抗HIV药物体外药效学研究关键技术的建立和应用
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摘要
对药物进行快速准确的抗HIV药物的药效学评价,是抗HIV药物研发的关键环节,是判断候选药物是否具有研发前景以及确定下一步研究方向的重要依据。HIV没有实用的动物模型,体外药效学研究的结果是评价其抗HIV活性、判断其能否进入临床研究的最重要标准。本研究在我们前期抗HIV药物药效学研究的基础上,对照美国FDA的标准和要求,建立了我国还不具备或不完善的5种抗HIV药物体外药效学研究关键技术,构建了10株耐药性HIV毒株。主要研究内容包括:
1、ATP法测定药物细胞毒性方法的建立和应用:基于细胞受到毒性影响后,其供能分子ATP活性下降的原理,建立了通过一步反应、测定荧光发光强度、推断并计算药物细胞毒性的方法,并与经典的MTT和XTT测定细胞毒性的方法进行了比较。用ATP、MTT和XTT方法测定了4种有机溶剂(二甲基亚砜、甲醇、二甲基甲酰胺、乙醇)对3种人T淋巴细胞系(MT-2、H9、Sup T1)的细胞毒性,3种方法测定的50%毒性浓度(TC50)两两组间比较,线性回归分析显示其相关系数均≥0.917,表明存在线性关系;主成分分析结果均>0.85;表明3种方法测定细胞毒性可以相互替代。利用ATP法反应步骤少、加液量少等优点,借助384孔细胞培养板、可进行微量移液自动化处理的液体工作站以及测定荧光强度的多功能读数仪,建立了ATP高通量法测定药物细胞毒性的方法,对HIV-1逆转录酶抑制剂多样性分子库中的53种化合物进行了细胞毒性测定,得出了53种化合物的TC50值,范围为17.22μM~ >2000μM。
2、抗HIV药物高通量筛选和评价系统的建立:引进了TZM bl细胞,利用这种细胞可以被各种HIV-1毒株感染,病毒感染后表达的Tat蛋白可以激活HIV-1 LTR控制下的报告基因的表达并可通过发光反应检测,且其光强度强度与感染的HIV-1的量成正比的原理,借助384孔细胞培养板、可进行微量移液自动化处理的液体工作站以及测定荧光强度的多功能读数仪,建立了体外抗HIV药物高通量筛选和评价方法。系列稀释的候选药物与HIV-1急性感染的MT-2细胞共同培养一定时间后,吸出部分上清加入含有TZM bl细胞的黑色384孔细胞培养板中,培养一定时间后,测定各孔的发光值,检测HIV-1复制的情况。利用这种方法,测定了3种候选化合物(SFT、JB25和JB26)和3种已上市药物对HIV-1的50%抑制浓度(IC50),分别是:47.02±1.15nM(AZT)、0.971±0.135 nM(EFV)、74.005±3.05 nM(SQV)、0.133±0.0367μg/ml(SFT)、4.036±0.651 nM(JB25)、3.408±0.265 nM(JB26)。对HIV-1逆转录酶抑制剂多样性分子库中的53种化合物进行了抗HIV活性测定,结果显示,其中13种化合物具有较强的抗HIV活性,治疗指数(TC50/ IC50)大于100,其中4种化合物的治疗指数大于1000,值得深入研究。
3、抗HIV药物的临床前耐药性研究:采用剂量增加体外加压培养的方法,将剂量逐渐增加的3种候选化合物(SFT、JB25、JB26)与HIV-1进行长期传代共培养,诱导耐药毒株,并对耐药基因型进行鉴定。(1)SFT的传代从其IC50附近开始,每传一代化合物的浓度都加倍,从第1代的0.005μg/ml直至第12代的10μg/ml,为起始浓度的2000倍,共传代79天。在第3代(浓度为0.02μg/ml),病毒的gp41基因出现A71S(GCT→TCT)突变,在第5代出现Q41K(CAG→AAG)突变,且两个突变同时存在。(2)JB25的传代从其2倍的IC50开始,每传一代化合物的浓度都加倍,从第1代的0.078nM直至第12代的160nM,为起始浓度的2048倍,共传代89天。在第6代出现L100I(TTA→ATA)突变,在第12代又变成100M(ATA→ATG),L100M是不常见的突变形式,其对HIV非核苷类逆转录酶抑制剂耐药的影响未见报道,在第10代还同时出现了对非核苷类逆转录酶抑制剂高度耐药的Y188C(TAT→TGT)突变。(3)JB26的传代从约2倍的IC50开始,每传一代化合物的浓度都加倍,从第1代的0.078nM直至第12代的160nM,为起始浓度的2048倍,共传代93天。在第8代出现K101E(AAA→GAA)突变,但在第10代又消失,仍然出现了L100I(ATA→ATG)突变。总的来看,JB26的耐药株出现的比JB25要慢。这些数据为这3种候选药物进入临床研究时,病例的选择、配伍药物的确定和病人的耐药性检测提供了重要依据。
4、联合用药抗HIV药效学研究:对3种候选化合物(SFT、JB25和JB26)与已上市的3种抗HIV药物(AZT:核苷类逆转录酶抑制剂;EFV:非核苷类逆转录酶抑制剂;SQV:蛋白酶抑制剂)联合用药的药效学进行了评价。将候选化合物的10个浓度梯度水平方向加入384孔细胞培养板,然后将7个梯度稀释的已上市药物垂直方向加入同一细胞培养板,同时设每一种药物的独立对照。然后加入HIV-1急性感染的MT-2细胞,与药物共培养3d,最后利用TZM bl细胞检测HIV的产量。利用MacSynergyⅡ软件对数据进行分析。
SFT与AZT、EFV以及SQV的平均协同/拮抗容量都小于50,没有显示出明显的协同抗病毒活性,但有相加抗病毒活性。而JB25,JB26与3种已上市药物均具有明显的协同抗病毒作用,其中以JB25与AZT的协同作用最为明显。平均三维空间表面图表明,协同作用通常集中在AZT浓度为3-10nM的区域,在此范围内很低浓度的JB25也显示出很强的抗HIV活性。而JB26在AZT浓度为3-10nM的区域也显示了很强的协同作用;同时在JB26的高浓度区域,AZT在低浓度下也显示出强大的协同抗病毒作用。
