Folate Metabolism in Mycobacterium tuberculosis Revisited: A Potential Drug Targe

描述（由申请人提供）：结核分枝杆菌（Mtb）的多药耐药（MDR）和广泛耐药（XDR）菌株在全球范围内的出现使当前的结核病（TB）流行严重复杂化。迫切需要新的结核病药物来对抗耐多药/广泛耐药结核病，并改善目前针对非耐药结核病的6个月药物治疗方案。叶酸生物合成途径一直是抗生素开发的有吸引力的目标，因为它在人类中不存在。本实验室利用转座子插入文库对M.斯美加已经鉴定了分枝杆菌中抗叶酸剂抗性的几种新的决定因素。一种抗叶酸剂敏感突变体编码真核细胞型蛋白激酶G（PknG）的同源物，最近被鉴定为可能的致病性分枝杆菌在巨噬细胞中的持久性调节剂。初步研究表明，PknG通过调节二氢新蝶呤三磷酸焦磷酸酶的活性来调节叶酸的从头生物合成，所述焦磷酸酶控制蝶呤部分流入叶酸途径。这种新的调节机制尚未被确定为从头叶酸的生物合成。PknG激酶活性的遗传中断和特异性化学抑制导致分枝杆菌不仅对抗叶酸药物而且对抗其他抗生素（包括一线TB药物如利福平和乙胺丁醇）的超敏感性。这分别是由于对从头叶酸生物合成的直接影响和通过改变细胞壁渗透性的间接影响。本申请的中心假设是，定义内在抗叶酸剂抗性的基因编码可被增效剂靶向的蛋白质，所述增效剂通过抑制抗性机制使Mtb对抗叶酸剂药物敏感。具体而言，PknG的药物失活可以使Mtb对抗叶酸剂和多种其他批准的药物敏感，目前它对这些药物具有抗性。设计了三个具体目标来检验这一假设。首先，使用非偏倚的方法，我们将确定和表征结核分枝杆菌的全基因组抗叶酸剂抗性决定簇（抗叶酸剂抗性组）。其次，我们将严格研究PknG调节Mtb中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效作用以及它们对抗耐药和非耐药Mtb的联合作用的功效。这些拟议的研究不仅将提供洞察到一个以前未知的调节机制从头叶酸在细菌中的生物合成，但也到Mtb的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点，并提供概念证明，即抑制结核分枝杆菌的内在耐药途径可用于提高现有抗生素的有效性。) 公共卫生相关性：由于叶酸在人体中的缺失，叶酸的从头生物合成为开发新型抗生素提供了一个有吸引力的靶点，有助于减少目前耐药细菌感染的流行，包括多重耐药和广泛耐药结核病（MDR/XDR TB）。除了其他目标，我们的研究确定了真核生物型蛋白激酶G（PknG）作为一种新的调节剂，控制从头叶酸在结核分枝杆菌的生物合成，结核病的病原体，通过调节酶的活性，转化蝶呤部分进入叶酸合成途径。叶酸生物合成的这种调控是新颖的，并且可以靶向增强抗叶酸药物的抗TB活性，从而提供治疗MDR/XDR TB的新方法;因此，我们的发现将与NIH的使命相关，并且将对正在开发新药以对抗细菌抗生素耐药性的工业和学术实体感兴趣。

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. ) PUBLIC HEALTH RELEVANCE: Because of its absence in humans, de novo folate biosynthesis provides an attractive target for development of novel antibiotics that help reduce the current epidemic of drug resistant bacterial infections, including the multidrug resistant and extensively drug resistant tuberculosis (MDR/XDR TB). Besides other targets, our research identified the eukaryotic-type protein kinase G (PknG) as a novel regulator that controls de novo folate biosynthesis in Mycobacterium tuberculosis, the causative agent of TB, by regulating activity of an enzyme that converts the pterin moiety for entry into the folate synthetic pathway. This regulatory control of folate biosynthesis is novel and could be targeted to potentiate anti-TB activity of antifolate drugs thus providing a new approach to the treatment for MDR/XDR TB; therefore our findings will be relevant to the mission of the NIH and will be of interest to both industrial and academic entities that are developing new drugs to combat bacterial antibiotic resistance.