摘要
艾滋病(AIDS)是一种由人类免疫性缺陷病毒(HIV)引起的一种后天性细胞免疫功能出现缺陷而导致严重随机感染及/或继发肿瘤并致命的一种疾病。HIV病毒分为两种：HIV-1, HIV-2。HIV-1的毒性与传染性均高于HIV-2,因此目前主要针对HIV-1进行大量的研究。HIV-1为带有Ⅰ型膜融合蛋白的包膜病毒,它进入靶细胞首先是利用它的包膜表面gp120亚基与靶细胞主要受体CD4和辅助受体CCR5或者CXCR4先后发生结合,导致它的跨膜亚基gp41构象发生改变,其N端融合肽插入到宿主细胞膜中,gp41形成gp41NHR三聚体形式的中间态,然后,gp41的NHR和CHR区域形成稳定的6-HB结构,引起病毒包膜与靶细胞膜的融合。在gp41NHR的沟槽内有一个深的疏水口袋区域,并且这个区域对于HIV-1病毒的膜融合和6-HB的稳定性非常关键,因此,这个疏水区被认为是发展HIV融合抑制剂的重要靶点。
利用计算机辅助设计,将gp41口袋区作为靶点,并结合本室建立以ELISA为手段的gp416HB抑制实验,Jiang等人成功地筛选出ADS-J1小分子化合物,它抑制HIV介导的细胞融合和HIV-1复制活性达到低微摩尔水平。机制研究表明,ADS-J1能够和一个可溶性杂交分子IQN17结合,并且能够显著抑制PIE7与IQN17口袋区特异性地结合。IQN17是由GCN4序列和形成疏水口袋区的N-多肽N17联接组成,它可以模拟形成gp41三聚体上的口袋区结构。PIE7是一个能与IQN17口袋区特异性地结合的短肽。另外,ADS-J1能够阻止衍生于病毒gp41NHR和CHR多肽所模拟的6-HB的形成。计算机模型分析表明,ADS-J1的磺酸基团能够与gp41口袋区的第574位带正电荷的赖氨酸(K574)结合,但是将带负电荷的氨基酸替代K574后则完全消除了ADS-J1与gp41口袋区的结合。
然而,Este等人认为ADS-J1既不作用于gp41口袋区,也不作用于gp41其它区域,而是靶向gp120的V3区域。这主要源于他们没有诱导出突变点位于gp41口袋区的ADS-J1抗性株,相反,他们诱导出了位于gp120V3区域的ADS-J1抗性株。然而,他们选用的HIV-1病毒株是AR177(靶向gp120的抑制剂)抗性株或者相关病毒株作为野生型毒株,在ADS-J1存在下,侵染MT-4细胞进行诱导突变。由于他们没有设计一个靶向gp41口袋区的肽或者小分子化合物作为对照,因此这些结果不是令人信服的。
因此,本课题主要以ADS-J1为研究对象,利用gp41口袋区的假病毒突变株和HIV-1T-2635耐药性病毒株,探讨了ADS-J1的耐受机制。另外,对ADS-J1进行了抑制T-20耐受株、HIV-1临床分离株的活性研究。同时,进行了将ADS-J1与不同机制的抗病毒药物联合应用时抑制HIV-1IIIB和HIV-1Bal的活性研究,评估了它们潜在的协同效应。
实验方法：
1)Gp41口袋区Q64和A67突变对ADS-J1抑制HIV-1病毒活性的影响。利用FuGENE6转染试剂转染的方法,对以前构建好的假病毒Q64A,Q64L, A67G和A67S进行病毒包装；测定标准p24蛋白的不同浓度与对应的A450值,绘制标准曲线,根据标准曲线计算出假病毒中p24蛋白的含量；将浓度为250ng p24/ml的假病毒与1×105/ml的TZM-B1细胞共同培养,测定假病毒感染性；将浓度为250ng p24/ml的假病毒,与不同浓度的ADS-J1,对照抑制剂C34、T20分别孵育30min,加入密度为1×105/ml的TZM-B1细胞,感染72h后,测定相应的荧光吸收值,采用Calcusyn软件计算IC50。
2)T-2635耐药株对ADS-J1抑制HIV-1病毒活性的影响。采用CaCl2转染的方法将T-2635耐药株质粒转染至293T细胞中,72h后收集T-2635耐药株上清病毒粒子,然后将其侵染MT-2细胞,进一步扩增病毒,根据CPE现象,在感染4-7天后收集病毒上清；根据标准曲线计算出T-2635耐药株p24蛋白的含量；在测定这些抗性株具有感染性后,将浓度为5ng p24/ml的T-2635耐药株与不同浓度的ADS-J1,对照抑制剂T2635、AZT分别孵育30min,加入密度为1×105/ml的TZM-B1细胞,感染72h后,测定相应的荧光吸收值,采用Calcusyn软件计算IC50。
3)N36Fd和它的突变体N36(Q64A)Fd, N36(Q64L)Fd, N36(A67G)Fd, N36(A67S)Fd, N36(Q66R)Fd构建、蛋白表达和纯化。采用PCR方法分别扩增出Fd片段、N36及其突变体片段,然后进行二次PCR将N36及其突变体分别和Fd片段连接上,构建出N36Fd和它的突变体；在0.1mM诱导剂IPTG存在下,采用原核表达的方法,表达出上述6种蛋白；利用Glutathione-Sepharose4B层析柱以及3KD、30KD浓缩管,进行蛋白的浓缩、纯化。
4)天然聚丙烯酰胺凝胶电泳(N-PAGE)检测ADS-J1对N36Fd及其突变体和C34相互结合形成的复合体6-HB的抑制作用。将N36Fd及其突变体分别加入PBS或者不同浓度的ADS-J1,孵育30min后,加入C34在共同孵育30min。N36Fd及其突变体蛋白、C34终浓度为10μM。孵育好的样品混合液与2×Native sample buffer等体积混匀,加样于预制的18%Tris-glycine gel孔中。恒压125V,室温下电泳2h。用染色剂考马斯亮蓝R250染色2h,脱色后用FluorChem8800凝胶成像仪拍照记录。
