CN115920003A - 脂蛋白LprG及其基因作为抗结核分枝杆菌药物靶点的应用 - Google Patents
脂蛋白LprG及其基因作为抗结核分枝杆菌药物靶点的应用 Download PDFInfo
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- CN115920003A CN115920003A CN202211053803.4A CN202211053803A CN115920003A CN 115920003 A CN115920003 A CN 115920003A CN 202211053803 A CN202211053803 A CN 202211053803A CN 115920003 A CN115920003 A CN 115920003A
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Abstract
本发明公开了脂蛋白LprG及其基因作为抗结核分枝杆菌药物靶点的应用。所述脂蛋白为结核分枝杆菌的LprG,以LprG或其同源蛋白或编码该蛋白的基因为靶点,敲除或突变其基因,或者开发阻断或下调该蛋白表达量或活性的药物,从而影响结核菌细胞壁的通透性。靶向LprG或其同源蛋白或者编码该蛋白的基因的药物与现有抗结核药物联用,能够显著提高杀菌效果,进而缩短治疗周期,实现快速治疗结核病的目标。
Description
技术领域
本发明涉及生物医药领域,特别涉及一种结核分枝杆菌脂蛋白LprG及其基因作为新的抗结核药物靶点的应用。
背景技术
结核病是结核分枝杆菌(Mycobacterium tuberculosis,MTB)感染所引起的一种传染性疾病,是全球十大死因之一。世界卫生组织(WHO)最新的全球结核病报告数据显示:2020 年全球新发结核病患者987万例,约有130万例患者死亡。与此同时,结核病治疗和管理在全球范围内仍面临着MTB多药耐药日益严峻的挑战。目前治疗结核病必须采用联合用药的方式,进而提高治疗疗效,降低耐药性产生。尽管已经研发出贝达喹啉(Bedaquiline,BDQ)、 pretomanid(PA-824)等新型药物,但结核病,特别是耐多药结核病的治疗疗程仍需长达数月时间,且有药物不良反应,病人依从性差,容易用药不规范,甚至提前终止用药,最终又导致结核菌耐药性增加。因此,迫切需要发展针对新靶标及具有新的作用机制的结核病药物,替代和改进目前的联合治疗方案,进而缩减目前的治疗周期。
结核菌与其他病原菌相比具有独特且复杂的细胞被膜结构(Cell Envelope),对分枝杆菌菌体构成极强的保护屏障。这种被膜结构不仅能够与宿主细胞互作,调节宿主的免疫应答,维持结核菌在宿主体内的存活,而且还赋予结核菌对多种抗结核药物天然的耐药性。因此,结核菌细胞被膜是开发新的抗结核药物的重要方向。
结核菌的细胞被膜主要是由外侧的外膜层(Outer Membrane),中间的细胞壁(Cell Wall) 以及最内侧的质膜(Cytoplasmic Membrane)构成。除此之外,外膜外还存在由多糖、蛋白质和少量脂质组成的荚膜(Capsule)。外膜层是由多种脂质、糖脂和分泌蛋白等组成。中间的细胞壁主要由分枝菌酸层(Mycolic Acid),多分支的阿拉伯半乳聚糖(Arabinogalactan,AG) 以及网状交联的肽聚糖(Peptidoglycan,PG)组成,即mAGP复合体,是结核菌细胞被膜的核心结构。其中,分枝菌酸层具有特殊的非对称性双层结构:内层主要由长链分枝菌酸组成,并与阿拉伯半乳聚糖-肽聚糖共价结合,外层主要由分枝菌酸以非共价连接的方式形成海藻糖分枝酸酯(Trehalose Monomycolate,TMM),磷脂酰肌醇甘露糖(Phosphatidylinositol Mannoside, PIM),海藻糖二分枝酸酯(Trehalose dimycolate,TDM)以及脂阿拉伯糖甘露聚糖 (Lipoarabinomannan,LAM)。
