Mechanism of HIV-1 Integrase Inhibition by Styrylquinoline Derivatives in Vitro

Abstract

Styrylquinoline derivatives (SQ) efficiently inhibit the 3Ј-processing activity of integrase (IN) with IC 50 values of between 0.5 and 5 M. We studied the mechanism of action of these compounds in vitro. First, we used steady-state fluorescence anisotropy to assay the effects of the SQ derivatives on the formation of IN-viral DNA complexes independently of the catalytic process. The IC 50 values obtained in activity and DNAbinding tests were similar, suggesting that the inhibition of 3Ј-processing can be fully explained by the prevention of IN-DNA recognition. SQ compounds act in a competitive manner, with K i values of between 400 and 900 nM. In contrast, SQs did not inhibit 3Ј-processing when IN-DNA complexes were preassembled. Computational docking followed or not by molecular dynamics using the catalytic core of HIV-1 IN suggested a competitive inhibition mechanism, which is consistent with our previous data obtained with the corresponding Rous sarcoma virus domain. Second, we used preassembled IN-preprocessed DNA complexes to assay the potency of SQs against the strand transfer reaction, independently of 3Ј-processing. Inhibition occurred even if the efficiency was decreased by about 5-to 10-fold. Our results suggest that two inhibitorbinding modes exist: the first one prevents the binding of the viral DNA and then the two subsequent reactions (i.e., 3Ј-processing and strand transfer), whereas the second one prevents the binding of target DNA, thus inhibiting strand transfer. SQ derivatives have a higher affinity for the first site, in contrast to that observed for the diketo acids, which preferentially bind to the second one.Integration of the HIV-1 DNA into the host genome ensures stable maintenance of the viral genome and perpetuation of the virus in the host organism. Therefore, this reaction, catalyzed by integrase (IN), is a key process in the life cycle of the virus. Inhibitors of therapeutic interest for AIDS exist. Inhibitors that act early in the replication cycle target reverse transcriptase and those that act later target protease. The high mutation rate of HIV means that drug resistance is emerging, making it necessary to develop new drugs with different targets (combined therapies). Thus, it is essential to find drugs that target alternative steps of the HIV-1 replication cycle. IN, the third enzyme of the Pol precursor protein, is then an attractive target for novel drugs because of its central role in the HIV-1 life cycle.IN is a 32-kDa protein (288 amino acids) that consists of three functional domains. The central domain (or core domain, residues 50 -210) contains the catalytic triad (DDE) that is essential for enzymatic activity. This domain is flanked by the N-terminal domain (which has a role in the assembly of an active multimeric form of the enzyme) and the C-terminal domain (which has a role in the nonspecific DNAbinding activity). These three domains, protein oligomerization, the integrity of the catalytic triad, and a cationic cofactor such as magnesium are all necessary fo...

Inhibition of Human Immunodeficiency Virus Type 1 Reverse Transcriptase, RNase H, and Integrase Activities by Hydroxytropolones

“…Biochemical data and modeling indicated that the IN active site adopts different conformations during the two catalytic steps of the integration process (6,18) and that it is possible to find step-specific IN inhibitors (12,25). Interestingly, our data showed that SP1 is a rather good inhibitor of the transfer step (IC 50 of 17 M) but a very poor inhibitor of 3Ј-end processing (Table 1).…”

“…To our knowledge, this is the first time that this strategy has been used to inhibit the polymerase activity of HIV-1 RT, and there has been only one published rational attempt to simultaneously target two metal ions in the RNase H active site (29). While this work was in progress, it became increasingly likely that several IN inhibitor families identified by "blind" or focused screening bind magnesium in the IN active site (12,23,39). However, it is not completely clear whether these inhibitors bind one or two metal ions (19,23).…”

Styrylquinolines, Integrase Inhibitors Acting Prior to Integration: a New Mechanism of Action for Anti-Integrase Agents

“…It was proposed that in vitro these molecules interfere with integrase DNA binding (17). In fact, these compounds strongly inhibit 3Ј processing and also the strand transfer step, even if the inhibition of the second reaction is not as efficient as inhibition of the first one.…”

“…After 2 h, RNAs were extracted and used for quantitative RT-PCR experiments. The reverse transcription step was done with a poly(T) [12][13][14][15][16][17][18] oligonucleotide as the primer, and the amount of cDNA was measured with ARNVϩ (5Ј-CTGTACT GGGTCTCTCTGGTTAGA-3Ј) and ARNVA2Ϫ (5Ј-TTTTTTTTTTTTTGAG CACTC-3Ј) as the primers and ARN SONDE (5Ј-6-carboxyfluorescein [FAM]-TCTGGCTAACTAGGGAACCCACTGCTTAA-6-carboxy tetramethylrhoda mine [TAMRA]-3Ј) as the Q-PCR probe.…”

“…Styrylquinolines (SQLs) are efficient in vitro integrase inhibitors that act both on 3Ј processing and strand transfer activities (52), probably interfering with long terminal repeat (LTR)/ integrase binding (37) through a competitive inhibition mechanism (17).…”