摘要
研究背景和目的
艾滋病(Acquired immunodeficiency syndrome, AIDS)是由人类免疫缺陷病毒(Human immunodeficiency virus, HIV)感染引起的疾病,严重威胁人类的生存,对人口健康和社会经济的发展产生巨大的影响,全世界估计有3400万人感染艾滋病。艾滋病病毒的基因组比已知任何一种病毒基因都复杂,而且变化多样。到目前为止,依然没有一种疫苗可以付诸大规模使用。在今后相当长的一段时期内,发展安全有效的抗艾滋病药物,依然是当前艾滋病预防和治疗的重点。
迄今为止已有24种化合物实体和至少9种组合物被批准用于临床。可分为逆转录酶抑制剂、蛋白酶抑制剂、整合酶抑制剂和病毒进入抑制剂四大类。前两类药物易诱导HIV耐药性突变,而且对人体有较大的毒副作用,后两类仅有
3种化合物实体被批准。
HIV进入抑制剂,尽管目前仅有Enfuvitide (T-20)和Maraviroc两种,但它们对耐受前两类抗HIV药物的病毒依然有效。并且,进入抑制剂是作用在HIV感染的早期阶段,能够阻止HIV与靶细胞的融合,被认为在艾滋病的防治上具有更好的应用前景。开发新的阻止病毒进入的抗HIV药物也成为目前抗艾滋病药物研究的热点。
HIV进入靶细胞的过程主要由包膜蛋白(Envelope Proteins, Env)介导。在HIV进入靶细胞过程中,首先是gp120与靶细胞上的CD4分子和辅助受体(趋化因子受体CCR5或CXCR4等)先后结合,随后跨膜亚基gp41的构型发生改变,介导病毒包膜与靶细胞膜的融合,完成病毒进入宿主细胞的感染过程。从病毒融合机制的角度,HIV进入抑制剂可分为三大类,一是干扰gp120与CD4受体结合；二是拮抗辅助受体,阻止其与gp120-CD4复合物结合；第三类是阻止gp41核心结构的形成,称为HIV融合抑制剂。目前,针对HIV进入抑制剂的研究主要集中在两个方面：一是针对gp120为作用靶点,因为HIV gp120与靶细胞上的CD4受体相结合是病毒进入靶细胞的第一步,如果能在病毒感染的第一步就进行抑制,理论上则可对人体的危害性降到最低,有更好的应用前景；二是针对gp41六螺旋束核心骨架的形成进行抑制。
作用于HIV gp120的进入抑制剂主要为大分子的抑制剂和小分子的抑制剂两大类。其中,大分子抑制剂主要为可溶性CD4及其衍生物以及靶向gp120的广谱中和抗体两大类；小分子的抑制剂主要以抑制gp120与CD4结合为靶点,或者以Phe43结合空腔为靶点。其化学结构主要为BMS-378806、BMS-488043和NBD-556、NBD-557及其类似物。
Zhou T等人从HIV感染者的血清中分离出两个抗体,VRC01和VRC02,它们能够抑制200多各种亚型HIV毒株中的90%,这是迄今为止分离得到的最广谱、最有效的中和抗体。通过VRC01-gp120共结晶的单晶x-射线衍射结构分析,VRC01的结构与CD4有一定的相似性,能部分地模拟CD4与gp120的结合。与CD4不同的是,VRC01在CD4与gp120结合方向上偏转了43°，并且离开了6A的距离。这使得VRC01既能结合于gp120上易受攻击的CD4作用位点,又能克服gp120表面糖基及构象的掩盖作用。VRC01上CDR H2的Arg61能插入到gp120上V5与p24围成的空腔中,并形成5个氢键的作用。Arg61结合靶穴可能是中和抗体具有广谱、高效活性的重要作用靶点。如果化合物能结合在这一结合靶点,则可能具有较好地抗HIV感染的活性。这项研究为抑制HIV的感染找到了座标,使得研制艾滋病疫苗的目标更加明确；同时,也为设计作用于gp120的小分子化合物找到了新的作用靶点,目前还未见报道作用于此靶点的小分子化合物。
小分子进入抑制剂研究方面：BMS-378806与BMS-488043结合在gp120上的CD4结合位点,从而阻止gp120与CD4的结合,其IC50为5nmol/L。 BMS-378806进入了Ⅰ期临床试验研究,但因其抗病毒谱太窄被中止了进一步的临床研究。BMS-488043进入了Ⅱ期临床研究,但因为剂量相对太大(1800mg)而被暂停。针对BMS-378806所做的类似物的结构改造工作并没有取得很大的突破,目前还没有药物上市。NBD-556、NBD-557是本课题组大规模筛选33000个化合物的基础上得到的两个小分子化合物,化学结构为N-苯基N'-哌啶基草酰胺衍生物。作用机制研究表明,NBD-556能抑制gp120与CD4的结合,并且是特异性地作用于gp120,而不是受体CD4分子。NBD-556能起到类似可溶性CD4的作用,结合到gp120上的CD4Phe43结合口袋位置,诱导gp120发生构象变化,使gp120形成辅助受体结合位点。NBD-556活性在μM级,如何进行结构改造,提高其抗HIV活性,以及对各种亚型HIV病毒株的广谱抑制活性,是这类小分子化合物研究的关键。
基于以上文献报道,我们拟从以下两方面进行研究：
一、基于Arg61结合靶穴的化合物库虚拟筛选,并对虚拟筛选打分较高的化合物进行病毒活性测试。对活性化合物进行作用机制研究,并对活性化合物进行结构修饰,以期得到活性更好的化合物。