5、药物与血浆蛋白结合及其对抗病毒活性影响的研究:(1)采用高效液相-质谱联用(HPLC-MS)的方法测定了4种已上市药物(3TC, AZT, EFV, IDV)和2种候选化合物(JB25、JB26)的血浆蛋白结合率,在高中低三个浓度,3TC血浆蛋白结合率存在着剂量依赖性,浓度越高结合率越低,分别为48.9%, 36.8%,20.4%;AZT血浆蛋白结合率在33.9-36.1%之间;EFV的血浆蛋白结合率很高,在90.6-99.5%之间;IDV的血浆蛋白结合率在51.8-60.4%之间。JP25、JP26的血浆蛋白结合率不存在剂量依赖关系,结合均率在85.2-91.3%之间。(2)测定药物在5%,10%,20%和40%人血清存在条件下的IC50,结果显示,IC50随着血清浓度的变化而变化,除SFT的IC50随着血清浓度的升高而降低之外,其余均升高。为了分析化合物与哪种蛋白结合,将化合物与两种蛋白分别作用,检测其IC50变化的情况,结果显示,SFT的IC50在白蛋白(HSA)浓度由11.25mg/ml增至45mg/ml时,IC50逐步下降,可见HSA的结合反而使SFT的作用增强。其它化合物的IC50与HSA的浓度呈线性关系,说明他们与HSA发生了结合。化合物EFV、SQV和JB26在α-1酸性糖蛋白(AAG)存在的条件下抗病毒作用也受到影响,一般IC50随AAG浓度的升高而升高,且二者之间呈线性关系,说明它们与AAG也发生了结合。另两种化合物(AZT、JB25)的IC50只是随AAG浓度变化上下波动,可能是因为它们与AAG不结合或者结合的量很小。
6、对非核苷类逆转录酶抑制剂和膜融合抑制剂耐药的病毒的构建:为了丰富抗HIV药物体外药效学研究的毒种库,为评价药物对耐药毒株的抑制活性提供必要的材料,以野生型pNL4.3质粒为模板,采用定点突变(site-directed mutagenesis)的方法,构建了针对非核苷类逆转录酶抑制剂和膜融合抑制剂耐药的病毒株。
以pNL4.3质粒为模板,分别扩增两端都带有酶切位点的、突变位点所在区域的DNA片段,将其连接到T载体。选取非核苷类逆转录酶抑制剂的耐药突变为100,103,181位点,膜融合抑制剂的耐药突变为36,38,42,43位点,将经过鉴定的带有突变位点的片段从T载体酶切下来,与经过同样处理的pNL4.3骨架进行连接,然后转染293T细胞,即得到含有目的突变的病毒,将培养上清液接种MT-2细胞,观察其感染和复制情况。
共得到4株含有非核苷类逆转录酶抑制剂耐药突变的病毒,含有的耐药突变分别是K103N,Y181C,L100I+K103N和K103N+Y181C;得到6株含有膜融合抑制剂耐药突变的病毒,分别含有D36G,V38A,V38A+N42D,N42T+N43S,N42S以及V38E+N42S突变,这些病毒均可感染MT-2细胞并出现细胞病变。对这些突变病毒的表型耐药性鉴定正在进行中。
上述5种技术的建立及10株耐药病毒的构建,使抗HIV药物体外药效学研究技术平台得到极大地完善,向着国际先进水平、技术全面、高通量快速的方向前进了一大步,可为我国抗HIV药物的研发提供有力的药效学技术支撑。对化合物库中的53种化合物和3种候选化合物的体外抗HIV活性进行了系统的评价,确定了进一步研究的目标和方向,促进了这些药物的尽早研发和应用。
Evaluating the anti-HIV efficiency quickly and accurately is the key point on the anti-retroviral agents’research and development, and is the most important foundation deciding the prospect of development and the direction of next step research. Until now, there are no practical HIV infected animal model, and the results gotten from the pharmacodynamics research is the most important criterion, on which a potential drug enter clinical trials is based. In this work, we established 5 key techniques of anti-HIV activity and constructed 10 drug-resistant HIV-1 strains based on our former researches of anti-HIV activity, the main contents of the work include:
1. Establishment and application of determination of cytotoxicity with ATP assay. Based on the principle of the most important chemical energy reservoir in cells, ATP concentration decrease when the cells are exposed to a harmful environment, we establish a one-step reaction to determine the luminescent signal and deduce the cytotoxicity of compounds, and then compare it with classical MTT and XTT colorimetric assay. We determined the cyottoxicity of 4 organic solvents (dimethyl sulphoxide, methanol, dimethyl