Folate Metabolism in Mycobacterium tuberculosis Revisited: A Potential Drug Targe

描述(由申请人提供)：全球范围内出现的结核分枝杆菌(Mtb)多药耐药(MDR)和广泛耐药(XDR)菌株使当前的结核病(TB)流行严重复杂化。迫切需要新的结核病药物来抗击耐多药/广泛耐药结核病，并改进目前针对非耐药结核病的6个月用药方案。叶酸生物合成途径一直是抗生素开发的一个有吸引力的目标，因为它在人类中是不存在的。我们实验室使用耻垢分枝杆菌转座子插入文库进行的初步研究已经确定了分枝杆菌中几个新的抗叶酸抗性决定因素。一个对叶酸敏感的突变体编码真核型蛋白激酶G(PKng)的同源物，最近被发现可能是致病分支杆菌在巨噬细胞中持续存在的调节因子。初步研究表明，pKNG通过调节二氢蝶呤三磷酸焦磷酸酶的活性来调节新叶酸的生物合成，该酶控制蝶呤部分进入叶酸途径。这一新的调节机制以前还没有被发现用于从头合成叶酸。基因中断和pKNG激酶活性的特异性化学抑制都会导致分枝杆菌不仅对抗叶酸药物，而且对其他抗生素，包括一线结核病药物，如利福平和乙胺丁醇，都具有高度敏感性。这分别是由于对新叶酸生物合成的直接影响和通过改变细胞壁通透性而产生的间接影响。这一应用的中心假设是，定义内在抗叶酸抵抗的基因编码的蛋白质可以被增强剂靶向，通过抑制耐药机制使结核分枝杆菌对抗叶酸药物敏感。具体地说，pKng的药物灭活可能会使结核分枝杆菌对抗叶酸盐和目前具有耐药性的多种其他批准药物敏感。为了检验这一假说，我们设计了三个具体目标。首先，使用一种无偏见的方法，我们将识别和表征结核分枝杆菌全基因组范围的抗叶酸决定簇(抗叶酸抵抗组)。其次，我们将严格研究pKNG调控Mtb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联合作用对耐药和非耐药结核分枝杆菌的疗效。这些研究不仅将深入了解以前未知的细菌中从头合成叶酸的调节机制，而且还将深入了解结核分枝杆菌对抗叶酸药物的内在耐药机制。在药物开发方面，这些研究将揭示新的靶点，并提供概念证据，证明抑制结核分枝杆菌的内在耐药途径可以用来提高现有抗生素的有效性。) 公共卫生相关性：由于在人类中不存在，新的叶酸生物合成为开发新的抗生素提供了一个有吸引力的目标，有助于减少目前耐药细菌感染的流行，包括耐多药和广泛耐药结核病(MDR/XDR TB)。除了其他靶点外，我们的研究还发现，真核型蛋白激酶G(PKNG)是一种新的调节因子，通过调节一种酶的活性来控制结核分枝杆菌的新叶酸合成，该酶能将蝶呤部分转化为进入叶酸合成途径。这种对叶酸生物合成的调控是新颖的，可以针对增强抗叶酸药物的抗结核活性，从而为耐多药/广泛耐药结核的治疗提供一种新的方法；因此，我们的发现将与NIH的任务相关，并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.