Enzymology of N5-CAIR Synthetase

N5-CAIR 合成酶的酶学

基本信息

批准号：
8231437
负责人：
Steven M Firestine
金额：
$ 31.24万
依托单位：
WAYNE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-01 至 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8231437
关键词：
Active Sites Address Amino Acids Anabolism Animal Model Anthrax disease Anti-Bacterial Agents Antibiotic Resistance Antifungal Agents Area Bacterial Infections Binding Binding Sites Biochemical Biological Assay Bioterrorism Books Carbon Carbon Dioxide Carboxyamidotriazole Catalysis Cessation of life Chemistry Communicable Diseases Complex Computer Simulation Coupled Data Development Disease Progression Drug Design Drug Industry Drug resistance Enzymatic Biochemistry Enzyme Inhibitor Drugs Enzyme Inhibitors Enzymes Foundations Fungal Antibiotic Resistance Genes Genetic Goals Grant Health Health Care Costs Homologous Gene Hospitalization Human Incidence Infection Kinetics Laboratories Lead Ligase Measures Methods MgADP MgATP Microbe Modeling Mus Mutation Mycoses Nucleotides Organism Outcome Pathway interactions Pharmaceutical Preparations Physicians Positioning Attribute Public Health Purines Refractory Reporting Research Ribonucleotides Risk Role Sequence Analysis Serum Site-Directed Mutagenesis Source Structure Surgeon Techniques Virulence Factors Work antimicrobial antimicrobial drug base carbonic acid, monoanhydride with phosphoric acid, ion(2-)computer studies design drug discovery drug resistant bacteria enzyme mechanism experience fungus grasp high throughput screening inhibitor/antagonist innovation member microbial microbial disease microorganism mortality novel protein structure function purine small molecule tool

项目摘要

DESCRIPTION (provided by applicant):The increasing risk of drug resistant bacterial and fungal infections indicate that there is a growing need for new and effective antimicrobial agents. One promising, but unexplored area in antimicrobial drug design is de novo purine biosynthesis. Recent research has shown that de novo purine biosynthesis is different in microbes than in humans. The differences in the pathways are centered around the synthesis of 4-carboxyaminoimidazole ribonucleotide (CAIR) which requires the enzyme N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) synthetase. Humans do not require this enzyme, and sequence analysis reveals that humans have no homologs for this enzyme. Genetic studies have shown that microorganisms deficient in this enzyme are unable to grow in minimal media, human serum or mice. Furthermore, the gene encoding this enzyme has been identified as a virulence factor and mutations that inactivate the enzyme render microbes non-pathogenic. Despite this biochemical rationale for the development of new antimicrobial agents, little additional biochemical studies have been conducted on N5-CAIR synthetase since its initial discovery, and no drug-like small molecule inhibitors of the enzyme have been reported. To remedy this problem, we will conduct detailed biochemical, mechanistic, structural, computational and drug discovery studies on N5-CAIR synthetase. Our preliminary mechanistic, structural and computational studies have begun to elucidate the mechanism of the enzyme and high-throughput screening has resulted in the identification of drug-like small molecule inhibitors. To continue our studies and accomplish the overall objectives of this application, we will pursue the following three specific aims: 1. Site-directed mutagenesis and pH-rate studies of critical residues proposed to be important in binding and catalysis. 2. Elucidating the mechanism of N5-CAIR synthetase. 3. Understanding, designing, and synthesizing inhibitors of N5-CAIR synthetase. At the end of these studies we expect to have obtained detailed biochemical and mechanistic information on N5-CAIR synthetase and to have developed selective and potent inhibitors of the enzyme. PUBLIC HEALTH RELEVANCE: The incidence of drug-resistant bacterial and fungal infections has increased considerably over the last 20 years and now poses a major challenge for physicians. Invasive fungal and antibiotic-resistant bacterial infections are associated with high mortality rates, increased hospitalizations, and higher health care costs. Unfortunately, methods to treat fungal infections are limited, and treatment options for antibiotic- resistant bacterial infections, while better, are becoming compromised at an alarming rate. This, coupled with the reduction of antimicrobial research within the pharmaceutical industry, highlights the critical need for the continued development of new antimicrobial agents. This grant seeks to address this significant public health problem by studying an enzyme involved in de novo purine biosynthesis. At the conclusion of this study, we anticipate that we will have obtained critical biochemical information about this enzyme and will have identified potent and selective inhibitors of this enzyme. This information will be invaluable for the development of new antimicrobial agents that are capable of treating microbial diseases refractory to existing treatments.

描述（由申请人提供）：耐药细菌和真菌感染的风险不断增加，表明对新型有效抗菌剂的需求不断增长。抗菌药物设计中一个有前景但尚未探索的领域是从头嘌呤生物合成。最近的研究表明，微生物中的嘌呤生物合成与人类中的不同。途径的差异集中在 4-羧基氨基咪唑核糖核苷酸 (CAIR) 的合成上，其需要 N5-羧基氨基咪唑核糖核苷酸 (N5-CAIR) 合成酶。人类不需要这种酶，序列分析表明人类没有这种酶的同源物。遗传学研究表明，缺乏这种酶的微生物无法在基本培养基、人血清或小鼠中生长。此外，编码这种酶的基因已被鉴定为毒力因子，而使该酶失活的突变使微生物变得非致病性。尽管有开发新抗菌药物的生化原理，但自最初发现 N5-CAIR 合成酶以来，几乎没有对其进行额外的生化研究，并且尚未报道该酶的药物样小分子抑制剂。为了解决这个问题，我们将对 N5-CAIR 合成酶进行详细的生化、机理、结构、计算和药物发现研究。我们的初步机制、结构和计算研究已开始阐明酶的机制，高通量筛选已鉴定出类药小分子抑制剂。为了继续我们的研究并实现本申请的总体目标，我们将追求以下三个具体目标： 1. 对被认为对结合和催化很重要的关键残基进行定点诱变和 pH 速率研究。 2. 阐明N5-CAIR合成酶的作用机制。 3. N5-CAIR合成酶抑制剂的理解、设计和合成。在这些研究结束时，我们期望获得有关 N5-CAIR 合成酶的详细生化和机制信息，并开发出该酶的选择性和有效抑制剂。公共卫生相关性：过去 20 年来，耐药细菌和真菌感染的发生率大幅增加，现在对医生构成了重大挑战。侵袭性真菌和耐抗生素细菌感染与高死亡率、住院率增加和医疗保健费用增加有关。不幸的是，治疗真菌感染的方法是有限的，并且抗生素耐药性细菌感染的治疗选择虽然更好，但正在以惊人的速度受到损害。再加上制药行业抗菌研究的减少，凸显了持续开发新抗菌药物的迫切需要。这笔赠款旨在通过研究一种参与嘌呤从头生物合成的酶来解决这一重大的公共卫生问题。在这项研究结束时，我们预计我们将获得有关该酶的重要生化信息，并将鉴定出该酶的有效和选择性抑制剂。这些信息对于开发能够治疗现有治疗方法难治的微生物疾病的新型抗菌药物具有无价的价值。