Biomolecular Structure and Mechanism, Structure-Based Drug Design

我们的结构分析目标是绘制选定生物大分子的反应轨迹或功能周期，我们的药物发现目标是设计，合成和表征新的抗癌和抗菌药物。迄今为止，我们已经描述了HPPK（微生物必需的叶酸途径酶，但在哺乳动物中不存在）、Era（一种将细胞生长与细胞分裂结合的必需GTPase）、RapA（一种循环RNA聚合酶的Swi2/Snf2蛋白）、RNase III家族的两个成员（dsrna特异性核糖核酸内切酶家族）和DDX3X（一种解绕短RNA双链的DEAD-box解旋酶）的反应轨迹和/或功能周期。其中，HPPK是新型抗菌药物的靶点，DDX3X是新型抗癌和抗hiv药物的靶点。基于结构的药物开发正在进行中。RNase III家族的代表成员包括原核RNase III和真核Rnt1p、Drosha和Dicer。它们在RNA加工和成熟、转录后基因沉默和防御病毒感染中发挥重要作用。在机理研究中，细菌酶是整个家族的一个有价值的模型系统。我们已经展示了RNase III内切酶结构域（RIIID）的二聚化如何在两个裂解位点所在的位置创建一个催化谷，催化谷如何以一种方式容纳dsRNA，使两条RNA链中的每一条都与两个裂解位点之一排列，每条链的水解如何涉及RIIID，以及RNase III如何使用两个裂解位点在其产物中创建2-核苷酸（2-nt） 3'悬垂。我们还展示了镁对于催化能力强的蛋白质- rna复合物的形成是必不可少的，两个镁离子的使用如何驱动每个磷酸二酯键的水解，以及底物和蛋白质的构象变化是组装催化复合物的关键因素。此外，我们还提供了RNase III执行磷酰转移反应的逐步轨迹。酵母Rnt1p与细菌酶一样，对RNase III的作用机制提供了丰富的信息，是真核生物RNase III酶的一个有价值的模型系统。与使用四条催化侧链的细菌酶不同，真核rna酶iii使用六条催化侧链。与细菌酶不同的是，每一个真核生物的RNase III都有一个n端延伸。更重要的是，Rnt1p具有严格的鸟嘌呤核苷酸特异性，这在RNase III酶中是独一无二的。我们已经展示了Rnt1p的底物结合模式如何与细菌RNase III不同，所有六个催化侧链如何参与裂解位点，Rnt1p的一个新的rna结合基元如何作为鸟嘌呤特异性钳，以及Rnt1p的dsrna结合域和n端结构域如何作为测量鸟嘌呤核苷酸与两个裂解位点之间距离的两个标子。这种不寻常的底物选择性机制代表了底物选择性进化的一个例子。RNase III家族的所有成员都通过2 - mg2 +离子催化推动RNA水解。我们还提供了Rnt1p执行磷酰转移反应的逐步轨迹。2 - mg2 +离子催化反应轨迹的结构基础是由在磷酸二酯键裂解后立即确定的裂解后复合物的晶体结构提供的，为此我们已经确定了细菌RNase III和酵母Rnt1p的高分辨率结构。这些裂解后结构揭示了原核和真核RNase III酶（包括Drosha和Dicer）催化2 - mg2 +离子的明显特征，但现有的结构不能提供2 - mg2 +离子催化的基础。DDX3X属于DEAD-box解旋酶家族，通过解绕短RNA双链来调节RNA加工和代谢。DDXs共享由两个reca样结构域（D1D2）组成的解旋酶核心，以atp依赖的非进程方式发挥作用。作为一个有吸引力的癌症和艾滋病治疗靶点，DDX3X及其同源物被广泛研究，产生了丰富的生化和生物物理数据，包括载脂蛋白D1D2和解绕后D1D2:ssRNA复合物的结构。然而，直到我们最近确定了具有2转RNA双链的D1D2核心复合物在预解绕状态下的晶体结构，才知道预解绕D1D2:dsRNA复合物的结构，表明两个ddx识别RNA双链。每个DDX主要识别单链，ATP结合引起的构象变化以合作的方式解开RNA双链。我们的新结构极大地改变了之前的三分子协同性模型。为了验证我们的新模型，我们目前正在使用位点定向诱变、rna解绕试验、atp水解试验、Hill协同性分析和结构研究来阐明DDX3X的功能循环。我们也开始基于现有的结构和机理信息开发DDX3X抑制剂。我们开展基于结构的药物开发主要是作为我们对具有抗癌和抗菌意义的生物分子系统的结构和机制的基础研究的延续。之前，我们设计了PABA/NO，一种酶激活的抗癌前药，通过释放一氧化氮从内部杀死癌细胞。我们还通过设计和合成连接嘌呤-蝶呤抑制剂以及测定其与酶配合物的晶体结构，在靶向HPPK的新型抗菌药物方面取得了重大进展。最近，我们进一步开发了PABA/NO和HPPK抑制剂。PABA/NO衍生物含有PARP抑制剂，因此它不仅产生一氧化氮，而且在同一室中同时释放PARP抑制剂，破坏DNA并抑制其修复（PMID: 24521039；美国专利号：9168266）。改进的HPPK抑制剂与催化配合物的过渡态非常相似，因此具有更高的效力（PMID: 33199204；美国专利号：11091509）。

Our goal of structural analysis is to map the reaction trajectory or functional cycle of selected biological macromolecules, and our goal of drug discovery is to design, synthesize, and characterize novel anticancer and antimicrobial agents. To date, we have characterized the reaction trajectory and/or functional cycle of HPPK (a folate pathway enzyme essential for microorganisms but absent in mammals), Era (an essential GTPase that couples cell growth with cell division), RapA (a Swi2/Snf2 protein that recycles RNA polymerase), two members of the RNase III family (a family of dsRNA-specific endoribonucleases), and DDX3X (a DEAD-box helicase that unwinds short RNA duplexes). Among these biomolecules, HPPK is a target for novel antibacterial agents, and DDX3X is a target of novel anticancer and anti-HIV agents. Structure-based drug development is in progress. Representative members of the RNase III family include prokaryotic RNase III and eukaryotic Rnt1p, Drosha, and Dicer. They play important roles in RNA processing and maturation, post-transcriptional gene silencing, and defense against viral infection. For mechanistic studies, bacterial enzyme is a valuable model system for the entire family. We have shown how the dimerization of the RNase III endonuclease domain (RIIID) creates a catalytic valley where two cleavage sites are located, how the catalytic valley accommodates a dsRNA in a manner such that each of the two RNA strands is aligned with one of the two cleavage sites, how the hydrolysis of each strand involves both RIIIDs, and how RNase III uses the two cleavage sites to create the 2-nucleotide (2-nt) 3' overhangs in its products. We have also shown how magnesium is essential for the formation of a catalytically competent protein-RNA complex, how the use of two magnesium ions can drive the hydrolysis of each phosphodiester bond, and how conformational changes in both the substrate and the protein are critical elements for assembling the catalytic complex. Moreover, we have provided a stepwise trajectory by which RNase