二、基于Phe43结合靶穴的化合物结构改造及活性筛选。主要是以NBD-556为先导物,对其进行结构分析,并设计合成系列类似物,对合成的类似物进行抗HIV活性测试。总结其构效关系,以期发现广谱、高效的小分子化合物。
方法
对文献报道的gp120-VRC01复合物的单晶结构进行分析。VRC01的广谱中和活性与其CDR H2的Arg61能插入到gp120上V5与β24序列围成的空腔(Arg61靶穴)有关,我们首先在曙光W580I桌面超级计算机上,采用分子动力学模拟,运用MM-PBSA方法计算gp120与Arg61(位点1)以及VRC01的58-61序列(位点2)的结合自由能。利用分子对接方法,从库容为40000的IBS天然产物数据库中虚拟筛选能与gp120上的位点1和位点2结合的化合物,并进一步计算gp120与这些化合物的结合自由能变化。对结合自由能降低较大的化合物进行抗HIV假病毒及真病毒的活性筛选,确定先导化合物。最终筛选得到化合物NC-2,并对其抑制病毒感染活性的机制进行研究。同时以NC-2为先导物,对其进行结构修饰,合成系列类似物,并进行活性筛选。
(1)配体数据库：ibs2009oct_nc:受体：3NGB-A；位点1Arg61,位点258到61号氨基酸序列。
(2)用Surflex-Dock中基于配体的方法生成Protomol文件,利用分子对接技术,对ibs2009oct_nc天然产物库进行虚拟筛选,得到同时与两个位点结合打分较高的化合物。
(3)计算打分较高的化合物与gp120结合的自由能变化情况,使用MM/PBSA的方法进行计算。并与△Ggp120-VRC01和△Ggp120-CD4进行比较。
(4)对结合自由能变化较大的化合物购买,并测试其抗HIV病毒感染的活性,得到小分子NC-2候选化合物。
(5)由于化合物NC-2有较好的抑制HIV假病毒与HIV-1mB实验株活性,所以对其作用机制进行研究。通过细胞-细胞融合实验,病毒-细胞融合实验,时间-过程实验,判断NC-2是作用于HIV的进入阶段；通过抑制gp120与CD4的结合实验,抑制gp41六螺旋束形成实验,研究其抑制HIV进入的作用靶点。
(6)以NC-2为先导物,进行结构改造,设计合成系列化合物,并进行化学合成及抗HIV活性测试。
目前文献报道的NBD-556类似物的改造,其活性并没有明显提高。对NBD-556化学结构进行分析,2,2,6,6-四甲基-哌啶基及中间的草酰胺连接骨架与其活性密切相关。因此,我们拟保留四甲基哌啶基,以及中间的草酰胺连接骨架,对其R取代基进行结构修饰,连接不同取代基的芳香环及杂环,并对合成的化合物抑制HIV感染的活性进行研究。
(1)以草酰二乙酯或草酰氯单乙酯为原料,通过酯的胺解反应及取代反应,合成系列NBD-556类似物。
(2)对合成的化合物,进行抗HIV活性测试。
结果
以Arg61结合靶穴为靶点进行虚拟筛选：分别以Arg61,58到61号氨基酸序列作为两个位点,对库容为40000的ibs2009oct_nc天然产物数据库进行筛选。先使用Surflex-Dock中基于配体的方法生成Protomol文件,使用12个CPU并行计算,其它参数使用默认值。对接得到位点1得分大于8.0的化合物113个,位点2得分大于8.0的化合物180个,其中有30个化合物既能与gp120上的位点1结合,也能与位点2结合,选取这30个化合物,进行结合自由能的计算。
体系中的结合自由能的计算采用MM/PBSA与MM/GBSA的方法,分别计算了候选的30个化合物与gp120结合后的自由能差,并与△Ggp120-VRC01和△Ggp120-CD4相比较。gp120与Arg61(位点1)结合自由能的变化为：PBTOT=-10.67,GBTOT=-13.89.gp120与58-61序列(位点2)的结合自由能,PBTOT=-18.37,GBTOT=-16.27。其中,gp120与58-61序列的结合自由能降低值要大于gp120与Arg61结合自由能的降低值,说明58-61序列与gp120的结合能力要强于单一的氨基酸残基Arg61与gp120的结合能力。gp120与CD4的结合自由能,PBTOT=-57.95,GBTOT=-23.77.gp120与VRC01的结合自由能,PBTOT=-113.62,GBTOT=-78.88。通过计算可知,△Ggp120-VRC01小于△Ggp120-CD4,说明gp120与抗体VRC01的结合作用要强于gp120与CD4受体的结合,这个计算结果也能说明为什么VRC01结构与CD4类似,却具有更强效的中和能力,能阻止gp120与CD4结合。
对侯选化合物与gp120结合自由能变化较大的19个化合物购买,并用假病毒体系进行抗HIV感染活性筛选。其中NC-2与NC-12有较好的抑制活性,并且这两个化合物与gp120结合自由能变化值也较大。NC-2抗HIV-1JRFL假病毒的IC50为10.58μM,抗HIV-1实验株HIV-1IIIB的IC50为1.95μM。而且NC-2对靶细胞的毒性较小,其对U87.CD4.CCR5细胞的CC50为121.25μM,对MT-2细胞的CC50为17.23μM。NC-12虽然也具有一定的抗病毒感染的活性,但其抑制病毒感染IC50和对靶细胞毒性CC50接近,因此NC-12对细胞毒性较大。其抗病毒活性可能是由于细胞毒性引起的。
