S. P. Korolev

Specific features of HIV-1 integrase inhibition by bisphosphonate derivatives

The integration of viral DNA into the cell genome is one of the key steps in the replication cycle of human immunodeficiency virus type 1 (HIV-1). Therefore, the viral enzyme integrase (IN) catalyzing this process is of great interest as a target for new antiviral agents. We performed a structural-functional analysis of five different series of methylenebisphosphonates (BPs), PO3H2-C(R)(X)-PO3H2, as IN inhibitors with the goal of assessing structural elements required for the inhibitory activity. We found that IN is inhibited only by BP bearing a chlorobenzyl substituent R at the bridging carbon of the P-C-P backbone. These BP inhibited both IN-catalyzed reactions with similar efficacies. They were also active toward some INs with mutations characteristic for HIV-1 strains resistant to strand transfer inhibitors. The study of the mechanism of the IN inhibition by various BP showed that it is effected by the nature of the second substituent (X) at the bridging carbon. Among the tested compounds, only the BP with the amino group bound directly to the BP bridging carbon was found to be a noncompetitive inhibitor and, hence, it can be promising for further studies as potential inhibitor of the IN activity within the preintegration complex.

Structure–activity relationship study for design of highly active covalent peroxidase-mimicking DNAzyme

We synthesized a series of conjugates of hemin and its aptamer EAD2, named covalent peroxidase-mimicking DNAzymes (PMDNAzymes), varying the length, rigidity and 5′-/3′-position of a linker between the oligonucleotide and hemin. Systemic structure–activity relationship study of these PMDNAzymes showed that covalent PMDNAzyme with hemin bound to the 5′-end of EAD2 via T10 spacer (PMDNAzyme(T10)) demonstrated the highest activity in luminol oxidation assay. Its activity was significantly higher in comparison to the non-covalent complex of hemin and aptamer EAD2 (non-covalent PMDNAzyme). Comparison of the detection limit values for the PMDNAzyme(T10) in the reactions of oxidation of luminol and ABTS, which were equal to 0.2 and 1.6 pM, respectively, showed that the chemiluminescent method of PMDNAzyme(T10) detection is preferred over the colorimetric one. Similarity of the detection limit values for the PMDNAzyme(T10) and horseradish peroxidase, whose activity was measured in an enhanced chemiluminescence reaction (0.25 pM), opens up very promising perspectives for the development of highly sensitive PMDNAzyme(T10)-based assays and devices.

DNA sequence-specific ligands: XIII. New dimeric Hoechst 33258 molecules, inhibitors of HIV-1 integrase in vitro

Dimeric Hoechst 33258 molecules [dimeric bisbenzimidazoles (DBBIs)] that, upon binding, occupy one turn of the B form of DNA in the narrow groove were constructed by computer simulation. Three fluorescent DBBIs were synthesized; they consist of two bisbenzimidazole units tail-to-tail linked to phenolic hydroxy groups via penta-or heptamethylene or tri(ethylene glycol) spacers and have terminal positively charged N,N-dimethylaminopropyl carboxamide groups in the molecule. The absorption spectra of the DBBIs in the presence of different DNA concentrations showed a hypochromic effect and a small shift of the absorption band to longer wavelengths, which indicated the formation of a complex with DNA. The presence of an isobestic point in the spectrum indicates the formation of one type of DBBI-DNA complexes. The interaction of DBBIs with DNA was studied by CD using a cholesteric liquid-crystalline dispersion (CLD) of DNA. The appearance of a positive band in the absorption region of ligand chromophores in the CD spectrum of the DNA CLD indicates the formation of a DBBI-DNA complex in which ligand chromophores are arranged at an angle close to 54° relative to the helix axis of DNA, which suggests the localization of the DBBI in the narrow groove of DNA. All the DBBIs were found to be in vitro inhibitors of HIV-1 DNA integrase in the 3′-processing reaction, and, of the three DBBIs, two dimers inhibit HIV-1 integrase even in submicromolar concentrations.

Modulation of HIV-1 Integrase Activity by Single-Stranded Oligonucleotides and their Conjugates with Eosin

Integration of the DNA copy of the genomic RNA into an infected cell genome is one of the key steps of the replication cycle of all retroviruses. It is catalyzed by the viral enzyme, integrase. We have shown that conjugates of short single-stranded oligonucleotides with eosin efficiently inhibit the catalytic activity of the HIV-1 integrase. In this article, we have found that the dependence of the integrase catalytic activity on the concentration of oligonucleotides has a bell-shaped pattern. The modulation of HIV-1 integrase activity correlated with the oligonucleotide length and was not associated with specific sequences. Moreover, a similar mode of the oligonucleotide action was found for integrase from the prototype foamy virus. This dual effect of the oligonucleotide and their conjugates with eosin might be explained by their binding with retroviral integrase in two different sites; the oligodeoxynucleotide binding in the first site results in integrase activation, whereas interactions with another one lead to inhibition of the enzyme activity. Eosin coupling to oligonucleotides did not change the mode of their action but enhanced their affinity to both binding sites. The affinity increase was found to be much more important for the site responsible for the integrase inhibition, thus explaining the high inhibitory potency of oligonucleotide-eosin conjugates.

