抗生素筛选方法

细菌耐药性发生率的升高,激励着人们去寻找更多的筛选抗生素的策略,进而发现了更多的抗生素作用机制.靶向策略、高通量筛选等方法已经被用于检测有潜力的抗生素.细菌的DNA复制、细胞分裂和蛋白合成的中间步骤已经成为了筛选的新靶点,双组分信号传递系统也成为了较为重要的药物新筛选靶点.微生物基因组学的发展,给新抗生素的发现带来了更大的希望,使人们可以发现更多的新作用靶点.     

近年抗生素研究进展

细菌耐药性问题的日益严重和临床应对手段的有限已引起全社会的极大关注,故研发能有效对付耐药菌感染的新抗生素迫在眉睫。近年来,科学家通过对现有抗生素(如氟喹诺酮、四环素、氨基糖苷和β-内酰胺等)的结构改造以及新靶点(如肽去甲酰酶)或代谢途径(如脂肪酸生物合成)的识别及其抑制剂的筛选,已发现若干具有开发前景的新化合物。本文按作用机制的不同对目前处于临床前后期及临床阶段的抗生素候选物的研究进展进行了较系统的综述。     

抗菌药物研发进展

随着抗菌剂的广泛使用甚至滥用，细菌耐药性不断增加，已成为目前临床上最棘手的问题之一，因此尽快研发能够有效对付耐药菌感染的新型抗菌药物迫在眉睫。在简述抗生素发展史和分析当前研发新型抗菌药物紧迫性的基础上，本文主要从传统抗菌药物（如β-内酰胺类、喹诺酮类、四环素类、大环内酯类等）的结构修饰物和新作用靶点（如HFab1、蛋氨酰-tRNA合成酶、MurI、RFaE、WaaC等）抑制剂2个方面比较全面地综述了近年来抗菌药物的最新研发进展，并进一步指出寻找新作用靶点抑制剂和新作用机制药物可能是解决细菌耐药性的潜在途径。     

基于核糖体结构和功能的新型抗生素研究进展

本文介绍了以核糖体为靶点的几种重要抗生素的作用机制,在此基础上对近几年来出现的此类药物研究的新方法新思路进行了综述。远程交互作用网络及诱导-契合机制的发现、抗生素抑制蛋白质合成的新靶点的发掘、利用计算机辅助药物设计技术及其他物理化学分析方法如分子建模技术、计算机动态模拟技术、分子对接技术、X-ray衍射技术进行新型抗生素研究,使我们对于药物与核糖体的结合方式及结合位点有了更加深入的了解,为科研人员对新型抗生素的开发和研究带来了无限的希望。     

抗生素开发的新靶点:核糖开关

核糖开关是位于mRNA区域的特定片段,被小分子配体激活后即可顺式调控基因表达,无需蛋白参与。经进化选择,大部分细菌内核糖开关采用必需代谢产物为配体,负责调控与配体生物合成或转运相关基因的表达。代谢产物类似物可以抑制核糖开关介导的基因表达,抑制细菌生长,甚至引起细菌死亡,因此核糖开关可以作为抗生素开发的新靶点。已经发现的几种先导化合物显示了以核糖开关为靶点的开发潜力。本文简要阐述了可以作为抗生素靶点的核糖开关及其作用机制,介绍了以核糖开关为靶点的新型抗生素高通量筛选及药物设计方法,并对可能遇到的困难进行了分析。     

非编码sRNA在细菌耐药机制方面的研究进展

近年来的研究发现,细菌应答抗生素压力时会产生特异的非编码小RNA(small RNA, sRNA)谱,进而可能调控下游基因的表达,帮助细菌克服抗生素压力。sRNA以各种方式调控细菌耐药相关基因(如抗生素转运蛋白、药物外排泵、细胞被膜的合成与修饰),参与细菌耐药网络。因此,sRNA及其相关因子(如Hfq)可能被用作抗菌治疗的靶标。本文将从sRNA应答抗生素压力并产生抗生素耐药及其作为药物靶点的前景等方面,综述sRNA在细菌耐药调控方面的研究进展。     

