HPPK

BIOMOLECULAR STRUCTURE AND MECHANISM, STRUCTURE-BASED DRUG DESIGN

The primary goals of our Section are to address the structure and mechanism of RNA-processing proteins, RNA polymerase-associated transcription factors, enzymes in the folate and shikimate pathways, and virus-neutralizing antibodies. Although our major effort is devoted to basic research, we seek opportunities to initiate or participate in translational research. Once a sufficient amount of structural information is available for a potential drug target, we attempt structure-based drug design.

The Mechanism of RNase III Action: How Dicer Dices. 

Members of the RNase III family (represented by bacterial RNase III and eukaryotic Rnt1p, Drosha, and Dicer) are double-stranded (ds) RNA-specific endoribonucleases, characterized by a signature motif in their active centers and a 2-nucleotide (nt) 3′ overhang in their products. Dicer functions as a dsRNA-processing enzyme, producing small interfering RNA (siRNA) of approx. 24 nt in length (approx. 20-basepair RNA duplex with a 2-nt 3′ overhang on each end). Bacterial RNase III functions not only as a processing enzyme, but also as a binding protein that binds dsRNA without cleaving it. As a processing enzyme, it produces siRNA-like RNA of approx. 13 nt in length (approx. 9-basepair duplex with a 2-nt 3′ overhang on each end) as well as various types of mature RNA. As a binding protein, it binds and stabilizes certain RNAs, thus suppressing the expression of certain genes. Dicer is structurally most complicated member of the family; bacterial RNase III is comparatively much simpler. Thus, for mechanistic studies, bacterial RNase III has been a valuable model system for the entire family. We have shown how the dimerization of the endonuclease domain of the enzyme creates a catalytic valley where two catalytic sites are located, how the catalytic valley accommodates a dsRNA in a manner such that each of the two RNA strands is aligned with one of the two catalytic sites, how the hydrolysis of each strand involves both subunits (residues from one subunit are involved in the selection of the scissile bond, while those from the partner subunit are involved in the cleavage chemistry), and how RNase III uses the two catalytic sites to create the 2-nt 3' overhangs in its products. We have also demonstrated how Mg2+ is essential for the formation of a catalytically competent protein-RNA complex, how the use of two Mg2+ ions can drive the hydrolysis of each phosphodiester bond, and how conformational changes in both the substrate and the protein are critical elements for assembling the catalytic complex. Moreover, we have modeled a protein-substrate complex and a protein-reaction intermediate (transition state) complex on the basis of our crystal structures. Together, our structures and models suggest a stepwise mechanism for the enzyme to execute the phosphoryl transfer reaction. The structural information of protein-dsRNA interactions and the mechanism of dsRNA processing by bacterial RNase III can be extrapolated to other family members.

Structure-Based Design of Anticancer Prodrug PABA/NO.

Glutathione S-transferase (GST) is a superfamily of detoxification enzymes, represented by GSTα, GSTμ, GSTπ, etc. GSTα is the predominant isoform of GST in human liver, playing important roles for our well being. GSTπ is overexpressed in many forms of cancer, thus presenting an opportunity for selective targeting of cancer cells. Our structure-based design of prodrugs intended to release cytotoxic levels of nitric oxide in GSTπ-overexpressing cancer cells has yieldedPABA/NO, which exhibits anticancer activity both in vitro and in vivo with a potency similar to that of cisplatin. The design is efficient because it is on the basis of the reaction mechanism and the structures of related GST isozymes at both the ground state and the transition state. The ground-state structures outline the shape and property of the substrate-binding site in different isozymes, and the structural information at the transition-state indicate distinct conformations of the Meisenheimer complex of prodrugs in the active site of different isozymes, providing guidance for the modifications of the molecular structure of the prodrug molecules. Two key alterations of a GSTα-selective compound lead to the GSTπ-selective PABA/NO.

Our collaborators include Drs. Donald Court (NCI), Dimiter Dimitrov (NCI), Ding Jin (NCI), Larry Keefer (NCI), Shivendra Singh (University of Pittsburgh),Joseph Tropea (NCI), David Waugh (NCI), and Honggao Yan (Michigan State University). We will continue this interdisciplinary approach by extending existing collaborative efforts and seeking new collaborations whenever appropriate.