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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

基本信息

批准号：
7732999
负责人：
XINHUA JI
金额：
$ 128.6万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/7732999
关键词：
ATP phosphohydrolase Binding Binding Sites Biological Assay Biological Models Catalytic Domain Cells Chemistry Cisplatin Complex DNA-Directed RNA Polymerase Development Dimerization Double-Stranded RNA Drug Design Drug Metabolic Detoxication Elements Enzymes Exhibits Family Family member Future Gene Expression General Transcription Factors Genetic Transcription Glutathione S-Transferase Glutathione Transferase mu Human Hydrolysis In Vitro Ions Isoenzymes Kinetics Length Liver Malignant Neoplasms Mediating Modeling Modification Nitric Oxide Nucleotides Object Attachment Personal Satisfaction Play Process Prodrugs Property Protein Isoforms Protein Overexpression Proteins RNA RNA Interference RNA Processing RNA polymerase sigma 70 Reaction Recycling Reporting Ribonuclease III Role Shapes Small Interfering RNA Structure anticancer activity antimicrobial drug base cancer cell cytotoxic design ds-DNA endonuclease enzyme mechanism human DICER1 protein in vivo member novel phosphodiester protein folding research study structural biology transcription factor

项目摘要

RNA interference is mediated by small interfering RNAs produced by members of the ribonuclease III (RNase III) family, including Dicer. For mechanistic studies, bacterial RNase III has been a valuable model system for the entire family. Previously, 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-nucleotide 3' overhangs in its products. Recently, we have 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 in a meaningful way. Together, the models and crystal structures 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, including eukaryotic Rnt1p, Drosha and Dicer. RapA, as abundant as sigma 70 in the cell, is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein with ATPase activity. It stimulates RNAP recycling during transcription. Recently, we reported the first structure of RapA, which is also the first full-length structure for the entire Swi2/Snf2 family. RapA contains seven domains, two of which exhibit novel protein folds. Our model of RapA in complex with ATP and double-stranded (ds) DNA suggests that RapA may bind to and translocate on dsDNA. Our kinetic template-switching assay shows that RapA facilitates the release of sequestered RNAP from a posttranscrption/posttermination complex (PTC) for transcription reinitiation. Our in vitro competition experiment indicates that RapA binds to core RNAP only but is readily displaceable by sigma 70. RapA is likely another general transcription factor, the structure of which provides a framework for future studies of this bacterial Swi2/Snf2 protein and its important roles in RNAP recycling during transcription. Glutathione S-transferase (GST) is a superfamily of detoxification enzymes, represented by GST-alpha, GST-mu, GST-pi, etc. GST-alpha is the predominant isoform of GST in human liver, playing important roles for our well being. GST-pi 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 GST-pi-overexpressing cancer cells yielded PABA/NO, which exhibited anticancer activity both in vitro and in vivo with a potency similar to that of cisplatin. The design was based on GST structures at both ground state and transition state. The ground-state structures outlined the shape and property of the substrate-binding site in different isozymes, and the structural information at the transition-state provided guidance for structural modifications of the prodrug molecules. Two key alterations of a GST-alpha-selective compound led to the GST-pi-selective PABA/NO.RNA interference is mediated by small interfering RNAs produced by members of the ribonuclease III (RNase III) family, including Dicer. For mechanistic studies, bacterial RNase III has been a valuable model system for the entire family. Previously, 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-nucleotide 3' overhangs in its products. Recently, we have 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 in a meaningful way. Together, the models and crystal structures 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, including eukaryotic Rnt1p, Drosha and Dicer. RapA, as abundant as sigma 70 in the cell, is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein with ATPase activity. It stimulates RNAP recycling during transcription. Recently, we reported the first structure of RapA, which is also the first full-length structure for the entire Swi2/Snf2 family. RapA contains seven domains, two of which exhibit novel protein folds. Our model of RapA in complex with ATP and double-stranded (ds) DNA suggests that RapA may bind to and translocate on dsDNA. Our kinetic template-switching assay shows that RapA facilitates the release of sequestered RNAP from a posttranscrption/posttermination complex (PTC) for transcription reinitiation. Our in vitro competition experiment indicates that RapA binds to core RNAP only but is readily displaceable by sigma 70. RapA is likely another general transcription factor, the structure of which provides a framework for future studies of this bacterial Swi2/Snf2 protein and its important roles in RNAP recycling during transcription. Glutathione S-transferase (GST) is a superfamily of detoxification enzymes, represented by GST-alpha, GST-mu, GST-pi, etc. GST-alpha is the predominant isoform of GST in human liver, playing important roles for our well being. GST-pi 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 GST-pi-overexpressing cancer cells yielded PABA/NO, which exhibited anticancer activity both in vitro and in vivo with a potency similar to that of cisplatin. The design was based on GST structures at both ground state and transition state. The ground-state structures outlined the shape and property of the substrate-binding site in different isozymes, and the structural information at the transition-state provided guidance for structur [summary truncated at 7800 characters]

项目成果

期刊论文数量（13）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Structure-Based Design of Anticancer Prodrug PABA/NO.

DOI：
10.2147/dddt.s3931
发表时间：
2008
期刊：
Drug design, development and therapy
影响因子：
0
作者：
Ji X;Pal A;Kalathur R;Hu X;Gu Y;Saavedra JE;Buzard GS;Srinivasan A;Keefer LK;Singh SV
通讯作者：
Singh SV

Structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody.

DOI：
10.1074/jbc.m600697200
发表时间：
2006-06-09
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Prabakaran P;Gan J;Feng Y;Zhu Z;Choudhry V;Xiao X;Ji X;Dimitrov DS
通讯作者：
Dimitrov DS

Structural mimicry of CD4 by a cross-reactive HIV-1 neutralizing antibody with CDR-H2 and H3 containing unique motifs.

通过具有交叉反应性的 HIV-1 中和抗体与含有独特基序的 CDR-H2 和 H3 来模拟 CD4。

DOI：
10.1016/j.jmb.2005.12.062
发表时间：
2006
期刊：
Journal of molecular biology
影响因子：
5.6
作者：
Prabakaran,Ponraj;Gan,Jianhua;Wu,You-Qiang;Zhang,Mei-Yun;Dimitrov,DimiterS;Ji,Xinhua
通讯作者：
Ji,Xinhua

The mechanism of RNase III action: how dicer dices.

RNase III 作用机制：切丁机如何切丁。