新抗菌靶点肽去甲酰基酶抑制剂的研究进展

综述新抗菌靶点肽去甲酰基酶在细菌蛋白合成中的作用、三维结构及其抑制剂的设计、发现与开发和耐药菌的产生。随着细菌耐药性的广泛发生以及多药耐药菌的出现,临床上急需对耐药菌有效的新型抗菌药,而寻找新型抗菌靶点为抗耐药菌药物的研究提供了新思路。     

抗生素耐药性现状透视

抗生素耐药性日益受到人们的重视,抗生素耐药机理包括酶对抗生素的修饰和破坏,减少抗生素向细菌内的摄入、增加抗生素的主动排出作用,新靶位的产生及药物作用靶位的过度表达。本文指出了目前临床存在的问题,并提出了解决方法,如减少抗生素的使用,开发新的抗生素,或开发治疗感染的新策略。     

以转糖基酶为作用靶标的新型抗生素的研究进展

由于抗生素的滥用,细菌耐药性问题愈发严重,研发抗菌新药迫在眉睫。细菌细胞壁是细菌维持正常生命活动所必需的结构,而肽聚糖是细菌细胞壁必不可少的组成部分,它的合成过程涉及到很多种酶,特别是转糖基酶和转肽酶,其在生物合成中起到非常重要的作用,可作为抗菌药物的有效靶点。以转肽酶为靶点的新药研发比较广泛,而针对转糖基酶的抑制剂研究较少。随着酶活性分析方法以及计算机辅助药物设计的发展,转糖基酶抑制剂的研发取得了重大进展。本文对近年来以转糖基酶为靶点的抗生素的研究进行综述。     

抗生素耐药性的主动外排机制

细菌对抗生素产生耐药性的机制多种多样，常见的有：细菌产生酶对抗生素的修饰与灭活；药物作用靶点发生改变；膜通透性下降；膜产生主动外排作用；生物被膜的形成。膜的主动外排机制是由各种外排蛋白系统介导的把抗生素等药物从细菌细胞内泵出的主动排出过程，外排系统具有能量依赖性、底物广泛性、系统多样性及功能复杂性的特点；与常用抗生素、合成抗菌药、染料、去污剂及金属离子的多重耐药性密切相关，尤其对四环素、氯霉素及氟喹诺酮类药物等广谱抗菌药的作用更为明显；其生理功能与正常物质转运和代谢有关。以利血平为代表的外排泵抑制剂能有效地对抗主动外排作用的产生，此类抑制剂将是防制细菌多重耐药性的有效措施之一。     

Bacterial targets to antimicrobial leads and development candidates

The unabated spread of drug-resistant bacterial pathogens continues to pose a significant threat to patient health. However, there are now several new and improved derivatives from established classes of antibiotics that have recently been approved or are in late-stage development. In drug discovery, screening of new targets derived from genomic-based discovery stratagems has resulted in a number of potent antibacterial leads. Further optimization and development of these promising compounds offer the opportunity to develop new classes of antimicrobials that act through modes of action that are unlikely to be affected by resistance mechanisms defined to date. In addition, new discoveries in biochemical mechanisms of antibiotic action continue to help identify new approaches to design novel antibiotic analogs.     

The role of genomics in the discovery of novel targets for antibiotic therapy

The emergence of antibiotic resistance and multi-drug resistance in bacterial pathogens underscores the need for the development of novel classes of antibiotics. The availability of complete genome sequence data from many important human pathogens provides a wealth of fundamental information. This allows us to define each gene and thus to better understand molecular pathogenesis. New techniques have enabled the identification and characterization of genes that are critical for bacterial growth and survival during infection. The combination of genome sequence data and new technologies make it possible to systematically explore the function of each open reading frame in a genome and identify any potential molecular targets for drug discovery. With particular emphasis on antibacterial therapy, this review discusses genome-based technologies and their important applications to anti-infective drug discovery.     