5)圆二色光谱(CD)检测ADS-J1对N36Fd及其突变体和C34相互结合形成的复合复合螺旋构象的干扰作用。将N36Fd及其突变体分别加入PBS或者不同浓度的ADS-J1,孵育30min后,加入C34在共同孵育30min。N36Fd及其突变体蛋白、C34终浓度为10μM,ADS-J1终浓度为50μM。参数设置：检测温度：4℃；样品池：0.1cm；波长扫描范围：180-300nm；波宽：5.0nm；狭缝0.1nm；时间常数4.0s；扫描速度：50]mm/min。将孵育好的样品加入CD特定比色皿中,进行CD波长扫描,保存CD信号[θ]222nm值,并计算出α-螺旋值。
6)等温滴定量热技术(ITC)检测ADS-J1对N36Fd和它的突变体的结合能力。将50μM的N36Fd或者它的突变体蛋白加入样品池中,将750μM ADS-J1逐滴滴入至样品池中。设置滴定间隔为200sec,滴定体积为10μL/滴,搅拌速度是350rpm。用软件Launch Nano Analyze计算热力学常数。
7) ADS-J1抑制HIV-1T-20耐药株活性检测。利用MT-2细胞扩增出HIV-1T-20耐药株后,根据标准曲线计算出p24蛋白的含量,按照Reed&Muench法计算病毒TCID50。将100倍50%组织感染浓度TCID50的HIV-1T-20耐药株,与不同浓度的ADS-J1,阳性对照T20分别孵育30min,加入密度为1×105/ml的MT-2细胞,感染4天后,观察细胞病变效应CPE,取培养上清,并加入等量5%Triton X-100裂解病毒,ELISA方法测定上清中p24抗原含量,采用Calcusyn软件计算IC50。
8) ADS-J1抑制HIV-1临床分离株活性检测。利用CEMx1745.25M7细胞扩增出HIV-1临床分离株后,根据标准曲线计算出p24蛋白的含量,按照Reed&Muench法计算病毒TCID50。将100倍50%组织感染浓度TCID50的HIV-1临床分离株与不同浓度的ADS-J1,阳性对照T20分别孵育30min,加入密度为5×105/mL的CEMx1745.25M7细胞后,培养7天后取培养上清,并加入等量5%Triton X-100裂解病毒,ELISA方法测定上清中p24抗原含量,采用Calcusyn软件计算IC50。
9) ADS-J1与不同机制的抗病毒药物联合应用时抑制HIV-1IIIB的活性检测。利用MT-2细胞扩增出HIV-1IIIB后,根据标准曲线计算出p24蛋白的含量,按照Reed&Muench法计算病毒TCID50。将不同浓度的ADS-J1和不同机制的抗病毒药物混合液倍比稀释,加入100倍50%组织感染浓度TCID50的HIV-1IIIB,孵育30min后加入密度为1×105/ml的MT-2细胞,感染4天后,观察细胞病变效应CPE,取培养上清,并加入等量5%Triton X-100裂解病毒,ELISA方法测定上清中p24抗原含量,采用Calcusyn软件计算IC50。
10) ADS-J1与不同机制的抗病毒药物联合应用时抑制HIV-1Bal的活性检测。利用CEMx1745.25M7细胞扩增出HIV-1Bal后,根据标准曲线计算出p24蛋白的含量,按照Reed&Muench法计算病毒TCID50。将不同浓度的ADS-J1和不同机制的抗病毒药物混合液倍比稀释,加入100倍50%组织感染浓度TCID50的HIV-1Bal,孵育30min后加入密度为5×105/mL的CEMx1745.25M7细胞,培养7天后取培养上清,并加入等量5%Triton X-100裂解病毒,ELISA方法测定上清中p24抗原含量,采用Calcusyn软件计算IC50。
实验结果：
1)Gp41口袋区带有突变点的假病毒侵染性降低,且这些假病毒对ADS-J1和C34具有高的抗性,但对T-20却相对敏感的。单次感染实验结果表明,与野生型相比,带有突变点病毒的感染性有显著性差异,并具有统计学意义(F=604, P<0.001), Q64A和Q64L的突变使HIV-1假病毒的侵染能力大概只有野生型病毒的34%和27%,A67G和A67S的突变使使HIV-1假病毒的侵染能力大概只有野生型病毒的57%和31%。所有的突变株假病毒对ADS-J1和含有口袋区的C肽C34产生高的抗性,其中对ADS-J1了30-91倍的抗性,对C34产生了103-244倍的抗性,而对不含口袋区的C肽T-20相对敏感的,以上结果表明ADS-J1可能主要作用于HIV-1gp41NHR的口袋区。
2)T-2635耐药株也对ADS-J1产生了抗性。我们选择了7株带有单突变点,4株带有双突变点,和2株带有多突变点的T2635耐药株对T2635, ADS-J1, AZT(逆转录酶抑制剂做为对照)的进行敏感性检测。实验结果表明,单突变株对T2635产生一定抗性,为4到7倍的抗性,双突变株对它产生了较高的抗性,为13到23倍,而多突变株对它产生了非常高的抗性,达到36到263倍,这些与以前报道一致。与T2635相似的是,随着突变点的增多,对ADS-J1产生的抗性也相应增加。相关性分析表明,这些病毒株对T2635和/SDS-Jl抗性具有相关性(r=0.946,P>0.001)。这些结果说明,ADS-J1和T2635可能有相似的作用机制,都是作用于gp41区域。
