Mechanism of anti-folate resistance in mycobacteria

分枝杆菌抗叶酸耐药机制

• 批准号: 10057777
• 负责人: Endang Purwantini
• 金额: $ 23.87万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10057777
• 关键词: Anabolism; Animals; Archaea; Bacteria; Bacterial Infections; Binding; C-terminal; Carbon; Cause of Death; Cell Wall; Cells; Chlamydia; Coenzymes; Complement; Data; Development; Dihydrofolate Reductase; Dihydropteroate Synthase; Disease; Dose; Drug resistance; Drug resistance in tuberculosis; Effectiveness; Enzyme Inhibitor Drugs; Enzymes; Escherichia coli; Ethambutol; FDA approved; Flavin Mononucleotide; Folate Biosynthesis Pathway; Folic Acid; Folic Acid Antagonists; Folylpolyglutamate synthase; Future; Generations; Genes; Genus Mycobacterium; Glutamates; Goals; Homologous Gene; Human; Hypersensitivity; In Vitro; Investigation; Laboratories; Ligase; Link; Modeling; Multi-Drug Resistance; Mycobacterium smegmatis; Mycobacterium tuberculosis; N-terminal; Organism; Oxidation-Reduction; Oxidoreductase; Pharmaceutical Preparations; Proteins; Pterins; Publishing; Reaction; Reporting; Research; Resistance; Rifampin; Side; Structure; Sulfamethoxazole; Sulfonamides; System; Testing; Tetrahydrofolates; Time; Toxic effect; Tuberculosis; adduct; base; combat; dihydrofolate; experimental study; folic acid metabolism; inhibitor/antagonist; inorganic phosphate; multiple drug use; mutant; mycobacterial; nitrosative stress; novel therapeutics; p-Aminosalicylic Acid; polyglutamate; success; tuberculosis drugs; tuberculosis treatment

The long-term goal of this project is to develop a co-drug that will increase the effectiveness of sulfonamides and para-aminosalicylic acid (PAS) in the treatment of tuberculosis (TB), which is caused by the bacterium Mycobacterium tuberculosis (Mtb). A continued increase in the number of multiple drug resistant (MDR) TB cases calls for the development of new TB drugs, which is a challenging endeavor. A viable alternative or parallel solution is to increase the effectiveness of FDA-approved TB-drugs that have become less attractive. Sulfonamides were used as TB drugs until the early 1950s, but, due to poor effectiveness and toxicity of their early forms, they were discontinued for the treatment of TB. Sulfamethoxazole (SMX), a sulfonamide that was approved in 1961 for the treatment of bacterial infections in humans and animals, is also well-tolerated by Mtb. PAS was first used as a TB drug in 1944. However, it is less effective than newer drugs and is required to be administered in a high dose. For these reasons PAS is no longer a first-line TB-drug but, instead, is a second- line drug used for MDR TB. Thus, an improvement in the effectiveness of SMX and PAS would bring major help in combating TB, especially the drug resistant forms of the disease. The proposed project will leverage one of our discoveries for making sulfonamides and PAS more effective in killing Mtb. Both of these compounds are anti-folates. Some of the folate synthesis enzymes activate these compounds, which in turn inhibit the folate biosynthesis system; sulfonamides inhibit even in their unmodified form. Our preliminary results suggest that Mtb use F420-gammaglutamyl-ligase (FbiB), a protein that is unrelated to folate biosynthesis, to counter the actions of SMX and PAS; consequently, a co-drug that inhibits FbiB will make SMX and PAS more effective TB-drugs. This protein, encoded by the fbiB gene, catalyzes the synthesis of the polyglutamate side chain of coenzyme F420. Both F420 and FbiB are found in all methanogenic archaea and certain bacteria including all mycobacteria, but are rarely found in eukarya and are absent in humans. A deletion of fbiB makes Mycolicibacterium smegmatis (Msmeg), a relative of Mtb, hypersensitive to SMX and PAS, and a complementation with the MtbfbiB gene restores the ability to tolerate high levels of these compounds. Based on preliminary analysis, we have developed two hypotheses: i) FbiB provides two alternate folate biosynthesizing enzymatic activities that do not activate PAS and are not sensitive to sulfonamides, PAS, and their activated forms. (ii) FbiB transforms or degrades these drugs into non-inhibitory compounds. In the proposed exploratory project, we will test these hypotheses through an investigation with the following specific aims. 1. To functionally and structurally characterize two folate biosynthesizing enzymatic activities of MtbFbiB and the effects of sulfonamides, PAS, and their activated forms on these activities. 2. To characterize, both functionally and structurally, FbiB's ability to transform or degrade the drugs. The resulting information will provide clearer hypotheses for detailed studies leading to an inhibitor of FbiB that will make SMX and PAS more effective TB-drugs.

该项目的长期目标是开发一种联合药物，将增加磺胺类药物的有效性， 对氨基水杨酸（PAS）在治疗结核病（TB），这是由细菌引起的 结核分枝杆菌（Mtb）。耐多药结核病的数量持续增加 新的结核病病例要求开发新的结核病药物，这是一项具有挑战性的奋进。可行的替代或平行 解决方案是增加FDA批准的结核病药物的有效性，这些药物已经变得不那么有吸引力。 磺胺类药物一直被用作结核病药物，直到20世纪50年代初，但由于其效果差和毒性， 早期的形式，他们停止了结核病的治疗。磺胺甲恶唑（SMX），一种磺胺， 1961年批准用于治疗人类和动物的细菌感染，也被Mtb良好耐受。 PAS于1944年首次用作结核病药物。然而，它比新的药物效果差，需要 以高剂量给药。由于这些原因，PAS不再是一线结核病药物，而是第二种- 用于耐多药结核病的一线药物。因此，提高工作人员-管理层交流和考绩制度的效力将带来重大帮助 在抗击结核病，特别是耐药性结核病方面。拟议的项目将利用以下其中一项 我们的发现使磺胺类药物和PAS更有效地杀死Mtb。这两种化合物都是 抗叶酸剂一些叶酸合成酶激活这些化合物，从而抑制叶酸 磺胺类药物甚至以其未修饰的形式也抑制生物合成系统。我们的初步结果表明，结核分枝杆菌 使用F420-γ-谷氨酰连接酶（FbiB），一种与叶酸生物合成无关的蛋白质， 因此，抑制FbiB的联合药物将使SMX和PAS更有效的TB药物。 这种由fbiB基因编码的蛋白质，催化辅酶A的聚谷氨酸侧链的合成。 F420 F420和FbiB都存在于所有产甲烷古菌和某些细菌中，包括所有分枝杆菌， 但在真核生物中很少发现，在人类中也不存在。fbiB的缺失使得分枝杆菌属 Mtb的近亲，对SMX和PAS超敏感，与MtbfbiB互补 基因恢复了耐受高水平这些化合物的能力。根据初步分析， 我提出了两个假设：i）FbiB提供了两种替代的叶酸生物合成酶活性， 激活PAS，对磺胺类、PAS及其激活形式不敏感。(ii)FbiB变换或 将这些药物降解成非抑制性化合物。在拟议的探索性项目中，我们将测试这些 通过具有以下具体目标的调查进行假设。1.从功能上和结构上 表征MtbFbiB的两种叶酸生物合成酶活性以及磺胺类、PAS、 以及它们的激活形式。2.为了从功能和结构上描述FbiB的能力， 转化或降解药物。由此产生的信息将为详细的研究提供更清晰的假设 导致FbiB的抑制剂，其将使SMX和PAS更有效的TB药物。