HIV-1整合酶去整合活性高通量检测方法的建立

HIV-1整合酶催化病毒DNA与宿主细胞基因组整合,是病毒复制所需的关键酶之一,也是抗病毒药物研发的重要靶点.IN及其核心结构域均能在体外催化去整合反应.本研究表达纯化了IN和IN-CCD蛋白,建立了一种检测IN和IN-CCD去整合活性的微孔板式高通量方法.设计了生物素和地高辛修饰的去整合DNA底物,运用链亲和素标记的珠子捕获反应产物,再通过酶标地高辛抗体及随后的酶联免疫吸附实验方法对地高辛定量以检测去整合.结果显示,IN和IN-CCD催化的去整合反应信号（A405）分别达到1.6和1.2,而背景信号值低于0.05;IN去整合反应更倾向于使用Mn2＋而不是Mg2＋作为金属辅助离子;研究还发现,已知的IN抑制剂baicalein是IN-CCD抑制剂.以上结果表明,本工作建立的检测方法能高通量、高灵敏度和高特异性地研究去整合反应,并能够应用于以IN为靶点,特别是以IN-CCD为靶点的HIV抑制剂的筛选.     

HIV-1整合酶四聚体结构模拟及其活性位点分析

HIV-1复制需要HIV-1整合酶将其环状DNA整合进宿主DNA中,这其中包括2个重要反应,即“3′-加工”和“链转移”,两者均由HIV-1整合酶催化完成.阻断其中的任一反应,都能达到抑制HIV-1复制的目的.因此,了解HIV-1整合酶的完整结构和聚合状态,对深入探讨其作用机理及设计新型抑制剂具有重要的指导作用.然而,迄今为止仅有HIV-1整合酶单独结构域的晶体结构可供参考,而其全酶晶体结构尚未获得解析.本研究利用分子模拟技术,通过蛋白质 蛋白质/DNA分子对接、动力学模拟等方法,构建了全长整合酶四聚体的结构模型、HIV-1 DNA与整合酶复合物的结构模型,进一步从理论上证实HIV-1整合酶是以四聚体形态发挥催化作用,明确“3′-加工”和“链转移”在HIV-1整合酶上的催化位点.同时,通过与作用机理相似的细菌转座子Tn5转座酶等的结构比对,推测HIV-1整合酶的核心结构域中应有第2个Mg^２＋存在,其位置螯合于Asp64与Glu152之间.在HIV-1整合酶结构研究的基础上,有望进一步设计出新的抗艾滋病药物.     

HIV-1整合酶核心区可溶性表达及抑制剂筛选

为了实现HIV-1整合酶蛋白核心区 (central core domain of integrase, IN-CCD) 的可溶性表达,并建立以IN-CCD为靶点的抑制剂体外筛选方法,从包含F185K突变HIV-1 IN基因的质粒中经PCR扩增得到含有F185K突变的IN-CCD基因,克隆到pET28b载体上构建重组质粒pIN-CCD,转化pIN-CCD至E. coli BL21 (DE3)中经IPTG诱导、表达,Ni-亲和层析纯化,获得IN-CCD蛋白。修饰DNA底物,以链亲和素包被的磁珠为载体捕获DNA产物,结合酶联免疫吸附测定法(ELISA)检测IN-CCD的去整合活性,并筛选以IN-CCD为靶点的抑制剂。结果表明重组蛋白IN-CCD实现了高效可溶性表达,纯化后蛋白纯度达95%。建立的ELISA可以检测IN-CCD的去整合活性,且方法特异性和灵敏度好,可以实现高通量抑制剂筛选。从100个样品中筛选得到5个具有初步抑制IN-CCD去整合活性的样品。     

分子信标方法研究HIV-1整合酶3'加工反应动力学

HIV-1整合酶催化病毒cDNA与宿主细胞基因组DNA的整合,是病毒在宿主细胞中增殖的一个关键酶.3'加工是整合酶催化整合过程的第一步反应,3'加工反应动力学的研究对整合酶催化机理研究和以整合酶为靶点的药物研发都具有重要意义.构建了野生型HIV-1整合酶重组质粒,在大肠杆菌BL21中诱导表达,通过对包涵体变性、复性,纯化得到了整合酶蛋白.基于分子信标原理,设计了荧光和淬灭基团标记的DNA底物,通过荧光信号实时监测3'加工反应,对酶反应的动力学性质进行研究.结果表明,纯化的整合酶蛋白具有较高的活性,酶反应表现出显著的Mg2+偏好性.酶动力学研究(Km=131.79 nmol/L,Kcat=0.0042 min-1)表明,该分子信标方法和设计的DNA底物可用于整合酶3'加工反应动力学研究以及酶反应性质的研究.     

