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整合酶核心区野生型和F185K突变型活性和溶解性的比较及分子动力学模拟分析

表达纯化了野生型(WT)及F185K突变型HIV-1整合酶核心区蛋白(IN_C),并对二者的溶解性和活性进行了比较．实验结果表明：F185K 突变后IN_C溶解性显著提高,活性有一定程度降低．对WT和F185K IN_C体系进行了1800 ps的分子动力学模拟．模拟结果表明：F185K IN_C功能loop区柔性和蛋白质整体运动性降低,使蛋白质活性降低,F185K突变后盐桥网络的变化驱动了IN_C局部构象改变,引起IN_C表面的部分疏水残基被包埋,亲水残基暴露,相对亲水溶剂可接近面积增大,同时,突变后IN_C与水之间形成氢键的数量增加,与水之间作用加强,以上变化使IN_C溶解性提高．分子动力学模拟与实验结果相吻合．为理解蛋白质溶解性和对蛋白质进行可溶性改造提供了一定的理论依据．     

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.     

HIV-1整合酶的功能及其抑制剂的研究进展

I型人类免疫缺陷病毒（human immunodeflciency virustype1,HIV-1）在宿主细胞内经逆转录得到的cDNA,由整合酶（integrase,ry）催化插入到宿主基因组DNA中,该过程称为整合过程。整合是HIV-1复制周期中不可缺少的步骤,对于病毒的复制至关重要,因此对整合酶的抑制能够有效地起到抗HIV的作用。该文综述了整合酶的结构与功能以及目前关于整合酶抑制剂的最新研究进展。     

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整合酶链转移反应抑制剂的荧光筛选方法

整合酶被认为是抗HIV-1药物研究的理想靶点之一。为了建立便捷高效的整合酶链转移反应抑制剂筛选方法,首先将HIV-1整合酶原核表达载体pNL-IN转化入大肠杆菌感受态细胞BL21(DE3)进行原核表达,并用镍琼脂糖凝胶进行亲和纯化,获得了纯度和活性均较高的整合酶重组蛋白;然后设计了生物素标记的供体DNA和FITC标记的靶DNA,用链霉亲和素磁珠捕获反应体系中的DNA产物;最后用荧光分析仪检测DNA产物的荧光信号,并计算待测样品的抑制率。用已知整合酶抑制剂S-1360和MK-0518对筛选方法进行了验证,测定结果与已有实验数据相当,表明本筛选方法能够有效应用于HIV-1整合酶链转移反应抑制剂的筛选。与现有的整合酶链转移反应抑制剂筛选方法相比,本筛选方法步骤更为简化、耗时更短、成本更低。     

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。     

Sp100与HIV-1整合酶互作并抑制其介导的病毒整合

Sp100是核颗粒ND10的组成蛋白,在哺乳动物细胞中广泛存在．Sp100参与多种细胞生理病理过程,如转录调控、细胞内抗病毒免疫等．利用酵母双杂交系统,我们发现了Sp100的互作蛋白HIV-1整合酶,免疫共沉淀实验进一步证实了Sp100与 HIV-1整合酶的互作,细胞内荧光共定位实验也证实了二者在细胞内部分共定位．此外,突变体实验表明,Sp100的C端300～480氨基酸和HIV-1的催化结构域是两个蛋白质的互作区域．利用siRNA降低细胞内Sp100的表达量,可以增加HIV-1整合酶介导的病毒的整合,反之,细胞内过表达Sp100则会降低HIV-1整合酶介导的病毒的整合．这是首次发现Sp100可以和HIV-1整合酶发生相互作用,并进而抑制病毒的整合．我们发现了Sp100作为HIV-1整合酶互作蛋白的新功能,并扩展了细胞防御病毒感染的相关研究．     

Constructing HIV-1 integrase tetramer and exploring influences of metal ions on forming integrase-DNA complex

HIV-1 integrase (IN) is essential for the replication of HIV-1 in human cells. At present, the complete structure of complex IN-DNA has not been resolved. In this paper, a HIV-1 IN tetramer model was built with homology modeling and molecular dynamics simulation approach, in which two Mg2+ ions were reasonably located in each catalytic core domain. Moreover, it was found that the AB and CD chains of HIV-1 IN tetramer were different in the structures and metal ions of HIV-1 IN tetramer might have great influences on DNA locating on IN. These findings may provide a more complete structural basis for guiding drug discovery and revealing integration mechanism.     