3)N36Fd和它的突变体的构建。通过两次PCR反应,构建了编码N36Fd和它的突变体基因片段。然后将其插入带有BamHI和Xhol酶切位点的pGEX-6P-1表达载体中,经测序鉴定后筛选出了具有正确序列的克隆载体N36Fd-pGEX6p-1, N36(Q64A)Fd-pGEX6p-1, N36(Q64L)Fd-pGEX6p-1N36(A67G)Fd-pGEX6p-1, N36(A67S)Fd-pGEX6p-1,和N36(Q66R)Fd-pGEX6p-1。利用原核表达蛋白的方法,表达出了N36Fd及其突变体蛋白,纯化后采用聚丙烯凝胶电泳(SDS-PAGE)的方法进行了鉴定。
4)与抑制N36Fd和C34形成6-HB的能力相比,ADS-J1抑制N36Fd突变体和C34形成6-HB的作用降低。N-PAGE实验结果显示,在ADS-J1存在下,C34能够和N36Fd以及它的突变体N36(Q64A)Fd, N36(Q64L)Fd, N36(A67G)Fd, N36(A67S)Fd和N36(Q66R)Fd形成6-HB结构,表明这些突变点没有显著影响6-HB的形成。但是,随着ADS-J1浓度增加,6-HB条带越来越弱,同时C34条带越来越强。对于N36Fd来说,当ADS-J1达到100μM时,能够完全阻止N36Fd和C34形成6-HB。相比而言,对于突变体N36(Q64A)Fd, N36(Q64L)Fd, N36(A67S)Fd和N36(Q66R)Fd,当ADS-J1达到200μM时,才能够完全阻止N36Fd和C34形成6-HB。另外,对于突变体N36(A67G)Fd,当ADS-J1达到400μM时,才能够完全阻止N36Fd和C34形成6-HB,这些结果表明,ADS-J1抑制突变体N36Fd和C34形成6-HB的能力减弱。
5)与干扰N36Fd和C34形成的二级构象相比,ADS-J1干扰N36Fd突变体和C34形成二级构象的作用降低。CD结果显示,Q64,A67,Q66三个位点突变后,能够影响N36Fd和C34形成的6-HB的构象和稳定性,野生型N36Fd和C34可以形成92.8%的α-螺旋含量,突变后的N36Fd和C34形成的α-螺旋含量降低至43.9%到59.1%。在加入ADS-J1后,野生型N36Fd和C34形成的α-螺旋含量从92.8%降低至56.2%。相比而言,ADS-J1的加入对于突变后的N36Fd和C34形成的α-螺旋含量没有显著影响,这说明突变后的N36Fd与ADS-J1结合力降低。
6)与N36Fd和ADS-J1的结合能力相比,N36Fd突变体和ADS-J1的结合能力减弱。ITC结果显示,野生型N36Fd与ADS-J1解离常数为1.91×10-7M,5个突变株与ADS-J1结合力降低2-10倍(解离常数为5.21×10-7-1.76×10-6M),滴定实验表明,ADS-J1与N36Fd或者它的突变体的化学比为1.5-2.7：1,而不是1：1,这说明大约两分子的ADS-J1可以与一分子的N36Fd或者它的突变体结合。
7) ADS-J1有效对抗T-20耐药株。病毒抑制实验结果表明,ADS-J1能够有效抑制T-20耐药株和T-20敏感株的感染,其ICso范围为0.85到1.98μM。
8) ADS-J1有效对抗HIV-1临床分离株。病毒抑制实验结果表明,ADS-J1能够有效的抑制A亚型-F亚型及O亚型的临床分离株,其IC50范围为0.64到2.26μM。
9) ADS-J1与不同机制的临床使用抗HIV-1药物联合应用抑制HIV-1IIIB(X4毒株)具有协同作用。将ADS-J1与不同机制的抗病毒药物联合应用抑制HIV-1IIIB的协同效果进行分析。利用Calcusyn软件计算协同指数(CI)的CI50。当CI50<0.1代表很强协同效应,CI50=0.1-0.3说明强协同效应,CI50=0.3-0.7说明具有协同效应,CI50=0.7-0.85明具有弱协同效应,而当CI50=0.85-0.90说明具有轻微协同效应。ADS-J1与两种融合抑制剂(T-20和SFT)能引起很强的协同作用或协同效应,其对抗HIV-1IIIB的协同作用指数CI50分别为0.085和0.342；与三种逆转录酶抑制剂(AZT、D4T和TMC120)能引起微弱协同作用或协同效应,其对抗HIV-1IIIB的协同作用指数CI50分别为0.926、0.489和0.322；与三种蛋白酶抑制剂(Kaletra、Invirase和Agenerase)能引起协同效应或强的协同作用,其对抗HIV-1IIIB的协同作用指数CI50分别为0.548、0.210和0.166；与整合酶抑制剂Raltegravir能引起协同效应,其对抗HIV-1IIIB的协同作用指数CIs0为0.462。
10) ADS-J1与不同机制的临床使用抗HIV-1药物联合应用抑制HIV-1Bal(R5毒株)具有协同作用。将ADS-J1与不同机制的抗病毒药物联合应用抑制HIV-1Bal的协同效果进行分析。ADS-J1与两种融合抑制剂(T-20和SFT)能引起协同效应或很强的协同作用,其对抗HIV-1Bal的协同作用指数CI50分别为0.673和0.128；与三种逆转录酶抑制剂(AZT、D4T和TMC120)能引起微弱协同作用或协同效应,其对抗HIV-1Bal的协同作用指数CI50分别为0.896、0.312和0.792；与三种蛋白酶抑制剂(Kaletra、Invirase和Agenerase)能引起强协同作用或弱协同作用,对抗HIV-1Bal的协同作用指数CI50分别为0.107、0.738和0.113；与整合酶抑制剂Raltegravir能引起协同效应,其对抗HIV-1Bal的协同作用指数CI50为0.491。
结论：
1)HIV-1假病毒中gp41的NHR上口袋区的Q64和A67两个位点的突变导致这些假病毒对ADS-J1产生抗性。
