STRUCTURAL CHEMISTRY OF BIOMOLECULAR SYSTEMS

我们的主要目标是解决具有抗癌和抗菌意义的生物分子系统的结构和功能，并探索设计药物/前药以靶向这些生物分子的可行性。在我们努力实现这些目标的过程中，我们在NIH内部以及与遗传学、分子生物学、蛋白质化学、酶学、致癌作用和药物化学方面的校外专家建立了合作关系。这些合作极大地扩展了我们的实验范围。谷胱甘肽S-转移酶：用于一氧化氮药理学递送的亲电二氮烯二醇盐的合理设计许多肿瘤通过过表达解毒酶谷胱甘肽S-转移酶（GST）而产生耐药性。在三种GST主要同种型中，u α、mu和pi u pi是癌细胞中的主要形式。我们正试图设计通过在GST-π的活性位点选择性地产生一氧化氮（NO）来克服这种耐药性的药物，这可以增加抗癌疗法的有效性。我们的活性位点和三种同工酶的过渡态类似物的比较揭示了一个潜在的策略，实现同工酶的选择性。这种策略的应用导致了pi-选择性NO供体。如果我们计划的细胞毒性研究显示该供体或随后的NO供体改善亲电子抗癌剂对过表达GST-pi的细胞的效力，则克服某些临床重要肿瘤类型中的耐药性的手段可能即将到来。焦磷酸转移酶的作用机制6-羟甲基-7，8-二氢蝶呤焦磷酸激酶（6-Hydroxymethyl-7，8-dihydropterin pyrophosphokinase，HPPK）是叶酸生物合成途径中的第一个酶，催化焦磷酸从ATP转移到6-羟甲基-7，8-二氢蝶呤（HP）。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取叶酸。相反，大多数微生物必须从头合成叶酸。因此，HPPK是开发新型抗菌药物的理想靶标，这是对抗全球抗生素耐药性危机的迫切需要。HPPK含有158个氨基酸残基，是热稳定的，这使得它成为一个很好的模型系统的焦磷酸转移机制的研究，其中知之甚少。我们测定了apo-HPPK（1.50 +）、与MgADP（1.50 +）的二元配合物和与HP和MgAMPCPP（1.25 +）的三元配合物的晶体结构。我们对这些结构的分析将为焦磷酸转移的反应机制提供必要的信息，并为新型抗菌分子的设计提供重要的知识。Era蛋白：GTP酶依赖的细胞周期调节剂Era是迄今为止在测序的每种细菌中发现的一种必需的GTP酶。高度保守的Era同源物也发现于真核生物中，例如小鼠和人类。Era同源物可能是肿瘤抑制因子的候选者，因为它位于杂合性丢失通常与各种类型的癌症相关的染色体区域。在细菌中，Era通过偶联细胞生长速率与胞质分裂在细胞周期控制中具有调节作用。当达到Era活性的阈值时，细胞分裂发出信号。牺牲性地减少Era的表达或损害Era的活性导致细菌细胞周期停滞在分裂前的两细胞阶段。这种停滞持续到Era活性积累到阈值水平，允许另一个细胞周期开始。由于Era本身的合成与生长速率正相关，因此细胞分裂速率得以协调维持。我们已经确定了Era的晶体结构，从大肠杆菌在2.4 +分辨率，这揭示了两个结构域的安排：一个N-末端结构域，类似于p21 Ras和一个独特的C-末端结构域，包含RNA结合基序。与GDP和GTP类似物复合的Era的晶体结构测定正在进行中。我们对这些结构的分析将提供深入了解GTP水解过程中蛋白质的构象变化，这可能是这种细胞周期调节剂的信号通路的一部分。

The primary goals of our Section are to address the structure and function of biomolecular systems with anticancer and antimicrobial significance and to explore the feasibility of designing drugs/prodrugs to target such biomolecules. In our efforts to achieve these goals, we have established collaborations within NIH as well as with extramural experts in genetics, molecular biology, protein chemistry, enzymology, carcinogenesis, and medicinal chemistry. These collaborations have greatly extended our range of experiments. Glutathione S-transferase: Rational Design of Electrophilic Diazeniumdiolates for Pharmacologic Delivery of Nitric OxideMany tumors become drug resistant by overexpressing the detoxification enzyme glutathione S-transferase (GST). Of the three GST major isoforms u alpha, mu, and pi u pi is the predominant form in cancer cells. We are attempting to design agents that will overcome this drug resistance by generating nitric oxide (NO) selectively in the active site of GST-pi, which could increase the effectiveness of anticancer therapies. Our comparison of the active sites and transition-state analogs of the three isozymes revealed a potential strategy for achieving isozyme selectivity. Application of this strategy has resulted in a pi-selective NO donor. If our planned cytotoxicity studies show that this donor or subsequent NO donors improve the potency of electrophilic anticancer agents toward cells overexpressing GST-pi, the means of overcoming drug resistance in some clinically important tumor types may be forthcoming.6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase: Mechanism of Pyrophosphoryl Transfer6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is the first enzyme in the folate biosynthetic pathway, catalyzing the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). Folate cofactors are essential for life. Mammals derive folates from their diets. In contrast, most microorganisms must synthesize folate de novo. Therefore, HPPK is an ideal target for the development of novel antimicrobial agents, which are urgently needed to fight the worldwide crisis of antibiotic resistance. HPPK contains 158 amino acid residues and is thermostable, which makes it an excellent model system for the study of the pyrophosphoryl transfer mechanism, of which little is known. We have determined the crystal structures of apo-HPPK at 1.50 +, of the binary complex with MgADP at 1.50 +, and of the ternary complex with both HP and MgAMPCPP at 1.25 +. Our analysis of these structures will provide essential information on the reaction mechanism of pyrophosphoryl transfer and critical knowledge for the design of novel antimicrobial molecules.Era Protein: GTPase-Dependent Cell Cycle RegulatorEra is an essential GTPase found in every bacterium sequenced to date. Highly conserved Era homologs are also found in eukaryotes, such as mouse and human. The Era homolog may be a candidate for a tumor suppressor, because it is located in a chromosomal region where loss of heterozygosity is often associated with various types of cancer. In bacteria, Era has a regulatory role in cell cycle control by coupling cell growth rate with cytokinesis. Cell division is signaled when a threshold of Era activity is reached. Artificially reducing the expression or impairing the activity of Era results in bacterial cell cycle arrest at a predivisional two-cell stage. The arrest lasts until Era activity accumulates to the threshold level, allowing another cell cycle to start. Because the synthesis of Era itself is positively correlated with growth rate, the cell division rate is thus coordinately maintained. We have determined the crystal structure of Era from Escherichia coli at 2.4 + resolution, which reveals a two-domain arrangement: an N-terminal domain that resembles p21 Ras and a unique C-terminal domain that contains an RNA-binding motif. The crystal structure determination of Era in complex with GDP and with a GTP analog is in progress. Our analysis of these structures will provide insight into the conformational changes of the protein during GTP hydrolysis, which may be part of the signaling pathway of this cell cycle regulator.