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.
Showing the most recent 10 out of 28 publications
