Laboratory of Nucleic Acid Chemistry, Department of Chemistry of Natural Compounds, Faculty of Chemistry

The Ku protein contains two subunits, Ku70 and Ku80, and is an important participant in the repair system of double-stranded breaks in DNA, where it binds to the ends of DNA. In addition to repair, it is involved in other cellular processes, where it can also bind nucleic acids. In order to characterize the effect of Ku on transcription, the interaction of Ku with the internal regions of DNA is studied. Approaches to the creation of inhibitors of Ku binding to DNA are also being developed, which is of interest for the treatment of oncological diseases. It has also been shown that the Ku protein is able to bind to certain RNA structures. In particular, it interacts with TAR RNA, which plays an important role in many stages of HIV-1 development, and 7SCRNA, which regulates the transcription of many genes carried out by RNA polymerase II.

Eng Investigation of the mechanism of action of one of the main enzymes of the human immunodeficiency virus (HIV-1) – integrase and the structure of the integrase complex with DNK On the basis of a detailed kinetic study of enzymatic reactions, it was shown for the first time that HIV-1 integrase is practically a single-turn enzyme. The stoichiometry of protein-nucleic binding has been studied and, using a set of modified viral DNA analogues, it has been established which DNA regions directly interact with certain lysine residues in the protein. The structure of the integrase complex with DNA and its cellular cofactor, the LEDGF/p75 protein, was studied using the methods of protein and DNA “crosslinking” and cryo–electron microscopy. In collaboration with the Laboratories of Biotechnology and Applied Genetic Pharmacology of the NCNI in Kashan and the Institute of Genetics and Molecular Biology in Strasbourg, France, a fundamentally new model of the interaction of integrase with viral DNA has been proposed. ​ For the first time, a systematic study of the enzymatic properties and drug resistance of variants of the consensus integrase of HIV-1 subtype A of the FSU-A strain and the new genetic variant of HIV-1 CRF63_02A1, widely distributed in Russia, was conducted. The effect of mutations causing virus resistance to therapeutic integration inhibitors on the activity of these integrases has been studied.Development of approaches to the creation of HIV-1 integrase and reverse transcriptase inhibitors The information obtained during the study of the structure of HIV-1 integrase has been used to develop new integration inhibitors based on compounds of various natures, including those based on short modified oligonucleotides. They have been shown to be allosteric integrase inhibitors: they interact with the integrase complex with viral DNA and cause its rapid and effective dissociation. Using the Lomonosov and Lomonosov-2 supercomputers, the binding of the most effective oligonucleotide inhibitor with integrase was simulated. It has been shown that some oligonucleotide inhibitors of HIV-1 integrase are capable of suppressing reverse transcription in infected cells. Several new classes of low molecular weight organic compounds with anti-integrase activity have also been discovered. The study of the role of cellular proteins in HIV-1 replication, in particular, the study of the effect of Ku and SFPQ proteins on post-integration repair and transcription processes, This area of work is extremely relevant both for a detailed understanding of the development of this dangerous virus and for the development of new approaches to combating it. The main focus is on studying the involvement of two proteins – Ku70 and SFPQ - in the early stages of the HIV-1 replication cycle: reverse transcription, integration, post-integration repair and transcription. Reporter systems have been developed to assess the effect of these proteins on only one stage of the HIV-1 replication cycle. The interaction of Ku70 and SFPQ proteins with viral enzymes: integrase and reverse transcriptase is analyzed. ​ For the first time, it was shown that DNA-dependent protein kinase (DNA-PK) participates in post-integration repair of cellular DNA, which is the main participant in the repair system of double-stranded breaks in DNA along the path of non-homologous end joining. DNA-PK consists of the Ku protein, which is a sensor of the free end of DNA, and a catalytic subunit (DNA-PKcs). It has been established that the Ku protein is able to bind to HIV-1 integrase, and amino acids in the integrase composition necessary for binding to Ku have been identified. Using a pseudovirus containing a mutant integrase unable to bind Ku, it was shown that post-integration repair begins with the attraction of the Ku protein to the site of integration precisely due to its binding to the viral integrase. Further, the formed DNA PC phosphorylates proteins that directly carry out the repair process. Thus, the integrase and Kim complex can be considered as a new potential target for anti-HIV drugs.

The information obtained during the study of the structure of HIV-1 integrase was used to develop new integration inhibitors based on compounds of various natures, including those based on short modified oligonucleotides. They have been shown to be allosteric integrase inhibitors: they interact with the integrase complex with viral DNA and cause its rapid and effective dissociation. Using the Lomonosov and Lomonosov-2 supercomputers, the binding of the most effective oligonucleotide inhibitor with integrase was simulated. It has been shown that some oligonucleotide inhibitors of HIV-1 integrase are capable of suppressing reverse transcription in infected cells. Several new classes of low molecular weight organic compounds with anti-integrase activity have also been discovered.

Eng Investigation of the mechanism of action of one of the main enzymes of the human immunodeficiency virus (HIV-1) – integrase and the structure of the integrase complex with DNK On the basis of a detailed kinetic study of enzymatic reactions, it was shown for the first time that HIV-1 integrase is practically a single-turn enzyme. The stoichiometry of protein-nucleic binding has been studied and, using a set of modified viral DNA analogues, it has been established which DNA regions directly interact with certain lysine residues in the protein. The structure of the integrase complex with DNA and its cellular cofactor, the LEDGF/p75 protein, was studied using the methods of protein and DNA “crosslinking” and cryo–electron microscopy. In collaboration with the Laboratories of Biotechnology and Applied Genetic Pharmacology of the NCNI in Kashan and the Institute of Genetics and Molecular Biology in Strasbourg, France, a fundamentally new model of the interaction of integrase with viral DNA has been proposed. ​ For the first time, a systematic study of the enzymatic properties and drug resistance of variants of the consensus integrase of HIV-1 subtype A of the FSU-A strain and a new genetic variant of HIV-1 CRF63_02A1, widely distributed in Russia, was conducted. The effect of mutations causing virus resistance to therapeutic integration inhibitors on the activity of these integrases has been studied.