Mechanism of anti-folate resistance in mycobacteria

分枝杆菌抗叶酸耐药机制

• 批准号: 10247084
• 负责人: Endang Purwantini
• 金额: $ 20万
• 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 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10247084
• 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; 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副毒品，该杂志将提高磺酰胺和 在结核病（TB）治疗的帕拉 - 氨基化酸（PAS），这是由细菌引起的 结核分枝杆菌（MTB）。多种药物抗药性（MDR）结核病的数量持续增加 案件要求开发新的结核病药物，这是一项艰巨的努力。可行的替代方案或平行 解决方案是提高FDA批准的TB-Strugs的有效性，而TB-drugs的吸引力降低了。 直到1950年代初，磺酰胺一直用作结核病药物，但由于其有效性和毒性不佳 早期形式，它们被停产用于治疗结核病。磺胺甲恶唑（SMX），磺酰胺 MTB于1961年批准用于治疗人类和动物的细菌感染。 PAS于1944年首次被用作结核病药物。但是，它的效率不如新药物，必须为 以高剂量给药。由于这些原因，PA不再是一线TB-grug 用于MDR TB的管道药物。因此，SMX和PA的有效性的提高将带来重大帮助 在打击结核病，尤其是疾病的抗药性形式时。拟议的项目将利用 我们制造磺酰胺和PA的发现在杀死MTB方面更有效。这两种化合物都是 抗粉状。一些叶酸合成酶激活这些化合物，进而抑制叶酸 生物合成系统；磺酰胺即使以未修饰的形式抑制。我们的初步结果表明MTB 使用F420-Gammagaglutamyl-ligase（FBIB），一种与叶酸生物合成无关的蛋白质来对抗这些作用 smx和pas;因此，抑制FBIB的共同药物将使SMX和PAS更有效地TB-Prugs。 该蛋白质由FBIB基因编码，催化辅酶的聚谷氨酸侧链的合成 F420。 F420和FBIB都在所有甲烷古细菌和某些细菌中都发现，包括所有分枝杆菌， 但在真主党很少发现，在人类中不存在。删除FBIB会导致霉菌杆菌 Smegmatis（MSMEG），MTB的亲戚，对SMX和PAS高度敏感，以及与MTBFBIB的补充 基因恢复了耐受高水平这些化合物的能力。基于初步分析，我们有 提出了两个假设：i）FBIB提供了两个不属于酶促活性的替代叶酸生物合成活性 激活PA，对磺酰胺，PA及其活化形式不敏感。 （ii）FBIB变换或 将这些药物降解为非抑制性化合物。在拟议的探索项目中，我们将测试这些 通过以下特定目的进行调查来假设。 1。在功能和结构上 表征MTBFBIB的两个叶酸生物合成酶活性以及磺酰胺，PAS，PAS， 以及他们在这些活动上激活的形式。 2。在功能和结构上表征FBIB的能力 转化或降解药物。最终的信息将为详细研究提供更清晰的假设 导致FBIB的抑制剂，这将使SMX和PAS更有效。