利用功能缺失型(Loss-of-function)策略的高通量筛选是目前开展功能基因筛选、药物靶点以及揭示信号通路等研究的主要策略。利用构建的联合CRISPR-KO敲除文库及CRISPRi 文库的高通量筛选平台进行药物筛选,通过增加细胞被膜结构通透性可促进抗结核药物的杀菌能力,也为以细胞被膜结构为靶点开发新药,以及利用新药与现有药物进行药物联用进而提高治疗方案的功效缩短治疗周期提供了理论依据。
发明内容
本发明利用前期构建的基于CRISPR系统的全基因组的功能基因组平台,该平台联合了 CRISPR-KO敲除文库和CRISPRi文库,用于结核分枝杆菌高通量功能缺失筛选。该功能基因组筛选平台在贝达喹啉(Bedaquiline,BDQ)药物筛选中发现MTB脂蛋白LprG可增加细胞通透性,经研究,lprG突变株在BDQ药物处理后导致胞内ATP水平比野生型经BDQ处理的ATP水平要低,且呈现出药物浓度水平依赖性。与此同时,lprG突变株针对BDQ的抑菌生长曲线(MIC)以及杀菌动力学均表现出其对BDQ更为敏感。针对lprG的小分子化合物 LB04-III在与BDQ联合使用,表现出更强的杀菌作用。此外,本发明发现lprG突变株对其他抗结核药物,如利福平(Rifampicin,Rif),德拉马尼(Delamanid,DLM)以及普瑞玛尼pretomanid(PA-824)均更敏感。因此本发明发现了LprG通过影响细胞壁通透性,进而能够作为潜在的抗结核药物新靶点,且以该蛋白为靶点的药物可以与现有的抗结核药物进行联用,提高杀菌效果进而缩短治疗周期。
一方面,本发明发现了增强结核分枝杆菌细胞通透性的脂蛋白LprG,该蛋白是具有序列表中SEQ ID NO:1所示氨基酸序列的蛋白质,或者是与之有至少80%氨基酸序列同源性且具有相同或相似生物学功能的蛋白质,都可以作为抗结核药物的靶点。
序列表中SEQ ID NO:1所示的脂蛋白LprG全长237个氨基酸,该脂蛋白与同一操纵子的Rv1410共同调控结核分枝杆菌三酸甘油酯(TAG)的水平,而TAG是结核分枝杆菌细胞壁结构的重要组分来源;且有研究表明,LprG能够结合胞壁的糖脂成分——脂阿拉伯糖甘露糖(LAM),将LAM从胞质的一侧运输到细胞表面,使LAM在结核分枝杆菌表面作为毒力因子发挥作用。
以LprG或其同源蛋白为靶点,获得对LprG或其同源蛋白进行阻断或下调干预的抗结核药物,包括对所述靶点的表达量和/或活性进行抑制或下调干预。
所述抗结核药物还可以以编码LprG或其同源蛋白的基因为靶点,从DNA水平敲除或突变所述基因,或者敲低所述基因mRNA的转录。
作为抗结核药物靶点的基因是编码序列表中SEQ ID NO:1所示氨基酸序列的多核苷酸,或者是与之有至少80%(优选至少85%,更优选至少90%)序列同源性且编码具有相同或相似的生物学功能的蛋白的多核苷酸。
编码所述脂蛋白LprG的一种基因序列如序列表中SEQ ID NO:2所示。本领域技术人员应当了解,基于密码子的简并性,脂蛋白LprG的编码基因不止SEQ ID NO:2;而且,在蛋白编码序列的基础上,其基因中可包含编码序列5’端和/或3’端的非编码序列、标签序列等。
靶向所述LprG或其同源蛋白或编码该蛋白的基因的小分子化合物、抗体、多肽或寡核苷酸都在本发明的保护范围内,例如siRNA、小分子抑制剂、抗体或其他多肽类药物。
将靶向LprG或其同源蛋白或者编码该蛋白的基因的基因工程载体或工程细胞应用于抗结核药物的制备也在本发明的保护范围内。
另一方面,本发明以脂蛋白LprG或其同源蛋白或编码该蛋白的基因作为抗结核药物的靶点,将其作为靶标开发其抑制剂可增强现有抗结核药物的杀菌作用,因此可作为新的联合用药方案。
本发明还提供了一种药物组合物,包含靶向所述LprG或其同源蛋白或编码该蛋白的基因的药物或其药学上可接受的盐,以及现有抗结核药物中的一种或多种,还可以包含药学上可接受的载体或赋形剂。所述药物组合物用于结核病的预防和/或治疗。所述现有抗结核药物包括但不限于贝达喹啉(BDQ)、利福平(Rif)、德拉马尼(DLM)以及普瑞玛尼pretomanid (PA-824)。