计算机辅助设计抗HIV-1整合酶抑制剂的研究进展

HIV-1整合酶是HIV-1生命周期中必不可少的酶之一,已成为目前最具潜力的抗HIV药物设计的靶点之一。2007年,Merk公司研发的MK-5108作为首个整合酶抑制剂药物被美国食品药物管理局批准上市,标志着整合酶抑制剂研究的重大突破,也激发了抗HIV-1整合酶抑制剂研究的新一轮高潮。计算机辅助药物设计(computer-aided drug design,CADD)具有效率高、成本低等特点,基于计算机辅助手段合理药物设计已取得了很大的进展。文章综述了近几年来计算机辅助设计抗HIV整合酶抑制剂及耐药机理方面的研究进展。     

Crystal structure of the HIV-1 integrase core domain in complex with sucrose reveals details of an allosteric inhibitory binding site

HIV integrase (IN) is an essential enzyme in HIV replication and an important target for drug design. IN has been shown to interact with a number of cellular and viral proteins during the integration process. Disruption of these important interactions could provide a mechanism for allosteric inhibition of IN. We present the highest resolution crystal structure of the IN core domain to date. We also present a crystal structure of the IN core domain in complex with sucrose which is bound at the dimer interface in a region that has previously been reported to bind integrase inhibitors.

Structured summary

MINT-7713125: IN (uniprotkb:P04585) and IN (uniprotkb:P04585) bind (MI:0407) by X-ray crystallography (MI:0114)     

Active site binding modes of the beta-diketoacids: a multi-active site approach in HIV-1 integrase inhibitor design

Predicting a bioactive conformation of a ligand is of paramount importance in rational drug design. The task becomes very difficult when the receptor site possesses a region with unusual conformational flexibility. Significant conformational differences are present in the active site regions in the available crystal structures of the core domains of HIV-1 integrase (IN). Among all reported IN inhibitors, the β-diketoacid class of compounds has proved to be of most promise and indeed S-1360 was the first IN inhibitor to enter clinical studies. With an aim to predict the bioactive (active site bound) conformation of S-1360, we performed extensive docking studies using three different reported crystal structures where the active site or partial active site region was resolved. For comparison we extended our studies to include 5CITEP (the first compound cocrystallized with IN core domain) and a bis-diketoacid (BDKA). We found that the conformation of S-1360 when bound in one of the active sites matches that of the experimentally observed results of IN escape mutants resistant to S-1360. Therefore, we propose that this active site conformation is the biologically relevant conformation and can be used for the future structure-based drug design studies selectively targeting IN.     

Molecular dynamics studies of the full-length integrase-DNA complex

We have carried out a molecular dynamics (MD) simulation of full-length HIV-1 integrase (IN) dimer complexed with viral DNA with the aim of gaining information about the enzyme motion and investigating the movement of the catalytic flexible loop (residues 140-149) thought to be essential in the catalytic mechanism of IN. During the simulation, we observed quite a different behavior of this region in the presence or absence of the viral DNA. In particular, the MD results underline the crucial role of the residue Tyr143 in the mechanism of integration of viral DNA into the host chromosome. The present findings confirm the experimental data (e.g., site-directed mutagenesis experiments) showing that the loop is involved in the integration reactions and its mobility is correlated with the catalytic activity of HIV-1 integrase.     

Anti-HIV-1 integrase diterpenoids from Dichrocephala benthamii

Five new clerodane diterpenoid dichrocephnoids A–E (1,2, 6–8) together with three known analogues were isolated from the whole herb of Dichrocephala benthamii C.B. Clarke. Their structures were elucidated on the basis of spectroscopic analyses (UV, IR, MS, 1D and 2D NMR). The relative configurations of the new compounds were established by NOESY correlations, and the absolute configuration of 1 was determined on the basis of CD methods. Dichrocephnoid A (1) possesses a very rare pentacyclic ring system with highly oxygenated functionalities. All of the isolated compounds exhibited inhibitory activity against HIV-1 integrase, however, dichrocephnoid C (6) showed the most promising inhibition of the enzyme with IC₅₀ value of 12.35 ± 1.27 μM.     