formamine and ethanol) in 3 human T lymphoma cell lines (MT-2, H9 and Sup T1) with ATP, MTT and XTT assay, respectively. A statistical comparison of the multiple intercorrelations between the assays was performed. Included were the 3 different cell lines, the 3 different cell viability assays, and the 4 cytotoxic agents for 3 pair wise regression analyses shown in Figure 4 A-C. All correlation coefficients were≥0.917, indicating linear relationships. Then the equality of the results was determined by principal component analysis, and the components were all more than 0.85, indicating the equality and compatibility of these 3 assays. Owing to fewer handling steps and less reagents added, we set up a high-throughput ATP assay to evaluate the cytotoxicity of candidate compounds by utilizing of 384-well plates, liquid handling workstation and multifunction plate reader, and with which we determine 53 compounds out of a diverse compounds library of NNRTIs. The TC50s of the 53 compounds were ranged from 17.22μM to >2000μM.
2. Establishment of the system of high-throughput screening and evaluation of anti-HIV agents. We introduced a new cell line called TZM bl, which contain reporter genes controlled by HIV-1 LTR. TZM bl cells can be infected by diverse HIV-1 strains and HIV-1 Tat proteins are produced, which activate the expression of the reporter genes. The activity of reporter genes can be measured by adding fluorogenic substrate, and the fluorescent signal is proportional to the amount of infectious HIV-1 particles. By using of 384-well plates, microplate pipetting system and microplate fluorescence reader, we established a platform of high-throughput screening of anti-HIV agents in vitro. Serially diluted candidate compounds co-cultured with HIV-1 infected MT-2 cells for 3days, then a portion of culture supernant were transferred to a new black 384-well plate containing TZM bl cells. After 24 hours, we detected the fluorescence and HIV-1 replication. Using this method, we determined the 50% inhibitory concentrations (IC50s) of 3 candidate compounds (SFT, JB25, JB26) and 3 proved drugs (AZT, EFV, SQV), which were 47.02±1.15nM (AZT), 0.971±0.135 nM (EFV), 74.005±3.05 nM (SQV), 0.133±0.0367μg/ml (SFT), 4.036±0.651 nM (JB25), 3.408±0.265 nM (JB26), respectively. At the same time, we evaluated the anti-HIV activity of the 53 compounds mentioned above, and 13 of them showed high anti-HIV activity and TIs were more than 100, especially TIs of 4 compounds were more than 1000, which deserved advanced research.
