HIV Inhibitor Screening And Evaluation Based On Rev-CRM1 Interaction

AIDS(acquired immunodeficiency syndrome),has caused millions of deaths since it was found in the 1980 s,raising serious public health and social problems.However,breakthrough in the vaccine against HIV is unlikely in near future.Therefore,development of anti-HIV drug remains a very important research filed.There are many effective antiretroviral therapy drugs,such as reverse transcriptase inhibitors,integrase inhibitors,protease inhibitors and HIV entry inhibitors.Combination of more than two different inhibitors could inhibit the replicatio n of HIV to undetectable levels.However,the drug-resistant mutations of HIV are being more and more frequent.So it is still very important to develop new anti-HIV drugs aiming at novel target in HIV life cycle.Rev is one of the regulatory proteins of HIV and one of the essential proteins of HIV.It plays an extremely important role in the life cycle of HIV.In the later stage of HIV life cycle,Rev can recruit host protein CRM1 and RanGTP,and HIV RNA(HIV genome and mRNA)containing RRE(Rev response element)generated by transcription,thus forming HIV RNA transport complex.The whole complex combines with the nuclear pore complex and is powered by the hydrolysis of GTP by RanGTP.The RNA can be transported out of the nucleus,and the RNA from the nucleus can be used for subsequent translation and virus packaging.Hence,Rev-CRM1 complex is considered as a potential target of new HIV inhibitors.Since the 1990s,the theory of computer aided drug design has become more and more perfect,and now it has become a major approach for drug discovery.It can simulate the interaction between receptor and ligand by computer simulation or design new and more effective drugs by the common structure of known active ligands.At present,molecular docking,pharmacophore,QSAR and other methods have been developed,especially molecular docking has been widely used in new drug discovery.We have obtained the structure of HIV Rev-CRM1 interaction in PDB database,and obtained two compound databases: China natural product database and NCI database.In this paper,the rigid docking and semi flexible docking strategies are used by molecular docking,and nine compounds,such as fukinolic acid,are finally selected and purchased from the two compound libraries through drug-likeness rules and toxicity prediction.After that,the activity of these nine compounds was evaluated at the cellular level.The results showed that three compounds(fukinolic acid;72722-20-4;32886-40-1)could inhibit the replication level of HIV in varying degrees.Among them,the activity of fukinolic acid was the best(EC50: 10.93 ? m;Si: 28.36).After that,we further confirmed the target of these drugs through DARTS(drug affinity response target stability)experiment,TOA(time of drug addition experiment)and biolayer interference experiment,and found that these three compounds all exert their anti-HIV function in the process of transportation of HIV RNA,and they all primarily bind to CRM1 but not Rev protein.Among them,fukinolic acid was confirmed to inhibit HIV rev-crm1 interaction at protein level.To sum up,we have obtained three small molecular compounds with HIV inhibitory activity for Rev-CRM1 target through this study.Compared with the three compounds previously reported,the selection coefficient of these three compounds has been significantly improved.These results will contribute to the development of anti HIV drugs based on Rev-CRM1 target.In the follow-up study,we will use QSAR method to optimize the structure of all the currently reported Rev-CRM1 inhibitors,and expect to get the compounds that have better activity and lower toxicity.