microorganisms and some of the enzymes in the two pathways are absent from mammals, offering ideal targets for the development of novel antimicrobial agents. For example, the molecular targets for both sulfonamides and trimethoprim are folate pathway enzymes. We have obtained a sufficient amount of structural information for 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydroneopterin aldolase (DHNA) in the folate pathway and of shikimate kinase and shikimate dehydrogenase in the shikimate pathway, which allowed us to derive the catalytic mechanism for these enzymes. These enzymes are not targets for any existing drugs and therefore are ideal targets for structure-based design of novel antibiotics. Glutathione S-transferase (GST) catalyzes glutathione conjugation with electrophilic compounds. In preneoplastic and neoplastic cells, specific forms of GST are expressed at high levels and to participate in the cells' resistance to anticancer drugs. Class pi GST (GSTP) is of particular importance in biological resistance to alkylating agents. A new family of GST-activated prodrugs has shown great potential, which function by releasing nitric oxide inside cancer cells. We have achieved GSTP specificity of a lead compound with two structural modifications. In addition, we have determined several GSTP structures containing inactivated glutathione molecules for structural characterization of GSTP in complex with prodrug molecules.

RNA干扰由核糖核酸酶III（RNase III）家族成员（包括Dicer）产生的小干扰RNA介导。对于机制研究，细菌RNase III一直是整个家族的有价值的模型系统。之前，我们已经展示了酶的核酸内切酶结构域的二聚化如何产生两个催化位点所在的催化谷，催化谷如何以使得两条RNA链中的每条与两个催化位点中的一个对齐的方式容纳dsRNA，每条链的水解如何涉及两个亚基（来自一个亚基的残基参与选择易断裂键，而来自配偶体亚基的残基参与切割化学），以及RNase III如何使用两个催化位点在其产物中产生2-核苷酸3'突出端。最近，我们已经证明了Mg 2+是如何形成催化活性蛋白质-RNA复合物所必需的，如何使用两个Mg 2+离子可以驱动每个磷酸二酯键的水解，以及底物和蛋白质的构象变化是组装催化复合物的关键因素。此外，我们已经模拟了蛋白质底物复合物和蛋白质反应中间体（过渡态）复杂的有意义的方式。总之，模型和晶体结构表明，逐步机制的酶执行磷酰基转移反应。蛋白质-dsRNA相互作用的结构信息和细菌RNase III处理dsRNA的机制可以外推到其他家族成员，包括真核生物Rnt 1 p，Drosha和Dicer。叶酸和莽草酸途径是微生物所必需的，并且这两种途径中的一些酶在哺乳动物中不存在，这为开发新型抗菌剂提供了理想的靶点。例如，磺胺类药物和甲氧苄啶的分子靶标都是叶酸途径酶。我们已经获得了足够数量的结构信息6-羟甲基-7，8-二氢蝶呤焦磷酸激酶（HPPK）和二氢新蝶呤醛缩酶（DHNA）的叶酸途径和莽草酸激酶和莽草酸脱氢酶的莽草酸途径，这使我们能够推导出这些酶的催化机制。这些酶不是任何现有药物的靶标，因此是基于结构设计新型抗生素的理想靶标。谷胱甘肽S-转移酶（GST）催化谷胱甘肽与亲电子化合物的结合。在肿瘤前和肿瘤细胞中，GST的特定形式以高水平表达并参与细胞对抗癌药物的抗性。π类GST（GSTP）在对烷化剂的生物抗性中特别重要。一个新的GST激活的前药家族已经显示出巨大的潜力，其通过在癌细胞内释放一氧化氮发挥作用。我们已经实现了GSTP特异性的两个结构修饰的先导化合物。此外，我们已经确定了几个GSTP结构含有失活谷胱甘肽分子的结构表征GSTP与前药分子的复合物。