Establishment And Application Of Key Techniques Of The Efficiency Evaluation Of Anti-retroviral Agents In Vitro

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.