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: 9343594
• 负责人: XINHUA JI
• 金额: $ 160.63万
• 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/9343594
• 关键词: 4-Aminobenzoic Acid; Anti-Bacterial Agents; Antibiotics; Antineoplastic Agents; Bacteria; Bacteriophage lambda; Basic Science; Binding; Biochemical; Biochemistry; Biological Models; Cell division; Cells; Cisplatin; Complex; Couples; Crystallography; DNA-Directed RNA Polymerase; Development; Dihydropteroate Synthase; Diphosphotransferases; Double-Stranded RNA; Drug Design; Escherichia coli; Exhibits; Family; Francisella tularensis; Gene Expression; Gene Structure; Genetic Transcription; Genome; Glutathione S-Transferase; Goals; Growth; Guanine Nucleotides; Guanosine Triphosphate Phosphohydrolases; In Vitro; International; Isoenzymes; Journals; Kinetics; Lead; Legal patent; Link; Maps; Measures; Mediating; Methods; Molecular; Mutation; N-terminal; Nitric Oxide; Positioning Attribute; Post-Transcriptional Regulation; Prodrugs; Proteins; Pterins; Publishing; Purines; RNA; RNA Processing; Reaction; Recycling; Research; Resistance; Ribonuclease III; Route; Saccharomyces cerevisiae; Site; Structure; Therapeutic; Therapeutic Agents; Tularemia; United States; Work; analog; anticancer activity; antimicrobial drug; antitermination; base; cell growth; design; drug development; ds-DNA; endonuclease; in vivo; inhibitor/antagonist; insight; invention; novel; novel anticancer drug; stem; structural biology; sulfa drug; 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) and Era (conserved GTPase that couples cell growth with cell division)] and RNA polymerase (RNAP)-associated transcription factors [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 and a breakthrough advance in the structure and functional cycle of Era. We also determined the crystal structure of RapA and a plectonemic RNA supercoil, and provided structural insights 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. Last year, our most significant accomplishment is the crystal structure of the Saccharomyces cerevisiae RNase III (Rnt1p) post-cleavage complex that explains why Rnt1p binds to RNA stems capped with an NGNN tetraloop. The structure shows specific interactions between a structural motif located at the end of Rnt1p dsRNA-binding domain (dsRBD) and the guanine nucleotide in the second position of the loop. Strikingly, structural and biochemical analyses indicate that the dsRBD and N-terminal domains (NTD) of Rnt1p function as two rulers that measure the distance between the tetraloop and the cleavage site. These findings provide a framework for understanding eukaryotic RNase IIIs, including Drosha and Dicer. This study is published as a featured research article in Molecular Cell and highlighted on the issue cover of the journal. This year, our most significant accomplishment is the structural and biochemical studies of RapA, revealing the allosteric activation of the only bacterial Swi2/Snf2 protein by RNA polymerase. This study is published in the Journal of Biological Chemistry. 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%. Last year, we have elucidated the binding and inhibitory activities of two HPPK inhibitors (HP-18 and HP-26) against Francisella tularensis (Ft) HPPK that is combined with dihydropteroate synthase (DHPS), determined the structure of FtHPPK-DHPS in complex with HP-26, and measured the kinetic parameters for the dual enzymatic activities of FtHPPK-DHPS. The biochemical analyses showed that HP-18 and HP-26 have significant isozyme selectivity and that FtHPPK-DHPS is unique in that the catalytic efficiency of its DHPS activity is only 1/260,000 that of Escherichia coli DHPS. Sequence and structural analyses suggest that HP-26 is an excellent lead for developing tularemia therapeutics and that the very low DHPS activity is due, at least in part, to the lack of a key residue that interacts with the substrate p-aminobenzoic acid (pABA). A BLAST search of 10 F. tularensis genomes indicated that the bacterium contains a single FtHPPK-DHPS. The marginal DHPS activity and the single copy existence of FtHPPK-DHPS in F. tularensis make this bacterium more vulnerable to DHPS inhibitors. Current sulfa drugs are ineffective against tularemia; new inhibitors targeting the unique pABA-binding pocket may be effective and less subject to resistance because mutation may make the marginal DHPS activity unable to support the growth of F. tularensis. This work is published in the FEBS Journal, the special issue celebrating the International Year of Crystallography 2014. This year, two United State patents have been issued, entitled "PARP inhibitor/NO donor dual prodrugs as anticancer agents" and "Linked purine pterin HPPK inhibitors useful as antibacterial agents," respectively.