本发明的有益效果:本发明提供了一种可作为潜在的抗结核药物的新靶点。以该蛋白或其基因为靶点研究的抑制剂或药物可以与现有药物进行联用,有望实现快速杀菌、缩短治疗疗程,最终实现快速治疗结核病的目标。
附图说明
图1.实施例2联合CRISPRi文库和CRISPR-KO敲除文库BDQ药物筛选结果统计,其中a为CRISPRi文库BDQ药物筛选基因差异分析(火山图),b为CRISPR-KO敲除文库BDQ 药物筛选基因差异分析(火山图),c为CRISPRi文库和CRISPR-KO敲除文库药物筛选差异基因的功能分类。
图2.实施例3中MIC及杀菌动力学验证筛选结果,其中a为不同突变株在BDQ处理下生长曲线差异,b为lprG杀菌动力学差异比较。
图3.实施例4的实验结果显示LprG是通过影响MTB细胞被膜结构的完整性而对BDQ敏感性增加,其中a为溴化乙锭通透性实验比较lprG突变株与野生型细胞被膜结构完整性差异,b为lprG突变株与野生型BDQ药物处理后细胞内ATP含量的差异。
图4.显示影响细胞被膜结构可对多种药物的敏感性增加,其中a为针对pretomanid药物的生长曲线差异,b为针对利奈唑胺(linezolid)药物的生长曲线差异,c为针对德拉马尼 (delamanid)药物的生长曲线差异,d为针对利福平(rifampicin)药物的生长曲线差异。
图5.显示影响细胞被膜通透性的小分子药物LB04-III以及已知抗结核药物EMB与BDQ 联合处理后存活统计,其中a为在H37Ra减毒菌株中进行药物处理的结果统计,b图为在H37Rv标准菌株进行药物处理的结果统计。**代表P<0.01,表明具有显著性差异。
具体实施方式
下面结合具体实施例,进一步阐明本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1、CRISPR-KO敲除文库和CRISPRi文库的构建
1)sgRNA文库的设计
根据NCBI上结核分枝杆菌的全基因组序列,对结核菌基因组中的基因、rRNA、tRNAs 以及非编码RNA区域分别设计靶向sgRNA序列。sgRNA的设计原则:每个基因至少设计8 个sgRNA;当sgRNA靶向基因的开放阅读框时,sgRNA序列尽量设计在基因的前50%区域,为兼顾CRISPR-KO及CRISPRi文库,sgRNA靶向序列设计包含模板链与非模板链;全基因组脱靶效应分析需考虑至少4个碱基错配且保证sgRNA的种子序列(seed sequence)与脱靶位点的匹配数尽可能低;同时靶向sgRNA序列尽量避免含有“TTTT”或“AAAA”序列。在结核分枝杆菌中需考虑sgRNA的5’碱基为G或A,以此提高其转化效率。
2)sgRNA质粒文库的构建
本发明选取来源于嗜热链球菌(Streptococcus thermophilus)的Cas9作为编辑工具。我们利用表达Cas9sth1和sgRNA的整合质粒pYC1446作为CRISPR-KO sgRNA质粒骨架,利用 pLJR965(参见Programmable transcriptional repression in mycobacteriausing an orthogonal CRISPR interference platform[J].Nat Microbiol,2017,2:16274.)作为CRISPRi sgRNA质粒骨架。依照上述设计原则,共设计出79863个sgRNA靶向序列,这其中包含了 90%的sgRNA靶向结核分枝杆菌已有注释的基因以及1272个非靶向sgRNA。利用 CustomArray芯片合成sgRNA oligo。合成好的sgRNA oligo经PCR扩增的方法加上接头,然后利用Gibson assembly的方法分别与BsmBI酶切后切胶回收的pYC1446或pLJR965载体片段进行连接。
将连接产物转化至大肠杆菌感受态中,复苏1小时后涂布到150mm×150mm的含有相应抗性的平板中。24小时后刮取平板上的菌落混合用无内毒素的大提质粒试剂盒提取CRISPR-KO sgRNA质粒文库和CRISPRi sgRNA质粒文库。
4)CRISPR-KO敲除突变体文库的构建
