异柠檬酸裂解酶的制备及其抑制剂筛选模型的建立

目的 制备结核分枝杆菌异柠檬酸裂解酶(isocitrate lyase,ICL)并建立ICL抑制剂筛选模型.方法 通过四步层析法或金属螯合层析法纯化大肠埃希菌BL21(DE3)表达的ICL,并以异柠檬酸为底物,用纯化的ICL裂解舁柠檬酸,生成乙醛酸可与苯肼反应生成苯腙,苯腙在324nm波长下产生光吸收峰,测定酶促反应体系在324nm波长下光吸收检测异柠檬酸裂解酶的活性,根据待测样品对酶活性的抑制程度,筛选异柠檬酸裂解酶抑制剂.结果 用金属螯和层析法得到纯度为90%、比活力为2.8U/mg的ICL,建立并优化ICL抑制剂筛选模型,模型的信噪比(S/N)远大于3,变异系数(CV)远小于10%.结论 通过金属螯合层析法获得了特异性高、稳定性好的ICL抑制剂筛选模型,该模型可有效的应用于异柠檬酸裂解酶抑制剂的高通量筛选.     

抗结核分枝杆菌持留菌的药物研究进展

持留态结核分枝杆菌具有对现有抗结核药物表型耐药的特征,是结核病病程迁延和复发的主要原因.因此,研发抗结核分枝杆菌持留菌的药物,对缩短抗结核治疗疗程,减少耐药性发生意义重大.本文就结核分枝杆菌持留菌的耐药机制、PA-824等新药的研究进展以及筛选评价模型进行简要综述,为抗结核分枝杆菌持留菌药物的研究提供参考.     

抗结核药物的研究进展（二）

结核病仍为最具破坏性的细菌性疾病之一，发病率和死亡率都很高。结核分枝杆菌能侵入宿主免疫系统，在肺肉芽肿中持留，致使目前的抗结核药物无法杀灭菌体。近年来药物耐受以及伴有HIV感染的结核病发病率急剧增加都给结核的控制带来了很大的困难，迫切需要深入了解目前抗结核药物的作用机制及耐药机制、病菌繁殖的分子机制，以指导开发对持留菌和耐药菌更加有效的新型药物。文中介绍了近年来从现有药物中发现的具有抗结核活性的化合物，以及一些有开发潜能的抗结核药物靶点。     

Progress in the research of antituberculous drugs(I)

结核病仍为最具破坏性的细菌性疾病之一，发病率和死亡率都很高。结核分枝杆菌能侵入宿主免疫系统，在肺肉芽肿中持留，致使目前的抗结核药物无法杀灭菌体。近年来药物耐受以及伴有HIV感染的结核病发病率急剧增加都给结核的控制带来了很大的困难，迫切需要深入了解目前抗结核药物的作用机制及耐药机制、病菌繁殖的分子机制，以指导开发对持留菌和耐药菌更加有效的新型药物。文中综述了近年来通过天然物筛选、新药设计合成以及对现有抗结核药物的再修饰等途径，发现的一些有抗结核活性的化合物，以及结核杆菌分子生物学和抗结核治疗靶点的研究进展。     

卷曲霉素作用机制和耐药机制的功能基因组学研究进展

随着一线抗结核药物的长期和广泛使用，结核病耐药性亦不断增加，其中耐多药结核分枝杆菌和持留菌感染已经成为结核病有效防治的关卡。寻找新型抗结核药物迫在眉睫。卷曲霉素被认为是较理想的二线结核病治疗药物，也是开发新的肽类结核病药物的模板。本文综述了从功能基因组学角度研究卷曲霉素的生物合成基因簇、转录组水平的作用机制和细菌耐药新机制，为合理使用卷曲霉素和开发新一代肽类抗生素提供借鉴。     

新氟喹诺酮类DC-159a体外抗分枝杆菌活性研究

喹诺酮类如氧氟沙星和左氧氟沙星现作为二线药物用于治疗多耐药结核病（multidrug-resistanttuberculosis，MDR-TB），其靶酶不同于一线治疗药物，而它们在临床应用中对结核分枝杆菌（Mycobacteriumtuberculosis，MTB）的抗菌活性较弱。     

参与我院合理使用抗结核病药物咨询的体会

结核病是由结核分枝杆菌复合群（Mycobacterium tuber-culosis complex,简称结核分枝杆菌或结核菌）引起的,经由呼吸道传播严重危害人民健康的慢性传染病,可累及全身多器官系统,在全球广泛流行。抗结核化学药物治疗对控制结核病起决定性的作用,合理的化疗可以使病灶消灭,最终达到痊愈。但是抗结核病药物使用不合理将影响患者的治疗效果,     

