Folate Metabolism in Mycobacterium tuberculosis Revisited: A Potential Drug Targe

描述（由申请人提供）：世界范围内出现的多药耐药（MDR）和广泛耐药（XDR）结核分枝杆菌（Mtb）菌株严重复杂化了当前的结核病（TB）流行。迫切需要新的结核病药物来对抗耐多药/广泛耐药结核病，并改进目前针对非耐药结核病的6个月服药方案。叶酸生物合成途径已成为抗生素开发的一个有吸引力的目标，因为它在人类中不存在。在我们的实验室使用转座子插入文库的初步研究耻垢分枝杆菌已经确定了几个新的决定因素抗叶酸耐药分枝杆菌。一种抗叶酸敏感突变体编码真核型蛋白激酶G （PknG）的同源物，最近发现它可能是巨噬细胞中致病性分枝杆菌持续存在的调节剂。初步研究表明，pkg通过调节二氢蝶呤三磷酸焦磷酸酶的活性来调节叶酸的新生生物合成，而二氢蝶呤三磷酸焦磷酸酶可控制蝶呤片段流入叶酸途径。这种新的叶酸生物合成调控机制以前没有被发现过。基因中断和pknk激酶活性的特异性化学抑制导致分枝杆菌不仅对抗叶酸药物敏感，而且对其他抗生素敏感，包括利福平和乙胺丁醇等一线结核病药物。这是由于对叶酸生物合成的直接影响和通过改变细胞壁通透性的间接影响。本应用的中心假设是，定义内在抗叶酸药物抗性的基因编码的蛋白质可以被增强剂靶向，增强剂通过抑制耐药机制使结核分枝杆菌对抗叶酸药物敏感。具体来说，PknG的药物失活可能使结核分枝杆菌对抗叶酸药物和其他多种已批准的药物敏感，结核分枝杆菌目前对这些药物具有耐药性。为了验证这一假设，设计了三个具体目标。首先，使用无偏见的方法，我们将识别和表征结核分枝杆菌的全基因组抗叶酸抗性决定因子（抗叶酸抗性组）。其次，我们将严格研究结核分枝杆菌中pkg调控的叶酸生物合成的分子机制。最后，我们将描述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Mtb的联合效应。这些拟议的研究不仅将为以前未知的细菌中叶酸生物合成的调控机制提供见解，而且还将为结核分枝杆菌对抗叶酸药物的内在耐药机制提供见解。在药物开发方面，这些研究将揭示新的靶点，并证明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。

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.