The strand transfer oligonucleotide inhibitors of HIV-integrase

Retroviral integrase participates in two catalytic reactions, which require interactions with the two ends of the viral DNA in the 3′processing reaction, and with a targeted host DNA in the strand transfer reaction. The 3′-hydroxyl group of 2′-deoxyadenosine resulting from the specific removing of GT dinucleotide from the viral DNA in the processing reaction provides the attachment site for the host DNA in a transesterification reaction. We synthesized oligonucleotides (ONs) of various lengths that mimic the processed HIV-1 U5 terminus of the proviral long terminal repeat (LTR) and are ended by 2′-deoxyadenosine containing a 3′-O-phosphonomethyl group. The duplex stability of phosphonomethyl ONs was increased by covalent linkage of the modified strand with its complementary strand by a triethylene glycol loop (TEG). Modified ONs containing up to 10 bases inhibited in vitro the strand transfer reaction catalyzed by HIV-1 integrase at nanomolar concentrations.     

Enzymatic capability of HIS-tagged HIV-1 integrase using oligonucleotide disintegration substrates

Disintegration, wherein a half-site integration substrate is resolved into separate viral and host DNA components via DNA strand transfer, is one of three well-established in vitro activities of HIV-1 integrase. The role of disintegration in the HIV-1 replicative cycle, however, remains a mystery. In this report, we describe the expression inEscherichia coli and purification of HIV-1 integrase as a fusion protein containing a 6×His tag at its amino terminus. Integrase resolved dumbbell and Y-substrates optimally at pH 6.8–7.2 in the presence of 2 mM MnCl₂. Substrate requirements for intramolecular disintegration included a 10 base pair viral U5 LTR arm and a CA dinucleotide located at the 3 end of the LTR. Disintegration was not sensitive to changes in the host DNA portion of the substrate. A dumbbell substrate with a 5 oligo-dA tail also underwent disintegration. The released LTR arm with an oligo-dA tail was utilized as a template primer by several DNA polymerases indicating that disintegration occurred via nucleophilic attack on the phosphodiester bond located immediately adjacent to the CA dinucleotide at the 3 end of the LTR. Coupled disintegration-DNA polymerase reactions provided a highly efficient and sensitive means of detecting disintegration activity. Integrase also catalyzed an apparently concerted disintegration-5-end joining reaction in which an LTR arm was transferred from one dumbbell substrate molecule to another.     

A novel short peptide is a specific inhibitor of the human immunodeficiency virus type 1 integrase

The retroviral encoded protein integrase (IN) is required for the insertion of the human immunodeficiency virus type 1 (HIV-1) proviral DNA into the host genome. In spite of the crucial role played by IN in the retroviral life cycle, which makes this enzyme an attractive target for the development of new anti-AIDS agents, very few inhibitors have been described and none seems to have a potential use in anti-HIV therapy. To obtain potent and specific IN inhibitors, we used the two-hybrid system to isolate short peptides. Using HIV-1 IN as a bait and a yeast genomic library as the source of inhibitory peptides (prey), we isolated a 33-mer peptide (I33) that bound tightly to the enzyme. I33 inhibited both in vitro IN activities, i.e. 3' end processing and strand transfer. Further analysis led us to select a shorter peptide, EBR28, corresponding to the N-terminal region of I33. Truncated variants showed that EBR28 interacted with the catalytic domain of IN interfering with the binding of the DNA substrate. Alanine single substitution of each EBR28 residue (alanine scanning) allowed the identification of essential amino acids involved in the inhibition. The EBR28 NMR structure shows that this peptide adopts an alpha-helical conformation with amphipathic properties. Additionally, EBR28 showed a significant antiviral effect when assayed on HIV-1 infected human cells. Thus, this potentially important short lead peptide may not only be helpful to design new anti-HIV agents, but also could prove very useful in further studies of the structural and functional characteristics of HIV-1 IN.