[Interaction of DNA Methyltransferase Dnmt3a with Phosphorus Analogs of S-Adenosylmethionine and S-Adenosylhomocysteine].

Enzymatic methyltransferase reactions are of crucial importance for cell metabolism. S-Adenosyl-L-methionine (AdoMet) is a main donor of the methyl group. DNA, RNA, proteins, and low-molecular-weight compounds are substrates of methyltransferases. In mammals, DNA methyltransferase Dnmt3a de novo methylates the C5 position of cytosine residues in CpG sequences in DNA. The methylation pattern is one of the factors that determine the epigenetic regulation of gene expression. Here, interactions with the catalytic domain of Dnmt3a was for the first time studied for phosphonous and phosphonic analogs of AdoMet and S-adenosyl-L-homocysteine (AdoHcy), in which the carboxyl group was substituted for respective phosphorus-containing group. These AdoMet analogs were shown to be substrates of Dnmt3a, and the methylation efficiency was only halved as compared with that of natural AdoMet. Both phosphorus-containing analogs of AdoHcy, which is a natural methyltransferase inhibitor, showed similar inhibitory activities toward Dnmt3a and were approximately four times less active than AdoHcy. The finding that the phosphonous and phosphonic analogs are similar in activity was quite unexpected because the geometry and charge of their phosphorus-containing groups differ substantially. The phosphorus-containing analogs of AdoMet and AdoHcy are discussed as promising tools for investigation of methyltransferases.

Synthesis of 1-[ω-(Bromophenoxy)alkyl]-3-naphthalenylmethyl Derivatives of Uracil and Their Analogues As Probable Inhibitors of Human Cytomegalovirus Replication.

The synthesis of a new series of 1-[ω-(bromophenoxy)alkyl]-uracil derivatives containing in position 3 naphthalen-1-yl-, naphthalen-2-yl-, 1-bromonaphthalen-2-ylmethyl, benzyl, and anthracene 9-methyl fragment was carried out. The antiviral properties of the synthesized compounds were studied against human cytomegalovirus. It was found that the compound that contained a bridge of five methylene groups has a high anti-cytomegalovirus activity in vitro.

New Analogues of Uridine as Possible Anti-Viral Agents Specific to SARS-CoV-2.

The development of specific drugs against SARS-CoV-2 infection is a major challenge facing global science and healthcare. Despite numerous attempts, there are still no truly effective drugs. Currently, the main approach in the creation of drugs against COVID-19 is repurposing, i.e., re-profiling existing drugs approved for medical use, for example, the use of a drug for the treatment of Ebola-Remdesivir, and the use of a drug for the treatment of influenza-Favipiravir. However, it is already obvious that these drugs are not specific enough nor effective enough. Another promising approach is the creation of new molecules, but it should be noted immediately that implementation requires much more time and costs. However, the search for new SARS-CoV-2 specific antiviral agents continues. The aim of our work was the creation of new 5-substituted uridine derivatives as potential inhibitors of coronavirus RNA-dependent RNA polymerase. The substances were obtained in high yields by the Suzuki‒Miyaura reaction and characterized using modern physicochemical methods. However, testing of their antiviral activity against SARS-CoV-2 did not reveal a significant inhibitory effect.

[New Analogues of Uridine as Possible Anti-Viral Agents Specific to SARS-CoV-2].

The development of specific drugs against SARS-CoV-2 infection is a major challenge facing global science and healthcare. Despite numerous attempts, there are still no truly effective drugs. Currently, the main approach in the creation of drugs against COVID-19 is repurposing, i.e., re-profiling existing drugs approved for medical use, for example, the use of a drug for the treatment of Ebola-Remdesivir, and the use of a drug for the treatment of influenza-Favipiravir. However, it is already obvious that these drugs are not specific enough nor effective enough. Another promising approach is the creation of new molecules, but it should be noted immediately that implementation requires much more time and costs. However, the search for new SARS-CoV-2 specific antiviral agents continues. The aim of our work was the creation of new 5-substituted uridine derivatives as potential inhibitors of coronavirus RNA-dependent RNA polymerase. The substances were obtained in high yields by the Suzuki-Miyaura reaction and characterized using modern physicochemical methods. However, testing of their antiviral activity against SARS-CoV-2 did not reveal a significant inhibitory effect.

Evaluation of the Antiviral Potential of Modified Heterocyclic Base and 5'-Norcarbocyclic Nucleoside Analogs Against SARS-CoV-2.

