Biomolecular Structure and Mechanism, Structure-Based Drug Design

我们一直在研究三种RNA加工蛋白：RNase III（启动RNA干扰的双链（ds）RNA特异性内切核酸酶家族），KsgA（一种普遍保守的甲基转移酶，催化小亚基核糖体RNA中两个相邻腺苷的二甲基化）和ERA（一种保守的GTdR，将细菌中的细胞分裂与细胞生长速率结合起来，是哺乳动物中潜在的肿瘤抑制因子）。之前，我们已经建立了RNase III执行dsRNA切割的逐步机制，这可以外推到家族的其他成员，包括Rnt 1 p，Drosha和Dicer。今年，我们在KsgA研究方面取得了重大进展，在ERA研究方面取得了突破性进展。ERA是一种重要的核糖体生物合成因子，由一个N-末端的GT3结构域和一个RNA结合的KH结构域组成。与16 S rRNA和30 S核糖体亚基结合。然而，它的RNA结合位点，两个结构域之间的功能关系，以及它在核糖体生物合成中的作用仍然不清楚。我们已经确定了ERA的两种晶体结构，一种是与GDP的二元复合物，一种是与GTP类似物和16 S rRNA 3端的1531 AUCACCUCCUUA 1542序列的三元复合物。在三元复合物中，12个核苷酸中的前9个被蛋白质识别。我们表明，GTP结合是RNA识别ERA的先决条件，RNA识别刺激其GTP水解活性。基于这些和其他数据，我们提出了ERA的功能周期，这表明该蛋白质作为一个伴侣的加工和成熟的16 S rRNA和30 S核糖体亚基组装的检查点。AUCA序列在细菌、古生菌和真核生物中高度保守，而CCUCC（称为抗Shine-Dalgarno序列）仅在非真核生物中保守。因此，这些数据表明，在所有三个生命王国中高度保守的ERA功能的共同机制是通过识别AUCA，而非真核ERA蛋白的扭曲也通过识别CCUCC。ERA存在于几乎所有细菌物种中，并且对于生长和分裂是必需的，这在细菌的所有其他已知蛋白质功能中是独特的。抑制细菌ERA功能可能会停止细菌核糖体的合成。因此，ERA是开发新型抗生素以应对全球抗生素耐药性危机的潜在目标。ERA的真核同源物（EARL 1）是一种有吸引力的肿瘤抑制剂候选物。它位于线粒体核糖体的小亚基上，与12 S rRNA相互作用，在线粒体核糖体组装和细胞活力中起重要作用。目前，正在进行人和小鼠ERAL 1蛋白的结构和功能研究。我们在基于结构的药物开发方面的努力主要集中在两个系统，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代表解毒酶的超家族，以GST-α、GST-μ、GST-π等为代表。GST-α是人体肝脏中GST的主要亚型，对我们的健康起着重要作用。GST-pi在许多形式的癌症中过表达，因此提供了选择性靶向癌细胞的机会。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。哺乳动物从它们的饮食中获得叶酸，而大多数微生物必须从头合成叶酸。因此，叶酸途径是开发抗微生物剂的理想靶点。此外，HPPK对于微生物是独特的，并且不是任何现有抗生素的靶标。因此，它是开发新型抗菌剂的理想靶标。以前，我们基于结构的设计的前药，旨在释放GST-π过表达的癌细胞中的细胞毒性水平的一氧化氮产生PABA/NO，其表现出在体外和体内的抗癌活性与顺铂的效力相似。今年，我们设计、合成并表征了一组基于结构的HPPK抑制剂，作为新型抗生素的先导化合物。这些抑制剂是连接的嘌呤蝶呤化合物。它们以高亲和力和特异性结合HPPK。提出了含有HPPK抑制剂的药物组合物和用一种或多种HPPK抑制剂治疗患者的细菌感染的方法。还提供了使用所述化合物指导开发另外的新型抗菌剂的方法。

We have been studying three RNA-processing proteins: RNase III [a family of double-stranded (ds) RNA-specific endonucleases that initiate RNA interference], KsgA (a universally conserved methyltransferase that catalyzes the dimethylation of two adjacent adenosines in small-subunit ribosomal RNA), and ERA (a conserved GTPase that couples cell division with cell growth rate in bacteria and is a potential tumor suppressor in mammals). Previously, we have established a stepwise mechanism for RNase III to execute dsRNA cleavage, which can be extrapolated to other members of the family, including Rnt1p, Drosha and Dicer. This year, we have made significant progress in KsgA research and breakthrough advances in ERA research. ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is an essential ribosome biogenesis factor. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the 1531AUCACCUCCUUA1542 sequence at the 3 end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in non-eukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a twist for non-eukaryotic ERA proteins by also recognizing the CCUCC. ERA is present in nearly every bacterial species and is essential for growth and division, which is unique among all other known protein functions of bacteria. Inhibition of bacterial ERA function will likely stop the synthesis of bacterial ribosome. Hence, ERA is a potential target for the development of novel antibiotics to fight the worldwide crisis of antibiotic resistance. The eukaryotic homologs of ERA (EARL1) 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. Our effort in structure-based drug development has been focused on two systems, Glutathione S-transferase (GST) and 6-hydroxymethyl-7,8-dihydroptein pyrophosphokinase (HPPK). GST represents a superfamily of detoxification enzymes, represented by GST-alpha, GST-mu, GST-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. Thus, the folate pathway is an ideal target for developing antimicrobial agents. In addition, HPPK is unique for microorganisms and is not the target for any existing antibiotics. Therefore, it is an ideal 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 had yielded PABA/NO, which exhibits anticancer activity both in vitro and in vivo with a potency similar to that of cisplatin. This year, we have designed, synthesized, and characterized a group of structure-based HPPK inhibitors as lead compounds for novel antibiotics. These inhibitors are linked purine pterin compounds. They bind to HPPK with high affinity and specificity. Pharmaceutical compositions containing HPPK inhibitors and methods of treating a bacterial infection in a patient with one or more of the HPPK inhibitors are proposed. Methods of using the compounds to guide the development of additional novel anti-bacterial agents are also provided.