中国抗生素杂志 第32卷1～12期（2007年）目次检索

进展、述评与专栏海洋新放线菌及其次级代谢产物研究进展王淑霞,朱天骄,卢圳域,等32(9):513海洋放线菌——新活性代谢物的重要来源(英文)姜怡,JuttaWiese,徐丽华,等32(12):705海洋放线菌活性代谢产物研究最新进展(英文)朱峰,刘晓红,林永成32(3):129以异柠檬酸裂解酶为靶点筛选抗持留结核分枝杆菌药物王,肖春玲32(7):391抗生素作用新靶点的发掘策略谢练武,李翔,欧阳永长,等32(11):641HIV-1整合酶抑制剂的研究进展闫世凤,赵桂森,孙健,等32(10):577链霉菌自动调控因子的研究进展吴杭,张部昌,马清钧32(12):712稀有放线菌质粒生物学的研究进…     

治疗结核病新药和药物作用靶位

尽管对结核病的治疗和免疫预防已取得了很大的进展,但结核病仍然是主要的传染疾病,虽然能治愈,但至少需6～9个月,这种长时间的治疗易产生患者的依从性差,并产生明显的毒性及耐药性.鉴于其耐药性的不断增加和持留态分枝杆菌的潜伏传染特性,急需开发对耐药菌具有抗菌活性的新的结核病治疗药物,更重要的是这些新药能杀死持留细菌,并缩短治疗所需的时间.本文介绍具有发展潜力的新的治疗结核的化学治疗药物.     

996例结核患者对利福平与异烟肼的耐药情况分析

耐多药结核病（MDR-TB）是造成全球结核病疫情回升的重要因素之一,也是结核病防治的难题,治疗难度大。为探讨门诊及住院MDR-TB患者的耐多药情况,指导临床选择有效的药物治疗。本文对996例结核患者结核分枝杆菌核酸检测阳性标本进行结核分枝杆菌耐药基因检测,初步观察其主要抗痨一线药利福平（RFP ）与异烟肼（IN H ）的耐药情况。现将我院2012年4月至2013年4月门诊及住院结核患者996例分枝杆菌核酸提取阳性标本进行RFP与IN H 耐药基因检测分析,以便探讨基因芯片法对 MDR-TB 快速诊断的临床意义。现报告如下。     

A high throughput screening approach to identify isocitrate lyase inhibitors from traditional Chinese medicine sources

Isocitrate lyase (ICL) catalyses the first step of the glyoxylate bypass pathway, which reversibly cleaves isocitrate into succinate and glyoxylate. This pathway occurs in a wide range of pathogens and plays a key role in the pathogenesis of Mycobacterium tuberculosis (MTB) suggesting that it may represent a drug target for the treatment of tuberculosis. ICL was cloned, expressed, and purified, and a high‐throughput screen (HTS) developed to screen active extracts derived from traditional Chinese medicines (TCMs) for inhibition of ICL. A colorimetric assay based on the formation of glyoxylate‐phenylhydrazone was used to measure ICL activity. The assay had signal to noise (S/N) of 12.74 and Z′ factor of 0.72, indicating that the assay was suitable for HTS. Screening of a collection of 465 extracts derived from TCMs resulted in the identification of two extracts from Illicium verum Hook.f (Illiciaceae, XHD‐1) and Zingiber officinale Rosc (Zingiberaceae, XHD‐2), which inhibited ICL with IC₅₀ values of 47.7 ± 16.9 and 18.2 ± 0.9 µg/ml, respectively. Drug Dev. Res. 67:818–823, 2006. © 2007 Wiley‐Liss, Inc.     

抗结核药物药效学非临床研究技术指南

摘要：研发新型抗结核药物极具挑战性,难度大,周期长,非临床研究的数据是开展临床试验的前提。全面充分的结核分 枝杆菌体外和在动物体内的药效学评价是临床试验前的重要步骤和关键内容。本技术指南主要适用于治疗由结核分枝杆菌引起 的肺结核病药物的研发,旨在为创新抗结核药物的临床前药效学研究提供参考。     

Optimization of phage heptapeptide library-screening process for developing inhibitors of the isocitrate lyase homologue from Mycobacterium tuberculosis

The aims of this study were to screen peptide inhibitors specific for isocitrate lyase (ICL) using a phage peptide library and computer molecular docking and to explore the relevant mechanisms. Using ICL as a target, the phage peptide library was screened to obtain peptides with specific binding affinity. Based on the three-dimensional crystal structure of ICL(pdb:1F8I), the obtained polypeptides were docked to the 1F8I using the computer-simulated molecular docking technique. The successfully docked polypeptides were synthesized using the Fmoc solid-phase synthesis method, and the ICL inhibition rate of these peptides was measured. Finally, the possible mechanism underlying the inhibition was explored by Binding Site Analysis. A total of 29 heptapeptides were obtained through screening the phage peptide library. We found that 12 out of the 29 peptides were successfully docked to the 1F8I, and all 12 peptides could obviously inhibit the ICL activity, of which three heptapeptides showed an inhibiting (extent of inhibition over 50 %), IC50 value of 126 μM. Structural analysis revealed that the ICL tetramer has a large cavity in the center, and the polypeptides bind to ICL at amino acid residue 119’s GLN of the ICL monomer. We successfully obtained peptide inhibitors specific for ICL, and analyzed the mechanism underlying the interaction between the peptides and ICL. Our study provides scientific evidence for the development of antituberculosis peptide drugs targeting ICL.     