对NC-2抑制HIV感染的作用机制进行研究。NC-2能抑制细胞-细胞融合,以及病毒-细胞融合,时间-过程实验进一步确认NC-2作用于HIV感染的进入阶段,且作用于HIV的包膜蛋白(统计学采用2因素析因分析方法,P<0.001)。NC-2体外能抗gp120与CD4的结合,不能抑制gp41六螺旋束的形成(统计学采用单因素方差分析,多重比较LSD方法,P<0.001)。说明NC-2可能是通过抑制HIV gp120与CD4的结合,从而具有抑制HIV感染的作用。
NC-2是天然产物中分离得到的,其来源有限,且活性在μμM级。需要对其进行结构优化,以期得到活性更好的小分子进入抑制剂。对NC-2化学结构进行分析,设计了两类化合物,并进行化学合成。其中,合成了2-(3,4,5-三甲氧基)苯甲酰基-5-(苯乙炔基)噻吩类似物9个。对其进行抗HIV假病毒活性测试,发现化合物7b和7c有抑制HIV-1JRFL假病毒感染的活性,其IC50分别为：21.36±0.78μM和15.14±6.27μM。
以草酸二乙酯或草酰氯单乙酯为原料,通过酯的胺解反应及取代反应,合成系列NBD-556类似物8个。对其抗HIV活性进行测试,发现经结构改造后的化合物部分具有抗HIV假病毒感染的活性,但与NBD-556相比,活性没有明显提高。
结论
以Arg61结合靶穴为靶点,通过计算机虚拟筛选辅助病毒活性测试的方法,得到小分子化合物NC-2具有较好的抗HIV假病毒与HIV-1ⅢB病毒的活性。经作用机制研究,发现NC-2是作用于HIV感染的进入阶段,且作用靶点为HIV包膜蛋白。进一步的机制研究表明,NC-2体外能抗gp120与CD4的结合,不能抑制gp41六螺旋束的形成。这与我们虚拟筛选的目的一致,说明Arg61结合靶穴是小分子化合物抗HIV感染的一个重要作用靶点。此筛选模型有一定的可信度,有可能用于其它化合物库的大规模虚拟筛选。
以NC-2为先导物,进行结构改造,己合成了2-(3,4,5-三甲氧基)苯甲酰基-5-(苯乙炔基)噻吩类似物9个。活性测试结果表明,化合物7b和7c有抑制HIV-1JRFL假病毒感染的活性,其IC50分别为：21.36±0.78μM和15.14±6.27μM。另一个系列的化合物的合成也已取得了突破性的进展。对其进一步的结构优化及构效关系研究,有可能得到活性更好的小分子化合物。
以NBD-556为先导物,进行结构优化,合成了NBD-556系列类似物8个,抗HIV假病毒感染的活性表明,结构改造后,化合物的活性没有明显提高。
Background and Objection
An estimated34million people are infected worldwide with human immuno-deficiency virus (HIV), the etiologic cause of acquired immunodeficiency syndrome (AIDS). The AIDS epidemic becomes more and more serious in the world. The successful development of a safe and effective HIV vaccine is still in the future. Therefore, research continues to focus on disease treatment by chemical anti-HIV agents.
Until now,24chemical entities and9compositions were approved in clinical. They consist of highly active antiretroviral therapy (HAART), protease inhibtor (PR), integrase inhibitor and virus entry inhibitor. On HAART and PR regimens, multiple drug therapies can lead to increased adverse effects and toxicities due to long-term use and drug-drug interactions. Only3chemical entities of last two classes were approved.