3. Preclinical drug resistance researches. Selection for resistant variants was performed by sequential passage of HIV-1 strain NL4.3 in escalating concentrations of 3 candidate compounds, and the phenotype of variants were sequenced, respectively. (1) For the initial passage, SFT was present at approximately the IC50 in MT-2 cells. For the subsequent passages, the concentrations were increased twofold compared with the precious passages, from 0.005μg/ml of first passage to 10μg/ml of twelfth, became 2000 times and occupied 79 days. At 3third passage, with concentration of 0.02μg/ml, there emerged A71S (GCT→TCT) mutation in pg41 region. At fifth passage, Q41K (CAG→AAG) mutation appeared, they presented at the same time. (2) For the initial passage, JB25 was present at approximately twofold IC50 in MT-2 cells. For the subsequent passages, the concentrations were increased twofold compared with the precious passages, from 0.078nM of first passage to 160nM of twelfth, became 2048 times of the first concentration and occupied 89 days. At sixth passage, there emerged L100I (TTA→ATA) mutation in RT region, then it changed to 100M (ATA→ATG) at twelfth passage, which was a rare mutation and never reported that association with NNRTIs. At tenth passage, another mutation Y188C (TAT→TGT) emerged, which was highly resistant to NNRTIs. (3) For the initial passage, JB26 was present at approximately twofold IC50 in MT-2 cells. For the subsequent passages, the concentrations were increased twofold compared with the precious passages, from 0.078nM of first passage to 160nM of twelfth, became 2048 times of the first passage concentration and occupied 93 days. At eighth passage, there emerged K101E (AAA→GAA). But at tenth passage, L100I (ATA→ATG) was presented and K101E disappeared. In a conclusion, the resistance to JB26 was harder than JB25. the data provided references to the case selection, drug co-administration and drug resistance test.
4. Anti-HIV activities of drug combinations. We evaluated the anti-HIV activity of 3 candidate compounds combined with 3 approved drugs, including AZT, EFV, and SQV, which were NRTI, NNRTI and PI, respectively. 10 serial dilutions of candidates were added horizontally to a 384-well microplate, then 7 serial dilutions of approved drugs were added vertically to the same one, then co-cultured with MT-2 cells for 3 days, and the HIV-1 replication were evaluated by using TZM bl cells. The data were processed by MacSynergyⅡsoftware. The experiment data that every drug used alone were needed.
The average Synergism/Antagonism capacities of SFT combined with AZT, EFV, and SQV were all less than 50, showing additive activity but no synergy. Both JB25 and JB26 showed significant synergy with the approved drugs, especially JB25 combined with AZT. The average 3 dimensional response surface showed significant synergy where concentrations of AZT was at 3-10nM. When concentrations of JB26 were in a high region, AZT showed strong anti-HIV activity with a lower concentration for synergy.
5. Plasma proteins binding of drugs and their effect on the anti-retroviral activity. (1) The ratios of 4 approved drugs and 2 candidates binding to the plasma proteins were determined by HPLC-MS methods. The binding ratio of 3TC was dose dependent, and the higher concentrations, the lower ratios, which were 48.9%, 36.8% ,20.4% according to a decreased concentrations. The binding ratio of AZT was at 33.9-36.1%. The ratio of EFV binding was highest up to 90.6-99.5% and 51.8-60.4% for IDV. The binding ratios of JB25 and JB26 were dose independent, varying from 85.2% to 91.3%. (2) We determined the IC50s of drugs in the presence of 5%,10%,20% and 40% human serum, and the results showed that IC50s of the drugs elevated corresponding to the plasma concentrations except SFT, a peptide fusion inhibitor, whose anti-HIV activity increased with higher plasma concentrations. To distinguish which protein the drugs bound to, we determine the IC50s in the presence of human serum albumin and AAG, respectively. We found that the IC50s of SFT decreased with elevated HSA concentrations. But IC50s of other drugs showed linear relationship with HSA concentrations, indicating that the bindings to HSA. EFV、SQV and JB26 showed elevated IC50s corresponding to AAG concentrations, indicating linear relationship and binding to AAG, too. AZT, SFT and JB25 have less maybe or no binding to AAG.
6. Construction of mutant virus resistant to NNRTIs and FIs. To obtain more drug-resistant virus for drug evaluation and enrich our virus library, we constructed mutated virus resistant to NNRTIs and FIs with pNL4.3 vector by site-directed mutagenesis.
The DNA fragments with enzymatic sites were amplified from pNL4.3 plasmid RT and gp41, recpectively, and then were ligated them to T vectors. The T vectors containing mutations to NNRTIs at 100, 103, 181sites and mutations to FIs at 36, 38, 42, 43 sites were amplified and digested, ligated to the pNL4.3 backbone with same digestion. After proliferation in E. coli, the plasmids were transfected with 293T cells, and the mutant virus stocks were obtained. We inoculated MT-2 cells and viruses were produced.
4 mutatant viruses resistant to NNRTIs respectively containing mutations K103N , Y181C , L100I+K103N and K103N+Y181C, and 6 resistant to FIs respectively containing mutations D36G, V38A, V38A+N42D, N42T+N43S, N42S and V38E+N42S.
