Biomolecular Structure and Mechanism, Structure-Based Drug Design

RNA干扰是由核糖核酸酶III （RNase III）家族成员（包括Dicer）产生的小干扰RNA介导的。对于机制研究，细菌RNase III已成为整个家族的有价值的模型系统。之前，我们已经展示了酶的内切酶结构域的二聚化如何在两个催化位点所在的地方创造一个催化谷，催化谷如何以一种方式容纳dsRNA，使两条RNA链中的每一条都与两个催化位点中的一个对齐，每条链的水解如何涉及两个亚基（一个亚基的残基参与了剪刀键的选择），而来自伴侣亚基的那些则参与了裂解化学)，以及RNase III如何利用这两个催化位点在其产物中产生2-核苷酸3'悬垂。最近，我们已经证明了Mg2+如何对催化能力强的蛋白质- rna复合物的形成至关重要，两个Mg2+离子如何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如何成为组装催化复合物的关键因素。此外，我们以一种有意义的方式模拟了蛋白质-底物复合物和蛋白质-反应中间（过渡态）复合物。综上所述，模型和晶体结构提示了酶执行磷酰转移反应的逐步机制。细菌RNase III蛋白与dsRNA相互作用的结构信息和加工dsRNA的机制可以外推到其他家族成员，包括真核生物Rnt1p、Drosha和Dicer。叶酸和莽草酸途径是微生物所必需的，哺乳动物缺乏这两种途径中的一些酶，为开发新型抗菌药物提供了理想的靶点。例如，磺胺类药物和甲氧苄啶的分子靶点都是叶酸途径酶。我们获得了叶酸途径中6-羟甲基-7,8-二氢蝶呤焦磷酸激酶（HPPK）和二氢蝶呤醛缩酶（hna）的结构信息，以及莽草酸途径中莽草酸激酶和莽草酸脱氢酶的结构信息，从而可以推导出这些酶的催化机制。这些酶不是任何现有药物的靶点，因此是基于结构的新型抗生素设计的理想靶点。谷胱甘肽s -转移酶（GST）催化谷胱甘肽与亲电化合物的偶联。在肿瘤前和肿瘤细胞中，特定形式的GST在高水平表达，并参与细胞对抗癌药物的抵抗。pi类GST （GSTP）在对烷基化剂的生物抗性方面尤为重要。一个新的gst激活前药家族显示出巨大的潜力，其功能是释放癌细胞内的一氧化氮。我们已经通过两个结构修饰实现了先导化合物的GSTP特异性。此外，我们已经确定了几种含有灭活谷胱甘肽分子的GSTP结构，用于GSTP与前药分子复合物的结构表征。

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.