Among these targets, viral entry is one of the most promising for HIV drug development. HIV entry inhibitor, although only2chemical entities were approved, was found effective for the virus resistant to HARRT and PR therapy. Enfuvirtide (T-20), which is a HIV fusion inhibitor, was the first drug with a target other than RT and PR to be approved by the US FDA. The success of enfuvirtide and maraviroc validates the clinical application of viral entry inhibitors as a new class of antiretroviral drugs. Continued effort in discovering new HIV entry inhibitors, especially potent, orally bioavailable small molecules, is still needed.
The progress that HIV entry into host cells involves at least three steps:(1) first, an attachment step, this step requires CD4receptor binding,(2) second, co-receptor binding,(3) third, a fusion process, HIV fuse with target cellular membrane. The envelope glycoprotein (Env) on the surface of virus mediates the entry progress. Virus can attach to target cells, followe by specific binding of HIV gp120to the CD4receptor on the cellular membrane. This binding induces a conformational change in gp120that opens up a high-affinity binding site located within the third variable loop (V3) and surrounding surfaces for the chemokine co-receptors (primarily CCR5and CXCR4). Co-receptor binding results in further conformational rearrangements of gp120that expose the fusion-peptide domain of gp41. The heptad repeat (HR) regions, HR1and HR2of the three subunits of gp41, fold and pack into a six-helix bundle, which brings the viral and cell membranes into juxtaposition and creates pores in the target cell membrane, enabling the release of viral capsid into the cytoplasm.
From the virus fuse target cells progress, different entry inhibitors can be divided into3classes:(1) first, gp120-CD4binding inhibitors,(2) second, co-receptor binding inhibitors, and (3) fusion inhibitors, especially inhibit gp41core structure formation. Each step of this sequential entry process has been suggested as a potential target for developing anti-HIV-1drugs. Among HIV entry into host cells progress, an attachment step that gp120binds to CD4was the first step. HIV entry inhibitors can target this first step can theoretically reduce the harmful to human.