分子信标方法研究HIV-1整合酶3'加工反应动力学

HIV-1整合酶催化病毒cDNA与宿主细胞基因组DNA的整合,是病毒在宿主细胞中增殖的一个关键酶。 3'加工是整合酶催化整合过程的第一步反应,3'加工反应动力学的研究对整合酶催化机理研究和以整合酶为靶点的药物研发都具有重要意义。构建了野生型HIV-1整合酶重组质粒,在大肠杆菌BL21中诱导表达,通过对包涵体变性、复性,纯化得到了整合酶蛋白。 基于分子信标原理,设计了荧光和淬灭基团标记的DNA底物,通过荧光信号实时监测3' 加工反应,对酶反应的动力学性质进行研究。 结果表明,纯化的整合酶蛋白具有较高的活性,酶反应表现出显著的Mg²⁺偏好性。 酶动力学研究 (K_m = 131.79 nmol/L,K_cat = 0.0042 min ^-1) 表明,该分子信标方法和设计的DNA底物可用于整合酶3'加工反应动力学研究以及酶反应性质的研究。     

HIV-1整合酶蛋白的可溶性表达及功能研究

HIV 1整合酶是HIV病毒复制中一个重要的酶,也是治疗艾滋病药物的一个重要靶点。为了开展以整合酶蛋白为靶点的抑制剂筛选,构建HIV 1整合酶重组质粒,在原核细胞中进行可溶性表达和功能研究。通过重叠PCR技术引入F185K和C280S突变于HIV 1 B亚型标准株的整合酶cDNA片段中,PCR扩增片段克隆到pET 28a(+)表达载体中,构建重组质粒,在E. coli中进行整合酶基因表达,SDS PAGE鉴定表达产物,亲和层析纯化蛋白,酶联免疫吸附实验方法测定整合酶的生物学活性。结果构建的重组质粒获得高效稳定的可溶性表达,ELISA实验证实该蛋白具有整合酶的3′切割DNA和5′链转移的活性。HIV 1整合酶蛋白的可溶性表达和活性研究为建立以整合酶为靶点的抗HIV药物筛选平台打下了基础。     

T66I突变提高HIV-1整合酶可溶性研究

在研究HIV-1整合酶（IN）抗药性突变T66I时,发现这一突变同时可以提高整合酶的溶解性。原核表达了IN1–288/T66I和野生型（WT）,取菌体破碎后的上清, SDS-PAGE和his标签蛋白质染色进行分析,结果表明IN1–288/T66I可溶性约是WT的2.4倍。600 ml培养基中诱导表达IN1–288/T66I/BL21,亲和层析纯化共收获蛋白质4.72 mg。用改进的ELISA方法测定IN1–288/T66I和IN1 288/F185K /C280S链转移催化活性,结果显示两种蛋白质活性基本相当。提供了有别于F185K /C280S突变的另外一种整合酶可溶性表达的途径,IN1–288/T66I重组蛋白还可以应用到整合酶抑制剂筛选中,以获取避开T66I抗药性突变的抑制剂。     

HIV-1整合酶及其抑制剂的研究进展

HIV-1整合酶是由HIV病毒pol基因编码的分子量为32KD的蛋白质,是HIV病毒复制的必需酶之一,它催化病毒DNA整合入宿主染色体DNA。人类细胞中没有HIV 整合酶的类似物[1],理论上抑制整合酶对人体副作用很小。因此HIV-1整合酶成为继HIV-1蛋白酶,逆转录酶后治疗艾滋病的富有吸引力和合理的靶标。本文综述了HIV整合酶结构,抑制剂的研究以及以HIV-1 整和酶为靶点治疗AIDS方法的最新研究进展。     

E92A是HIV-1整合酶耐药突变N155S的活性回复突变

HIV-1整合酶是目前抗艾滋病药物研发的重要靶点之一,整合酶的耐药突变是导致整合酶抑制剂类药物治疗失败的主要原因,但突变产生耐药性的机理仍不清楚.本工作通过人工构建突变型整合酶,测试其活性和耐药性,对整合酶的耐药机理进行初步探索.构建整合酶的突变型包括E92A、N155S两种单突变及E92A/N155S双突变.通过基因工程操作引入突变、构建质粒、表达纯化得到整合酶蛋白.用基于磁珠的整合酶链转移ELISA测试整合酶的链转移活性,用S-1360和Raltegravir两种抑制剂测试整合酶的耐药性.另外,用Autodock软件做了S-1360和整合酶核心区(包括野生型和突变型)的分子对接.结果表明,N155S突变使整合酶链转移活性下降约80%,而E92A/N155S双突变仅使活性下降约42%,这表明N155S突变基础上的E92A突变可使整合酶的活性大幅回复.E92A和E92A/N155S对不同的抑制剂可产生不同的耐药性,它们对Raltegravir的耐药性强于对S-1360.突变对整合酶活性和耐药性的影响主要是通过改变整合酶活性中心结构实现的,E92A突变可能导致其与周围残基静电相互作用减弱,间接影响到D64和D116残基,产生活性回复作用.     

