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: 8348975
• 负责人: XINHUA JI
• 金额: $ 135.04万
• 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/8348975
• 关键词: 4-Aminobenzoic Acid; Affinity; African Trypanosomiasis; Aldehydes; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Antimalarials; Antineoplastic Agents; Aromatic Compounds; Bacteria; Bacteriophage lambda; Binding; Binding Sites; Biogenesis; Biological Models; Carboxylic Acids; Cardiovascular Diseases; Cell Survival; Cell division; Cisplatin; Complex; Couples; DNA-Directed RNA Polymerase; Data; Development; Diet; Diphosphotransferases; Double-Stranded RNA; Drug Design; Drug Metabolic Detoxication; Enzymes; Escherichia coli; Esters; Exhibits; Family; Folate; Folic Acid Antagonists; Gene Expression; Genetic Transcription; Glutathione S-Transferase; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; Human; In Vitro; Investigation; KH Domain; Lead; Life; Link; Liver; Malignant Neoplasms; Mammals; Maps; Mediating; Methods; Methyltransferase; Mitochondria; Modeling; Mus; Nitric Oxide; Nitric Oxide Synthase; Nucleotides; Pathway interactions; Phenotype; Play; Prodrugs; Protein Isoforms; Proteins; Pterins; Purines; RNA; RNA Processing; RNA Recognition Motif; RNA, ribosomal, 12S; RNA-Protein Interaction; Reaction; Recycling; Relative (related person); Research; Ribonuclease III; Ribosomal RNA; Ribosomes; Role; Route; Site; Specificity; Starvation; Structure; System; Therapeutic Agents; Tumor Suppressor Proteins; Work; anticancer activity; antimicrobial drug; antitermination; antitermination factor; base; cancer cell; cell growth; cofactor; cytotoxic; design; drug development; endonuclease; fighting; glutathione S-transferase alpha; glutathione S-transferase pi; human DICER1 protein; in vivo; inhibitor/antagonist; insight; microorganism; novel; overexpression; protein function; pteridine reductase; purine; structural biology; transcription factor; translocase

We have been studying 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). Previously, we made pioneering contribution to the mechanism of RNase III action, significant progress in KsgA-RNA interactions, and a breakthrough advance in the structure and function of Era. This year, we have completed our structural studies of bacterial Era. Era, essential for bacterial cell viability, is composed of a GTPase domain and an RNA-binding KH domain. We have characterized the functional cycle of Era, providing structural basis for its essential roles in the maturation of 16S rRNA and assembly of 30S ribosomal subunit. We have shown that Era recognizes 10 nucleotides (nt 1530-1539, GAUCACCUCC in Escherichia coli) near the 3' end of 16S rRNA, and that this recognition stimulates the GTP-hydrolyzing activity of the protein. The GAUCA sequence and the upstream helix 45 (h45, nt 1506-1529) are highly conserved in all three kingdoms of life. We have shown that Era also binds h45. Among the 10 nt, however, G1530 does not stimulate Era's GTPase activity. Rather, A1531 and A1534 are most important for stimulation and h45 further contributes to the stimulation. Although G1530 does not contribute to the GTPase activity, its interaction with Era is essential for the protein to function, leading to the discovery of a cold-sensitive phenotype of the protein. Present in nearly every bacterial species and essential for both cell growth and cell division, Era is unique among all other known protein functions of bacteria. Inhibition of Era function will likely stop the synthesis of bacterial ribosome. Therefore, Era is a potential target for the development of novel antibiotics to fight the worldwide crisis of antibiotic resistance. The eukaryotic homologs of Era (EraL1) is an attractive candidate for a tumor suppressor. It is located in the small subunit of mitochondrial ribosome and interacts with the 12S rRNA, playing important roles in mitochondrial ribosome assembly and cell viability. Currently, structural and functional studies of human and mouse EraL1 proteins are undertaken. We have been working on 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 determined the crystal structure of SspA, RapA, and NusG. This year, we have provided structural insight into phage lambda N protein-mediated transcription antitermination. Processive transcription antitermination requires the assembly of a complete antitermination complex, which is initiated by the formation of the ternary NusB-NusE-BoxA RNA complex. We have elucidated the crystal structure of this complex, demonstrating that BoxA is composed of eight nt that are recognized by the NusB-NusE heterodimer. Functional data support the structural observations and establish the relative significance of key protein-protein and protein-RNA interactions. Further crystallographic investigation of a NusB-NusE-dsRNA complex reveals a heretofore unobserved dsRNA-binding site, which is contiguous with BoxA-binding site. We propose that the observed dsRNA represents the BoxB RNA, as both single-stranded BoxA and double-stranded BoxB components are present in the classical lambda antitermination site. Combining these data with known interactions amongst antitermination factors suggests a specific model for the assembly of the complete antitermination complex. Our effort in structure-based drug development has been focused on Glutathione S-transferase (GST) and 6-hydroxymethyl-7,8-dihydroptein pyrophosphokinase (HPPK). GST represents a superfamily of detoxification enzymes, represented by GST-alpha, -mu, -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. 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. In addition, HPPK is unique for microorganisms and is not the target for any existing antibiotics. Therefore, it is an attractive target for developing novel antimicrobial agents. Previously, our structure-based design of prodrugs intended to release cytotoxic levels of nitric oxide in GST-pi-overexpressing cancer cells yielded PABA/NO, which exhibits anticancer activity both in vitro and in vivo with a potency similar to that of cisplatin. We also designed, synthesized, and characterized a group of HPPK inhibitors as lead compounds for novel antibiotics. These inhibitors are linked purine pterin compounds. They bind to HPPK with high affinity and specificity. This year, we have optimized the synthetic route of HPPK inhibitors, leading to the invention of a novel intermediate (6-carboxylic acid ethyl ester-7,7-dimethyl-7,8-dihydropterin) and a new method for the synthesis of a known intermediate (6-aldehyde-7,7-dimethyl-7,8-dihydropterin) with a yield of 95%. The derivatives of these two compounds can be used as antifolate agents (antibacterials, antimalarials, and anticancer drugs), as nitric oxide synthase activators for the treatment of cardiovascular diseases, and as pteridine reductase inhibitors targeting African sleeping sickness and the Leishmaniases. The method can also be applied to other systems from methyl heteroaromatic and aromatic compounds to carboxylic ester heteroaromatic and aromatic compounds.

