Biomolecular Structure and Mechanism, Structure-Based Drug Design

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RNT1P、DROSHA和DICER为例的一系列dsRNA特异性内切酶的模型系统)和Era(将细胞生长与细胞分裂结合在一起的保守GTPase)]和RNA聚合酶(RNAP)相关的转录因子[RapA(在转录过程中循环RNAP的依赖于ATP的dsDNA转位酶)和利用N的物质A、B、E和G(NusA、NusB、NUSE和NusG)]。此前，我们在RNaseIII的作用机制方面做出了开创性的贡献，在Era的结构和功能循环方面取得了突破性进展。我们还测定了RAPA和阳离子RNA超螺旋的晶体结构，并通过测定NusB-NUSE-Boxa和NusB-NUSE-dsRNA三元复合体的晶体结构，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。去年，我们最重要的成就是酿酒酵母RNaseIII(Rnt1p)裂解后复合体的晶体结构，这解释了为什么Rnt1p与带有NGNN四环的RNA茎结合。该结构显示了位于Rnt1p dsRNA结合域(DsRBD)末端的结构基序与环第二位置的鸟核苷酸之间的特异性相互作用。值得注意的是，结构和生化分析表明，RNT1P的dsRBD和N-末端结构域(NTD)起着两个尺子的作用，它们测量四元环到切割位点之间的距离。这些发现为理解真核RNaseIII，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的结构和生化研究，揭示了RNA聚合酶对唯一的细菌Swi2/Snf2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主要集中在谷胱甘肽S转移酶激活的一氧化氮释放的抗癌前药和用作抗菌药物的6-羟甲基-7，8-二氢磷酸肌醇双底物类似物(HPPK)。以前，我们的基于结构的前药设计产生了PABA/NO，它在体外和体内都表现出抗癌活性，其效力类似于顺铂。我们还设计、合成和表征了一组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，从而发明了一种新的中间体和一种合成已知中间体的新方法，产率为95%。去年，我们已经阐明了两种HPPK抑制剂(HP-18和HP-26)与二氢蝶形酸合酶(DHPS)结合对土拉方杆菌(Francisella tularensis，Ft)HPPK的结合和抑制活性，测定了FtHPPK-DHPS与Hp-26形成的复合体的结构，并测定了FtHPPK-DHPS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，而FtHPPK-DHPS的催化效率仅为大肠杆菌DHPS的1/26万。序列和结构分析表明，HP-26是开发兔热病治疗药物的极佳先导分子，DHPS活性非常低，至少部分原因是缺乏与底物对氨基苯甲酸(PABA)相互作用的关键残基。对10个图拉氏丝虫基因组的BLAST搜索表明，该细菌含有单一的FtHPPK-DHPs。DHPS的边缘活性和FtHPPK-DHPS在图拉氏酵母中的单拷贝存在使该细菌更容易受到DHPS抑制剂的影响。目前的磺胺类药物对图拉氏症无效；针对独特的PABA结合口袋的新的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的DHPS活性无法支持图拉氏菌的生长。这项研究发表在FEBS期刊上，这是庆祝2014年国际结晶学年的特刊。今年，已经颁发了两项美国专利，分别名为“PARP抑制剂/NO供体双前药作为抗癌剂”和“可用作抗菌剂的联用嘌呤蝶呤HPPK抑制剂”。

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.