Practical applications and feasibility of efflux pump inhibitors in the clinic--a vision for applied use

The world of antibiotic drug discovery and development is driven by the necessity to overcome antibiotic resistance in common Gram-positive and Gram-negative pathogens. However, the lack of Gram-negative activity among both recently approved antibiotics and compounds in the developmental pipeline is a general trend despite the fact that the plethora of covered drug targets are well-conserved across the bacterial kingdom. Such intrinsic resistance in Gram-negative bacteria is largely attributed to the activity of multidrug resistance (MDR) efflux pumps. Moreover, these pumps also play a significant role in acquired clinical resistance. Together, these considerations make efflux pumps attractive targets for inhibition in that the resultant efflux pump inhibitor (EPI)/antibiotic combination drug should exhibit increased potency, enhanced spectrum of activity and reduced propensity for acquired resistance. To date, at least one class of broad-spectrum EPI has been extensively characterized. While these efforts indicated a significant potential for developing small molecule inhibitors against efflux pumps, they did not result in a clinically useful compound. Stemming from the continued clinical pressure for novel approaches to combat drug resistant bacterial infections, second-generation programs have been initiated and show early promise to significantly improve the clinical usefulness of currently available and future antibiotics against otherwise recalcitrant Gram-negative infections. It is also apparent that some changes in regulatory decision-making regarding resistance would be very helpful in order to facilitate approval of agents aiming to reverse resistance and prevent its further development.     

Novel targets for antibiotics

The rapid and extensive emergence of antibiotic resistant bacteria has resulted in a clear cut need to discover new antibiotics. Because of the many years of extensive screening, it is likely that most of the easy discoveries have been made and, therefore, new targets for antibiotics and new screening strategies for their discovery need to be developed. The approaches described in this overview are divided into several categories that are associated with different probabilities for a successful discovery. Approaches that are more likely to be successful include a continuation of classical discovery tactics including the chemical modification of extant structures, the use of new screens for classical targets (for example, the use of the enzyme DNA gyrase to discover new 4-fluoroquinolones), and the development of novel methods of drug delivery. These approaches, however, are likely to yield small incremental advances. More novel approaches should yield radically new chemical structures, however, the likelihood for a successful discovery will be lower than the classical approaches. The novel approaches include rational drug design, the discovery of new essential targets for antibiotics and using them for the purpose of drug screening, and the intervention in pathways necessary for pathogenesis. A middle of the road approach is to discover new agents that interfere with mechanisms of antibiotic resistance. Implicit in this overview is the need to develop new methods that result in real technologic advances. This may require a complete re-thinking of how antibiotics are discovered including the restricted use of live microbe killing assays as a primary screening tool.     

Novel antibacterials: a genomics approach to drug discovery

The appearance of antibiotic resistant pathogens, including vancomycin resistant Staphylococcus aureus, in the clinic has necessitated the development of new antibiotics. The golden age of antibiotic discovery, in which potent selective compounds were readily extracted from natural product extracts is over and novel approaches need to be implemented to cover the therapeutic shortfall. The generation of huge quantities of bacterial sequence data has allowed the identification of all the possible targets for therapeutic intervention and allowed the development of screens to identify inhibitors. Here, we described a number of target classes in which genomics has contributed to its identification. As a result of analyzing sequence data, all of the tRNA synthetases and all of the two-component signal transduction systems were readily isolated; which would not have been easily identified if whole genome sequences were not available. Fatty acid biosynthesis is a known antibacterial target, but genomics showed which genes in that pathway had the appropriate spectrum to be considered as therapeutic targets. Genes of unknown function may seem untractable targets, but if those that are broad spectrum and essential are identified, it becomes valuable to invest time and effort to determine their cellular role. In addition, we discuss the role of genomics in developing technologies that assist in the discovery of new antibiotics including microarray gridding technology. Genomics can also increase the chemical diversity against which the novel targets can be screened.     

Riboflavin Biosynthetic and Regulatory Factors as Potential Novel Anti-Infective Drug Targets

Riboflavin (vitamin B₂) is the direct precursor of redox enzyme cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are essential for multiple cell physiology. The riboflavin biosynthetic pathway is regarded as a rich resource for therapeutic targets for broad spectrum antibiotics. Enzymatic pathways, regulatory factors of the riboflavin biosynthesis, and relevant drug discovery are summarized in this review. The novel riboswitch regulatory mechanism of riboflavin metabolism is also described. A compendium of chemical modulators of riboflavin biosynthesis and regulatory networks is listed and such demonstrates the promise of riboflavin biosynthesis and regulatory mechanisms as potential therapeutic targets for novel antibiotic drug discovery.     

