In Vitro Inhibitory Activity Of New Non-nucleoside Reverse Transcriptase Inhibitors And Co-receptor Inhibitors Against HTV

BackgroundHuman immunodeficiency virus (HIV) is the international known mutations faster in the study of the virus. In the mass of hughly active antiretroviral therapy (HAART), the development of drug resistance to antiviral treatment failure. Therefore it is neceddary to research and development of new drugs, the design development of drugs can inhibit the drug-resistant virus.The anti-hiv drugs are divided into class nucleoside reverse transcriptase inhibitors, non-nuclear nucleoside reverse transcriptase inhibitors, proteolytic enzyme inhibitors, fusion inhibitors, auxiliary receptor inhibitors, integrase inhibitors, etc. Most of the new drug is based on the design of these drugs compounds for continuous improvement. Due to high variability of HIV drugs with different targets, different design to the sensitivity of different subtypes of HIV inhibitory activity is different, the inhibition for drug-resistant strains is endless also and same. Drugs in vitro inhibitory activity of detection is a new drug for HIV inhibitory effect evaluation index, the most intuitive use of China’s main HIV epidemic strains of pharmacological inhibition of the activity detection can detect the inhibitory activity of widely, follow-up for drug research and development to provide important reference data.Objective1. By text of drugs in vitro inhibitory activity for standard laboratory strains HIV-1SF33, HIV-1BAL and HIV-lA17, preliminary screen and evaluate the inhibitory activity on drugs resistance to HIV, explore HIV drugs inhibitory activity on the predominant strains;2. Study on drug resistance mechanism of novel HIV inhibitors by in vitro drug pressure test.MethodsStudy subjects:the study of nucleoside reverse transcriptase inhibitors was designed by college of Peking University health science synthesis of 11 kinds of thymus uracil compound Numbers for W1, W2, W3, W4, W5, W6, W7, project.it, W9, W23 and W24. And 12 kinds of pyridine ketone compound Numbers for W12, W13, W14, W15, W16, W25, W26, W27, W40, W41, W42 and W43. Auxiliary receptor inhibitor is provided by the basic medical school of fudan university two auxiliary CCR5 receptor inhibitor XLLN-148-40 and XLLN-148-42. Respectively choosing standard laboratory strains and clinical separation in China the HIV virus in vitro screening antiviral activity of these inhibitors.Methods:1. HIV epidemic strains preparation:whole blood used to test CD4+T cell count, plasma isolated from whole blood used for detection of viral load and virus subtype. PBMCs were isolated using gradient centrifugation and used for growing the virus co-cuLtured with healthy PBMCs. After infecting healthy people of PBMCs cuLtivation, virus cuLture supernatant were collected followed by P24 antigen test and virus titer determination. Isolated virus used in the detection of drug resistance to HIV inhibitory activity in vitro.2. In vitro inhibitory activity of HIV candidate inhibitors:after continuous gradient dilution, candidate HIV inhibitors were respectively reacted with TZM-B1, PBMCs and MT4, half lethal concentration of inhibitor cells (CC50) was alcuLated by live cell staining OD value. Candidate HIV inhibitors inhibitory the same amount of HIV virus were carried out on TZM-B1, PBMC and MT4 after continuous gradient dilution, half inhibitory concentration (EC50) inhibitors were calcuLated by detecting cell light emission or concentration of P24 antigen. By calcuLated CC50/EC50, got the drug selective index SI.3.In vitro induced resistant HIV-1:use drugs to inhibit the replication of the virus on MT4 cells, continuous cuLtivation of 14 weeks with time and increasing drug dosage, the cuLture