Biomolecular Structure and Mechanism, Structure-Based Drug Design

我们的基础研究主要集中在RNA加工蛋白[RNase III（细菌RNase III和真核生物Rnt1p， Drosha和Dicer等dsrna特异性内切酶家族的模型系统），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）]和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的结构和功能周期方面取得了突破性进展。我们还测定了SspA、RapA和NusG的晶体结构，并通过测定三元NusB-NusE-BoxA RNA和NusB-NusE-dsRNA复合物的晶体结构，为噬菌体lambda N蛋白介导的转录抗终止提供了结构上的见解。今年，我们最重要的发现是血小板RNA超级线圈的晶体结构。基因组包装是一个基本的管家过程，在几乎所有的生物适当的存储和维护遗传信息。虽然包装的程度和机制各不相同，但这个过程涉及到核酸上层结构的形成。DNA盘绕线圈的晶体结构表明，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，RNA的这种超结构尚未被发现。我们已经确定了RNA超级线圈的晶体结构，它比先前报道的DNA螺旋线圈结构显示出更高水平的分子组织。在水蛭NusB蛋白存在的情况下，两个互锁的RNA卷曲的双链RNA， a ？一卷一卷的线圈？形成电磁超级线圈。分子动力学模拟表明，蛋白质-RNA相互作用是超卷曲RNA稳定性所必需的。晶格中的超卷曲RNA的核酸密度为42 bp/100 nm3。有趣的是，dsRNA病毒的平均基因组包装密度为40 bp/100 nm3。这项研究为核酸的高阶包装机制提供了结构性的见解。此外，由于风茄NusB蛋白与超卷曲RNA的序列无关的相互作用，可能被用于促进其他核酸超结构的形成和/或结晶，或用于构建新的纳米结构。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）激活、一氧化氮释放、抗癌前药和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。在此之前，我们基于结构的前药设计产生了PABA/NO，其在体内和体外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，并优化了HPPK抑制剂的合成路线，从而发明了一种新型中间体和一种已知中间体的新合成方法，收率为95%。今年，我们合成并表征了另一种HPPK先导抑制剂，它与HPPK具有不同的结合方式，代表了进一步发展的新方向。

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.