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）和Era（将细胞生长与细胞分裂偶联的保护性GTPase）]和RNA聚合酶（RNAP）相关转录因子[RapA （atp依赖性dsDNA转位酶，在转录过程中循环RNAP）和利用物质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，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超级线圈的晶体结构，并通过确定三元NusB-NusE-BoxA RNA和NusB-NusE-dsRNA复合物的晶体结构，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研究了酿酒酵母RNase III （Rnt1p）切割后复合体的晶体结构，这解释了为什么Rnt1p会与带有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的第二个位置的鸟嘌呤核苷酸之间的特定相互作用。引人注目的是，结构和生化分析表明，Rnt1p的dsRBD和n端结构域（NTD）是测量四环和切割位点之间距离的两个标尺。这些发现为理解包括Drosha和Dicer在内的真核RNase iii提供了一个框架。这项研究作为一篇专题研究文章发表在Molecular Cell杂志上，并在该杂志的问题封面上突出显示。今年，我们最重要的成就是RapA的结构和生化研究，揭示了唯一的细菌Swi2/Snf2蛋白被RNA聚合酶变构激活。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（GST）激活、一氧化氮释放的抗癌前药和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，这些抑制剂可作为抗菌药物。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%。去年，我们研究了两种HPPK抑制剂（HP-18和HP-26）对土拉弗朗西斯菌（Francisella tularensis, Ft） HPPK与二氢蝶酸合成酶（DHPS）结合的结合和抑制活性，确定了FtHPPK-DHPS与HP-26配合物的结构，并测量了FtHPPK-DHPS双酶活性的动力学参数。生化分析表明，HP-18和HP-26具有显著的同工酶选择性，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26万分之一。序列和结构分析表明，HP-26是开发兔热病治疗药物的极好先导，DHPS活性极低至少部分是由于缺乏与底物对氨基苯甲酸（pABA）相互作用的关键残基。对10个土拉菌基因组的BLAST搜索表明，该细菌含有单个FtHPPK-DHPS。土拉菌的边际DHPS活性和FtHPPK-DHPS的单拷贝存在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；针对独特的paba结合口袋的新抑制剂可能有效且不易产生耐药性，因为突变可能使边际DHPS活性无法支持土拉菌的生长。这项研究发表在2014年国际晶体学年特刊《FEBS杂志》上。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“连接嘌呤-蝶呤- HPPK抑制剂作为抗菌剂”。

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.