我们一直在研究rna加工蛋白RNase III (dsrna特异性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，作为核糖体生物发生因子）和Era（保守的GTPase，结合细胞生长和细胞分裂）。在此之前，我们在RNase III的作用机制方面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的结构和功能方面取得了突破性进展。今年，我们完成了细菌纪元的结构研究。Era是细菌细胞生存所必需的，由GTPase结构域和rna结合KH结构域组成。我们对Era的功能周期进行了表征，为其在16S rRNA成熟和30S核糖体亚基组装中的重要作用提供了结构基础。我们已经证明Era识别16S rRNA 3'端附近的10个核苷酸（nt 1530-1539，大肠杆菌中的GAUCACCUCC），并且这种识别刺激了蛋白质的gtp水解活性。GAUCA序列和上游螺旋45 （h45, nt 1506-1529）在所有三种生物中都高度保守。我们已经证明Era也能结合h45。然而，在10个nt中，G1530不刺激Era的GTPase活性。相反，A1531和A1534对刺激最重要，h45对刺激有进一步的作用。虽然G1530不参与GTPase的活性，但它与Era的相互作用对于该蛋白的功能至关重要，从而发现了该蛋白的冷敏感表型。Era存在于几乎所有细菌物种中，对细胞生长和细胞分裂都是必不可少的，在所有已知的细菌蛋白质功能中，Era是独一无二的。抑制Era功能可能会阻止细菌核糖体的合成。因此，Era是开发新型抗生素以对抗全球抗生素耐药性危机的潜在靶点。Era的真核同系物（EraL1）是一个有吸引力的肿瘤抑制因子候选物。它位于线粒体核糖体的小亚基上，与12S rRNA相互作用，在线粒体核糖体组装和细胞活力中发挥重要作用。目前，正在进行人类和小鼠EraL1蛋白的结构和功能研究。我们一直致力于RNA聚合酶（RNAP）相关转录因子SspA（严格饥饿蛋白A）， RapA （atp依赖的dsDNA转位酶，在转录过程中循环RNAP）和n利用物质A， B， E和G （NusA, NusB， NusE和NusG）。在此之前，我们确定了SspA、RapA和NusG的晶体结构。今年，我们提供了噬菌体lambda N蛋白介导的转录抗终止的结构见解。过程转录抗终止需要一个完整的抗终止复合物的组装，这是由三元NusB-NusE-BoxA RNA复合物的形成发起的。我们已经阐明了该复合物的晶体结构，证明BoxA是由8个被NusB-NusE异二聚体识别的nt组成的。功能数据支持结构观察，并确立了关键蛋白-蛋白和蛋白- rna相互作用的相对重要性。对NusB-NusE-dsRNA复合物的进一步晶体学研究揭示了一个迄今为止未观察到的dsrna结合位点，该位点与boxa结合位点相邻。我们认为观察到的dsRNA代表BoxB RNA，因为单链BoxA和双链BoxB成分都存在于经典的lambda抗终止位点。将这些数据与已知的反终止因子之间的相互作用相结合，提出了完整反终止复合物组装的特定模型。我们在基于结构的药物开发方面的努力主要集中在谷胱甘肽s -转移酶（GST）和6-羟甲基-7,8-二氢蛋白焦磷酸激酶（HPPK）。GST是一个解毒酶的超家族，以GST- α、-mu、-pi等为代表。GST- α是人类肝脏中GST的主要亚型，对我们的健康起着重要的作用。GST-pi在许多形式的癌症中过度表达，从而为选择性靶向癌细胞提供了机会。HPPK是叶酸生物合成途径中的关键酶。叶酸辅助因子是生命所必需的。哺乳动物从饮食中摄取叶酸，而大多数微生物必须从头合成叶酸。此外，HPPK对微生物是独特的，不是任何现有抗生素的靶点。因此，它是开发新型抗菌药物的一个有吸引力的靶点。在此之前，我们基于结构设计的前药旨在释放gst -pi过表达的癌细胞中一氧化氮的细胞毒性水平，产生PABA/NO，其在体外和体内都具有与顺铂相似的抗癌活性。我们还设计、合成并表征了一组HPPK抑制剂作为新型抗生素的先导化合物。这些抑制剂是嘌呤-蝶呤类化合物。它们以高亲和力和特异性与HPPK结合。今年，我们优化了HPPK抑制剂的合成路线，发明了一种新的中间体（6-羧酸乙酯-7,7-二甲基-7,8-二氢蝶呤）和一种已知中间体（6-醛-7,7-二甲基-7,8-二氢蝶呤）的合成方法，收率为95%。这两种化合物的衍生物可用作抗叶酸剂（抗菌药、抗疟药和抗癌药物），用作治疗心血管疾病的一氧化氮合酶激活剂，以及用于治疗非洲昏睡病和利什曼病的蝶啶还原酶抑制剂。该方法也可应用于从甲基杂芳烃和芳香烃化合物到羧酸酯杂芳烃和芳香烃化合物的其他体系。