The 5 key techniques we established and 10 HIV-1 resistant strains can improve the platform of evaluation of anti-HIV activity in vitro, access to advanced, comprehensive, high throughput and fast orientation and support anti-HIV drug research and development of our country.
1、ATP法测定药物细胞毒性方法的建立和应用:基于细胞受到毒性影响后,其供能分子ATP活性下降的原理,建立了通过一步反应、测定荧光发光强度、推断并计算药物细胞毒性的方法,并与经典的MTT和XTT测定细胞毒性的方法进行了比较。用ATP、MTT和XTT方法测定了4种有机溶剂(二甲基亚砜、甲醇、二甲基甲酰胺、乙醇)对3种人T淋巴细胞系(MT-2、H9、Sup T1)的细胞毒性,3种方法测定的50%毒性浓度(TC50)两两组间比较,线性回归分析显示其相关系数均≥0.917,表明存在线性关系;主成分分析结果均>0.85;表明3种方法测定细胞毒性可以相互替代。利用ATP法反应步骤少、加液量少等优点,借助384孔细胞培养板、可进行微量移液自动化处理的液体工作站以及测定荧光强度的多功能读数仪,建立了ATP高通量法测定药物细胞毒性的方法,对HIV-1逆转录酶抑制剂多样性分子库中的53种化合物进行了细胞毒性测定,得出了53种化合物的TC50值,范围为17.22μM~ >2000μM。
2、抗HIV药物高通量筛选和评价系统的建立:引进了TZM bl细胞,利用这种细胞可以被各种HIV-1毒株感染,病毒感染后表达的Tat蛋白可以激活HIV-1 LTR控制下的报告基因的表达并可通过发光反应检测,且其光强度强度与感染的HIV-1的量成正比的原理,借助384孔细胞培养板、可进行微量移液自动化处理的液体工作站以及测定荧光强度的多功能读数仪,建立了体外抗HIV药物高通量筛选和评价方法。系列稀释的候选药物与HIV-1急性感染的MT-2细胞共同培养一定时间后,吸出部分上清加入含有TZM bl细胞的黑色384孔细胞培养板中,培养一定时间后,测定各孔的发光值,检测HIV-1复制的情况。利用这种方法,测定了3种候选化合物(SFT、JB25和JB26)和3种已上市药物对HIV-1的50%抑制浓度(IC50),分别是:47.02±1.15nM(AZT)、0.971±0.135 nM(EFV)、74.005±3.05 nM(SQV)、0.133±0.0367μg/ml(SFT)、4.036±0.651 nM(JB25)、3.408±0.265 nM(JB26)。对HIV-1逆转录酶抑制剂多样性分子库中的53种化合物进行了抗HIV活性测定,结果显示,其中13种化合物具有较强的抗HIV活性,治疗指数(TC50/ IC50)大于100,其中4种化合物的治疗指数大于1000,值得深入研究。
3、抗HIV药物的临床前耐药性研究:采用剂量增加体外加压培养的方法,将剂量逐渐增加的3种候选化合物(SFT、JB25、JB26)与HIV-1进行长期传代共培养,诱导耐药毒株,并对耐药基因型进行鉴定。(1)SFT的传代从其IC50附近开始,每传一代化合物的浓度都加倍,从第1代的0.005μg/ml直至第12代的10μg/ml,为起始浓度的2000倍,共传代79天。在第3代(浓度为0.02μg/ml),病毒的gp41基因出现A71S(GCT→TCT)突变,在第5代出现Q41K(CAG→AAG)突变,且两个突变同时存在。(2)JB25的传代从其2倍的IC50开始,每传一代化合物的浓度都加倍,从第1代的0.078nM直至第12代的160nM,为起始浓度的2048倍,共传代89天。在第6代出现L100I(TTA→ATA)突变,在第12代又变成100M(ATA→ATG),L100M是不常见的突变形式,其对HIV非核苷类逆转录酶抑制剂耐药的影响未见报道,在第10代还同时出现了对非核苷类逆转录酶抑制剂高度耐药的Y188C(TAT→TGT)突变。(3)JB26的传代从约2倍的IC50开始,每传一代化合物的浓度都加倍,从第1代的0.078nM直至第12代的160nM,为起始浓度的2048倍,共传代93天。在第8代出现K101E(AAA→GAA)突变,但在第10代又消失,仍然出现了L100I(ATA→ATG)突变。总的来看,JB26的耐药株出现的比JB25要慢。这些数据为这3种候选药物进入临床研究时,病例的选择、配伍药物的确定和病人的耐药性检测提供了重要依据。
4、联合用药抗HIV药效学研究:对3种候选化合物(SFT、JB25和JB26)与已上市的3种抗HIV药物(AZT:核苷类逆转录酶抑制剂;EFV:非核苷类逆转录酶抑制剂;SQV:蛋白酶抑制剂)联合用药的药效学进行了评价。将候选化合物的10个浓度梯度水平方向加入384孔细胞培养板,然后将7个梯度稀释的已上市药物垂直方向加入同一细胞培养板,同时设每一种药物的独立对照。然后加入HIV-1急性感染的MT-2细胞,与药物共培养3d,最后利用TZM bl细胞检测HIV的产量。利用MacSynergyⅡ软件对数据进行分析。
SFT与AZT、EFV以及SQV的平均协同/拮抗容量都小于50,没有显示出明显的协同抗病毒活性,但有相加抗病毒活性。而JB25,JB26与3种已上市药物均具有明显的协同抗病毒作用,其中以JB25与AZT的协同作用最为明显。平均三维空间表面图表明,协同作用通常集中在AZT浓度为3-10nM的区域,在此范围内很低浓度的JB25也显示出很强的抗HIV活性。而JB26在AZT浓度为3-10nM的区域也显示了很强的协同作用;同时在JB26的高浓度区域,AZT在低浓度下也显示出强大的协同抗病毒作用。
