Biochemistry and Modeling of Human ITPase Substrate Specificity Mutants

人 ITPase 底物特异性突变体的生物化学和建模

• 批准号: 8773454
• 负责人: Nicholas Earl Burgis
• 金额: $ 29.8万
• 依托单位: EASTERN WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8773454
• 关键词: Advanced Development; Adverse effects; Affect; Age; Amino Acids; Bacteria; Basic Science; Biochemical; Biochemistry; Biological Assay; Cardiac; Cardiac Function Study; Cardiomyopathies; Cell Cycle Progression; Cells; Chromosome abnormality; Collaborations; Complement; Complex; Data; Defect; Discrimination; Docking; Drug toxicity; Engineering; Enzyme Kinetics; Enzymes; Experimental Models; Future; Generations; Genes; Genetic Polymorphism; Genome; Genomic Instability; Goals; Guanosine Triphosphate; High Pressure Liquid Chromatography; Homeostasis; Human; Inosine; Kinetics; Link; Literature; Maintenance; Malignant Neoplasms; Measurement; Measures; Modeling; Molecular; Molecular Models; Mus; Nucleotides; Orthologous Gene; Outcome; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Population; Prodrugs; Protein Dynamics; Proteins; Protocols documentation; Publishing; Purines; Reaction; Research; Ribavirin; Roentgen Rays; Role; Sarcomeres; Site-Directed Mutagenesis; Specificity; Structure; Substrate Specificity; System; Testing; Toxic effect; Validation; Variant; Weaning; Work; Yeasts; base; drug metabolism; enzyme substrate complex; gene therapy; improved; inosine triphosphatase; interest; molecular dynamics; molecular modeling; mouse model; mutant; novel; nucleoside triphosphate; offspring; oxidative damage; programs; protein structure; public health relevance; purine; research study; simulation; small molecule; therapy development; thiopurine; tool; tripolyphosphate

DESCRIPTION (provided by applicant): Human ITPase (encoded by the ITPA gene) is thought to exclude noncanonical (deoxy)nucleoside triphosphates ((d)NTPs) from (d)NTP pools. Noncanonical purine NTPs arise spontaneously in cells from the purine salvage pathway and oxidative damage of canonical (d)NTPs. The critical role for ITPase orthologs in the sanitization of dNTP pools has been demonstrated in bacteria, yeast and murine systems where ITPase deficiencies result in sensitivity to noncanonical purines, a delay in cell cycle progression and chromosomal abnormalities including double-strand breaks. ITPase deficiency in mice is lethal due to cardiomyopathy which is thought to result from disorganization of sarcomere structure. This indicates that a defect in the maintenance of quality ATP pools leads to a phenotype that is consistent with mouse models of human cardiomyopathy. About 5% of the human population has been identified as having decreased ITPase activity, and ITPA status has been linked to altered outcomes for patients treated with either thiopurines or ribavirin. For patients undergoing thiopurine therapy, toxicity is associated with ITPA polymorphism, but for patients undergoing ribavirin treatment, ITPA polymorphism is associated with improved outcomes. Both of these drugs are prodrugs and are bioactivated to form NTPs. Thiopurine nucleoside triphosphates are substrates for ITPase, and it has been hypothesized that the nucleoside triphosphate form of ribavirin would be a substrate for ITPase. Despite the fact that ITPase has been studied for over 40 years, little is known about how ITPase discriminates between canonical and noncanonical (d)NTPs. This proposed research program aims to investigate the amino acid residues of human ITPase that are important for substrate specificity. We hypothesize that alteration of the amino acids implicated in substrate discrimination will result in an enzyme with altered specificity for noncanonical (d)NTPs. We have previously constructed 10 novel substrate specificity mutants. In this study we identified three mutants with enhanced selectivity for noncanonical (d)NTPs. We rationalize that our specific aims will provide a basis for developing small molecules and gene therapy treatments to modulate ITPase activity in an effort to reverse drug toxicity and provide tools to better understand cardiac function regarding noncanonical purines. The specific aims for this proposal include: (1) directly determine kinetic constants for canonical and noncanonical NTPs with select existing substrate specificity ITPase mutants, (2) perform molecular modeling of substrate specificity for ITPase mutants, and (3) engineer and test additional substrate specificity ITPase mutants based on existing and future data. Realization of our specific aims will provide a basis for developing small molecules and gene therapy treatments to modulate ITPase activity in an effort to reverse drug toxicity, provide tools to better understand cardiac function regarding noncanonical purines, and enhance our understanding of ITPase biochemistry.