T66I突变提高HIV-1整合酶大肠杆菌表达可溶性研究

在研究HIV-1整合酶(IN)抗药性突变T66I时,发现这一突变同时可以提高整合酶的溶解性。原核表达了IN1?288/T66I和野生型(WT),取菌体破碎后的上清,SDS-PAGE和his标签蛋白质染色进行分析,结果表明IN1?288/T66I可溶性约是WT的2.4倍。600ml培养基中诱导表达IN1?288/T66I/BL21,亲和层析纯化共收获蛋白质4.72mg。用改进的ELISA方法测定IN1?288/T66I和IN1-288/F185K/C280S链转移催化活性,结果显示两种蛋白质活性基本相当。提供了有别于F185K/C280S突变的另外一种整合酶可溶性表达的途径,IN1?288/T66I重组蛋白还可以应用到整合酶抑制剂筛选中,以获取避开T66I抗药性突变的抑制剂。     

Four novel bis-(naphtho-gamma-pyrones) isolated from Fusarium species as inhibitors of HIV-1 integrase

Integration of viral DNA into host cell DNA is an essential step in retroviral (HIV-1) replication and is catalyzed by HIV-1 integrase. HIV-1 integrase is a novel therapeutic target and is the focus of efforts to identify effective inhibitors that will prevent/or cure HIV infections. Four novel naphtho-gamma-pyrones, belonging to the chaetochromin and ustilaginoidin family, were discovered as inhibitors of HIV-1 integrase from the screening of fungal extracts using a recombinant in vitro assay. These compounds inhibit both the coupled and strand transfer activity of HIV-1 integrase with IC(50) values of 1-3 and 4-12 microM, respectively. The discovery, structure elucidation, chemical modification and the structure-activity relationship of these compounds are described.     

Homogeneous high-throughput screening assays for HIV-1 integrase 3beta-processing and strand transfer activities

HIV-1 integrase (HIV-IN) is a well-validated antiviral drug target catalyzing a multistep reaction to incorporate the HIV-1 provirus into the genome of the host cell. Small molecule inhibitors of HIV-1 integrase that specifically target the strand transfer step have demonstrated efficacy in the suppression of virus propagation. However, only few specific strand transfer inhibitors have been identified to date, and the need to screen for novel compound scaffolds persists. Here, the authors describe 2 homogeneous time-resolved fluorescent resonance energy transfer-based assays for the measurement of HIV-1 integrase 3'-processing and strand transfer activities. Both assays were optimized for high-throughput screening formats, and a diverse library containing more than 1 million compounds was screened in 1536-well plates for HIV-IN strand transfer inhibitors. As a result, compounds were found that selectively affect the enzymatic strand transfer reaction over 3beta processing. Moreover, several bioactive molecules were identified that inhibited HIV-1 reporter virus infection in cellular model systems. In conclusion, the assays presented herein have proven their utility for the identification of mechanistically interesting and biologically active inhibitors of HIV-1 integrase that hold potential for further development into potent antiviral drugs.     

Virtual screening application of a model of full-length HIV-1 integrase complexed with viral DNA

To address the absence of experimental data on the full-length structure of HIV-1 integrase and the way it binds to viral and human DNA, we had previously [Karki, R. G.; Tang, Y.; Burke, T. R., Jr.; Nicklaus, M. C. J. Comput. Aided Mol. Des.2004, 18, 739] constructed models of full-length HIV-1 integrase complexed with models of viral and human DNA. Here we describe the discovery of novel HIV-1 integrase strand transfer inhibitors based on one of these models. Virtual screening methods including docking and filtering by predicted ADME/Tox properties yielded several microM level inhibitors of the strand transfer reaction catalyzed by wild-type HIV-1 integrase.     

Substrate features important for recognition and catalysis by human immunodeficiency virus type 1 integrase identified by using novel DNA substrates.