5、药物与血浆蛋白结合及其对抗病毒活性影响的研究:(1)采用高效液相-质谱联用(HPLC-MS)的方法测定了4种已上市药物(3TC, AZT, EFV, IDV)和2种候选化合物(JB25、JB26)的血浆蛋白结合率,在高中低三个浓度,3TC血浆蛋白结合率存在着剂量依赖性,浓度越高结合率越低,分别为48.9%, 36.8%,20.4%;AZT血浆蛋白结合率在33.9-36.1%之间;EFV的血浆蛋白结合率很高,在90.6-99.5%之间;IDV的血浆蛋白结合率在51.8-60.4%之间。JP25、JP26的血浆蛋白结合率不存在剂量依赖关系,结合均率在85.2-91.3%之间。(2)测定药物在5%,10%,20%和40%人血清存在条件下的IC50,结果显示,IC50随着血清浓度的变化而变化,除SFT的IC50随着血清浓度的升高而降低之外,其余均升高。为了分析化合物与哪种蛋白结合,将化合物与两种蛋白分别作用,检测其IC50变化的情况,结果显示,SFT的IC50在白蛋白(HSA)浓度由11.25mg/ml增至45mg/ml时,IC50逐步下降,可见HSA的结合反而使SFT的作用增强。其它化合物的IC50与HSA的浓度呈线性关系,说明他们与HSA发生了结合。化合物EFV、SQV和JB26在α-1酸性糖蛋白(AAG)存在的条件下抗病毒作用也受到影响,一般IC50随AAG浓度的升高而升高,且二者之间呈线性关系,说明它们与AAG也发生了结合。另两种化合物(AZT、JB25)的IC50只是随AAG浓度变化上下波动,可能是因为它们与AAG不结合或者结合的量很小。
6、对非核苷类逆转录酶抑制剂和膜融合抑制剂耐药的病毒的构建:为了丰富抗HIV药物体外药效学研究的毒种库,为评价药物对耐药毒株的抑制活性提供必要的材料,以野生型pNL4.3质粒为模板,采用定点突变(site-directed mutagenesis)的方法,构建了针对非核苷类逆转录酶抑制剂和膜融合抑制剂耐药的病毒株。
以pNL4.3质粒为模板,分别扩增两端都带有酶切位点的、突变位点所在区域的DNA片段,将其连接到T载体。选取非核苷类逆转录酶抑制剂的耐药突变为100,103,181位点,膜融合抑制剂的耐药突变为36,38,42,43位点,将经过鉴定的带有突变位点的片段从T载体酶切下来,与经过同样处理的pNL4.3骨架进行连接,然后转染293T细胞,即得到含有目的突变的病毒,将培养上清液接种MT-2细胞,观察其感染和复制情况。
共得到4株含有非核苷类逆转录酶抑制剂耐药突变的病毒,含有的耐药突变分别是K103N,Y181C,L100I+K103N和K103N+Y181C;得到6株含有膜融合抑制剂耐药突变的病毒,分别含有D36G,V38A,V38A+N42D,N42T+N43S,N42S以及V38E+N42S突变,这些病毒均可感染MT-2细胞并出现细胞病变。对这些突变病毒的表型耐药性鉴定正在进行中。
上述5种技术的建立及10株耐药病毒的构建,使抗HIV药物体外药效学研究技术平台得到极大地完善,向着国际先进水平、技术全面、高通量快速的方向前进了一大步,可为我国抗HIV药物的研发提供有力的药效学技术支撑。对化合物库中的53种化合物和3种候选化合物的体外抗HIV活性进行了系统的评价,确定了进一步研究的目标和方向,促进了这些药物的尽早研发和应用。
Evaluating the anti-HIV efficiency quickly and accurately is the key point on the anti-retroviral agents’research and development, and is the most important foundation deciding the prospect of development and the direction of next step research. Until now, there are no practical HIV infected animal model, and the results gotten from the pharmacodynamics research is the most important criterion, on which a potential drug enter clinical trials is based. In this work, we established 5 key techniques of anti-HIV activity and constructed 10 drug-resistant HIV-1 strains based on our former researches of anti-HIV activity, the main contents of the work include:
1. Establishment and application of determination of cytotoxicity with ATP assay. Based on the principle of the most important chemical energy reservoir in cells, ATP concentration decrease when the cells are exposed to a harmful environment, we establish a one-step reaction to determine the luminescent signal and deduce the cytotoxicity of compounds, and then compare it with classical MTT and XTT colorimetric assay. We determined the cyottoxicity of 4 organic solvents (dimethyl sulphoxide, methanol, dimethyl