Screening And Identification Of The Transcription Regulatory Compounds Targeting HIV-1 Tat Protein And The LTR

Objective:Acquired immunodeficiency syndrome（AIDS）is a widespread and incurable disease caused by the human immunodeficiency virus（HIV）,which can gradually destroys the immune system of its host.With the continuous development of new antiretroviral drugs,the antiretroviral therapy（ART）can suppress the plasma viral load of AIDS patients to undetectable levels,greatly reducing HIV-related morbidity and mortality.However,because of the presence of viral reservoirs,interrupting the ART can cause the virus to bounce back quickly,therefore,patients need to take medication for life.At the same time,this will lead to the problem of drug resistance.The main targets of ART are three enzymes in HIV-1 encoded protein:reverse transcriptase,integrase and protease,and the resistance sites on current popular drug-resistant strains are also mainly located in the gene coding region of these three enzymes.Based on this,finding new targets with therapeutic potential,especially those enzymes or proteins that are specific to HIV-1 and play an important role in its life cycle,and developing new drugs with different mechanisms from existing drugs to enrich clinical treatment means,is the best way to solve the current drug resistance problem.HIV-1 Tat（transactivator of transcription）is a transcription regulatory protein,which is specific to retroviruses and encoded by virus genes,and generally consists of 86-102 amino acids.It transactivates transcriptional promoters on the 5’long terminal repeat（LTR）of HIV-1and turns on transcription,which is critical for viral infection,replication and reactivation.In addition,Tat proteins are the first to be synthesized after the virus infects cells.Even in uninfected cells,Tat proteins can transactivate genes in a paracrine manner,leading to HIV-1infection.Blocking Tat protein function can greatly inhibit HIV-1 replication,and has little effect on host cells because it only activates virus-specific transcriptional promoters.Therefore,it is of great academic value and potential clinical significance to find a novel Tat protein inhibitor.This study attempts to screen out compounds that can specifically inhibit Tat protein from the compound library,thus inhibiting HIV-1 replication and providing new ideas for the treatment and prevention of AIDS.Methods:First of all,this study established a high-throughput screening platform for HIV transcriptional regulatory factors targeting Tat protein by using monoclonal cell lines of statically expressed B subtype and CRF01＿AE recombination Tat protein,and verified its stability.Using this platform,small molecule compounds with regulatory effects on Tat-LTR of B subtype and CRF01＿AE recombination were screened from a diverse parent library containing 4208 small molecule compounds,and the cytotoxicity of the regulatory candidate was verified by ATP activity assay.Secondly,MT2 cells were infected with HIV-1 B subtype strain NL4-3 and CRF01＿AE recombinant strain GX002 in vitro.The selected regulatory candidate was subjected to in vitro virus culture system,and the P24 content and viral load in the supernatant of virus culture were detected by ELISA and q RT-PCR.To verify whether WAY-297131 and WAY-235035 inhibit HIV-1 replication mediated by Tat.The infected cells were treated with different concentrations of compounds.48h after virus infection,the supernatant was transferred into TZM-BL cells stably transfected with LTR-luciferase reporter gene.The dose-response curve of the compound on virus inhibition was drawn using fluorescence values as indicators.The half maximal inhibitory concentration(IC₅₀)and therapeutic index(TI=TC₅₀/IC₅₀)of the drug against the virus were calculated to further test the antiviral effect of the regulatory candidate.Finally,for the selected candidates,we further explored the mechanism of regulatory factor candidates’inhibition of Tat-mediated transcriptional activation of HIV-1 by simple western,molecular docking,bio-layer interferometry technique,protease and lysosome inhibitor inhibition experiments and other methods.Results:In this study,a high-throughput screening platform was used to screen the selected parent library of small molecule compounds.From 4208 compounds,two small molecule compounds,WAY-297131 and WAY-235035,were screened to effectively inhibit HIV-1 replication.The specific results are as follows:1.WAY-297131 and WAY-235035 inhibited the function of B subtype and CRF01＿AE recombination Tat proteins in a dose-dependent manner,but they did not inhibit the function of B subtype and CRF01＿AE recombination LTR and showed no cytotoxicity at experimental concentrations.2.In vitro viral infection experiments showed that WAY-297131 and WAY-235035inhibited Tat-mediated replication of HIV-1 B subtype strain NL4-3 and CRF01＿AE recombination strain GX002 in a dose-dependent manner.The IC50 values of WAY-297131 for NL4-3 and GX002 are 0.0799μM and 0.0099μM,respectively.The IC50 of WAY-235035 for NL4-3 and GX002 is 6.573μM and 0.0082μM,respectively.3.Molecular docking analysis showed that WAY-297131 and WAY-235035 had clear binding sites and low binding energy with B subtype and CRF01＿AE recombination Tat proteins,indicating that they are highly likely to have high binding ability.4.Affinity experiments showed that WAY-297131 and WAY-235035 inhibited HIV-1replication by decreasing the affinity of Tat protein to LTR.5.Inhibition effects of proteasome and lysosome inhibitors showed that WAY-297131 and WAY-235035 could also promote the degradation of B subtype and CRF01＿AE recombination Tat proteins through the proteasome pathway,which may be one of the mechanisms of their inhibition of HIV-1 replication.Conclusion:In this study,two small molecule compounds against HIV-1 virus,WAY-297131 and WAY-235035,were screened.They can directly act on Tat protein,reduce its affinity with LTR,and promote the degradation of Tat protein through the proteasome pathway to inhibit the replication of HIV-1.In vitro experiments have shown a low IC₅₀,which is close to the IC₅₀ of FDA-approved anti-HIV drugs,and is a promising candidate for anti-HIV-1 infection.