2)T2635耐药株中gp41区域内的单位点突变,双位点突变,多位点突变也会导致这些病毒对ADS-J1产生抗性。
3)天然聚丙烯酰胺凝胶电泳(N-PAGE)实验结果分析确定,N36Fd在口袋区突变会造成ADS-J1抑制C34和N36Fd三聚体形成的6-HB的活性降低。
4)圆二色光谱(CD)实验结果分析确定,N36Fd在口袋区突变会造成ADS-J1干扰C34和N36Fd三聚体作用形成复合螺旋构象的能力降低。
5)等温滴定量热技术(ITC)实验结果分析确定,N36Fd在口袋区突变会造成ADS-J1与N36Fd三聚体结合能力减弱。
6) ADS-J1能够有效抑制T-20耐药株和HIV-1临床分离株。
7) ADS-J1与目前临床应用的HIV-1融合抑制剂,逆转录酶抑制剂,蛋白酶抑制剂,整合酶抑制剂合用都能产生协同效应抑制X4型(IIIB) R5型(Bal) HIV-1毒株。
8)作用于gp41口袋区的小分子HIV融合抑制剂是一种新型抗HIV药物,可以利用ADS-J1作为先导化合物来开发用于治疗那些对当前药物已产生耐受作用的艾滋病病人。
Acquired immunodeficiency syndrome (AIDS) is a disease caused by human immunodeficiency virus (HIV), resulting in serious immunity deficiency in the patients. HIV can be divided into two major types, HIV type1(HIV-1) and HIV type2(HIV-2). Virulence and infectivity of HIV-1is higher than HIV-2. Therefore, most researches have focused on HIV-1. HIV-1is an enveloped virus with class I membrane fusion protein. Its entry into the target cell is initiated by binding of its Env protein surface subunit gp120to the primary receptor CD4and coreceptor, CCR5or CXCR4, triggering a series of confirmation changes of its Env protein transmembrane subunit gp41. Such changes involve insertion of the fusion peptide into the target cell membrane, and formation of the gp41NHR-trimer as the transit fusion-intermediate state and the6-HB between the gp41NHR and CHR domains as the fusion core, bringing the viral and target cell membranes into close proximity for fusion. A deep hydrophobic pocket in the groove on the gp41NHR-trimer plays an important role in stabilization of the gp416-HB formation and gp41-mediated membrane fusion. Therefore, this pocket has been recognized as an attractive target for developing HIV fusion inhibitors.
Using the gp41pocket as the target in a computer-aided virtual screening method, together with an ELISA-based gp416-HB inhibition assay, Jiang et al identified a small molecule compound, ADS-J1, which inhibited HIV-Env-mediated cell-cell fusion and HIV-1replication at low μM level. The mechanism studies have shown that ADS-J1binds to IQN17, a trimeric peptide containing the gp41pocket region, by conjugating the GCN4trimerization motif (IQ) with a17-aa gp41pocket-forming sequence, and inhibited binding of PIE7, a short D peptide, with the gp41pocket on IQN17. In addition, binding of ADS-J1to the pocket-containing NHR-peptide can block6-HB formation between the NHR-and CHR-peptides. Computer modeling analysis indicates that the negatively charged sulfonic acid group of ADS-J1could interact with the positively charged side chain of K574in the pocket region, but that the mutation of K574D resulte in the abrogation of ADS-J1binding to the gp41pocket region.