将含有NHEJ元件表达质粒的结核分枝杆菌制备感受态,并将CRISPR-KO sgRNA质粒文库以每1mL感受态细胞转入500ng质粒的比例与感受态细胞进行混合,轻轻吹打混匀后以200μL分装并进行电击。为了尽可能覆盖到所有sgRNA,共进行了100次转化。转化后立即加入7H9+OADC培养基,随后将富集的培养液置于滚瓶中培养48小时,使细菌在液体培养的条件中模拟稳定期转态,尽可能提高编辑效率。2天后将细菌培养液离心浓缩后涂在 150mm×150mm的含有卡那霉素、博来霉素和100ng/mL的7H10+OADC的平板上;与此同时取适量菌液进行稀释涂板以确定文库的转化效率。37℃培养21天后,平板上有清晰可见的菌苔,利用涂布棒收集菌体到液体培养基中,并利用全自动组织处理器(gentle MACS Dissociator,Miltenyi Biotec#130095937)将菌体温和地打散,获得均质的细菌培养液。对该菌液稀释,调整OD600为1.0,分装成每管10mL进行冻存,即为初始的CRISPR-KO敲除突变体文库。
5)CRISPRi文库的构建
将结核分枝杆菌野生型制备感受态,并将CRISPRi sgRNA质粒文库以每1mL感受态细胞转入200ng质粒的比例与感受态细胞进行混合,轻轻吹打混匀后以200μL分装并进行电击。为了尽可能覆盖到所有sgRNA,共进行了50次转化。转化后立即加入7H9+OADC培养基,将富集的培养液置于滚瓶中培养24小时,随后将细菌培养液离心浓缩后涂在150mm×150 mm的含有卡那霉素的平板上;与此同时取适量菌液进行稀释涂板以确定文库的转化效率。 37℃培养18天后,平板上有清晰可见的菌苔,利用涂布棒收集菌体到液体培养基中,并利用全自动组织处理器(gentle MACS Dissociator,Miltenyi Biotec#130095937)将菌体温和地打散,获得均质的细菌培养液。对该菌液稀释,调整OD600为1.0,分装成每管10mL进行冻存,即为初始的CRISPRi文库。
实施例2、联合CRISPR-KO敲除文库和CRISPRi文库进行BDQ药物筛选
1)BDQ筛选
将实施例1中获得的CRISPR-KO敲除文库和CRISPRi文库冻存液置于培养瓶中复苏培养,待复苏菌液长到OD600为1.0后进行转接,初始OD600为0.02。其中CRISPRi文库中加入100ng/mL ATc诱导dCas9表达进而抑制sgRNA靶向基因的表达。待OD600长到1.0后将CRISPR-KO敲除文库和CRISPRi文库分别分装到二个滚瓶中,二个滚瓶中分别加入DMSO (对照组),1000ng/mL BDQ后继续培养6天。6天后,对经过处理的CRISPR-KO敲除文库和CRISPRi文库菌液以3000×g离心10分钟,丢弃上清。PBST洗菌后再以3000×g离心10 分钟,丢弃上清,最终用收菌时等体积的培养基重悬起来,随后再转接到新鲜培养基中扩培 16代,最终收集约5mL菌液提取基因组DNA以进行后续扩增子测序。
基因组DNA提取方法具体如下:
2)扩增子建库及测序
利用PCR扩增sgRNA区域。每组共计扩增8管,每管为50μL体系;扩增体系如下:
I-5TM2×Hi-Fi PCR Master Mix(MCLAB),25μL
正向引物:2.5μL
反向引物:2.5μL
基因组DNA:50ng
加H2O至50μL
PCR正向引物序列为5’-CTCTGACCAGGGAAAATAGCCC-3’(SEQ ID NO:3)
PCR反向引物序列为5’-GCCATTGATAATGCTCTTCATCCC-3’(SEQ ID NO:4)
对扩增sgRNA区域的PCR产物使用胶回收试剂盒进行纯化,并进行核酸定量。随后参照illumina试剂盒进行文库构建。具体流程如下:每组样品取>50ng的纯化产物通过EndPrep Enzyme Mix进行末端修复(包括5’末端磷酸化和3’末端加A),随后通过TA连接在两端加接头。再使用DNA Clean Beads纯化片段,而后每组样品用引物进行扩增。最终PCR产物使用Qseq100生物分析仪(Bioptic,China)检测文库质量,并且通过Qubit 3.0检测文库浓度。DNA文库混合后,按Illumina Novaseq(Illumina,San Diego,CA,USA)仪器使用说明书进行2×150bp双端测序(PE),由Novaseq自带的Novaseq Control Software(NCS)+OLB +GAPipeline-1.6读取序列信息。
