Biomolecular Structure and Mechanism, Structure-Based Drug Design

生物分子结构与机制、基于结构的药物设计

• 批准号: 10702336
• 负责人: XINHUA JI
• 金额: $ 198.2万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702336
• 关键词: 4-Aminobenzoic Acid; Acquired Immunodeficiency Syndrome; Anti-Bacterial Agents; Anti-HIV Agents; Basic Science; Binding; Biochemical; Biogenesis; Biological; Biological Assay; Biological Models; Biophysics; Catalysis; Cell division; Closure by clamp; Complex; Couples; Crystallization; DNA Damage; DNA-Directed RNA Polymerase; Data; Dimerization; Double-Stranded RNA; Drug Design; Elements; Endoribonucleases; Enzymes; Evolution; Exhibits; Family; Folic Acid; Goals; Guanine; Guanine Nucleotides; Guanosine Triphosphate Phosphohydrolases; Hydrolysis; Ions; Legal patent; Link; Magnesium; Malignant Neoplasms; Mammals; Maps; Measures; Modeling; Molecular Conformation; N-terminal; Nitric Oxide; Nucleotides; Orthologous Gene; Play; Prodrugs; Proteins; Pterins; Purines; RNA; RNA Binding; RNA Interference; RNA Processing; RNA metabolism; Reaction; Research; Resolution; Ribonuclease III; Ribonucleases; Role; Side; Site; Site-Directed Mutagenesis; Specificity; Structure; System; Virus Diseases; Yeasts; anti-cancer; antimicrobial; antimicrobial drug; base; cancer cell; cell growth; design; drug development; drug discovery; endonuclease III; enzyme mechanism; enzyme pathway; helicase; improved; inhibitor; macromolecule; magnesium ion; member; microorganism; novel; novel anticancer drug; phosphodiester; repaired; structural biology; targeted agent; therapeutic development; transcription factor

Our goal of structural analysis is to map the reaction trajectory or functional cycle of selected biological macromolecules, and our goal of drug discovery is to design, synthesize, and characterize novel anticancer and antimicrobial agents. To date, we have characterized the reaction trajectory and/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 of novel anticancer and anti-HIV agents. Structure-based drug development is in progress. 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 mechanistic studies, bacterial enzyme is a valuable model system for the entire family. We have shown how the dimerization of the RNase III endonuclease domain (RIIID) creates a catalytic valley where two cleavage 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 cleavage sites, how the hydrolysis of each strand involves both RIIIDs, and how RNase III uses the two cleavage sites to create the 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, how the use of two magnesium 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 provided a stepwise trajectory by which RNase III executes the phosphoryl transfer reaction. As informative as the bacterial enzyme for the mechanism of RNase III action, yeast Rnt1p is a valuable model system for eukaryotic RNase III enzymes. Unlike bacterial enzymes that use four catalytic side chains, eukaryotic RNase IIIs use six. It is also distinguished from bacterial enzymes that every eukaryotic RNase III has an N-terminal extension. What is more, Rnt1p exhibits a strict guanine nucleotide specificity, which is unique among RNase III enzymes. We have shown how the substrate-binding mode of Rnt1p is distinct from that of bacterial RNase III, how all six catalytic side chains are engaged in the cleavage site, how a new RNA-binding motif of Rnt1p functions as a guanine-specific clamp, and how the dsRNA-binding domain and N-terminal domain of Rnt1p function as two rulers measuring the distances between the guanine nucleotide to the two cleavage sites. This unusual mechanism of substrate selectivity represents an example of the evolution of substrate selectivity. All members of the RNase III family propel RNA hydrolysis by two-Mg2+-ion catalysis. We have also provided a stepwise trajectory by which Rnt1p executes the phosphoryl transfer reaction. The structural basis for the reaction trajectory of two-Mg2+-ion catalysis is provided by the crystal structure of a postcleavage complex determined at a stage immediately after the cleavage of the phosphodiester bond, for which we have determined high-resolution structures for both bacterial RNase III and yeast Rnt1p. These postcleavage structures reveal distinct features of two-Mg2+-ion catalysis by prokaryotic and eukaryotic RNase III enzymes, including Drosha and Dicer, for which the available structures fail to provide the basis of two-Mg2+-ion catalysis. 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 new 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 have also started to develop DDX3X inhibitors based on available structural and mechanistic information. We carry out structure-based drug development primarily as a continuation of our basic research on the structure and mechanism of biomolecular systems with anticancer and antimicrobial significance. Previously, we designed PABA/NO, an enzymatically activated anticancer prodrug that kills cancer cells from within by releasing nitric oxide. We also made significant progress toward novel antibacterial agents targeting HPPK by the design and synthesis of linked purine pterin inhibitors and the determination of their crystal structures in complex with the enzyme. Recently, we have further developed both the PABA/NO and HPPK inhibitors. The PABA/NO derivative contains a PARP inhibitor and so it not only generates nitric oxide but also release a PARP inhibitor simultaneously in the same compartment, which damage DNA and inhibit its repair (PMID: 24521039; US Patent Number: 9168266). The improved HPPK inhibitors mimic closely the transition state of the catalytic complex and thereby have much higher potency (PMID: 33199204; US Patent Number: 11091509).