Structural Chemistry of Biomolecular Systems by X-ray Di

谷胱甘肽S转移酶激活的新型抗癌药物 GST代表一个II相解毒酶家族，其中PI类GST(GSTP)在许多肿瘤中高水平表达。我们参与了一系列GST激活的NO释放剂的基于机理的开发(美国专利6,610,660 B1；澳大利亚专利45957/97)。其中一种化合物JS-K已被证明对HL-60细胞有抑制增殖作用，其IC50为0.2-0.5 mm。JS-K对实体瘤细胞株的生长也有抑制作用，但抑制程度较小。利用基于结构的方法，我们设计了一种GSTP选择性的NO释放前药PABA/NO，用于在不损害正常组织的情况下直接靶向癌细胞。PABA/NO在人卵巢癌模型中产生了抗肿瘤作用。 6-羟甲基-7，8-二氢蝶呤焦磷酸酶(HPPK)反应轨迹 HPPK催化依赖于镁离子的焦磷酰化从ATP到6-羟甲基-7，8-二氢蝶呤(HP)的转移，反应产物为AMP和HP焦磷酸盐(HPPP)。HPPK是叶酸生物合成途径中的一个关键酶，对微生物是必不可少的，但在哺乳动物中是不存在的。HPPK不是任何现有抗生素的靶标，是开发新型抗菌剂的理想系统。大肠杆菌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制的理想模型。根据不同催化阶段大肠杆菌酶的五种晶体结构，我们绘制了HPPK催化焦磷酸转移的反应轨迹，为反应机理和基于结构的药物设计提供了必要的信息。这五种晶体结构包括apo-HPPK(1.50)、HPPK-MgATPanalog(1.50)、HPPK-MgATPanalog-HP(1.25)、HPPK-AMP-HPPP(1.56)和HPPK-HPPP(1.35) 核糖核酸酶III(RNaseIII)的结构，双链RNA(DsRNA)切割的机制，以及对RNA干扰(RNAi)的影响。 RNaseIII代表一个dsRNA内切酶家族。最简单的细菌酶含有一个内切酶结构域(EndND)和一个dsRNA结合域(DsRBD)。RNaseIII可以通过两种方式影响RNA结构和基因表达：一种是作为dsRNA处理酶来裂解dsRNA，另一种是作为dsRNA结合蛋白来结合但不裂解dsRNA。我们测定了Aquifex aeolicus RNaseIII(AA-RNaseIII)无配体形式和与Mn(II)或Mg(II)的络合物的晶体结构，并模拟了全长AA-RNaseIII与dsRNA的催化络合物，推测了dsRNA的切割机制。我们还测定了全长AA-RNaseIII与dsRNA的络合物的晶体结构，揭示了非催化组装。与dsRNA结合的两种功能形式的RNaseIII之间的主要区别是蛋白质的构象和dsRNA的方向和位置。EndND和dsRBD之间具有遗传重要性的连接子的灵活性使这两种形式之间的转换成为可能。DsRNA的切割参与了RNA的成熟、RNA的衰变、RNAi和转录后基因沉默。我们的结构和模型研究揭示了这些过程的机制。

Novel Anticancer Agents Activated by Glutathione S-Transferase (GST) GST represents a family of phase II detoxification enzymes, among which the Pi-class GST (GSTP) expresses at high levels in many tumors. We participated in the mechanism-based development of a family of GST-activated NO-releasing agents (US Patent 6,610,660 B1; Australia Patent 45957/97). One of these compounds, JS-K, has been demonstrated to be an antiproliferative agent in HL-60 cells, with an IC50 of 0.2-0.5 mM. JS-K also inhibited the growth of solid tumor cell lines but to a lesser extent. Using a structure-based approach, we designed a GSTP-selective NO-releasing prodrug, PABA/NO, for direct cancer-cell targeting without damaging normal tissues. PABA/NO has produced antitumor effects in a human ovarian cancer model. Reaction Trajectory of 6-Hydroxymethyl-7,8-Dihydropterin Pyrophosphokinase (HPPK) HPPK catalyzes the Mg(II)-dependent pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP); the products of the reaction are AMP and HP pyrophosphate (HPPP). HPPK is a key enzyme in the folate biosynthetic pathway, essential for microorganisms but is absent in mammals. Without being a target for any existing antibiotics, HPPK is an ideal system for the development of novel antimicrobial agents. HPPK from Escherichia coli contains 158 amino acid residues and is thermostable, which makes it an excellent model for the study of pyrophosphoryl transfer mechanism. With five crystal structures of the E. coli enzyme at different catalytic stages, we have mapped out the reaction trajectory of HPPK-catalyzed pyrophosphoryl transfer, providing essential information for the reaction mechanism and structure-based drug design. The five crystal structures include apo-HPPK (1.50 ), HPPK-MgATPanalog (1.50 ), HPPK-MgATPanalog-HP (1.25 ), HPPK-AMP-HPPP (1.56 ), and HPPK-HPPP (1.35 ) Structures of Ribonuclease III (RNase III), Mechanism of Double-stranded RNA (dsRNA) Cleavage, and Implications for RNA Interference (RNAi). RNase III represents a family of dsRNA endonucleases. The simplest bacterial enzyme contains an endonuclease domain (endoND) and a dsRNA-binding domain (dsRBD). RNase III can affect RNA structure and gene expression in either of two ways: as a dsRNA-processing enzyme that cleaves dsRNA, or as a dsRNA-binding protein that binds but does not cleave dsRNA. We determined the crystal structure of the endoND of Aquifex aeolicus RNase III (Aa-RNase III) in its ligand-free form and in complex with Mn(II) or Mg(II) and modeled a catalytic complex of full-length Aa-RNase III with dsRNA, which suggested a mechanism of dsRNA cleavage. We also determined the crystal structure of a full-length Aa-RNase III in complex with dsRNA, which revealed a non-catalytic assembly. The major differences between the two functional forms of RNase III with bound dsRNA are the conformation of the protein and the orientation and location of the dsRNA. The flexibility of a genetically important linker between the endoND and the dsRBD enables the transition between these two forms. The cleavage of dsRNA is involved in RNA maturation, RNA decay, RNAi, and posttranscriptional gene silencing. Our structural and modeling studies shed light on the mechanisms for these processes.