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 drug design targeting 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: Structure-based Design of Electrophilic Diazeniumdiolates for Pharmacologic Delivery of Nitric Oxide Many tumors become drug resistant by overexpressing the detoxification enzyme glutathione S-transferase (GST). Of the three major isoforms, alpha, mu, and pi, 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 anti-cancer therapies. 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 planned cytotoxicity studies show that this donor or subsequent NO donors improve the potency of electrophilic anticancer agents toward GST-pi-overexpressing cells, a means of overcoming drug resistance in some clinically important tumor types may be forthcoming. 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase: Mechanism of Pyrophosphoryl Transfer and Structure-based Design of Novel Antimicrobial Agents 6-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. At atomic resolutions (up to 0.89 Angstrom), we have determined the crystal structures of ligand-free enzyme as well as various well-chosen complexes. Our analysis of these structures has provided essential information on the reaction mechanism of pyrophosphoryl transfer and critical knowledge for the design of novel antimicrobial molecules. Of particular importance is the structure of HPPK in complex with HP and MgAMPCPP at 1.25-Angstrom resolution, which mimics most closely the ternary complex of the enzyme and reveals the atomic details of the catalytic assembly, and therefore has been the basis of our structure-based inhibitor design effort. We have carried out the design, synthesis, biochemical, and crystallographic studies of three bisubstrate-mimicking analogs, each of which consists of a pterin, an adenosine moiety, and a linker composed of 2-4 phosphoryl groups. Era Protein: GTPase-dependent Cell Cycle Regulator Era 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-Angstrom 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.
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