However, Este and colleagues argued that ADS-J1targeted neither the pocket region nor any other regions in gp41. Instead, they hypothesized that it targets the V3loop region of gp120because it failed to induce resistant mutations in the gp41pocket region, while it could induce resistant mutations in V3loop regions of gp120.These results were based on passage of an HIV-1strain resistant to AR177, an anti-HIV-1polyanionic oligonucleotide with primary target in gp120, in MT-4cells for more than8months in the presence of ADS-J1or the related HIV-1strains. However, we are not convinced by their results because they did not use a peptide or small molecule compound that mainly targets the gp41pocket as a control in their experiments.
Therefore, in the present study, we investigated the drug-resistance mechanism of ADS-J1by using the pseudoviruses with mutations in the gp41pocket region and T2635-resistant HIV-1clones. In addition, we detected the inhibitory activity of ADS-J1on infection by T-20resistant strains, and primary HIV-1isolates, and evaluated the potential synergistic effect of the combinations of ADS-Jl with the clinically used anti-HIV drugs with different mechanism of actions.
Methods:
1) Effect of mutations at positions64and67in the gp41pocket region on the inhibitory activity of ADS-J1on infection by HIV-1. The mutations (Q64A, Q64L, A67G, and A67S) pseudoviruses were packaging by using FuGENE6reagents; Ap24protein standard curve was set up according to different p24protein concentration and the corresponding absorbance at450nm, and the content of p24protein in pseudoviruses was calculated by the standard curve; The infectivity of HIV-1pseudovirus was determined by using the pseudovirus (250ng p24/ml) coculture with TZM-B1cells (1×105/ml); An HIV-1pseudovirus and its mutants (250ng p24/ml) were preincubated with ADS-J1, C34or T20at indicated concentration at37℃for30min, then the mixture was added to TZM-B1cells (1×105/ml), luciferase activity (relativelight units, RLU) was measured after culturing for72h. IC50was calculated using Calcusyn software.