HIV-1 integrase inhibition by dimeric bisbenzimidazoles with different spacer structures

HIV-1 integrase is responsible for one of the key stages in virus replication, namely, integration of viral cDNA into the host cell genome. Integration inhibition leads to a complete block of the virus replication. We studied the integration inhibition by dimeric bisbenzimidazoles DBBI(7) with heptamethylene and DBBI(8) with tri(ethylene glycol) spacers and found that IC50 for DBBI(7) was approximately 0.03 μM and for DBBI(8) it was approximately 10 μM. Cross-linking assays demonstrated that both compounds interfered with a proper positioning of the DNA substrate in the active centre of integrase. To clarify the inhibition mechanism, dissociation constants were determined for the complexes between DBBI and integrase DNA substrate. Calculated Kd values for the complexes formed by DBBI(7) and DBBI(8) were 270 and 140 nM, respectively. Thus, the integration inhibition is not directly connected with DBBI binding to DNA. The dependence of initial enzymatic reaction rate on DNA substrate concentration in the presence of different concentrations of inhibitors was found, and inhibition constants were determined. These data suggest that different inhibition activity of DBBI(7) and DBBI (8) is determined by different mechanisms underlying their action, namely, competitive inhibition of integrase by DBBI(7) and a more complex mechanism assumed for DBBI(8).

Fusion to Flaviviral Leader Peptide Targets HIV-1 Reverse Transcriptase for Secretion and Reduces Its Enzymatic Activity and Ability to Induce Oxidative Stress but Has No Major Effects on Its Immunogenic Performance in DNA-Immunized Mice

Reverse transcriptase (RT) is a key enzyme in viral replication and susceptibility to ART and a crucial target of immunotherapy against drug-resistant HIV-1. RT induces oxidative stress which undermines the attempts to make it immunogenic. We hypothesized that artificial secretion may reduce the stress and make RT more immunogenic. Inactivated multidrug-resistant RT (RT1.14opt-in) was N-terminally fused to the signal providing secretion of NS1 protein of TBEV (Ld) generating optimized inactivated Ld-carrying enzyme RT1.14oil. Promotion of secretion prohibited proteasomal degradation increasing the half-life and content of RT1.14oil in cells and cell culture medium, drastically reduced the residual polymerase activity, and downmodulated oxidative stress. BALB/c mice were DNA-immunized with RT1.14opt-in or parental RT1.14oil by intradermal injections with electroporation. Fluorospot and ELISA tests revealed that RT1.14opt-in and RT1.14oil induced IFN-γ/IL-2, RT1.14opt-in induced granzyme B, and RT1.14oil induced perforin production. Perforin secretion correlated with coproduction of IFN-γ and IL-2 (R = 0,97). Both DNA immunogens induced strong anti-RT antibody response. Ld peptide was not immunogenic. Thus, Ld-driven secretion inferred little change to RT performance in DNA immunization. Positive outcome was the abrogation of polymerase activity increasing safety of RT-based DNA vaccines. Identification of the molecular determinants of low cellular immunogenicity of RT requires further studies.

Hydrophobic-core PEGylated graft copolymer-stabilized nanoparticles composed of insoluble non-nucleoside reverse transcriptase inhibitors exhibit strong anti-HIV activity

Benzophenone-uracil (BPU) scaffold-derived candidate compounds are efficient non-nucleoside reverse transcriptase inhibitors (NNRTI) with extremely low solubility in water. We proposed to use hydrophobic core (methoxypolyethylene glycol-polylysine) graft copolymer (HC-PGC) technology for stabilizing nanoparticle-based formulations of BPU NNRTI in water. Co-lyophilization of NNRTI/HC-PGC mixtures resulted in dry powders that could be easily reconstituted with the formation of 150-250 nm stable nanoparticles (NP). The NP and HC-PGC were non-toxic in experiments with TZM-bl reporter cells. Nanoparticles containing selected efficient candidate Z107 NNRTI preserved the ability to inhibit HIV-1 reverse transcriptase polymerase activities with no appreciable change of EC50. The formulation with HC-PGC bearing residues of oleic acid resulted in nanoparticles that were nearly identical in anti-HIV-1 potency when compared to Z107 solutions in DMSO (EC50=7.5±3.8 vs. 8.2±5.1 nM). Therefore, hydrophobic core macromolecular stabilizers form nanoparticles with insoluble NNRTI while preserving the antiviral activity of the drug cargo.

Nitrobenzofuroxane derivatives as dual action HIV-1 inhibitors

Human immunodeficiency virus type 1 (HIV-1) causes one of the most dangerous diseases, HIV infection and AIDS. The search for new inhibitors of the virus still remains an urgent task. One of approaches to suppress the HIV infection is the use of dual action HIV-1 inhibitors, i.e. inhibitors targeting two stages of the viral life cycle. The catalytic domain of HIV-1 integrase shares similar structural organization with the ribonuclease (RNase H) domain of HIV-1 reverse transcriptase, and therefore an approach aimed at creation of dual action inhibitors which would simultaneously inhibit HIV-1 integrase and RNase H seems to be very promising. In this work we have synthesized a series of 6-nitrobenzofuroxane derivatives and studied their inhibitory activity towards two HIV-1 enzymes, integrase and RNase H.

Oligonucleotide inhibitors of HIV-1 integrase efficiently inhibit HIV-1 reverse transcriptase

The impact of conjugates of 11-mer 2′-О-methyl oligoribonucleotides with eosin and 6-carboxy-4,7,2′,4′,5′,7′-hexachlorofluorescein on the functioning of HIV-1 reverse transcriptase (RT) was studied. These compounds were shown to inhibit the activity of RT RNase H domain. The inhibition efficiency was higher for eosin conjugates and did not depend on the oligonucleotide primary structure. The eosin conjugates were also found to block the RT polymerase activity including the mutant proteins resistant to nonnucleoside inhibitors. Since the conjugates are efficient inhibitors of HIV-1 integrase, we can assume that they belong to a new class of HIV-1 dual acting inhibitors, potentially capable of blocking several initial stages of the viral replication cycle.