3)二代测序数据分析
Illumina测序结果采用MAGeCK分析方法(版本v0.5.9.4)中的Robust Rankanalysis进行分析。首先将在对照组中的sgRNA读数小于10的去除,随后利用非靶标sgRNA的读数对处理组(250ng/mL BDQ,1000ng/mL BDQ)与对照组进行均一化,而后基因水平上的倍数差异(Log2FC)以该基因中的所有sgRNA的倍数差异的中位数按照“阿尔法中位数”方法进行计算。最终分析结果如图1所示。
BDQ是近40多年来第一个上市的抗结核新药,属于二芳基喹啉类化合物。BDQ通过与 5’-三磷酸腺苷酶合成酶结合从而抑制三磷酸腺苷的合成。由火山图可知,目前已知与BDQ 耐药或敏感性相关的基因均被筛选出来。例如,编码外排泵的基因mmpS5-mmpL5是在负向筛选中较为明显的基因(图1),当外排BDQ药物的基因mmpS5-mmpL5被敲除或敲低后,导致细菌内BDQ药物增多,从而增加了结核菌对BDQ的敏感性;反之,rv0678是mmpS5-mmpL5 操纵子的负向调控因子,rv0678突变可导致编码MmpS5-MmpL5外排泵的基因表达上调,从而使结核菌对BDQ产生耐药。如图1所示,rv0678在CRISPR-KO敲除文库和CRISPRi文库的正向筛选中均为富集程度最高的基因。上述结果表明基于CRISPR的筛选平台可用于化学- 基因相互作用的分析。
CRISPRi文库筛选的一个关键优势是能够确定出某个必需基因对杀菌的协同作用。如图 1所示,许多参与三磷酸腺苷合成的必需基因,如atpB、atpC和atpH均在CRISPRi文库的负向筛选中富集,而未能够在CRISPR-KO文库筛选出来。与此同时,CRISPRi文库还筛选出基因pks13,而已有文献报道靶向pks13的药物TAM16与BDQ联用时有协同作用。综上,这些结果表明,基于CRISPR的筛选平台可用于发现与已知药物有协同作用潜在药物作用靶点。
与CRISPRi文库相比,CRISPR-KO文库是通过基因敲除发挥作用,使得某些表型比基因敲降更加显著。如表1所示,许多非必需基因在CRISPR-KO中筛选出来,而未能在CRISPRi 文库中筛选出来,如lprG,mmaA4,rv1410c,rip等基因。而这些基因突变后也在其他文献报道的转座子文库中被确认是对多种药物敏感,如利福平(rifampicin,Rif)、万古霉素 (vancomycin,VAN)以及美罗培南(meropenem,MER)。如表1、2所示,对BDQ筛选获得的耐药/易感基因的功能类别进行划分可发现,固有耐药主要由参与细胞壁合成和细胞过程的基因决定,这一结果也印证了“细胞通透性降低是结核分枝杆菌耐药的主要原因”这一主流观点。综上,我们可以发现联合CRISPR-KO敲除文库以及CRISPRi文库的高通量筛选平台能够克服各自方法的缺点从而为我们从多角度、立体化且更加全面地研究结核分枝杆菌生物学过程提供了一个强大的功能基因组学工具。
表1.CRISPRi文库在BDQ筛选富集的基因列表
a表示该基因在Tn-seq方法的不同药物筛选中有被确定为显著差异基因,具体筛选药物标注如上。 (PMID:28893793)
表2.CRISPR-KO敲除文库在BDQ筛选富集的基因列表
a表示该基因在Tn-seq方法的不同药物筛选中有被确定为显著差异基因,具体筛选药物标注如上。 (PMID:28893793)
实施例3、验证CRISPR-KO文库在BDQ中的筛选结果
CRISPR-KO文库在BDQ中的筛选结果中显示lprG突变株在负向筛选中富集,即该突变株对BDQ更加敏感。因此,我们构建lprG突变株并利用抑菌生长曲线(MIC)以及杀菌动力学来验证筛选结果。
1)抑菌生长曲线
