Folate Metabolism in Mycobacterium tuberculosis Revisited: A Potential Drug Targe

重新审视结核分枝杆菌中的叶酸代谢：潜在的药物目标

• 批准号: 8445317
• 负责人: Liem Duy Nguyen
• 金额: $ 36.53万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-15 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8445317
• 关键词: Antibiotic Resistance; Antibiotics; Antimycobacterial Agents; Antitubercular Agents; Bacteria; Bacterial Antibiotic Resistance; Bacterial Infections; Cell Wall; Chemicals; Clinical; Complement; Cyclic GMP-Dependent Protein Kinases; Development; Drug Resistant Tuberculosis; Drug resistance; Drug resistance in tuberculosis; Drug-sensitive; Effectiveness; Enzymes; Epidemic; Ethambutol; Extreme drug resistant tuberculosis; Folate; Folate Biosynthesis Pathway; Folic Acid Antagonists; Genes; Genetic; Genus Mycobacterium; Homologous Gene; Human; In Vitro; Interruption; Knowledge; Laboratories; Libraries; Metabolism; Mission; Molecular; Multi-Drug Resistance; Mycobacterium smegmatis; Mycobacterium tuberculosis; Natural Resistance; Pathway interactions; Permeability; Pharmaceutical Preparations; Pharmacologic Substance; Predisposition; Proteins; Pterins; Regimen; Regulation; Research; Resistance; Rifampin; Role; Testing; Tuberculosis; United States National Institutes of Health; cGMP-dependent protein kinase Ibeta; combat; combinatorial; design; drug development; drug efficacy; efficacy testing; enzyme activity; folic acid metabolism; genome-wide; improved; inhibitor/antagonist; insight; interest; isoniazid; macrophage; mutant; novel; novel strategies; novel therapeutic intervention; p-Aminosalicylic Acid; pre-clinical; public health relevance; pyrophosphatase; resistance mechanism; screening; tripolyphosphate; tuberculosis drugs

DESCRIPTION (provided by applicant): The worldwide emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) is severely complicating the current tuberculosis (TB) epidemic. New TB drugs are urgently needed to combat MDR/XDR TB and to improve the current 6-month drug regimens for non-resistant TB. The folate biosynthetic pathway has been an attractive target for antibiotic development since it is absent in humans. A preliminary study in our laboratory using a transposon insertion library in M. smegmatis has identified several novel determinants of antifolate resistance in mycobacteria. One antifolate sensitive mutant encodes a homolog of the eukaryotic-type protein kinase G (PknG), recently identified as possible regulator of persistence of pathogenic mycobacteria in macrophages. Preliminary studies reveal that PknG regulates de novo folate biosynthesis by modulating the activity of a dihydroneopterin triphosphate pyrophosphatase that controls the influx of pterin moiety into the folate pathway. This novel regulatory mechanism has not been previously identified for de novo folate biosynthesis. Both genetic interruption and specific chemical inhibition of PknG kinase activity result in hyper-susceptibility of mycobacteria not only to antifolate drugs but also other antibiotics, including frontline TB drugs such as rifampicin and ethambutol. This is due to a direct effect on de novo folate biosynthesis and an indirect effect by altering cell wall permeability, respectively. The central hypothesis of this application is that genes defining intrinsic antifolate resistance encode proteins that can be targeted by potentiators that sensitize Mtb to antifolate drugs by inhibiting the resistance mechanisms. Specifically, pharmaceutical inactivation of PknG could sensitize Mtb to antifolates and multiple other approved drugs, to which it is currently resistant. Three specific aims are designed to test this hypothesis. First, using a non-biased approach, we will identify and characterize the entire genome-wide antifolate resistant determinants (the antifolate resistome) of Mtb. Secondly, we will rigorously investigate the molecular mechanisms of PknG-regulated folate-biosynthesis in Mtb. Lastly, we will characterize the potentiating effects of PknG inhibitors on antifolate drugs and the efficacy of their combined effect against drug-resistant and non-resistant Mtb. These proposed studies will not only provide insight into a previously unknown regulatory mechanism of de novo folate biosynthesis in bacteria but also into the mechanisms of intrinsic resistance of Mtb to antifolate drugs. In terms of drug development, these studies will reveal novel targets and provide proof of concept that inhibition of intrinsic resistance pathways in Mtb can be used to improve the effectiveness of already available antibiotics. )

描述（由申请人提供）：全球抗药性（MDR）和广泛的耐药性（XDR）菌株的结核分枝杆菌（MTB）的出现严重使当前的结核病（TB）菌株变得严重复杂化。迫切需要新的结核病药物来对抗MDR/XDR TB并改善当前的6个月药物方案，用于无抗性结核病。叶酸生物合成途径一直是抗生素发育的有吸引力的靶标，因为它在人类中不存在。使用Smegmatis的转座子插入文库在我们的实验室进行的初步研究已经确定了分枝杆菌中抗叶酸耐药性的几种新型决定因素。一个抗叶酸敏感的突变体编码真核型蛋白激酶G（PKNG）的同源物，最近被确定为巨噬细胞致病性分枝杆菌持久性的可能调节剂。初步研究表明，PKNG通过调节三磷酸三磷酸二磷酸二磷酸二磷酸二磷酸酶的活性来调节从头叶酸的生物合成，从而控制翼龙部分的涌入叶酸途径。以前尚未确定这种新型调节机制用于从头叶酸生物合成。遗传中断和对PKNG激酶活性的特异性化学抑制作用都导致分枝杆菌对抗生物药物的过度敏感性不仅对抗叶酸药物，而且还导致其他抗生素，包括前线TB药物，例如利福平和乙糖醇。这是由于对从头叶酸的生物合成的直接影响和分别通过改变细胞壁渗透性而产生的间接作用。该应用的中心假设是，定义固有性抗卵酸耐药性的基因编码蛋白质，这些蛋白可以由增强剂靶向，这些蛋白可以通过抑制耐药机制来敏感MTB对抗叶酸药物的敏感性。具体而言，PKNG的药物灭活可能会使MTB对抗染料和其他批准的药物敏感，目前具有抗性。三个特定的目标旨在检验这一假设。首先，使用非偏见的方法，我们将识别并表征MTB的整个全基因组抗抗基质决定簇（抗叶酸抗性）。其次，我们将严格研究MTB中PKNG调节的叶酸生物合成的分子机制。最后，我们将表征PKNG抑制剂对抗叶酸药物的增强作用，以及它们对耐药和非耐药性MTB的综合作用的功效。这些提出的研究不仅将洞悉以前未知的细菌中从头叶酸生物合成的调节机制，而且还可以介绍MTB对抗叶酸药物的内在耐药性的机制。在药物开发方面，这些研究将揭示新的靶标，并提供概念证明，即MTB中内在抗性途径的抑制可用于提高已经可用的抗生素的有效性。 ）