supernatant of the resuLting gene sequencing, produce resistance loci.4. The preparation of infectious clone virus:use the QuikChange Site-Directed Mutagenesis Kit confrontation grain for site-directed Mutagenesis. Main point mutations, introduced by the PCR method, to get before resistance loci on infectious clone point mutation detection of inhibitors inhibit activity, is used to validate the generation of drug resistance.ResuLts1.11 kinds of thymus sf33 uracil compounds for HIV-1 strain in vitro inhibitory activity of EC50 0.002 uM 2.071 uM, selection index SI50 between 39-38215.12 sf33 pyridine ketone compounds for HIV-1 strain of in vitro inhibitory activity EC50 0.003 uM 0.103 uM, SI50 selection index between 386-28760. After HIV-1 a17 resistance standard strain screening, W6, thymus uracil compounds project.it W12, W9, W24 and pyridine ketone compounds, W25, W26, W42 has inhibitory activity, on the in vitro inhibitory activity EC50 were 0.091 uM 2.543 uM and 0.090 uM 0.090 uM. Selection index SI respectively in 48-between 1080 and 73-1068.Of compounds W6 sf33 strain of HIV-1 in vitro inhibitory activity of EC50 of 0.002 uM.2. The thymus W6 uracil compounds to our country main popuLar CRF07_BC HIV subtypes inhibitory activity EC50 0.003 uM 0.006 uM, restrain subtype B ’active EC50 0.001 uM 0.004 uM, subtype of CRF01_AE inhibitory activity EC50 0.001 uM 0.006 uM, SI50 selection index in 12735-40933,18021-between 76410 and 22967-76410.3. The use of compound W6 sf33 inhibiting HIV-1 on MT4 replication. Continuous cuLture 14 weeks and compound dosage increasing, the cuLture supernatant after gene sequencing and alignment Y188L and V106I drug resistance mutations.4. By building contains resistance loci Y188L and V106I cloning virus in vitro inhibitory activity detection and do further verify the effectiveness of the compound W6 resistance loci for Y188L and V106I.5. CCR5 receptor inhibitor XLLN-148-40, XLLN-148-42 sf33 for HIV-1 double addicted to sex balccr5 laboratory standard strains and HIV-1 single compuLsive standard strains and HIV-1 a17 non-nuclear nucleoside reverse transcriptase inhibitory activity drug-resistance standard EC50 were 0.871 uM and 14.959 uM and 8.641 uM and 0.006 uM and 0.059 uM and 0.078 uM, SI50 selection index were 66.8, 1148.1,115.7,16949.2,16666.7 and 35714.3. XLLN-148-40 major epidemic CRF07BC subtypes of HIV inhibitory activities of our country EC50 0.898 uM 12.246 uuM, restrain subtype B ’active EC50 2.368 uM 47.052 uM, subtype of CRF01_AE inhibitory activity EC50 0.362 uM 163.137 uM, antimicrobial selection index SI50 between 6.1 5181.3, XLLN-148-42 to our country main popuLar CRF07_BC HIV subtypes inhibitory activity EC50 0.003 uM 0.527 uM, restrain subtype B’ active EC50 0.0002 uM 0.602 uM, subtype of CRF01_AE inhibitory activity EC50 0.031 uM 0.793 uM, inhibitory activity EC50 for selecting index SI50 between 1661-5000000.ConclusionIn this study, thymus uracil compound, pyridone compound and CCR5 coreceptor inhibitors exist varying degrees of inhibitory activity against HIV-1SF33 amphotropic laboratory standard strains. In thymus uracil, compound W6 has the strongest inhibitory activity, as well as has a wide inhibition to China’s major HIV epidemic, but may appear predominantly Y188L and V106I mutations after long-time use. In pyridine ketone compounds, W42 has a relatively stronge inhibitory activities both for laboratory standard strains. CCR5 coreceptor inhibitors XLLN-148-40 and XLLN-148-42 have have different degrees of inhibition for double-tropic HIV-1SF33 laboratory standard strain, HFV-1BAL CCR5 ecotropic standard strains, standard strains of drug-resistant HIV-1A17 and major epidemic of HIV strains, inhibitory activity of the compound XLLN-148-42 is significantly higher than XLLN-148-40.