Research On Anti-HIV Target And Drug Resistance Of Indole Derivative RJQ-1

Objectives:The human immunodeficiency virus(HIV)causes acquired immunodeficiency syndrome(AIDS),by attacking human immune cells and damaging the human immune barrier.AIDS is a serious threat for human health and social and economic development,and it is also a cross-century problem that seriously threatens human public health.According to preliminary statistics,from the end of October 2019,China has identified a total of about 958,000 HIV carriers,an increase of 131,000 cases compared to the previous year,and 284,000 deaths have been reported.In the last 20years,HA ART has been widely used in HIV-infected patients,greatly reducing the mortality rate of AIDS patients.More and more researchers are exploring the role of reverse transcriptase inhibitors in inhibiting virus replication.NNRTIs occupies an important part of HA ART therapy.They are a class of hydrophobic small molecule compounds with diverse structures and usually only inhibit HIV-1 RT.With the widespread application of NNRTIs in HIV-1 infected persons,the gradual emergence of drug-resistant strains reduces the activity of the drug.Moreover,the increasing number of HIV-1 infections worldwide has prompted us to develop NNRTIs that are sensitive to drug-resistant strains and have low toxicity.In this study,a newly discovered indole derivative RJQ-1 was used to determine its effective antiviral activity.and then qPCR was used to explore its role in the replication process of the HIV-1 virus.Through phenotype analysis and genotype identification of related drug resistance mutations,and comparison and analysis with existing mutation sites,it further helps us understand the mechanism of action between the compound and HIV-1 reverse transcription,and provides a higher barrier for the development of drug resistance.Less toxic non-nucleoside reverse transcription inhibitors(NNRTIs)provide research and development ideas.Methods1.In vitro drug-resistant strain induction method is used to induce drug-resistant strains of compound RJQ-1 in vitro.If the EC50 of compound R JQ-1 to induce drug-resistant strains by itself rises above 100 times,it indicates that the induction of drug-resistant strains is successful.2.We detect the EC50 value of compound RJQ-1 to induce drug-resistant strains by using CPE and ELISA.3.We detect the genotype of drug-resistant virus strains by using the method of gene cloning.4.We use the software MEGA7 to perform sequence alignm ent and find out whether there are new mutation sites.5.We perform a single point mutation on the plasmid p NL4-3 by using a single point mutation kit.send it to the company for sequencing,and then compare the sequence.6.The plasmid with the correct single point mutation was transfected into 293T cells,and the supernatant was collected and stored at-80℃ after 48 hours.7.The activity of compounds RJQ-1 and EFV against single point mutation virus strains was tested.8.We detect the inhibitory effect of compound RJQ-1 on the reverse transcription process of virus HIV-1 by using qRT-PCR method.Results1.In vitro drug-resistant strain induction method was used to induce drug-resistant strains of compound RJQ-1 in vitro,and the EC so of compound RJQ-1 induced drug-resistant strains by CPE and ELISA methods were 138.086 μg/mL and>200 μg/mL respectively,the EC50 of the compound RJQ-1 to induce drug-resistant strains by itself increased to more than 1 00 times,indicating that the induction of drug-resistant strains was successful.2.We detect the genotype of the drug-resistant virus strain by using gene cloning method,and use the software MEGA7 to compare the sequence of the sequencing results to find a new mutation site Y181C.3.We test the activity of compounds RJQ-1 and EFV against single-point mutation virus strains,and the EC50 was 74.22 μg/mL,2.99 nM,respectively.4.We detect the inhibitory effect of compound RJQ-1 on the reverse transcription process of viral HIV-1 by using q-PCR method,it was found that.the compound is likely to inhibit the synthesis of the initial R/U5 of the reverse transcription process of viral HIV-1.ConclusionsIn this study,we use a newly discovered indole derivative RJQ-1,and then uses q-PCR method to study its inhibitory effect on the HIV-1 virus replication process,and found that the compound is likely to inhibit the initial stage of the viral HIV-1 reverse transcription process and R/U5 synthesis.Through phenotype analysis and genotype identification of related drug-resistant mutations,and a comparative analysis with existing mutation sites,it was found that a new mutation site Y181C appeared in the amino acid sequence of the drug-resistant strain HIV-1ⅢB/RJQ-1.This point is consistent with the common drug resistance mutation sites of NNRTIs pointed out in existing research reports[1],which further helps us understand the interaction mechanism between the compound and HIV-1 reverse transcription,and also provides a higher barrier for the development of drug resistance.Less toxic non-nucleoside reverse transcription inhibitors(NNRTIs)provide research and development ideas.