The Structural Investigation Of The HIV-1 Integrase Holoenzyme And The Screening Of Its Inhibitors

Human immunodeficiency virus is the pathogen which provokes the spreading of acquired immunodeficiency syndrome(AIDS) belonging to the subgenus of primate immunodeficiency virus of ribodeoxy virus and lentivirus. From 1981 when the first AIDS was discovered in America to now, there have been about 56,000,000 HIV victims and infectors over the world, and it has caused 13,900,000 people to death while about 16,000 infectors are adding everyday.HIV replicative cycle can be divided into the early stage including the absorbing, penetrating, uncoating, reverse transcriping, integrasing and the later stage of viral genome replication, advanced transcription, and so on. All these steps are so crucial to the infection that become the significant targets to the antivirus. Until August in 2006, there are 29 anti-HIV drug approved by FDA which include the nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, protease inhibitors and fusion inhibitors. Although all of them have been successfully used to cure AIDS, the severe drug resistance makes it still ugernt to search for more new drugs in clinical which effect on different targets.In recently years, the HIV-1 integrase becomes an attractive target to design new drugs. Different from protease and reverse transcriptase, the integrase has no counterpart in mammalian body, thus it has a better selectivity. But compared to the protease inhibitors and reverse transcriptase inhibitors, the designment of HIV-1 integrase inhibitors is relatively slow. As the inhibitor GS-9137 enters phaseⅠclinical in 2006 and raltegravir(MK-0518) was approaved to be the first new drug of integrase inhibitors in 2007, a significant breakthrough has been made in this field.To further develop the designment of HIV-1 integrase inhibitors, it is necessary to deeply understand the structure and the function of HIV-1 integrase. At present, there are 26 crystal structures of HIV-1 integrase in the protein data bank, which include single domain or two domains of it, but none of them is the holoenzyme structure of HIV-1 integrase. Thus, it is not sure that what multimer the HIV-1 integrase may adopt to play a catalytic role, and the understanding about the structure and the function of HIV-1 integrase is still ambiguous.This research makes full use of the structures of HIV-1 integrase and its inhibitors in the biological information database and chemical information database. Under the circumstances of the absent informations of the HIV-1 integrase holoenzyme, this acticle comprehensively use the molecular simulation technologies and theories of computer aided drug design(CADD) to carry out the investigation in two aspects. On one hand, constructing the pharmacophore of HIV-1 integrase inhibitors is accomplished based on the existing inhibitors, on the other hand building the HIV-1 integrase holoenzyme and the complex of HIV-1 integrase-viral DNA on the basis of the existing crystal structures of HIV-1 integrase. Besides, a docking pocket in the core domain of HIV-1 integrase is constructed to fulfill the docking test between HIV-1 integrase and its different inhibitors, then the docking results may offer useful informations which can provide important basis to develop the desigment of HIV-1 integrase inhibitors and the virtual screening based on the pharmacophore model and the docking pocket. Designment and screening of new HIV-1 integrase inhibitors also have importment significance to promote the combination therapy of curing AIDS.Objective: To simulate the heloenzyme structure of HIV-1 integrase, clarify the catalytic mechanism and active site of HIV-1 integrase,and then to screen new integrase inhibitors based on it.Methods: 1)The complete dimer of HIV-1 integrase was builded through structure comparison and homology modeling, and then the corresponding tetramer model was constructed to ascertain the multimer of HIV-1 integrase while it worked. 2) The complex of integrase-viral DNA was built through macromolecule docking and dynamics simulation to determine the active site and significant residues in the integrase. 3) To construct the pharmacophore of HIV-1integrase inhibitors based on the existing inhibitors. 4) To construct the docking pocket using the existing crystal structures of HIV-1 integrase. 5) Screening the Traditional Chinese Medicine Database to search for the suitable compounds based on the pharmacophore model and the docking pocket. 6) Through the molecular dynamics simulation, the binding informations is investigated between the HIV-1 integrase and the ligands. 7) Pharmacological tests of anti-HIV activity in vitro to the compounds.Results: The tetramer model of HIV-1 integrase and the complex of its dimer with viral DNA have been constructured. A pharmacophore of the strand transfer inhibitors of HIV-1 integrase with three accept atoms and a hydrophobic center has been constructed. Based on the pharmacophore model and the docking model, 9 suitable ligands from TCMD have been received after screening. They were glucobrassicin, glucocheirolin, glucoerysolin, glucoiberin, glucoraphanin, glucotropaeolin, sinigrin, neoglucobrassicin, sinalbin which had similar structures belonging to the thioglycosides. Besides, the molecular dynamics results truely showed the stubborn binding between the catalytic domain of HIV-1 integrase and these ligands.Conclusion:1 The existing research shows that the HIV-1 integrase may play a catalytic role as its dimer or tetramer. In this article we believe that the HIV-1 integrase is possible to play a catalytic role as its tetramer.2 It is possible that there are two Mg2+ existing in the catalytic domain of integrase. In other words, the integrase catalyze the whole reactions with a two ions mechanism.3 Two sinificant binding sites in the HIV-1 integrase of the 3′-processing(the viral DNA binding site) and the strand transfer(the human DNA binding site) are approximate but different, and the catalytic site of 3′-processing occupy the same position as the inhibitor of HIV-1 integrase in the crystal structure of 1QS4.4 The docking results show that chelating the important cofactor Mg2+ may be the inhibition mechanism of the strand transfer inhibitors.5 The pharmacophore model constructed with three accept atoms and a hydrophobic center may represent the significant features of most strand transfer inhibitors which are proved to be effective.6 The 9 compounds obtained through virtual screening are belonging to the thioglycosides, most of which are extracted from the cruciferae plants, such as isatis indigotica, folium isatidis. Plenty of the extractions from these plants with antibiosis, antivirus, antitumor and immunity improvement have been discovered through the pharmacological tests. Thus, the 9 compounds obtained after screening are possible to play a role of anti- HIV.