①本实验中用96孔板测定H37Ra及突变株对BDQ的敏感性差异。将待测菌株培养至对数期,用新鲜的7H9培养基稀释至1×106CFU/mL,然后接种到96孔板中;
②每个孔加入终浓度为0.00390625-1μg/mL的贝达喹啉并设置3组重复;
③盖上96孔板盖子,放在一个塑封袋中,37℃静置培养。7天后利用96孔平板读板仪读取OD600,而后利用GraphPad Prism 9软件绘制曲线并得出MIC50
2)杀菌实验
对野生型以及lprG突变株培养至对数期,一组转接到初始OD600为0.05,加入1×MIC BDQ 进行处理;另一组对培养至OD为1.0的野生型以及lprG突变株分别加入20×MICBDQ进行处理;处理不同天数后,分别洗菌进行稀释涂板,大约生长21天后,CFU计数。
如图2所示,已知参与外排BDQ药物的mmpS5-mmpL5以及lprG的突变株均导致MIC50减小,即对BDQ更敏感;针对对数期以及稳定期的lprG突变株都表现出更为敏感,从而验证了我们的筛选结果。
实施例4、LprG影响MTB细胞被膜结构的完整性进而提高药物的摄入
LprG是目前研究较多的细胞被膜脂蛋白,LprG一方面能够调控三酸甘油酯(TAG)的水平,另一方面能够装载脂阿拉伯糖甘露糖(LAM),将LAM从胞质的一侧运输到外膜层且更好地定位,从而形成完整的外膜结构。因此lprG突变株对BDQ敏感性增加可能是由于细胞被膜完整性受损导致BDQ药物进入的增加。为了验证该猜测,我们通过小分子溴化乙锭通透性实验以及测定胞内ATP含量来进行验证。
1)溴化乙锭通透性实验
将野生型菌株和lprG突变株在7H9培养基中培养至OD600为0.6-0.8,3000×g离心10分钟,丢弃上清。随后用含有0.05%Tween 80的PBS洗菌,再以3000×g离心10分钟,弃上清,最终用含0.4%葡萄糖的PBS重悬菌体并将OD600调整为0.8。在96孔板中的每孔加入100μL 菌液,随后每孔加入等体积的含有2μg/mL的含0.4%葡萄糖的PBS液体。在多功能酶标仪(Tecan Infinite 200pro)按照激发波长为530nm,发射波长为590nm,37℃下每90s读取溴化乙锭荧光值。
2)测定胞内ATP水平
用BacTiter-GloTM细胞活力测定试剂盒进行ATP含量的检测(Promega;#G8230)。具体操作如下:野生型菌株和lprG突变株在7H9培养基中培养至OD600为0.6-0.8后进行转接,初始OD600为0.05,随后加入不同浓度BDQ处理。24小时后,将样品与2倍体积的Tris-EDTA 试剂(100mM Tris,4mM EDTA,pH 7.75)混合,并在100℃孵育5分钟,随后立即置于冰上。混合液以5000×g离心10分钟后,将上清移到新的试管中,并取出50μL移至96孔平板中,随后加入等体积BacTiter-GloTM试剂避光混合5分钟。最终利用微孔板读数仪记录荧光值。
如图3所示,相较于野生型,lprG突变株对于小分子溴化乙锭的通透性增加。为了排除 lprG突变株胞内溴化乙锭的积累增加是由于外排作用降低导致的。我们在菌液中加入了外排抑制剂维拉帕米(verapamil),如图3中A所示,当加入维拉帕米后,野生型胞内溴化乙锭的积累量相较于不加入维拉帕米的只有少量增加;而lprG突变株相较于野生型则有1倍多的胞内溴化乙锭的增加。该结果表明lprG突变株中溴化乙锭积累增加与维拉帕米敏感的外排机制无关,可能是由于细胞被膜通透性增加导致。另一方面,我们对经过BDQ处理24小时的野生型与lprG突变株测定了胞内ATP的含量。结果发现,野生型与lprG突变株的胞内ATP 变化均呈现出梯度依赖性且lprG突变株胞内ATP含量随BDQ浓度的增加,其ATP水平降低更快,这从侧面验证了lprG突变株菌内的BDQ含量更多,从而使ATP水平降低更快。综上结果表明LprG影响MTB细胞被膜结构的完整性进而提高药物的摄入,从而促使BDQ更好地杀菌。
实施例5、lprG突变株对多种抗结核药物敏感