2) Effect of T2635-resistant mutations on ADS-J1-mediated inhibition of HIV-1infection. The plasmids of T2635-resistant clones were transfected into HEK293T cells by using the calcium phosphate method. Supernatants containing T2635-resistant viruses were harvested72h post-transfection. Then the virions in supernatants were further expanded by infecting MT-2cells. Cytopathic effect (CPE) was observed, and the supernatants were collected at days4to7; The content of p24protein in T2635-resistant clones was calculated by the standard curve; The infectivity of T2635-resistant clones was determined by using the virus (5ng p24/ml) coculture with TZM-B1cells (1×105/ml); T2635-resistant clones (5ng p24/ml) were preincubated with ADS-J1, T2635or AZT at indicated concentration at37℃for30min, then the mixture was added to TZM-B1cells (1×105/ml), luciferase activity (relativelight units, RLU) was measured after culture for72h. IC50was calculated using Calcusyn software.
3) Construction, protein expression and purification of N36Fd and its mutants N36(Q64A)Fd, N36(Q64L)Fd, N36(A67G)Fd, N36(A67S)Fd and N36(Q66R)Fd. The fragments of Fd, N36and its mutants were amplified by first PCR. Then the two overlapping fragments were mixed and used as templates for another PCR reaction. And N36Fd and its mutants were cloned into a pGEX6p-1vector. In the presence of0.1mM IPTG, the six proteins were expressed by prokaryotic expression. These proteins were purified and concentrated by Glutathione-Sepharose4B column,3kDa and30kDa Ultra-15Centrifugal Filter Device, respectively.
4) Detection of inhibitory activity of ADS-J1on6-HB core formation between N36Fd, N36Fd mutants and C34by N-PAGE. The mixture of an N36Fd, or its mutant (40μM), and ADS-J1at an indicated concentration was incubated at37℃for30min, followed by addition of the C peptide C34(40μM). After incubation at37℃for30min, the mixture was loaded onto the18%Tris-glycine gel. Gel electrophoresis was carried out at125V constant voltages at room temperature for2h. The gel was stained with Coomassie Blue and imaged with a FluorChem8800imaging system.
5) Detection of inhibitory activity of ADS-Jl in blocking interaction between N36Fd, N36Fd mutants and C34by CD. The N36Fd, or its mutants (10μM), were incubated with PBS or ADS-J1(50μM) at37℃for30min, followed by addition of C34(10μM). After further incubation at37℃for30min, the samples were cooled to room temperature. Thespectra of each sample were acquired on a spectropolarimeter at room temperature, using a5.0nm bandwidth,0.1nm resolution,0.1cm path length,4.0sec response time, and50nm/min scanning speed. The spectra were then corrected by subtraction of a background corresponding to the solvent. A [0]222value was taken to correspond to100%α-helical content.
6) Detection of the binding affinity of ADS-Jl to N36Fd and its mutants by ITC. ADS-J1(750μM) was injected into the ITC cell containing50μM N36Fd, or its mutants. The experiments were carried out at37℃. Data acquisition and analysis were performed using Launch NanoAnalyze software.
7) Detection of the inhibitory activity of ADS-J1on infection by T-20resistant strains. After T-20resistant strains were propagated by using MT-2cells, the content of p24protein in T-20resistant strains was calculated by the standard curve, as well as the TCID50of these virus were calculated by Reed&Muench. T20resistant strains (100×TCID50) were preincubated with ADS-J1and T20at indicated concentration at37℃for30min, then the mixture was added to MT-2cells (1×105/ml), Cytopathic effect (CPE) was observed, and the supernatants were collected and followed add5%triton at days4. The content of p24protein was detected by ELISA and IC50was calculated using Calcusyn software.
8) Detection of the inhibitory activity of ADS-J1on infection by HIV-1primary isolates. After HIV-1primary isolates were propagated by using CEMx1745.25M7, the content of p24protein in HIV-1primary isolates was calculated by the standard curve, as well as the TCID50of these virus were calculated by Reed&Muench. HIV-1primary isolates (100x TCID50) were preincubated with ADS-J1and T20at indicated concentration at37℃for30min, then the mixture was added to MT-2cells (1×105/ml), and the supernatants were collected and followed by addition of5%triton at days7. The content of p24protein was detected by ELISA and IC50was calculated using Calcusyn software.
9) Detection of the inhibitory activity of the combination of ADS-J1and other anti-HIV drugs with different mechanism on infection by HIV-1IIIB. After HIV-1IIIB was propagated by using MT-2cells, the content of p24protein in HIV-1IIIB was calculated by the standard curve, as well as the TCID50of these viruses were calculated by Reed&Muench. HIV-1IIIB (100x TCID50) was preincubated with the mixture of ADS-J1and other anti-HIV drugs at indicated concentration at37℃for30min, then the mixture was added to MT-2cells (1×105ml), Cytopathic effect (CPE) was observed, and the supernatants were collected and followed by addition of5%triton at days4. The content of p24protein was detected by ELISA and IC50was calculated using Calcusyn software.
