STRUCTURAL CHEMISTRY OF BIOMOLECULAR SYSTEMS

本研究组的主要目标是研究具有抗癌和抗微生物意义的生物分子系统的结构和功能，并探索设计针对这些生物分子的药物/前药的可行性。为了实现这些目标，我们在NIH内部以及与遗传学、分子生物学、蛋白质化学、酶学、癌症发生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：用于一氧化氮药理传递的亲电重氮二醇酸盐的合理设计许多肿瘤通过过度表达解毒酶谷胱甘肽S转移酶而产生耐药性。在三种主要的GST异构体中，uα、u u和p u pi是癌细胞中的主要形式。我们正试图设计一种药物，通过在GST-pi的活性部位选择性地产生一氧化氮(NO)来克服这种耐药性，这可能会增加抗癌治疗的有效性。我们对这三种同工酶的活性位点和过渡态类似物进行了比较，揭示了实现同工酶选择性的潜在策略。这一策略的应用导致了pi-选择性的NO供体。如果我们计划的细胞毒性研究表明这个供体或随后的NO供体提高了亲电抗癌药物对高表达GST-pi的细胞的效力，那么克服某些临床重要肿瘤类型的耐药性的手段可能会随之而来。6-羟甲基-7，8-二氢蝶呤焦磷酸激酶：焦磷转运蛋白的机制6-羟甲基-7，8-二氢蝶呤焦磷酸激酶(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得叶酸。相反，大多数微生物必须从头合成叶酸。因此，HPPK是开发新型抗菌剂的理想靶点，而新型抗菌剂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，具有热稳定性，是研究焦磷酰化转移机理的良好模型体系。我们测定了apo-HPPK的晶体结构为1.50，与MgADP的二元配合物的晶体结构为1.50，与HP和MgAMPCPP的三元配合物的晶体结构为1.25。我们对这些结构的分析将为焦磷酰化转移的反应机理提供必要的信息，并为设计新型抗菌分子提供关键知识。Era蛋白：依赖GTP酶的细胞周期调节器Era是一种重要的GTP酶，迄今在每个细菌中都有发现。在真核生物中也发现了高度保守的Era同源物，如小鼠和人类。Era同源基因可能是肿瘤抑制基因的候选基因，因为它位于染色体区域，杂合性缺失通常与各种类型的癌症相关。在细菌中，ERA通过将细胞生长速度与胞质分裂相结合，在细胞周期控制中发挥调节作用。当达到ERA活动的阈值时，细胞分裂就被发出信号。人为地减少ERA的表达或削弱ERA的活性会导致细菌细胞周期停滞在分裂前的两个细胞阶段。这种抑制持续到ERA活性累积到阈值水平，允许另一个细胞周期开始。由于Era本身的合成与生长速率呈正相关，因此细胞分裂速率被协调地维持。我们在2.4的分辨率下测定了大肠杆菌Era的晶体结构，揭示了一个两个结构域的排列：一个类似于p21Ras的N-末端结构域和一个独特的包含RNA结合基序的C-末端结构域。Era与GDP络合物和GTP类似物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对GTP水解过程中蛋白质构象变化的洞察，这可能是该细胞周期调节因子信号通路的一部分。

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.