我们的基础研究一直集中在RNA加工蛋白[RNase III（以细菌RNase III和真核生物Rnt 1 p为例的dsRNA特异性内切核酸酶家族的模型系统，Drosha，和切丁）和纪元（将细胞生长与细胞分裂偶联的保守的GT3）]和RNA聚合酶（RNAP）相关的转录因子[RapA（在转录期间使RNAP重排的ATP依赖性dsDNA移位酶）和N利用物质A、B、E和G（NusA、NusB、NusE和NusG）]。此前，我们对RNase III的作用机制做出了开创性贡献，并在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RapA和plectonemic RNA超螺旋的晶体结构，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。去年，我们最重要的成就是酿酒酵母核糖核酸酶III（Rnt 1 p）切割后复合物的晶体结构，这解释了为什么Rnt 1 p与NGNN四环封端的RNA茎结合。该结构显示了位于Rnt 1 p dsRNA结合结构域（dsRBD）末端的结构基序与环第二位的鸟嘌呤核苷酸之间的特异性相互作用。引人注目的是，结构和生物化学分析表明，dsRBD和N-末端结构域（NTD）的Rnt 1 p功能作为两个标尺，测量之间的距离的四环和切割位点。这些发现为理解真核生物RNase III（包括Drosha和Dicer）提供了一个框架。这项研究作为一篇专题研究文章发表在《分子细胞》上，并在该杂志的封面上突出显示。今年，我们最重要的成就是RapA的结构和生物化学研究，揭示了RNA聚合酶对唯一细菌Swi 2/Snf 2蛋白的变构激活。这项研究发表在生物化学杂志上。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可用作抗菌剂的6-羟甲基-7，8-二氢蛋白焦磷酸激酶（HPPK）的双底物类似物抑制剂上。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%。去年，我们已经阐明了两种HPPK抑制剂（HP-18和HP-26）对与二氢蝶酸合酶（DHPS）结合的土拉弗朗西斯菌（Ft）HPPK的结合和抑制活性，确定了与HP-26复合的FtHPPK-DHPS的结构，并测量了FtHPPK-DHPS的双酶活性的动力学参数。生化分析表明，HP-18和HP-26具有明显的同工酶选择性，FtHPPK-DHPS的独特之处在于其DHPS活性的催化效率仅为大肠杆菌DHPS的1/260，000。序列和结构分析表明，HP-26是一个很好的领导开发兔热病治疗和非常低的DHPS活性是由于，至少部分是由于缺乏一个关键的残基，与底物对氨基苯甲酸（pABA）的相互作用。BLAST检索10个F.土拉热菌基因组表明该细菌含有单个FtHPPK-DHPS。FtHPPK-DHPS在F.土拉热使这种细菌更容易受到DHPS抑制剂的影响。目前的磺胺类药物对兔热病无效;靶向独特的pABA结合口袋的新抑制剂可能是有效的，并且较少受到耐药性的影响，因为突变可能使边缘DHPS活性无法支持F的生长。土拉热。这项工作发表在FEBS杂志上，这是庆祝2014年国际晶体学年的特刊。今年，已经颁发了两项美国专利，分别题为“PARP抑制剂/NO供体双重前药作为抗癌剂”和“用作抗菌剂的连接嘌呤蝶呤HPPK抑制剂”。