The pandemic caused by the novel betacoronavirus SARS-CoV-2 has already claimed more than 3.5 million lives. Despite the development and use of anti-COVID-19 vaccines, the disease remains a major public health challenge throughout the world. Large-scale screening of the drugs already approved for the treatment of other viral, bacterial, and parasitic infections, as well as autoimmune, oncological, and other diseases is currently underway as part of their repurposing for development of effective therapeutic agents against SARS-CoV-2. In this work, we present the results of a phenotypic screening of libraries of modified heterocyclic bases and 5'-norcarbocyclic nucleoside analogs previously synthesized by us. We identified two leading compounds with apparent potential to inhibit SARS-CoV-2 replication and EC50 values in a range of 20-70 µM. The structures of these compounds can be further optimized to develop an antiviral drug.

Synthesis and Antiviral Properties of 1-Substituted 3-[ω-(4-Oxoquinazolin-4(3H)-yl)alkyl]uracil Derivatives.

A series of uracil derivatives containing a 4-oxoquinazoline fragment bound to the nitrogen atom N3 of the pyrimidine ring by a short methylene bridge was synthesized to search for new antiviral agents. Some compounds in this series are shown to exhibit high inhibitory activity against human cytomegalovirus and the varicella zoster virus in a HEL cell culture.

5-Arylaminouracil Derivatives as Potential Dual-Action Agents.

Several 5-aminouracil derivatives that have previously been shown to inhibit Mycobacterium tuberculosis growth at concentrations of 5-40 µg/mL are demonstrated to act also as noncompetitive non-nucleoside inhibitors of HIV-1 reverse transcriptase without causing toxicity in vitro (MT-4 cells) and ex vivo (human tonsillar tissue).

2-(2,4-Dioxy-1,2,3,4-Tetrahydropyrimidin-1-yl)-N-(4-Phenoxyphenyl)-Acetamides as a Novel Class of Cytomegalovirus Replication Inhibitors.

A series of novel uracil derivatives, bearing N-(4-phenoxyphenyl)acetamide moiety at N3 of a pyrimidine ring, has been synthesized. Their antiviral activity has been evaluated. It has been found that the novel compounds possess high inhibitory activity against replication of human cytomegalovirus (AD-169 and Davis strains) in HEL cell cultures. In addition, some of the derivatives proved to be inhibitory against varicella zoster virus.

Carbocyclic analogues of inosine-5'-monophosphate: synthesis and biological activity.

9-(4'-Phosphonomethoxy-2'-cyclopenten-1'-yl)hypoxanthine and 9-(4'-phosphonomethoxy-2',3'-dihydroxycyclopenten-1'-yl)hypoxanthine were synthesized as isosteric carbocyclic analogues of inosine-5'-monophosphate. The synthesized compounds were shown to be capable of inhibiting the activity of human type II inosine-5'-monophosphate dehydrogenase (IMPDH II) (IC(50 )= 500 µM) and to have no significant effects on the growth ofMycobacterium tuberculosis.

1,2,4-Triazoloazine derivatives as a new type of herpes simplex virus inhibitors.

A new class of inhibitors of herpes simplex virus replication was found. The compounds under study are derived from condensed 1,2,4-triazolo[5,1-c][1,2,4]triazines and 1,2,4-triazolo[1,5-a]pyrimidines, structural analogues of natural nucleic bases. Antiherpetic activity and cytotoxicity of the compounds were studied. The corresponding triphosphates of several active compounds were prepared and tested as inhibitors of DNA synthesis catalyzed by herpes simplex virus polymerase. The potential mechanism of their action is blocking of DNA dependent DNA polymerase, a key enzyme of viral replication.

[Structure-functional analysis of interactions of terminal deoxynucleotidyl transferase with new non-nucleoside substrates].

New non-nucleoside esters of phosphoric acid containing various hydrophobic groups, namely (1) N-(2-tripticencarbonyl)-4-aminobutyl; (2) 5-phenylsubstituted N-(2,4-dinitrophenyl)-4-aminobutyl; (3) N-(4-phenylbenzoyl)- and N-(4-(N-benzylamino)benzoyl)-2-aminoethyl groups, as well as (4) diphenylmethyl and fluorenyl groups were synthesized and studied as substrates of terminal deoxynucleotidyl transferase. With the exception of the two latter derivatives, all the analogues displayed substrate properties and could incorporate into the deoxyoligonucleotide 3'-end. As it was shown in biochemical experiments and by computer modeling, a linker joining the triphosphate and hydrophobic fragments of the molecule was necessary for these compounds to display substrate properties.

