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RNA特异性内切酶家族]，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GTPase，在细菌中将细胞分裂与细胞生长速率结合，在哺乳动物中是一种潜在的肿瘤抑制因子）。之前，我们已经建立了RNase III执行dsRNA切割的分步机制，该机制可以外推到该家族的其他成员，包括Rnt1p、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发生因子，由一个n端GTPase结构域和一个rna结合的KH结构域组成。它结合16S rRNA和30S核糖体亚基。然而，其rna结合位点、两个结构域之间的功能关系及其在核糖体生物发生中的作用尚不清楚。我们确定了ERA的两种晶体结构，即具有GDP的二元配合物和具有gtp类似物的三元配合物，以及16S rRNA 3端的1531AUCACCUCCUUA1542序列。在三元复合物中，12个核苷酸中的前9个被蛋白质识别。我们发现GTP结合是ERA识别RNA的先决条件，RNA识别刺激其GTP水解活性。基于这些数据和其他数据，我们提出了ERA的功能周期，表明该蛋白作为16S rRNA加工和成熟的伴侣蛋白和30S核糖体亚基组装的检查点。AUCA序列在细菌、古细菌和真核生物中高度保守，而CCUCC，被称为anti-Shine-Dalgarno序列，仅在非真核生物中保守。因此，这些数据表明，通过识别AUCA，在所有三个生命领域中都存在高度保守的ERA功能的共同机制，而对于非真核ERA蛋白，也可以识别CCUCC。ERA几乎存在于每一种细菌中，对细菌的生长和分裂至关重要，这在所有已知的细菌蛋白质功能中是独一无二的。抑制细菌的ERA功能可能会阻止细菌核糖体的合成。因此，ERA是开发新型抗生素以对抗全球抗生素耐药性危机的潜在目标。ERA （EARL1）的真核同源物是一个有吸引力的肿瘤抑制因子候选物。它位于线粒体核糖体的小亚基上，与12S rRNA相互作用，在线粒体核糖体组装和细胞活力中发挥重要作用。目前，正在进行人类和小鼠ERAL1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是一个解毒酶超家族，以GST-alpha、GST-mu、GST-pi等为代表。GST- α是人类肝脏中GST的主要亚型，对我们的健康起着重要的作用。GST-pi在许多形式的癌症中过度表达，从而为选择性靶向癌细胞提供了机会。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。哺乳动物从饮食中摄取叶酸，而大多数微生物必须从头合成叶酸。因此，叶酸途径是开发抗菌药物的理想靶点。此外，HPPK对微生物是独特的，不是任何现有抗生素的靶点。因此，它是开发新型抗菌药物的理想靶点。之前，我们基于结构的前药设计旨在释放gst -pi过表达癌细胞中的一氧化氮的细胞毒性水平，已经产生了PABA/NO，其在体外和体内都具有与顺铂相似的抗癌活性。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。这些抑制剂是嘌呤-蝶呤类化合物。它们以高亲和力和特异性与HPPK结合。提出了含有HPPK抑制剂的药物组合物和用一种或多种HPPK抑制剂治疗患者细菌感染的方法。还提供了使用所述化合物指导其它新型抗菌药物开发的方法。

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.