Timofey S. Zatsepin

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.

Characterization of HIV-1 integrase interaction with human Ku70 protein and initial implications for drug targeting

Human Ku70/Ku80 protein is known to influence HIV-1 replication. One of the possible reasons may be the protection of integrase from proteasomal degradation by Ku70 subunit. We demonstrated that recombinant HIV-1 integrase and Ku70 form a stable complex, while no interaction of Ku70 with integrase from prototype foamy virus was observed. By analyzing protein subdomains we determined two binding sites in the structure of both Ku70 and integrase: the 51–160 a.a. region of integrase interacts with residues 251–438 of Ku70, whereas Ku70 N-terminal domain (1–250 a.a.) contacts an α6-helix in the 200–220 a.a. integrase region. Single substitutions within integrase (E212A or L213A) block the interaction with Ku70 thus indicating that the binding site formed by the 200–220 a.a. integrase region is crucial for complex formation. E212A/L213A substitutions decreased the integrase capacity to bind Ku70 in HEK293T cells. A conjugate of 2′-ОMe-GGUUUUUGUGU oligonucleotide with eosin is shown by molecular modeling to shield integrase residues E212/L213 and is effective in blocking complex formation of Ku70 with integrase what makes the complex between α6-helix and Ku70(1–250) a possible target for drug development.

Synthesis and biological evaluation of novel mono- and bivalent ASGP-R-targeted drug-conjugates

Asialoglycoprotein receptor (ASGP-R) is a promising biological target for drug delivery into hepatoma cells. Nevertheless, there are only few examples of small-molecule conjugates of ASGP-R selective ligand equipped by a therapeutic agent for the treatment of hepatocellular carcinoma (HCC). In the present work, we describe a convenient and versatile synthetic approach to novel mono- and multivalent drug-conjugates containing N-acetyl-2-deoxy-2-aminogalactopyranose and anticancer drug - paclitaxel (PTX). Several molecules have demonstrated high affinity towards ASGP-R and good stability under physiological conditions, significant in vitro anticancer activity comparable to PTX, as well as good internalization via ASGP-R-mediated endocytosis. Therefore, the conjugates with the highest potency can be regarded as a promising therapeutic option against HCC.