The binding of HIV-1gp120to the cellular receptor CD4is critical for HIV-1 entry into cells and has also been suggested as a potential target for developing anti-HIV-1therapy. HIV entry inhibitors that block gp120-CD4interactions mainly consist of two types of compounds, large molecular compounds and small molecular compounds. Large molecular inhibitors were sCD4and neutralization antibodies and small molecular inhibitors consist of BMS-378806and NBD-556analogues. The Phe43cavity has been suggested as the putative binding site of BMS-378806and NBD-556, potent entry inhibitors. This discovery is fundamentally important and raised hopes that small molecule inhibitors are capable of disrupting protein-protein interactions when targeted to the so-called "hot spots" such as the cavity.
Zhou et al isolated two antibodies, which have broad neutralization of diverse viral strains from immunoglobulin genes from individual cells. They were the mostly broad neutralization antibodies which can neutralize over90%of circulating HIV-1isolates. The crystal structure of VRC01in complex with a human immunodeficiency virus HIV-1gp120core showed that VRC01partially mimics CD4interaction with gp120. A shift from the CD4-defined orientation, however, focuses VRC01onto the vulnerable site of initial CD4attachment, allowing it to overcome the glycan and conformational masking that diminishes the neutralization potency of most CD4-binding-site antibodies. V5loop is wedged in the gap formed by the heavy and light chains of VRC01. Arg61in the CDR H2penetrates into the cavity formed by gp120V5and β24, locking V5into a less flexible conformation. In contrast, CD4only interacts with the "front side" of V5. Heavy chain Arg61goes behind the V5and provides5hydrogen bonds to the cavity formed by gp120V5and β24. These results provide a foundation for rational vaccine design that is based not only on the particular mode of antibody-antigen interaction but also on defined relationships between genomic antibody precursors, somatic hypermutation, and interface-recognition element. It also built a new target for designing anti HIV gp120 compounds. Until now, there is no report that small molecular compounds bind to this hot-spot.
BMS-378806is the most potent small molecule inhibitor reported so far that blocks the interaction between gp120and CD4with IC505nmol/L. BMS-378806enter clinical I research and the further clinical investigation was suspended because of its narrow antiviral range. BMS-488043enters clinical II, and it was also suspended because of large dose. Until now, there are no BMS-378806analogues to be approved.
We screened a drug-like small molecule chemical library consisting of approximately33,000compounds using a syncytium formation assay and identified two N-phenyl-N-piperidin-4-yl-oxalamide analogs which significantly inhibited syncytium formation. Both NBD-556and NBD-557showed potent inhibitory activity against gp120-CD4interaction. These two compounds interfere with the gpl20-CD4interaction by binding to unliganded gp120core, resulting in inhibiting HIV-1particles from attaching to the cellular receptor, the first step of HIV entry into target cells. NBD-556and NBD-557did not inhibit gp41six-helix bundle formation, nor interact with co-receptors. Development of a new HIV-1entry inhibitor is important
Based on the above reported in the literature, we begin our research from the following two parts:
(1) Virtual screening method was used for screening the compound library based on Arg61. Then, we calculate the difference of the difference binding free energy of gp120with higher score compounds. The compounds with higher difference of binding free energy were purchased and their anti HIV activities were also examined. The lead compound NC-2was obtained, and its mechanism of action was also examined. The structure of active compounds were also been modified in order to get more active compounds.
(2) Synthesis and activity of compounds which block the gp120-CD4interaction using NBD-556as lead compound were studied.
Methods and materials
The crystal structure of VRC01in complex with a human immunodeficiency virus HIV-1gp120core showed that its broad neutralization had a relationship with its complex structure, especially with the hydrogen bonds that Arg61penetrating into the V5and β24cavity. We use MM/PBSA method to calculate the difference binding free energy of gp120and Arg61or58-61amino acid residues. Virtual screening method was used for screening the compound library based on binding site1and2. Then, we calculate the difference of the difference binding free energy of gp120and higher score compounds. The compounds with higher difference of binding free energy were purchased and their anti HIV activities were also examined. The lead compound NC-2was obtained, and its mechanism of action was also examined.
(1) Ligand database, ibs2009oct_nc
Receptor,3NGB-A
Binding site, site1, Arg61, site258to61amino acid residues
(2) Generate protomol files with surflex-dock. Virtual screen the ibs2009oct_nc compound library.
(3) Calculate the difference of free binding energy of high scored compounds and binding site using MM/PBSA method.
(4) Purchase the compounds of larger difference of free binding energy for activity test.