formamine and ethanol) in 3 human T lymphoma cell lines (MT-2, H9 and Sup T1) with ATP, MTT and XTT assay, respectively. A statistical comparison of the multiple intercorrelations between the assays was performed. Included were the 3 different cell lines, the 3 different cell viability assays, and the 4 cytotoxic agents for 3 pair wise regression analyses shown in Figure 4 A-C. All correlation coefficients were≥0.917, indicating linear relationships. Then the equality of the results was determined by principal component analysis, and the components were all more than 0.85, indicating the equality and compatibility of these 3 assays. Owing to fewer handling steps and less reagents added, we set up a high-throughput ATP assay to evaluate the cytotoxicity of candidate compounds by utilizing of 384-well plates, liquid handling workstation and multifunction plate reader, and with which we determine 53 compounds out of a diverse compounds library of NNRTIs. The TC50s of the 53 compounds were ranged from 17.22μM to >2000μM.
2. Establishment of the system of high-throughput screening and evaluation of anti-HIV agents. We introduced a new cell line called TZM bl, which contain reporter genes controlled by HIV-1 LTR. TZM bl cells can be infected by diverse HIV-1 strains and HIV-1 Tat proteins are produced, which activate the expression of the reporter genes. The activity of reporter genes can be measured by adding fluorogenic substrate, and the fluorescent signal is proportional to the amount of infectious HIV-1 particles. By using of 384-well plates, microplate pipetting system and microplate fluorescence reader, we established a platform of high-throughput screening of anti-HIV agents in vitro. Serially diluted candidate compounds co-cultured with HIV-1 infected MT-2 cells for 3days, then a portion of culture supernant were transferred to a new black 384-well plate containing TZM bl cells. After 24 hours, we detected the fluorescence and HIV-1 replication. Using this method, we determined the 50% inhibitory concentrations (IC50s) of 3 candidate compounds (SFT, JB25, JB26) and 3 proved drugs (AZT, EFV, SQV), which were 47.02±1.15nM (AZT), 0.971±0.135 nM (EFV), 74.005±3.05 nM (SQV), 0.133±0.0367μg/ml (SFT), 4.036±0.651 nM (JB25), 3.408±0.265 nM (JB26), respectively. At the same time, we evaluated the anti-HIV activity of the 53 compounds mentioned above, and 13 of them showed high anti-HIV activity and TIs were more than 100, especially TIs of 4 compounds were more than 1000, which deserved advanced research.