III executes the phosphoryl transfer reaction. As informative as the bacterial enzyme for the mechanism of RNase III action, yeast Rnt1p is a valuable model system for eukaryotic RNase III enzymes. Unlike bacterial enzymes that use four catalytic side chains, eukaryotic RNase IIIs use six. It is also distinguished from bacterial enzymes that every eukaryotic RNase III has an N-terminal extension. What is more, Rnt1p exhibits a strict guanine nucleotide specificity, which is unique among RNase III enzymes. We have shown how the substrate-binding mode of Rnt1p is distinct from that of bacterial RNase III, how all six catalytic side chains are engaged in the cleavage site, how a new RNA-binding motif of Rnt1p functions as a guanine-specific clamp, and how the dsRNA-binding domain and N-terminal domain of Rnt1p function as two rulers measuring the distances between the guanine nucleotide to the two cleavage sites. This unusual mechanism of substrate selectivity represents an example of the evolution of substrate selectivity. All members of the RNase III family propel RNA hydrolysis by two-Mg2+-ion catalysis. We have also provided a stepwise trajectory by which Rnt1p executes the phosphoryl transfer reaction. The structural basis for the reaction trajectory of two-Mg2+-ion catalysis is provided by the crystal structure of a postcleavage complex determined at a stage immediately after the cleavage of the phosphodiester bond, for which we have determined high-resolution structures for both bacterial RNase III and yeast Rnt1p. These postcleavage structures reveal distinct features of two-Mg2+-ion catalysis by prokaryotic and eukaryotic RNase III enzymes, including Drosha and Dicer, for which the available structures fail to provide the basis of two-Mg2+-ion catalysis. DDX3X belongs to the family of DEAD-box helicases that regulate RNA processing and metabolism by unwinding short RNA duplexes. Sharing a helicase core composed of two RecA-like domains (D1D2), DDXs function in an ATP-dependent, non-processive manner. As an attractive target for cancer and AIDS treatment, DDX3X and its orthologs are extensively studied, yielding a wealth of biochemical and biophysical data, including structures of apo-D1D2 and post-unwound D1D2:ssRNA complex. However, the structure of a pre-unwound D1D2:dsRNA complex was not available until we have recently determined the crystal structure of a D1D2 core in complex with a 2-turn RNA duplex at the pre-unwound state, showing that two DDXs recognize the RNA duplex. Each DDX mainly recognizes a single strand, and conformational changes induced by ATP binding unwinds the RNA duplex in a cooperative manner. Our new structure has significantly altered a previous model of three-molecule cooperativity. To validate our new model, we are currently elucidating the functional cycle of DDX3X using site-directed mutagenesis, RNA-unwinding assay, ATP-hydrolyzing assay, Hill cooperativity analysis, and structural studies. We have also started to develop DDX3X inhibitors based on available structural and mechanistic information. We carry out structure-based drug development primarily as a continuation of our basic research on the structure and mechanism of biomolecular systems with anticancer and antimicrobial significance. Previously, we designed PABA/NO, an enzymatically activated anticancer prodrug that kills cancer cells from within by releasing nitric oxide. We also made significant progress toward novel antibacterial agents targeting HPPK by the design and synthesis of linked purine pterin inhibitors and the determination of their crystal structures in complex with the enzyme. Recently, we have further developed both the PABA/NO and HPPK inhibitors. The PABA/NO derivative contains a PARP inhibitor and so it not only generates nitric oxide but also release a PARP inhibitor simultaneously in the same compartment, which damage DNA and inhibit its repair (PMID: 24521039; US Patent Number: 9168266). The improved HPPK inhibitors mimic closely the transition state of the catalytic complex and thereby have much higher potency (PMID: 33199204; US Patent Number: 11091509).