(5) Lead compound NC-2was obtained. It has good inhibitory acitivities on the infection of pseudotyped HIV-1JRFL and HIV-1mB virus. The mechanism of action was also studied.
(6) Analogues based on NC-2were synthesized, and the activities were examined.
The activities of reported NBD-556analogues did not significantly improve compare to NBD-556. We designed and synthesized NBD-556analogues and examined their inhibition activities againt HIV infection. The residues of2,2,6,6-tetramethyl-piperidine and oxalamine were remained. The substituent was introduced on R position; it maybe substituted aromatic group or aromatic heterocyclic compound. The synthetic route is the addition of substituted aniline to ethyl chlorooxoacetate in the presence of base or amidation reaction using aniline and diethyloxalate. Analogues based on NBD-556were synthesized, and the activities were examined.
Results
Virtual screening the ibs2009oct_nc natural compound library with Arg61and58to61amino acid residues as two binding sites was proceeded. Firstly, generate protomol files with surflex-dock using12CPU calculate, other parameters were default values.113compounds with docking scores greater than8.0which bind site1were obtained.180compounds with docking scores greater than8.0which bind site2were obtained. Among them,30compounds can bind both site1and site2. We choose these30compounds and calculate the difference of free binding energy.
We calculated the difference of free binding energy of high scored compounds and gp120with MM/PBSA method. Compare them with ΔGgP120-VRC01and ΔGgp120-CD4. ΔGgp120-Arg61, PBTOT=-10.67, GBTOT=-13.89.ΔGgp120-(58-61), PBTOT=-18.37, GBTOT=-16.27。ΔGgp120-(58-61) smaller than ΔGgp120-Arg61showed that the affinity of gp120bind to58-61amino acid residues is stronger than that of gp120with Arg61. ΔGgp120-VRC01, PBTOT=-113.62, GBTOT=-78.88. ΔGgp120-CD4, PBTOT=-57.95, GBTOT=-23.77. The results of ΔGgp120-VRC01smaller than ΔGgp120-CD4also showed that the affinity of gp120bind to VRC01is stronger than that of gp120bind to CD4. So VRC01has good neutralization ability.
Purchase the compounds with higher difference of binding free energy and examine their anti HIV activities. A Total of19molecules that showed higher difference of binding free energy were screened from40000molecules. Among them, NC-2showed anti-HIV-1activities against the infection of HIV-1pseudotyped virus and laboratory-adapted HIV-1. The IC50values of NC-2for inhibiting infection against HIV-1ⅢB and pseudotyped HIV-1JRFL were1.95±0.44μM and10.58±0.13μM, respectively. The CC50values of NC-2for inhibit U87.CD4.CCR5cell and MT-2cell were121.25μM and17.23μM, respectively. NC-12has a similar IC50and CC50values, so it inhibiting ability may be caused by its cytotoxicity.
NC-2could block HIV-1envelope mediated cell-cell fusion. The results of ELISA suggested that NC-2could inhibit the binding of HIV-1gp120to CD4but it could not block the formation of gp41six-helix bundle in vitro.
We designed and synthesized two types of analogues based on NC-2.9analogues were obtained. Synthesis of another series of compounds is ongoing.
The synthetic route is the addition of substituted aniline to ethyl chlorooxoacetate in the presence of base or amidation reaction using aniline and diethyloxalate.8analogues were obtained, and the inhibition activities against HIV infection showed that the activities of compounds did not greatly improve after structural modification.
Conclusion
This computer virtual screening with virus inhibition activity testing method was used for screening small molecular against the infection of HIV. A small molecular NC-2was obtained. The results of mechanism of action showed that NC-2was a HIV entry inhibitor and its binding target was HIV Env. NC-2can block the binding of HIV-1gp120and CD4, but it could not block the formation of gp41six-helix bundle in vitro. The Arg61binding site may be an important target of small molecular compounds against HIV infection. This method may be used for large scale screening HIV-1entry inhibitors targeting gp120.
The researches on analogues synthesized base on NC-2are ongoing.9of analogues were obtained. Another series of analogues also had breakthrough progress.
The structure of NBD-556analogues was optimized.8analogues were synthesized. The inhibition activities against HIV infection showed that the activities of compounds did not greatly improve after structural modification.
引文
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