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: 8552665
• 负责人: XINHUA JI
• 金额: $ 156.58万
• 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/8552665
• 关键词: 4-Aminobenzoic Acid; Anti-Bacterial Agents; Antibiotics; Antineoplastic Agents; Bacteriophage lambda; Basic Science; Binding; Biogenesis; Biological Models; Cell division; Cisplatin; Complex; Couples; Crystallization; DNA Structure; DNA-Directed RNA Polymerase; Development; Diphosphotransferases; Double Stranded RNA Virus; Double-Stranded RNA; Drug Design; Enzymes; Exhibits; Family; Gene Expression; Genetic; Genetic Transcription; Genome; Glutathione S-Transferase; Goals; Guanosine Triphosphate Phosphohydrolases; Housekeeping; In Vitro; Lead; Maintenance; Maps; Mediating; Methods; Methyltransferase; Molecular; Nanostructures; Nitric Oxide; Nucleic Acids; Organism; Process; Prodrugs; Proteins; RNA; RNA Processing; RNA-Protein Interaction; Reaction; Recycling; Reporting; Research; Ribonuclease III; Route; Starvation; Structure; Therapeutic Agents; analog; anticancer activity; antimicrobial drug; antitermination; base; cell growth; density; design; drug development; endonuclease; human DICER1 protein; in vivo; inhibitor/antagonist; insight; molecular dynamics; novel; small molecule; structural biology; transcription factor; translocase

Our basic research has been focused on RNA-processing proteins [RNase III (model system for a family of dsRNA-specific endonucleases exemplified by bacterial RNase III and eukaryotic Rnt1p, Drosha, and Dicer), KsgA (universally conserved methyltransferase that functions as a ribosomal biogenesis factor), and Era (conserved GTPase that couples cell growth with cell division)] and RNA polymerase (RNAP)-associated transcription factors [SspA (stringent starvation protein A), RapA (ATP-dependent dsDNA translocase that recycles RNAP during transcription), and N-utilizing substances A, B, E, and G (NusA, NusB, NusE, and NusG)]. Previously, we made pioneering contribution to the mechanism of RNase III action, significant progress in KsgA-RNA interactions, a breakthrough advance in the structure and functional cycle of Era. We also determined the crystal structure of SspA, RapA, and NusG, and provided structural insight into the phage lambda N protein-mediated transcription antitermination by determining crystal structures of the ternary NusB-NusE-BoxA RNA and NusB-NusE-dsRNA complexes. This year, our most significant discovery is the crystal structure of a plectonemic RNA supercoil.Genome packaging is an essential housekeeping process in virtually all organisms for proper storage and maintenance of genetic information. Although the extent and mechanisms of packaging vary, the process involves the formation of nucleic-acid superstructures. Crystal structures of DNA coiled coils indicate that their geometries can vary according to sequence and/or the presence of stabilizers such as proteins or small molecules. However, such superstructures have not been revealed for RNA. We have determined the crystal structure of an RNA supercoil, which displays one level higher molecular organization than previously reported structures of DNA coiled coils. In the presence of the NusB protein from Aquifex aeolicus, two interlocking RNA coiled coils of double-stranded RNA, a ?coil of coiled coils?, form a plectonemic supercoil. Molecular dynamics simulations suggest that protein-RNA interaction is required for the stability of the supercoiled RNA. The supercoiled RNA in the crystal lattice has a nucleic acid density of 42 bp/100 nm3. Intriguingly, the average genome packing density of dsRNA viruses is 40 bp/100 nm3. This study provides structural insight into higher-order packaging mechanisms of nucleic acids. Furthermore, the A. aeolicus NusB protein, given its sequence-independent interactions with supercoiled RNA, could potentially be utilized to promote the formation and/or crystallization of other nucleic-acid superstructures, or in the construction of novel nanostructures.Our effort in structure-based drug development has been focused on Glutathione S-transferase (GST)-activated, nitric oxide-releasing, anticancer prodrugs and bisubstrate analog inhibitors of 6-hydroxymethyl-7,8-dihydroptein pyrophosphokinase (HPPK) useful as antibacterial agents. Previously, our structure-based design of prodrugs yielded PABA/NO, which exhibits anticancer activity both in vitro and in vivo with potency similar to that of cisplatin. We also designed, synthesized, and characterized a group of HPPK inhibitors as lead compounds for novel antibiotics, and optimized the synthetic route of HPPK inhibitors, leading to the invention of a novel intermediate and a new method for the synthesis of a known intermediate with a yield of 95%. This year, we have synthesized and characterized another lead inhibitor of HPPK, which exhibits a distinct binding mode to the enzyme and represents a new direction for further development.

