ИСТИНА |
Войти в систему Регистрация |
|
ИСТИНА ПсковГУ |
||
Today, the treatment of HIV infection mostly relies on a combination of inhibitors of viral enzymes (HAART) and as a result, drug resistant viral strains are eventually generated. However, a new approach for HIV drug design is being currently developed that is focused on the disruption of functional interactions between viral enzymes and their cellular co-factors. Disruption of the interaction between HIV-1 integrase (IN) and its cellular co-factor LEDGF inhibited viral replication in cell culture [Christ F. et al. Nat. Chem. Biol. 2010]. Recently, few reports showed that a human protein Ku70, a component of the DNA-PK complex, prevents HIV-1 IN from degradation probably by direct interaction [Zheng Y. et al. J. Biol. Chem., 2011]. It is known, that in cells depleted of Ku70, levels of viral replication are significantly lowered. We have studied formation of a complex between recombinant HIV-1 IN and Ku70 purified in E. coli by pull down experiments, and have shown that these proteins formed a stable complex with Kd about 70 nM. Moreover, we have found that the region within IN catalytic core essential for binding with Ku70 lies within a.a. 160-230 [Anisenko A. et al. FEBS Journal, 2014]. With the aim to develop inhibitors of the Ku70-IN interaction, we studied the effect of a 2’-O-methylated 11-mer GGUUUUUGUGU conjugated to eosin (11-OM-E) on the formation and stability of the Ku70-IN complex. This conjugate 11-OM-E has been shown to inhibit HIV-1 IN by disrupting its complex with DNA [Agapkina J. et al. ACS Med. Chem. Lett., 2011]. To study the 11-OM-E effect on the complex formation, the conjugate (10 nM-10 uM) was mixed with both proteins, and the mixture was incubated at 25oC for 1h. In order to evaluate the conjugate influence of the complex stability, 11-OM-E was added to a pre-formed Ku70-IN complex. We have found that the conjugate 11-OM-E inhibits the complex formation with IC50 about 30 nM, whereas 10-fold higher concentration is necessary to produce the complex disruption (IC50 about 300 nM). The same experiments were carried out with the oligonucleotide without eosin (11-OM), and no inhibitory effect was detected. For comparison, we also tested the 11-OM-E influence of the integrity of IN-LEDGF complex. The conjugate did not influence the complex entirety, however, the catalytic activity of IN within its complex with LEDGF was inhibited (IC50 = 50 nM). Thus we have demonstrated that 11-OM-E efficiently inhibits interactions of HIV-1 IN with its cellular partner Ku70, and the presence of eosin is crucial for the inhibitory effect of the conjugate. This work was supported by RSF grant 14-24-00061.