Mechanism of anti-folate resistance in mycobacteria

该项目的长期目标是开发一种联合药物，以提高磺胺类药物和 对氨基水杨酸(PAS)治疗由细菌引起的结核病 结核分枝杆菌(Mtb)耐多药结核病(MDR)数量持续增加 病例需要开发新的结核病药物，这是一项具有挑战性的努力。可行的替代方案或平行方案 解决办法是提高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了。 直到20世纪50年代初，磺胺类药物一直被用作结核病药物，但由于其有效性和毒性较差， 在早期形式中，它们因治疗结核病而停止使用。磺胺甲恶唑(SMX)，一种磺胺类 1961年被批准用于治疗人类和动物的细菌感染，结核分枝杆菌也对其耐受性良好。 PAS于1944年首次被用作结核病药物。然而，它的效果不如新药，并被要求 大剂量地给药。出于这些原因，PAS不再是治疗结核病的一线药物，而是第二种药物-- 用于耐多药结核病的一线药物。因此，提高工作人员-管理层和考绩制度的效力将带来重大帮助 在防治结核病方面，特别是在抗药性方面。拟议的项目将利用其中一个 我们的发现使磺胺类药物和苯丙胺类抗生素在杀死结核杆菌方面更有效。这两种化合物都是 抗叶酸。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；磺胺类药物即使在未经修饰的形式下也会产生抑制作用。我们的初步结果表明结核分枝杆菌 使用与叶酸生物合成无关的蛋白质F420-γ-谷氨酰基连接酶(FbiB)来对抗这些作用 因此，抑制FbiB的联合药物将使SMX和PAS更有效。 该蛋白由fbiB基因编码，催化辅酶多聚谷氨酸侧链的合成。 F420。F420和FbiB在所有产甲烷古菌和包括所有分枝杆菌在内的某些细菌中都存在， 但在真核生物中很少发现，在人类中也不存在。FbiB的缺失使分枝杆菌 污垢杆菌(Msmeg)是Mtb的近亲，对SMX和PAS高度敏感，是MtbfbiB的补充 基因恢复了对高水平这些化合物的耐受能力。根据初步分析，我们有 提出了两个假设：i)FbiB提供了两种交替的叶酸生物合成酶活性，而不是 活化PAs，对磺胺类、PAs及其活化形式不敏感。(Ii)FbiB转换或 将这些药物降解成非抑制性化合物。在拟议的勘探项目中，我们将测试这些 通过调查得出的假设有以下具体目的。1.从功能和结构上 MtbFbiB的两种叶酸生物合成酶活性及磺胺类、PAS、MtbFbiB的影响 以及它们在这些活动上的激活形式。2.从功能和结构上表征FbiB的能力 转化或降解药物。由此得到的信息将为详细研究提供更清晰的假设 导致了一种FbiB的抑制剂，将使SMX和PAS更有效地治疗结核病。

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.