10) Detection of the inhibitory activity of the combination of ADS-Jl and other anti-HIV drugs with different mechanism on infection by HIV-1Bal. After HIV-1Bal were propagated by using CEMx1745.25M7, the content of p24protein in HIV-1Bal was calculated by the standard curve, as well as the TCID50of these virus were calculated by Reed&Muench. HIV-1Bal (100×TCID50) were preincubated with ADS-J1and other anti-HIV drugs at indicated concentration at37℃for30min, then the mixture was added to MT-2cells (1×105/ml), and the supernatants were collected and followed by addition of5%triton at days7. The content of p24protein was detected by ELISA and IC50was calculated using Calcusyn software.
Results:
1) Pseudoviruses with mutations in the pocket region of gp41exhibited reduced infectivity and high resistance to ADS-J1and C34, but relatively sensitive to T20. The infectivity of pseudoviruses with Q64A, A67L, A67G and A67S mutatations in gp41was about34%27%,57%and31%, respectively, of that of the wild-type pseudovirus (WT)(F=604, P<0.001). All the mutant pseudoviruses were highly resistant to ADS-J1(30-to91-fold increase of IC50value) and to C34, the PBD-containing CHR peptide (102-to244-fold increase of IC50value), while they were relatively sensitive to T20, the CHR peptide without PBD. Similar to C34, these results suggest that ADS-J1may mainly target the pocket region in the HIV-1gp41NHR-trimer.
2) T2635-resistant HIV-1clones are also resistant to ADS-J1. We selected7,4, and2variants with single, double and multiple mutations, respectively, and the wild-type (WT) HIV-1LAI strain to compare their sensitivity to T2635, ADS-J1, and AZT (an HIV-1reverse transcriptase inhibitor as a control). The results showed that all the variants with single mutations exhibited moderate resistance to T2635(4-to7-fold), while the four variants with double mutations had higher resistance (13-to23-fold) than those with single mutation. Those with multiple mutations were highly resistant to T2635(36-to263-fold), which are consistent with the previous report. Similarly, the variants with single, double and multiple mutations exhibited low, middle and high resistance to ADS-J1, respectively. According to the fold of resistance, resistance of viruses with single, double and multiple mutations against T2635was closely correlated with their resistance against ADS-J1(r=0.946, P<0.001). These results suggest that ADS-J1and T2635may share a similar mechanism of action, as well as the same target site in gp41.
3) N36Fd and its mutant fragments are constructed. Genes coding N36Fd and its mutants were amplified by two times PCR reaction. Then they were cloned into a pGEX6p-1vector with BamHl and Xholl restriction enzymes sites. After DNA sequencing, N36Fd-pGEX6p-1, N36(Q64A)Fd-pGEX6p-1, N36(Q64L)Fd-pGEX6p-1, N36(A67G)Fd-pGEX6p-1, N36(A67S)Fd-pGEX6p-1and N36(Q66R)Fd-pGEX6p-1were confirmed. N36Fd and its mutant proteins were expressed by prokaryotic expression. After purification, these proteins were analyzed by SDS- PAGE.
4) ADS-J1is less effective in inhibiting6-HB formation between C34peptide and the mutated N36Fd than wild-type N36Fd. N-PAGE analysis indicated that C34was able to form6-HB with N36Fd trimer and its mutants, including N36(Q64A)Fd, N36(Q64L)Fd, N36(A67G)Fd, N36(A67S)Fd and N36(Q66R)Fd in the absence of ADS-J1, suggesting that these mutations do not significantly affect the formation of6-HB. However, in the presence of increasing concentration of ADS-J1, the density of the6-HB bands became weaker and weaker, while the C34peptide bands became stronger and stronger. For the control N36Fd, ADS-J1could completely block the interaction between N36Fd and C34at100μM binding to the mutant N36Fd and less potent in blocking, while for mutant peptide, ADS-J1could fully inhibit6-HB formation at200or400μM. These results suggest that ADS-J1is less effective in blocking6-HB formation between the C34peptide and the mutated N36Fd than wild-type N36Fd.
5) ADS-J1is less effective in interfering with the interaction between C34and N36Fd mutants than wild-type N36Fd. The result from CD analysis demonstrated that mutations of the residues Q64, A67, and Q66could affect the conformation and stability of the6-HB formed by N36Fd and C34. The a-helicity content of the6-HBs formed between wild-type N36Fd and C34was92.8%, while the a-helicity content of the6-HBs formed between mutated N36Fd and C34was43.9%to59.1%. ADS-J1could interfere with the interaction between C34and N36Fd, as shown by the reduction of the a-helicity of the6-HB formed between C34and N36Fd from92.8%to56.2%after the addition of ADS-J1. However, addition of ADS-J1to the mixture of C34and mutated N36Fd caused no significant reduction of a-helicity, suggesting that the binding of ADS-J1to N36Fd was reduced because of mutations in N36Fd.