全球结核病药物研发联盟研发的PA-824与BDQ和利奈唑胺(Linezolid)组成的BPaL 方案将治疗耐多药结核病的治疗时间缩短至6-9个月,也减少了片剂用量,有助于提高治愈率。此外,德拉马尼(Delamanid)也是最近批准的治疗耐多药结核病药物。于是,我们验证了lprG突变株、embA和topA KD(Knockdown)菌株对利福平(Rifampicin)、德拉马尼、利奈唑胺和pretomanid疗效的影响。如图4所示,lprG突变株对所有药物均表现出敏感性。已知药物乙胺丁醇的作用底物阿拉伯糖基转移酶EmbA是参与AG与LAM的合成,从而影响细胞壁的完整性,如图3所示,embA KD突变株同样对待测的菌株增加了敏感性,反之拓扑异构酶topA KD突变株则没有变化;以上结果表明细胞被膜结构的完整性可能影响多种药物的杀菌功效;且在lprG突变株在BPaL这三种药物中均表现出敏感性增加,这也说明了lprG 可作为药物靶点开发新药且能与现有药物进行联用,进一步缩短治疗疗程,提高治愈率。
实施例6、针对LprG的小分子抑制剂LB04-III具有增强BDQ的作用
上述实验已验证了lprG突变株在BPaL这三种药物中均表现出敏感性增加,表明其可作为药物靶点参与到联合用药的过程中,开发出新的联合用药方式。为了验证其可靠性,我们利用他人研究发现的针对LprG的小分子化合物LB04-III进行联合杀菌处理,验证LB04-III 是否能够促进BDQ的杀菌作用,于此同时我们也利用同样能够增强细胞被膜通透性的乙胺丁醇EMB与BDQ同时杀菌。如图5所示,将LB04-III与BDQ同时处理时,其杀菌作用相较于单独使用BDQ增强了约5倍,而靶向同样具有改变细胞被膜通透性的embA的已知药物乙胺丁醇EMB在与BDQ联合使用时,杀菌作用没有显著变化。综上表明,针对LprG作为药物靶点可作为一种新的联合用药方式来增强现有药物的杀菌作用,进一步提高治疗效果。
以上对本发明的具体实施例进行了描述。需要理解的是,本发明并不局限于上述特定实施方式,本领域技术人员可以在权利要求的范围内做出各种变化或修改,这并不影响本发明的实质内容。本发明的保护范围由权利要求书所界定。
Claims (10)
1.LprG或其同源蛋白在制备抗结核药物中的应用,其特征在于,以结核分枝杆菌的脂蛋白LprG或其同源蛋白为靶点,获得对LprG或其同源蛋白进行阻断或下调干预的药物。
2.如权利要求1所述的应用,其特征在于,所述LprG或其同源蛋白选自:
(1)具有序列表中SEQ ID NO:1所示氨基酸序列的蛋白;
(2)与(1)所述蛋白有至少80%氨基酸序列同源性的蛋白,且具有与(1)所述蛋白相同或相似的生物学功能。
3.编码LprG或其同源蛋白的基因在制备抗结核药物中的应用,其特征在于,所述抗结核药物以编码结核分枝杆菌的脂蛋白LprG或其同源蛋白的基因为靶点,从DNA水平敲除或突变所述基因,或者敲低所述基因mRNA的转录。
4.如权利要求3所述的应用,其特征在于,所述编码LprG或其同源蛋白的基因选自:
(i)编码序列表中SEQ ID NO:1所示氨基酸序列的多核苷酸;
(ii)具有与(i)所述多核苷酸至少80%序列同源性的多核苷酸,且其编码的蛋白与(i)所述多核苷酸编码的蛋白具有相同或相似的生物学功能。
5.如权利要求4所述的应用,其特征在于,所述基因序列如序列表中SEQ ID NO:2所示。
6.靶向LprG或其同源蛋白或者编码该蛋白的基因的基因工程载体或工程细胞在制备抗结核药物中的应用。
7.一种药物组合物,包含靶向LprG或其同源蛋白或者编码该蛋白的基因的抗结核药物或其药学上可接受的盐,所述抗结核药物对LprG或其同源蛋白进行阻断或下调干预,或者从DNA水平敲除或突变所述基因,或者敲低所述基因mRNA的转录。
8.如权利要求7所述的药物组合物,其特征在于,靶向LprG或其同源蛋白或者编码该蛋白的基因的抗结核药物为小分子化合物、抗体、多肽和/或寡核苷酸。
9.如权利要求7所述的药物组合物,其特征在于,所述药物组合物还包含一种或多种现有抗结核药物。
10.如权利要求9所述的药物组合物,其特征在于,所述现有抗结核药物包括贝达喹啉、利福平、德拉马尼和普瑞玛尼。
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