Using microarray gene signatures to elucidate mechanisms of antibiotic action and resistance

Microarray analyses reveal global changes in gene expression in response to environmental changes and, thus, are well suited to providing a detailed picture of bacterial responses to antibiotic treatment. These responses are represented by patterns of gene expression, termed expression signatures, which provide insight into the mechanism of action of antibiotics as well as the general physiological responses of bacteria to antibiotic-related stresses. The complexity of such signatures is challenging the notion that antibiotics act on single targets and this is consistent with the concept that there are multiple targets coupled with common stress responses. A more detailed knowledge of how known antibiotics act should reveal new strategies for antimicrobial drug discovery.     

Targeting virulence for antibacterial chemotherapy: identifying and characterising virulence factors for lead discovery

The antibacterial drug discovery industry is fast losing participants; at the same time it is facing the challenge of developing new antibiotics that are effective against frequently occurring and multiply resistant organisms. One intriguing approach is to target bacterial virulence, and the last decade or so has seen a focus on bacterial pathogenesis along with the development of reagents and strategies that could make this possible. Several processes utilised by a range of bacteria to cause infection may be conserved enough to make attractive targets; indeed it is known that mammalian cells can affect bacterial gene expression and vice versa. Interesting targets involving virulence include type III secretion systems, two-component signal transduction systems, quorum sensing, and biofilm formation. In order to better understand these systems and strategies, investigators have developed novel strategies of their own, involving negative selections, surrogate models of infection, and screens for gene induction and antigenicity. Inhibitors of such targets would be unlikely to adversely affect patients, be cross-resistant to existing therapies, or cause resistance themselves. It might be the case that virulence target-based therapies would not be powerful enough to clear an existing infection alone, but if they are instead considered as adjunct therapy to existing antibiotics, or potentiators of the host immune response, they may show efficacy in a non-traditional way.     

Highly sensitive target-based whole-cell antibacterial discovery strategy by antisense RNA silencing

Examples of drug-resistant bacteria are increasing while the discovery of new antibiotics with new mechanisms of action has been essentially nonexistent. The antisense-based sensitization of bacterial targets in Staphylococcus aureus is one of the new approaches that provides increased sensitivity for the detection of target-specific antibiotics and whole-cell screening assays based on differential sensitivity of target-depleted strains. The screening of natural product extracts using this type of assay designed for condensing enzyme (FabH/FabF) targets of the fatty acid biosynthesis pathway led to the discovery of a number of target-specific inhibitors including the novel antibiotic platensimycin, which has displayed activity against various drug-resistant bacteria. The antisense-based discovery strategy, rationale and design of screening assays, and the application of such assays for screening of natural product extracts and the discovery of fatty acid condensing enzyme inhibitors are reviewed in this article.     

细菌整体代谢与新抗生素及抗生素增效剂靶标

摘要：抗菌药物的滥用加速了细菌耐药的产生与传播,每年因耐药细菌导致的人口死亡和医疗成本耗费都极为惊人。针对 耐药细菌的新型抗生素研制十分缓慢,自1987年以来没有一类新型抗生素上市。研究抗生素杀菌机制以开发新药或抗生素佐剂 是一种应对耐药细菌的良好策略。目前普遍认同活性氧(ROS)介导细胞死亡是抗生素杀菌的共享途径,抗生素作用于靶标介导 产生原发初级损伤,诱使ROS生成,ROS造成次级细胞损伤并刺激更多ROS生成,形成一种恶性循环,最终ROS累积超过细胞 氧化应激极限、致使细菌死亡。细菌胞内具有用以消除氧化应激压力的专能系统,针对性抑制这些系统可能是快速杀伤细菌的 一个好策略。然而,设计靶向细菌某些代谢节点的新型抗生素或相关佐剂很可能更为简单、有效,生物体代谢是一个巨大的相 互协作网络,关键节点的扰动很容易引起代谢通路的剧烈波动,能引发细菌整体氧化应激状态改变的代谢节点有望成为潜在新 抗生素或抗生素增效剂的靶标。