6) ADS-J1enhibited reduced binding affinity to N36Fd mutants. The result of ITC showed that the dissociation constant of wild-type N36Fd is1.91×10-7M, the dissociation constant of ADS-J1to the five mutants was reduced about2-to10-fold (5.21×10-7-1.76×10-6M). The titration experiments revealed that the stoichiometry of the binding of ADS-J1with N36Fd and its mutants was1.5-2.7:1, rather than1:1, indicating that about two small molecules interact with one N36Fd or its mutants.
7) ADS-J1is effective against T-20-resistant HIV-1strains. The result from viral inhibition assay demonstrated that ADS-J1was effective in inhibiting infection by T-20-resistant and T20-sensitive HIV-1strains with IC50in a range of0.85to1.98μM.
8) ADS-J1is effective against primary HIV-1isolates. The result from viral inhibition assay using primary HIV-1isolated suggested that ADS-J1could effectively inhibited infection by primary isolates, including subtypes A to F and group O with IC50ranging from0.64to2.26μM.
9) Combination of ADS-Jl with clinically used anti-HIV drugs with different mechanism exhibited synergistic effect against HIV-1IIIB (X4virus) infection. The potential synergistic effect of the combination of ADS-J1with the clinically used anti-HIV drugs on HIV-IIIB infection was analysized. The combination index (CI) at IC50level (CI50) was calculated with CalcuSyn program. CI50<0.1,0.1-0.3,0.3-0.7, and0.7-0.85, and>0.85indicate very strong synergism, strong synergism, synergism, weak synergism, and slight synergism, respectively. The combination of ADS-J1and entry inhibitor T-20or Sifuvirtide exhibited very strong synergism or synergism, with the CI50value at0.085and0.342, respectively. After the combination of ADS-J1with RTIs AZT, D4T or TMC120, slight synergism or synergism waw observed, with CI50values of0.926,0.489and0.322, respectively. The combination of ADS-J1with protease inhibitor Kaletra, Invirase or Agenerase exhibited synergism or strong synergism, with CI50values at0.548,0.210and0.166, respectively. The combination of ADS-J1with integrase inhibitors Raltegravir exhibited synergism, with CI50value at0.462.
10) Combination of ADS-Jl with clinically used anti-HIV drugs with different mechanism exhibited synergistic effect against HIV-1Bal (R5virus) infection. The potential synergistic effect of the combination of ADS-J1with the clinically used anti-HIV drugs on HIV-1Bal infection was analysized. The combination of ADS-J1with entry inhibitor T-20or Sifuvirtide exhibited synergism or strong synergism, with CI50values at0.673and0.128, respectively. After the combination of ADS-J1with RTIs AZT, D4T or TMC120, slight synergism or synergism were observed with CI50 values at0.896,0.312and0.792, respectively. The combination of ADS-J1wtih protease inhibitor Kaletra, Invirase or Agenerase exhibited strong synergism or light synergism, with CI50values at0.107,0.738and0.113, respectively. The combination of ADS-J1with integrase inhibitors Raltegravir exhibited synergism, with CI50value at0.491.
Conclusions:
1) Mutations residue Q64and A67in the pocket region of the gp41NHR domain of HIV-1pseudoviruses cause their resistance to ADS-J1.
2) Single, double and multiple mutations in gp41of T2635-resistant HIV-1clones cause their resistance to ADS-J1.
3) Mutations in the pocket region of the N36Fd attenuate the inhibitory activity of ADS-J1in blocking the6-HB formation between the C34peptide and N36Fd trimer, as shown by N-PAGE analysis.
4) Mutations in the pocket region of the N36Fd reduce the capacity of ADS-J1in interfering with the interaction between the C34peptide and N36Fd trimer, as shown by N-PAGE analysis.
5) Mutations in the pocket region of the N36Fd result in decreased binding affinity N36Fd trimer, as demonstrated by ITC analysis.
6) ADS-J1is highly effective against T-20-resistant strains and HIV-1primary isolates.
7) Combination of ADS-J1with clinically used anti-HIV drugs, including HIV entry inhibitor, RTIs, protease inhibitor and integrase inhibitor, exhibit synergistic effect against both X4(IIIB) and R5(Bal) HIV-1strains.
8) ADS-J1can be used as a lead compound for developing small molecule HIV fusion inhibitors targeting the gp41pocket, a new class of anti-HIV drug, for treatment of HIV-infected patients who fail to respond to the current anti-HIV drugs.
引文
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