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: 7965248
• 负责人: XINHUA JI
• 金额: $ 123.58万
• 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/7965248
• 关键词: Adenosine; Antibiotics; Archaea; Bacteria; Binding; Binding Sites; Biogenesis; Biological; Cell division; Cisplatin; Complex; Couples; DNA-Directed RNA Polymerase; Data; Development; Diet; Diphosphotransferases; Double-Stranded RNA; Drug Design; Drug Metabolic Detoxication; Enzymes; Eukaryota; Evolution; Exhibits; Family; Family member; Folate; GTP Binding; Gene Expression; Glutathione S-Transferase; Glutathione Transferase mu; Goals; Guanosine Triphosphate Phosphohydrolases; Human; Hydrolysis; In Vitro; Isoenzymes; KH Domain; Lead; Life; Ligands; Liver; Malignant Neoplasms; Mammals; Maps; Methyltransferase; Modification; Molecular Chaperones; Molecular Conformation; N-terminal; Nitric Oxide; Nucleotides; Pathway interactions; Play; Process; Prodrugs; Protein Isoforms; Proteins; RNA; RNA Binding; RNA Interference; RNA Processing; Reaction; Research; Ribonuclease III; Ribosomal RNA; Ribosomes; Role; Solubility; Specificity; Structure; Subarachnoid Hemorrhage; System; Therapeutic Agents; Tumor Suppressor Proteins; analog; anticancer activity; antimicrobial drug; base; cancer cell; cell growth; cofactor; cytotoxic; design; drug development; endonuclease; flexibility; human DICER1 protein; improved; in vivo; macromolecule; microorganism; novel; overexpression; structural biology; transcription factor

<P>We have been studying three RNA-processing proteins: RNase III [a family of double-stranded (ds) RNA-specific endonucleases that initiate RNA interference], KsgA (a universally conserved methyltransferase that catalyzes the dimethylation of two adjacent adenosines in small-subunit ribosomal RNA), and ERA (a conserved GTPase that couples cell division with cell growth rate in bacteria and is a potential tumor suppressor in mammals). Previously, we have established a stepwise mechanism for RNase III to execute dsRNA cleavage, which can be extrapolated to other members of the family, including Rnt1p, Drosha and Dicer. This year, we have made significant progress in KsgA research and breakthrough advances in ERA research.</P> <P>Among methyltransferases, KsgA and the reaction it catalyzes are conserved throughout evolution. However, the specifics of substrate recognition by the enzyme remain unknown. We have determined structures of KsgA, in its ligand-free form, in complex with RNA, and in complex with both RNA and S-adenosylhomocysteine (SAH, reaction product of cofactor S-adenosylmethionine), providing the first pieces of structural information on KsgA-RNA and KsgA-SAH interactions. Moreover, the structures show how conformational changes that occur upon RNA binding create the cofactor-binding site. There are nine conserved functional motifs (motifs I-VIII and X) in KsgA. Prior to RNA binding, motifs I and VIII are flexible, each exhibiting two distinct conformations. Upon RNA binding, the two motifs become stabilized in one of these conformations, which is compatible with the binding of SAH. Motif X, which is also stabilized upon RNA binding, is directly involved in the binding of SAH.</P> <P>ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is an essential ribosome biogenesis factor. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the 1531AUCACCUCCUUA1542 sequence at the 3′ end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in non-eukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a twist for non-eukaryotic ERA proteins by also recognizing the CCUCC.</P> <P>Our effort in structure-based drug development has been focused on two systems, Glutathione S-transferase (GST) and 6-hydroxymethyl-7,8-dihydroptein pyrophosphokinase (HPPK). GST represents a superfamily of detoxification enzymes, represented by GST-alpha, GST-mu, GST-pi, etc. GST-alpha is the predominant isoform of GST in human liver, playing important roles for our well being. GST-pi is overexpressed in many forms of cancer, thus presenting an opportunity for selective targeting of cancer cells. Previously, our structure-based design of prodrugs intended to release cytotoxic levels of nitric oxide in GST-pi-overexpressing cancer cells had yielded PABA/NO, which exhibits anticancer activity both in vitro and in vivo with a potency similar to that of cisplatin. This year, we have proposed structural modifications to PABA/NO for improved stability, solubility, and isozyme specificity. HPPK is a key enzyme in the folate biosynthetic pathway. Folate cofactors are essential for life. Mammals derive folates from their diet, whereas most microorganisms must synthesize folates de novo. Thus, the folate pathway is an ideal target for developing antimicrobial agents. In addition, HPPK is unique for microorganisms and is not the target for any existing antibiotics. Therefore, it is an ideal target for developing novel antimicrobial agents. Structure-based design and characterization of lead compounds are in progress.</P>

<我们一直在研究三种RNA加工蛋白： [启动RNA干扰的双链（ds）RNA特异性核酸内切酶家族]， KsgA（一种普遍保守的甲基转移酶，催化两个甲基化， 小亚基核糖体RNA中相邻的腺苷）和ERA（一种保守的GT3， 细胞分裂与细胞生长率在细菌中，是一种潜在的肿瘤抑制剂， 哺乳动物）。在此之前，我们已经建立了RNase III执行dsRNA的分步机制， 切割，可以外推到该家族的其他成员，包括Rnt 1 p，Drosha 还有切丁今年，我们在KsgA研究和突破方面取得了重大进展， 研究的进展& lt;/P P在 甲基转移酶KsgA及其催化的反应在整个进化过程中是保守的。 然而，底物识别的酶的细节仍然未知。我们有 确定KsgA的结构，以其无配体形式，与RNA复合，以及与RNA复合， 与RNA和S-腺苷高半胱氨酸（SAH，辅因子的反应产物 S-腺苷甲硫氨酸），提供了关于KsgA-RNA的第一段结构信息， KsgA-SAH相互作用。此外，这些结构显示了构象变化是如何发生的， 在RNA结合时产生辅因子结合位点。有9个保守的功能基序 （基序I-VIII和X）在KsgA中。在RNA结合之前，基序I和VIII是柔性的，各自 呈现出两种截然不同的构象在RNA结合后，两个基序变得稳定， 这些构象之一，与SAH的结合相容。Motif X， 也在RNA结合时稳定，直接参与 SAH.& lt;/P P ERA，由一个N-末端GTP酶组成 结构域，随后是RNA结合KH结构域，是一种必需的核糖体生物合成因子。它 与16 S rRNA和30 S核糖体亚基结合。然而，它的RNA结合位点，功能 这两个结构域之间的关系及其在核糖体生物发生中的作用仍不清楚。我们 测定了ERA的两种晶体结构，一种是与GDP的二元配合物，另一种是三元配合物 具有GTP类似物和16 S的3 #8242;末端的1531 AUCACCUCCUUA 1542序列 rRNA。在三元复合物中，12个核苷酸中的前9个被 蛋白我们发现GTP结合是ERA识别RNA的先决条件， 识别刺激其GTP水解活性。根据这些数据和其他数据，我们建议 ERA的一个功能周期，表明该蛋白质作为一个伴侣处理 以及16 S rRNA的成熟和30 S核糖体亚基组装的检查点。的 AUCA序列在细菌、古细菌和真核生物中高度保守，而CCUCC， 称为抗Shine-Dalgarno序列，仅在非真核生物中保守。因此，我们认为， 这些数据表明，在所有三种基因中，ERA功能高度保守， 通过识别AUCA，与非真核生物ERA蛋白的扭曲， 承认CCUCC。lt;/P P我们的努力 基于结构的药物开发一直集中在两个系统，谷胱甘肽S-转移酶 (GST)和6-羟甲基-7，8-二氢喋呤焦磷酸激酶（HPPK）。消费税代表 解毒酶超家族，以GST-α、GST-μ、GST-π等为代表。 GST-α是人肝脏中GST的主要亚型，在我们的研究中起着重要作用。 健康GST-pi在许多形式的癌症中过表达，因此提供了一个机会， 用于选择性靶向癌细胞。以前，我们基于结构的前药设计 在GST-π过表达的癌细胞中释放细胞毒性水平的一氧化氮， 产生PABA/NO，其在体外和体内均表现出抗癌活性， 类似于顺铂。今年，我们提出了PABA/NO的结构修饰， 以改善稳定性、溶解性和同工酶特异性。HPPK是一种关键酶， 叶酸生物合成途径叶酸辅助因子是生命所必需的。哺乳动物产生叶酸 而大多数微生物必须从头合成叶酸。因此，叶酸 途径是开发抗微生物剂的理想靶点。此外，HPPK还具有独特的 对于微生物来说，它不是任何现有抗生素的目标。因此，它是一个 是开发新型抗菌剂的理想目标。基于结构的设计和 铅化合物的特性鉴定正在进行中。lt;/P>