描述（由申请方提供）：认为人ITPA（由ITPA基因编码）可从（d）NTP样本池中排除非规范（脱氧）核苷三磷酸（（d）NTP）。非典型嘌呤NTP在细胞中自发产生于典型（d）NTP的嘌呤补救途径和氧化损伤。在细菌、酵母和小鼠系统中，ITlR同源物在dNTP池消毒中的关键作用已得到证实，其中ITlR缺陷导致对非规范嘌呤的敏感性、细胞周期进展延迟和染色体异常（包括双链断裂）。由于心肌病，小鼠中的ITR缺乏是致命的，心肌病被认为是由肌节结构的紊乱引起的。这表明在维持高质量ATP池方面的缺陷导致与人类心肌病小鼠模型一致的表型。约5%的人群已被确定为ITPA活性降低，ITPA状态与接受硫嘌呤或利巴韦林治疗的患者的结局改变有关。患者的 在硫嘌呤治疗中，毒性与ITPA多态性相关，但对于接受利巴韦林治疗的患者，ITPA多态性与改善的结果相关。这两种药物都是前药，并且被生物活化以形成NTP。硫嘌呤核苷三磷酸是ITR的底物，并且已经假设利巴韦林的核苷三磷酸形式将是ITR的底物。尽管ITNTPs已经被研究了40多年，但人们对ITNTPs如何区分典型和非典型（d）NTPs知之甚少。 这项研究计划的目的是调查的氨基酸残基的人ITR是重要的底物特异性。我们假设，改变涉及底物歧视的氨基酸将导致酶对非典型（d）NTPs的特异性改变。我们以前已经构建了10个新的底物特异性突变体。在这项研究中，我们确定了三个突变体的非典型（d）NTPs的选择性增强。我们合理地解释说，我们的具体目标将为开发小分子和基因治疗提供基础，以调节ITR活性，努力逆转药物毒性，并提供工具，以更好地了解心脏功能的非经典嘌呤。 具体目标包括：（1）直接测定反应动力学常数， 典型和非典型NTP与选择的现有底物特异性ITB1突变体，（2）进行ITB1突变体的底物特异性的分子模拟，和（3）基于现有和未来的数据工程化和测试另外的底物特异性ITB1突变体。我们具体目标的实现将为开发小分子和基因治疗提供基础，以调节ITR活性，从而逆转药物毒性， 为了更好地了解心脏功能的非经典嘌呤，并提高我们的理解ITR生物化学。

项目成果

Structural dynamics of inosine triphosphate pyrophosphatase (ITPA) protein and two clinically relevant mutants: molecular dynamics simulations.

肌苷三磷酸焦磷酸酶 (ITPA) 蛋白和两个临床相关突变体的结构动力学：分子动力学模拟。

• DOI: 10.1080/07391102.2020.1727363
• 发表时间: 2021
• 期刊: Journal of biomolecular structure & dynamics
• 影响因子: 4.4
• 作者: Houndonougbo,Yao;Pugh,Bethany;VanWormer,Kandise;April,Caitlin;Burgis,Nicholas
• 通讯作者: Burgis,Nicholas

Arginine-178 is an essential residue for ITPA function.

Arginine-178 是 ITPA 功能的重要残基。

• DOI: 10.1016/j.abb.2023.109700
• 发表时间: 2023
• 期刊: Archives of biochemistry and biophysics
• 影响因子: 3.9
• 作者: Burgis,NicholasE;April,Caitlin;VanWormer,Kandise
• 通讯作者: VanWormer,Kandise