| Highly active anti-retroviral therapy(HAART)effectively restrains HIV and contributes to prevent the infection of others,which saves millions of people with AIDS.However,this kind of treatment does not remove latent viruses in patients,who need lifelong medication.Once the treatment stops,HIV will soon rebound in the body forming viremia.HIV is prone to mutation,and lifelong use of drugs can lead to high accumulation of resistance and multidrug resistance,which greatly limits the availability of anti-retroviral therapy and ultimately leads to no drug options.In recent years,how to achieve "functional cure" has become a hot topic in the field of AIDS research,and a variety of technologies have emerged,such as gene editing technology,early detection and treatment of HIV infection,"Shock and kill" strategy and strong anti-retroviral drug research and development."Shock and kill" strategy involves activating the latent virus reservoir first,then using the immune system and antiviral drugs to kill it,in order to achieve a complete elimination of the HIV reservoir.Among them,bromodomain-containing Protein 4(Brd4)inhibitor can activate latent HIV-1.virus.However,there are some common problems such as low activity and high concentration.In this paper,full-length target protein Brd4 was expressed in vitro and the immobilized protein was used to make a screening chip,Single concentration activity screening was carried out on 240 natural products to discover natural product compounds that interact with Brd4 based on Biacore drug screening technology,24 compounds have strong response values and dynamic fitting well;Using NH1,NH2,J-lat A2 and 2D 10 cell lines which contain HIV-1 latent activation gene reporter(Luciferase and GFP),it was found that 8 compounds could activate HIV-1 latent activation cell model well.Among them,#6 has nanomolar level activated latent HIV-1 gene transcription at the cellular level;The high activation rate was 76.3%at 10 nM;the expression level of target protein Brd4 was down-regulated in cells treated with#6 for 24 hours;The effect of#6 on the whole protein expression profile of HeLa modified F1C2 cells was studied by using SILAC-MS,and the total proteins of the cells after drug treatment were segmened and analyzed by mass spectrometry.A total of 7230 proteins were identified,of which 6821 had quantitative information.Find#6 role in F1C2 cells after the main cause the protein expression difference of cytoplasm,nucleus,cell membrane and Golgi,In the cell,it mainly undertakes the molecular biological functions such as protein binding,ATP binding,protein kinase activity,serine/tyrosine protein kinase activity,protein kinase binding,phosphatidylinositol binding and ubiquitin-protein transferase activity,Mainly involved in the positive regulation of NF-κB transcription factor activity,regulation of TNF mediated signaling pathways,positive regulation of IκB kinase/NF-κB signaling,protein phosphorylation and positive regulation of transcription from RNA polymerase Ⅱ promoter and other biological processes.It was also found that ELL and ELL2 proteins in the SEC complex were up-regulated(up by 2.0 and 5.1).In conclusion,quantitative proteomics based on mass spectrometry was used to quantitatively analyze the protein expression of cell model under#6 drug treatment.It was found that#6 drug could effectively activate HIV-1 LTR transcription mainly through NF-κB and ELL regulatory pathways,which could provide new clues for understanding the mechanisms of HIV-1 letency. |
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