Hepatitis C virus helicase/NTPase: an efficient expression system and new inhibitors.

A method has been developed for obtaining a full-length protein NS3 of hepatitis C virus with the yield of 6.5 mg/liter of cell culture, and conditions for measuring its NTPase and helicase activities have been optimized. The helicase reaction can proceed in two modes depending on the enzyme and substrate concentration ratio: it can be non-catalytic in the case of enzyme excess and catalytic in the case of tenfold substrate excess. In the latter case, helicase activity is coupled with NTPase and is stimulated by ATP. A number of NTP and inorganic pyrophosphate analogs were studied as substrates and/or inhibitors of NS3 NTPase activity, and it was found that the structure of nucleic base and ribose fragment of NTP molecule has a slight effect on its inhibitory (substrate) properties. Among the nucleotide derivatives, the most efficient inhibitor of NTPase activity is 2 -deoxythymidine 5 -phosphoryl-beta,gamma-hypophosphate, and among pyrophosphate analogs imidodiphosphate exhibited maximal inhibitory activity. These compounds were studied as inhibitors of the helicase reaction, and it was shown that imidodiphosphate efficiently inhibited the ATP-dependent helicase reaction and had almost no effect on the ATP-independent duplex unwinding. However, the inhibitory effect of 2 -deoxythymidine 5 -phosphoryl-beta,gamma-hypophosphate was insignificant in both cases, which is due to the possibility of helicase activation by this ATP analog.

Aryl-containing esters of triphosphoric acid as substrates of terminal deoxynucleotidyl transferase.

A new group of terminal deoxynucleotidyltransferase (TDT) substrates, namely, non-nucleoside triphosphates (NNTP) bearing 5-substituted 2,4-dinitrophenyl fragments instead of nucleoside residues was synthesized.

Telomerase-dependent reactivation of DNA synthesis in macrophages implies alteration of telomeres.

In previous work we demonstrated that various types of cultured cells with a limited life span could not reactivate DNA synthesis in the nuclei of mouse peritoneal macrophages in heterokaryons. We now investigate the role of telomerase in the process of the macrophage nucleus reactivation in heterokaryons with immortal telomerase-positive 3T3 Swiss mouse fibroblasts and human fibroblasts with introduced hTERT gene. We report that introduction of the hTERT gene into human diploid fibroblasts results in emergence of telomerase activity in these cells and the ability to induce the reactivation of DNA synthesis in the macrophage nuclei in heterokaryons. Inhibition of telomerase activity in heterokaryons by reverse transcriptase inhibitors (azidothymidine and guanosine polyphosphonate analogues) and by a 2'-O-methyl-RNA oligonucleotide anti-sense to the template region of telomerase RNA, block reactivation of DNA synthesis in macrophage nuclei without inhibiting DNA synthesis in the nuclei of fibroblasts. Our results suggest alterations (shortening or damage) in the macrophage telomere structure. As far as we know, heterokaryons with macrophages are the first cellular model for rapid investigation of the effects of telomerase inhibitors.

Modified dinucleoside tetraphosphonates, new potential inhibitors of HIV reverse transcriptase.

New gamma-substituted analogues of dNTP were synthesized and their enzymatic stability and antiviral properties were evaluated.

Anti-HIV activity of novel phosphonate derivatives of AZT, d4T, and ddA.

Anti-HIV activity and cytotoxicity were tested for novel phosphonate derivatives of AZT, d4T and ddA. For d4T phosphonate derivatives the most active was 2',3'-Dideoxy-2',3'-didehydrothymidine 5'-isopropylphosphite and among the AZT phosphonate derivatives highest activity was shown by 2',3'-Dideoxy-3'-azidothymidine 5'-cyclohexylphosphite.

P-(alkyl)-nucleoside 5'-hydrogenphosphonates as depot forms of antiviral nucleotide analogues.

P-(Alkyl)esters of AZT 5'-hydrogenphosphonate were synthesized and their stabilities in the phosphate buffer and human serum were evaluated. The esters bearing residues of primary and secondary alcohols were degraded to give AZT, whereas those containing tertiary alkyl groups yielded AZT 5'-hydrogenphosphonate. The corresponding derivatives of d2A and d4T showed the same properties.