Screening And Identification Of Inhibitors On HIV-1 Integrase

Integration is a critical step in the retroviral life cycle. HIV-1 integrase is involved in the integration of HIV-1 DNA into host chromosomal DNA. It has become an attractive and rational target for selective anti-AIDS therapy. A random heptapeptide phage displayed library was panned on the recombinant HIV-1 integrase. After five rounds of panning, 13 positive phage clones were selected and sequenced. Two consensus peptides (TPSHSSR and HPERATL) were chemically synthesized. The non-radioactive ELISA-based HIV-1 integrase assay showed that the synthetic peptides TPSHSSR and HPERATL were able to inhibit the 3'cleavage or strand transfer activity of HIV-1 integrase to some extent (IC₅₀=54.56±5.18 μM, IC₅₀=28.29±1.32 μM, respectively). These heptapeptides could be used for developing new anti-HIV drug candidates, as well as for mechanism studies of the integration.1. Expression, purification and activity of recombinant HIV-1integraseA culture of Escherichia coli BL21 (DE3) cells, which contained the plasmid pT7-7-His-TX-WT-IN was used to expressed His-tagged HIV-1 integrase. Theculture was grown at 37 ℃ until OD₆₀₀ reached 0.6-0.8. Expression was induced by 1 mM IPTG, collected the cells after 5 h incubation at 30℃ by centrifugation. Cells were resuspended and the lysate was sonicated and centrifuged. The supernant was loaded onto a NTA resin column. The protein was eluted with buffer A containing 200 mM imidazole. Purified integrase filtered on membranes (0.22 μm) and was kept at -20 ℃. Both the integration and disintegration activity was assayed. The integration assay was performed by a non-radioactive ELISA-based method. And the disintegration products were analyzed on a 20% polyacrylamide gel.2. Panning of a peptide library and analysis of the selected peptidesfor integrase by ELISAIn our experiment, a phage display library of randomized linear heptamer peptides was used. The 96-well microtiter plates were coated with the integrase, and then blocked with 5% BSA. After five rounds of binding, elution, neutralization, titer and amplification, the selected phage was enriched approximate 700-fold. In order to identify the positive clones, we amplified the individual clones randomly from the fifth round of panning products. Assaying the different interaction of individual phage clones with the integrase and BSA, we determined the phage clones which specifically band to the integrase but not BSA as the positive ones. Using this method, we identified 13 phage clones from the linear heptamer peptides library which displayed peptides specifically band to the integrase.3. Sequence analysis of the positive clonesSequencing the single stranded DNA encoding the peptides displayed on the positive phages surface, we deduced the amino acid sequence of the selected heptapeptides. Scanning the amino acid sequence, two of the seven sequences have the same amino acid residues Thr, Pro and Ser at position one to three; another two sequences have the same amino acid residues Ser, His and Gly at position one, two and four. Besides, most of the seven amino acid sequences contain His, Arg, Pro and Ser.4. Competitive ELISA experimentsThe relative affinity of the two synthetic peptides was measured by their ability to compete for the integrase binding with their corresponding peptide-displaying phage using a competitive ELISA assay. This experiment was carried out on the 96-well immunosorbent plates coated with the integrase. The coated integrase was incubated with a solution containing a serially diluted synthetic peptide solution and a constant concentration of the corresponding phage. The phage binding rate was calculated by the formula: Phage binding rate = [ 1-(A_450-A'450/A₄₅₀]× 100%, where A₄₅₀ and A '₄₅₀ represent without and with the corresponding peptide, respectively. During the concentration of the synthetic peptides increased, the phage binding rate dropped. Thiese results further support the specific nature of the interaction between HIV-1 integrase and the selected peptides.5. Integration and disintegration inhibition experimentsThe inhibit effect of the synthetic peptides was investigated on the two in vitro activities of integrase (integration and disintegration) corresponding to different substrates. Inhibition experiment was performed as integrase activity assay expect that increasing concentrations of synthetic peptides were preincubated with the integrase. In our experiments, we found that the peptides TPSHSSR and HPERATL have the ability to inhibit both integration and disintegration reactions. Interestingly, the catalytic domain (or core domain) alone is capable of performing disintegration reaction while all three integrase domains (the core domain, N-terminal domain and C-terminal domain) are required for integration activity. These results indicate that the peptides TPSHSSR and HPERATL may act on the central catalytic domain. Of course, they also may have an effect on the DNA substrates.