我们的基础研究一直集中在RNA加工蛋白[RNase III（dsRNA特异性核酸内切酶家族的模型系统，例如细菌RNase III和真核生物Rnt1p、Drosha和Dicer），KsgA（普遍保守的甲基转移酶，其作为核糖体生物发生因子起作用），和时代（将细胞生长与细胞分裂偶联的保守GT3）]和RNA聚合酶（RNAP）相关转录因子[SspA（严格饥饿蛋白A）、RapA（在转录期间使RNAP水解的ATP依赖性dsDNA移位酶）和N利用物质A、B、E和G（NusA、NusB、NusE和NusG）]。在此之前，我们对RNase III的作用机制做出了开创性的贡献，在KsgA-RNA相互作用方面取得了重大进展，在Era的结构和功能循环方面取得了突破性进展。我们还确定了SspA，RapA和NusG的晶体结构，并通过确定三元NusB-NusE-BoxA RNA和NusB-NusE-dsRNA复合物的晶体结构，提供了对噬菌体λ N蛋白介导的转录抗终止的结构见解。今年，我们最重要的发现是plectonemic RNA超螺旋的晶体结构。基因组包装是几乎所有生物体中正确存储和维护遗传信息的基本管家过程。尽管包装的程度和机制各不相同，但该过程涉及核酸超结构的形成。DNA卷曲螺旋的晶体结构表明它们的几何形状可以根据序列和/或稳定剂如蛋白质或小分子的存在而变化。然而，这样的超结构还没有被揭示的RNA。我们已经确定了一个RNA超螺旋的晶体结构，它显示一个更高的水平比以前报道的DNA卷曲螺旋结构的分子组织。在存在的NusB蛋白从Aquifex aeolicus，两个互锁的RNA卷曲螺旋的双链RNA，一个？一卷又一卷？形成一个plectonemic超螺旋。分子动力学模拟表明，蛋白质-RNA相互作用是超螺旋RNA稳定所必需的。晶格中的超螺旋RNA具有42 bp/100 nm 3的核酸密度。有趣的是，dsRNA病毒的平均基因组包装密度为40 bp/100 nm 3。这项研究提供了更高阶的核酸包装机制的结构洞察。此外，A. aeolicus NusB蛋白，由于其与超螺旋RNA的序列无关的相互作用，可以潜在地用于促进其他核酸超结构的形成和/或结晶，或用于构建新型纳米结构。我们在基于结构的药物开发方面的努力一直集中在谷胱甘肽S-转移酶（GST）激活的、释放一氧化氮的抗癌前药和6-羟甲基-7，用作抗菌剂的8-二氢蛋白焦磷酸激酶（HPPK）。以前，我们基于结构的前药设计产生PABA/NO，其在体外和体内均表现出抗癌活性，其效力与顺铂相似。我们还设计、合成和表征了一组作为新型抗生素先导化合物的HPPK抑制剂，并优化了HPPK抑制剂的合成路线，从而发明了一种新的中间体和一种新的合成已知中间体的方法，产率为95%。今年，我们已经合成并表征了另一种HPPK的先导抑制剂，它与酶具有独特的结合模式，代表了进一步发展的新方向。