Biomolecular Structure and Mechanism, Structure-Based Drug Design

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By structural analysis, we map the reaction trajectory or functional cycle of selected biological macromolecules. By structure-based development, we design, synthesize, and characterize novel anticancer and antimicrobial agents. We carry out structure-based drug development as a continuation of our basic research on the structure and mechanism of biomolecular systems with anticancer and antimicrobial significance. To date, we have described the reaction trajectory or functional cycle of HPPK (a folate pathway enzyme essential for microorganisms but absent in mammals), Era (an essential GTPase that couples cell growth with cell division), RapA (a Swi2/Snf2 protein that recycles RNA polymerase), two members of the RNase III family (a family of dsRNA-specific endoribonucleases), and DDX3X (a DEAD-box helicase that unwinds short RNA duplexes). Among these biomolecules, HPPK is a target for novel antibacterial agents, and DDX3X is a target for novel anticancer and anti-HIV agents. Structure-based drug development is in progress. We have made significant progress toward novel antibacterial agents targeting HPPK, closely mimicking the reaction intermediate and thereby exhibiting high potency. Representative members of the RNase III family include prokaryotic RNase III and eukaryotic Rnt1p, Drosha, and Dicer. They play important roles in RNA processing and maturation, post-transcriptional gene silencing, and defense against viral infection. For structural and mechanistic studies, bacterial RNase III and yeast Rnt1p are valuable model systems for prokaryotes and eukaryotes, respectively. For both RNase III and Rnt1p, we have shown how the dimerization of their endonuclease domain (RIIID) creates a catalytic valley where two cleavage sites are located, how the catalytic valley accommodates a dsRNA substrate in a manner such that each of the two RNA strands is aligned with one of the two cleavage sites, how the hydrolysis of each strand involves both RIIIDs, and how RNase III uses the two cleavage sites to create 2-nucleotide (2-nt) 3' overhangs in its products. We have also shown how magnesium is essential for the formation of a catalytically competent protein-RNA complex, and how the use of magnesium ions can drive the hydrolysis of each phosphodiester bond. Moreover, we have described a stepwise model by which RNase III and Rnt1p execute the phosphoryl transfer reaction. All members of the RNase III family propel RNA hydrolysis by two-Mg2+-ion catalysis, which exhibits distinct features, however, by prokaryotes and eukaryotes. As revealed by our structures, prokaryotic RNase IIIs require a third magnesium ion in catalysis, whereas eukaryotic RNase IIIs employ two additional amino acid side chains. DDX3X belongs to the family of DEAD-box helicases that regulate RNA processing and metabolism by unwinding short RNA duplexes. Sharing a helicase core composed of two RecA-like domains (D1D2), DDXs function in an ATP-dependent, non-processive manner. As an attractive target for cancer and AIDS treatment, DDX3X and its orthologs are extensively studied, yielding a wealth of biochemical and biophysical data, including structures of apo-D1D2 and post-unwound D1D2:ssRNA complex. However, the structure of a pre-unwound D1D2:dsRNA complex was not available until we have recently determined the crystal structure of a D1D2 core in complex with a 2-turn RNA duplex at the pre-unwound state, showing that two DDXs recognize the RNA duplex. Each DDX mainly recognizes a single strand, and conformational changes induced by ATP binding unwinds the RNA duplex in a cooperative manner. Our structure has significantly altered a previous model of three-molecule cooperativity. To validate our new model, we are currently elucidating the functional cycle of DDX3X using site-directed mutagenesis, RNA-unwinding assay, ATP-hydrolyzing assay, Hill cooperativity analysis, and structural studies. We are also developing DDX3X inhibitors based on available structural and mechanistic information.