The integrase encoded by human immunodeficiency virus type 1 (HIV-1) is required for integration of viral DNA into the host cell chromosome. In vitro, integrase mediates a concerted cleavage-ligation reaction (strand transfer) that results in covalent attachment of viral DNA to target DNA. With a substrate that mimics the strand transfer product, integrase carries out disintegration, the reverse of the strand transfer reaction, resolving this integration intermediate into its viral and target DNA parts. We used a set of disintegration substrates to study the catalytic mechanism of HIV-1 integrase and the interaction between the protein and the viral and target DNA sequence. One substrate termed dumbbell consists of a single oligonucleotide that can fold to form a structure that mimics the integration intermediate. Kinetic analysis using the dumbbell substrate showed that integrase turned over, establishing that HIV-1 integrase is an enzyme. Analysis of the disintegration activity on the dumbbell substrate and its derivatives showed that both the viral and target DNA parts of the molecule were required for integrase recognition. Integrase recognized target DNA asymmetrically: the target DNA upstream of the viral DNA joining site played a much more important role than the downstream target DNA in protein-DNA interaction. The site of transesterification was determined by both the DNA sequence of the viral DNA end and the structure of the branched substrate. Using a series of disintegration substrates with various base modifications, we found that integrase had relaxed structural specificity for the hydroxyl group used in transesterification and could tolerate distortion of the double-helical structure of these DNA substrates.     

Development of an AlphaScreen-based HIV-1 integrase dimerization assay for discovery of novel allosteric inhibitors

In recent years, HIV-1 integrase (IN) has become an established target in the field of antiretroviral drug discovery. However, its sole clinically approved inhibitor, the integrase strand transfer inhibitor (INSTI) raltegravir, has a surprisingly low genetic barrier for resistance. Furthermore, the only two other integrase inhibitors currently in advanced clinical trials, elvitegravir and dolutegravir, share its mechanism of action and certain resistance pathways. To maintain a range of treatment options, drug discovery efforts are now turning toward allosteric IN inhibitors, which should be devoid of cross-resistance with INSTIs. As IN requires a precise and dynamic equilibrium between several oligomeric species for its activities, the modulation of this equilibrium presents an interesting allosteric target. We report on the development, characterization, and validation of an AlphaScreen-based assay for high-throughput screening for modulators of HIV-1 IN dimerization. Compounds identified as hits in this assay proved to act as allosteric IN inhibitors. Additionally, the assay offers a flexible platform to study IN dimerization.     

Equivalent inhibition of half-site and full-site retroviral strand transfer reactions by structurally diverse compounds.

In vitro assay systems which use recombinant retroviral integrase (IN) and short DNA oligonucleotides fail to recapitulate the full-site integration reaction as it is known to occur in vivo. The relevance of using such circumscribed in vitro assays to define inhibitors of retroviral integration has not been formerly demonstrated. Therefore, we analyzed a series of structurally diverse inhibitors with respect to inhibition of both half-site and full-site strand transfer reactions with either recombinant or virion-produced IN. Half-site and full-site reactions catalyzed by avian myeloblastosis virus and human immunodeficiency virus type 1 (HIV-1) IN from virions are shown to be equivalently sensitive to inhibition by compounds which inhibit half-site reactions catalyzed by the recombinant HIV-1 IN. These studies therefore support the utility of using in vitro assays employing either recombinant or virion-derived IN to identify inhibitors of integration.     

Rational design of 2-pyrrolinones as inhibitors of HIV-1 integrase

HIV-1 integrase is an essential enzyme for viral replication and a validated target for the development of drugs against AIDS. With an aim to discover new potent inhibitors of HIV-1 integrase, we developed a pharmacophore model based on reported inhibitors embodying structural diversity. Eight compounds of 2-pyrrolinones fitting all the features of the pharmacophore query were found through the screening of an in-house database. These candidates were successfully synthesized, and three of them showed strand transfer inhibitory activity, in which, one compound showed antiviral activity. Further mapping analysis and docking studies affirmed these results.     

Structural insights into the retroviral DNA integration apparatus

Retroviral replication depends on successful integration of the viral genetic material into a host cell chromosome. Virally encoded integrase, an enzyme from the DDE(D) nucleotidyltransferase superfamily, is responsible for the key DNA cutting and joining steps associated with this process. Insights into the structural and mechanistic aspects of integration are directly relevant for the development of antiretroviral drugs. Recent breakthroughs have led to biochemical and structural characterization of the principal integration intermediates revealing the tetramer of integrase that catalyzes insertion of both 3' viral DNA ends into a sharply bent target DNA. This review discusses the mechanism of retroviral DNA integration and the mode of action of HIV-1 integrase strand transfer inhibitors in light of the recent visualization of the prototype foamy virus intasome, target DNA capture and strand transfer complexes.