3. Preclinical drug resistance researches. Selection for resistant variants was performed by sequential passage of HIV-1 strain NL4.3 in escalating concentrations of 3 candidate compounds, and the phenotype of variants were sequenced, respectively. (1) For the initial passage, SFT was present at approximately the IC50 in MT-2 cells. For the subsequent passages, the concentrations were increased twofold compared with the precious passages, from 0.005μg/ml of first passage to 10μg/ml of twelfth, became 2000 times and occupied 79 days. At 3third passage, with concentration of 0.02μg/ml, there emerged A71S (GCT→TCT) mutation in pg41 region. At fifth passage, Q41K (CAG→AAG) mutation appeared, they presented at the same time. (2) For the initial passage, JB25 was present at approximately twofold IC50 in MT-2 cells. For the subsequent passages, the concentrations were increased twofold compared with the precious passages, from 0.078nM of first passage to 160nM of twelfth, became 2048 times of the first concentration and occupied 89 days. At sixth passage, there emerged L100I (TTA→ATA) mutation in RT region, then it changed to 100M (ATA→ATG) at twelfth passage, which was a rare mutation and never reported that association with NNRTIs. At tenth passage, another mutation Y188C (TAT→TGT) emerged, which was highly resistant to NNRTIs. (3) For the initial passage, JB26 was present at approximately twofold IC50 in MT-2 cells. For the subsequent passages, the concentrations were increased twofold compared with the precious passages, from 0.078nM of first passage to 160nM of twelfth, became 2048 times of the first passage concentration and occupied 93 days. At eighth passage, there emerged K101E (AAA→GAA). But at tenth passage, L100I (ATA→ATG) was presented and K101E disappeared. In a conclusion, the resistance to JB26 was harder than JB25. the data provided references to the case selection, drug co-administration and drug resistance test.
4. Anti-HIV activities of drug combinations. We evaluated the anti-HIV activity of 3 candidate compounds combined with 3 approved drugs, including AZT, EFV, and SQV, which were NRTI, NNRTI and PI, respectively. 10 serial dilutions of candidates were added horizontally to a 384-well microplate, then 7 serial dilutions of approved drugs were added vertically to the same one, then co-cultured with MT-2 cells for 3 days, and the HIV-1 replication were evaluated by using TZM bl cells. The data were processed by MacSynergyⅡsoftware. The experiment data that every drug used alone were needed.
The average Synergism/Antagonism capacities of SFT combined with AZT, EFV, and SQV were all less than 50, showing additive activity but no synergy. Both JB25 and JB26 showed significant synergy with the approved drugs, especially JB25 combined with AZT. The average 3 dimensional response surface showed significant synergy where concentrations of AZT was at 3-10nM. When concentrations of JB26 were in a high region, AZT showed strong anti-HIV activity with a lower concentration for synergy.
5. Plasma proteins binding of drugs and their effect on the anti-retroviral activity. (1) The ratios of 4 approved drugs and 2 candidates binding to the plasma proteins were determined by HPLC-MS methods. The binding ratio of 3TC was dose dependent, and the higher concentrations, the lower ratios, which were 48.9%, 36.8% ,20.4% according to a decreased concentrations. The binding ratio of AZT was at 33.9-36.1%. The ratio of EFV binding was highest up to 90.6-99.5% and 51.8-60.4% for IDV. The binding ratios of JB25 and JB26 were dose independent, varying from 85.2% to 91.3%. (2) We determined the IC50s of drugs in the presence of 5%,10%,20% and 40% human serum, and the results showed that IC50s of the drugs elevated corresponding to the plasma concentrations except SFT, a peptide fusion inhibitor, whose anti-HIV activity increased with higher plasma concentrations. To distinguish which protein the drugs bound to, we determine the IC50s in the presence of human serum albumin and AAG, respectively. We found that the IC50s of SFT decreased with elevated HSA concentrations. But IC50s of other drugs showed linear relationship with HSA concentrations, indicating that the bindings to HSA. EFV、SQV and JB26 showed elevated IC50s corresponding to AAG concentrations, indicating linear relationship and binding to AAG, too. AZT, SFT and JB25 have less maybe or no binding to AAG.
6. Construction of mutant virus resistant to NNRTIs and FIs. To obtain more drug-resistant virus for drug evaluation and enrich our virus library, we constructed mutated virus resistant to NNRTIs and FIs with pNL4.3 vector by site-directed mutagenesis.
The DNA fragments with enzymatic sites were amplified from pNL4.3 plasmid RT and gp41, recpectively, and then were ligated them to T vectors. The T vectors containing mutations to NNRTIs at 100, 103, 181sites and mutations to FIs at 36, 38, 42, 43 sites were amplified and digested, ligated to the pNL4.3 backbone with same digestion. After proliferation in E. coli, the plasmids were transfected with 293T cells, and the mutant virus stocks were obtained. We inoculated MT-2 cells and viruses were produced.
4 mutatant viruses resistant to NNRTIs respectively containing mutations K103N , Y181C , L100I+K103N and K103N+Y181C, and 6 resistant to FIs respectively containing mutations D36G, V38A, V38A+N42D, N42T+N43S, N42S and V38E+N42S.
The 5 key techniques we established and 10 HIV-1 resistant strains can improve the platform of evaluation of anti-HIV activity in vitro, access to advanced, comprehensive, high throughput and fast orientation and support anti-HIV drug research and development of our country.
引文
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