Drug susceptibility of the Mycobacterium genus: in vitro tests and clinical implications

This review describes the mechanisms of drug resistance of the most clinically relevant mycobacteria and the methods that have been used for studying drug susceptibility (pnenotype, genotype and in vivo tests) and it describes the more important resistance mechanisms to the drugs. Also, this review describes the relationship between microbiological and pharmacological data and the importance of latency -stationary phase- in mycobacteria. Current clinical guidelines on the treatment of tuberculosis (populations of Mycobacterium tuberculosis within the host, drugs and duration, importance of HIV infection in the treatment of tuberculosis, and treatment of latent tuberculosis) and other diseases caused by mycobacteria (specially associated a Mycobacterium kansasii, Mycobacterium avium complex, Mycobacterium fortuitum and Mycobacterium chelonei) are commented in view of drug resistance information, including the more commonly accepted treatments to these diseases. In addition, the impact of pharmacological studies in predicting response to therapy is reviewed. Finally, it describes the new methods of susceptibility testing and the new antituberculous drugs.     

Synthesis and evaluation of hydroquinone derivatives as inhibitors of Isocitrate Lyase

Isocitrate lyase (ICL) is envisaged as an attractive drug target for the development of antimicrobial agents. We have prepared a series of hydroquinone derivatives on the basis of the structure of halisulfates, a naturally occurring inhibitor of ICL. The obtained derivatives were evaluated against ICL of C. albicans. The preliminary structure-activity relationships and the minimal structural requirements for potency were established through structural modifications.     

异烟肼耐药耻垢分枝杆菌的建立及药物敏感性的测定

耐药结核病的涌现,使发展新型抗耐药结核药物变得尤为迫切。本研究选择生长快且无致病性的耻垢分枝杆菌为研究对象,探索快速评价药物抗异烟肼耐药结核分枝杆菌的能力。inhA是异烟肼的作用靶点,由于inhA的突变或者过表达可以引起结核分枝杆菌对异烟肼耐药性的产生。通过将inhA克隆入pMV261中,构建过表达inhA的耻垢分枝杆菌。结果显示,过量表达inhA的耻垢分枝杆菌对异烟肼的敏感性下降了100倍以上。建立了利用刃天青为指示剂的抗异烟肼耐药株的快速药效评价方法,可快速对药物的活性进行定性或定量评价,为进行新型抗结核药物的高通量筛选和药效评价奠定基础。     

5-tert-butyl-N-pyrazol-4-yl-4,5,6,7-tetrahydrobenzo[d]isoxazole-3-carboxamide derivatives as novel potent inhibitors of Mycobacterium tuberculosis pantothenate synthetase: initiating a quest for new antitubercular drugs

Pantothenate synthetase (PS) is one of the potential new antimicrobial targets that may also be useful for the treatment of the nonreplicating persistent forms of Mycobacterium tuberculosis. In this Letter we present a series of 5- tert-butyl- N-pyrazol-4-yl-4,5,6,7-tetrahydrobenzo[ d]isoxazole-3-carboxamide derivatives as novel potent Mycobacterium tuberculosis PS inhibitors, their in silico molecular design, synthesis, and inhibitory activity.     

New anti-Mycobacterium agents: recent advances in patent literature

Mycobacterium tuberculosis is the greatest single infectious cause of mortality worldwide. In addition, disseminated infection with Mycobacterium avium complex (dMAC) is an increasingly frequent complication in advanced HIV infection. Among the different classes of anti-Mycobacterium compounds studied in the recent years, those of oxazolidinones and imidazole derivatives seem the most likely to provide new useful drugs for the clinical treatment of Mycobacterium infections in the future. Although the most important improvement in the therapy of tuberculosis infections will be obtained using antisense oligonucleotides and vaccines, the research of new ‘conventional’ antituberculous drugs will also remain an important challenge.     

武冈市158株结核杆菌耐药性分析

目的：了解武冈市结核病患者结核杆菌的耐药状况,为结核病的防治提供实验依据。方法：连续收集我院2009年2月～2010年3月肺结核患者的结核杆菌158株,采用WHO/IUATLD推荐的比例法,对异烟肼、利福平、链霉素和乙胺丁醇4种抗结核药物进行药敏试验。结果：158株结核分枝杆菌中,91例对4种抗结核药物全部敏感,占57.59%;67例耐药,耐药率为42.41%,以耐链霉素为最高,占34.18%。结论：武冈市结核病患者结核杆菌耐药严重,必须进一步加强对耐药结核杆菌的监控。     

某区322株结核杆菌的耐药性分析

目的了解惠州市结核病防治院322株痰培养阳性结核杆菌的耐药情况,为临床合理用药提供理论依据。方法以我院322株痰培养阳性的结核杆菌为研究对象,用比例法进行一线结核用药的药敏实验。结果 322株结核分枝杆菌对4种抗结核药物全部耐药者36例,占10.84%;以耐链霉素为最高,占35.8%。结论惠州市结核病耐药情况仍然十分严峻,应加强结核病临床治疗的管理,制定合理化疗方案,防止耐药菌传播。