Favipiravir and Its Structural Analogs: Antiviral Activity and Synthesis Methods.

1,4-Pyrazine-3-carboxamide-based antiviral compounds have been under intensive study for the last 20 years. One of these compounds, favipiravir (6-fluoro-3-hydroxypyrazine-2-carboxamide, T-705), is approved for use against the influenza infection in a number of countries. Now, favipiravir is being actively used against COVID-19. This review describes the in vivo metabolism of favipiravir, the mechanism of its antiviral activity, clinical findings, toxic properties, and the chemical synthesis routes for its production. We provide data on the synthesis and antiviral activity of structural analogs of favipiravir, including nucleosides and nucleotides based on them.

A Cascade of Thermophilic Enzymes As an Approach to the Synthesis of Modified Nucleotides.

We propose a new approach for the synthesis of biologically important nucleotides which includes a multi-enzymatic cascade conversion of D-pentoses into purine nucleotides. The approach exploits nucleic acid exchange enzymes from thermophilic microorganisms: ribokinase, phosphoribosylpyrophosphate synthetase, and adenine phosphoribosyltransferase. We cloned the ribokinase gene from Thermus sp. 2.9, as well as two different genes of phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and the adenine phosphoribosyltransferase (APR-transferase) gene from Thermus thermophilus HB27 into the expression vectors, generated high-yield E. coli producer strains, developed methods for the purification of the enzymes, and investigated enzyme substrate specificity. The enzymes were used for the conversion of D-pentoses into 5-phosphates that were further converted into 5-phospho-α-D-pentofuranose 1-pyrophosphates by means of ribokinase and PRPP-synthetases. Target nucleotides were obtained through the condensation of the pyrophosphates with adenine and its derivatives in a reaction catalyzed by APR-transferase. 2-Chloro- and 2-fluoroadenosine monophosphates were synthesized from D-ribose and appropriate heterobases in one pot using a system of thermophilic enzymes in the presence of ATP, ribokinase, PRPP-synthetase, and APR-transferase.

[Optimization of modern conservative therapy of micropenis in hypogonadal men].

The study was aimed to the optimization of conservative therapy of micropenis in hypogonadal men using combination of traction therapy and androgen replacment therapy (ART) with injections of prolonged testosterone undecanoate (Nebido) and to evaluatiom of the safety of ART in terms of the risk of prostate cancer against the background of combined treatment of micropenis by both methods within 12 months. The study included 16 men aged 22-62 years with micropenis and hypogonadism. 10 men were diagnosed with primary hypogonadism, 6 men were diagnosed with secondary hypogonadism without reserve gonadal function; therefore, all 16 patients were treated with testosterone undecanoate 1000 mg intramuscularly according to the scheme: the second injection 6 weeks after the first injection, then each injection once a 12 weeks, the course of 12 months. During the first 3 months of ART, hypogonadism in all men was eliminated, but only at 6 month of ART, the length of the penis in the flaccid state at maximum extension increased from 5.8±1.2 to 8.3±1.2 cm (p<0.05), and the length of the erect penis - from 6.8±1.1 to 11.8±0.9 (p<0,05). At the next stage, from the 6th to the 12th month of ART, traction therapy was simultaneously carried out. At the end of the treatment, the length of the penis in the flaccid state at maximum extension increased by 58% of the original length, and in a state of erection - by 114% (p<0.05). During the 12 months of treatment, prostate volume in all men increased from 3.4±1.2 to 16.3±1.2 (p<0.05), which corresponds to the size of the prostate in healthy men. Total blood PSA level increased from 0.72±0.03 to 1.4±0.05 ng/ml (p<0.05), but it was in the acceptable range of reference values for healthy men during whole period of ART in all patients. Start therapy with prolonged testosterone undecanoate for 6 months significantly increases the efficiency of traction therapy in men with hypogonadism and micropenis, but for maintenance of the effect, ART should be continued during all period of treatment.

[The structure of urological disorders in athletes].

According to the data of preventive medical examinations since the end of 2009 to April 2012, data on the urological incidence in 654 athletes surveyed during this period were collected and analyzed. Among the diseases identified in athletes, the main place is occupied by varicocele (5.35%), urethritis (5.04%), urolithiasis without clinical manifestation of acute inflammation (1.37%).

[Nucleosides of 1,2,4-triazole: potentialities and restrictions of chemo-enzymatic method for synthesis].

The potentialities and restrictions of chemoenzymatic approach to the synthesis of new structural analogues of antiviral drug Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) have been determined. Syntheses of various amides of 1H-1,2,4-triazole-3-carboxylic acid and its 5-substituted analogues, prospective substrates of purine nucleoside phosphorylase (PNP), have been reported. The comparative effectiveness of the methods for obtaining amides aforementioned and also the methods for introducing functional groups to the C5 position of the heterocyclic system has been studied. New Ribavirin analogues bearing various substituents in the carboxamide group have been synthesized. The biotechnological method for the preparation of 1-beta-D-ribofuranosyl- 1,2,4-triazole-3-carbonitrile used as the intermediate in the synthesis of Viramidine, a contemporary Ribavirin analogue, has been developed.

[The arsenolysis reaction in the biotechnological method of synthesis of the ribavirin. Inhibition of reproduction of influenza A virus with the combination of ribavirin and ozeltamivir in experiments in vitro and in vivo].

Improved biotechnological method of receiving the antiviral drug ribavirin by the reaction of transglycosilation by addition of catalytic amounts of sodium arsenate in the reaction mixture. Such approach allows to hydrolyze the amount of the excess natural nucleoside donor--ribose and, as a consequence, to simplify the composition of the reaction mixture and the process of separation of ribavirin. The effect of ribavirin and ozeltamivir carboxylate and their combination on the reproduction of the virus of the influenza A in cell culture and in experiments on laboratory animals (mouse Balb/C). The greatest anti-influenza effect is observed when using a combination of drugs, as compared to each of them taken separately.

[In vitro activity of Russian ribavirin on the models of Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Tahyna and Dhori viruses].

The high activity of ribavirin made by effective biotechnology in Russia was established in in vitro experiments using the models Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Tahyna and Dhori viruses, which suggests that it is promising in using the drug in the treatment of infection with these viruses.

[Reprogramming of nuclear proteasomes in K562 cells undergoing apoptosis. II. Effect of anticancer drug doxorubicin].

The induction of apoptosis in K562 cells by doxorubicin (DR) was used as a model to investigate changes in the subunit composition, phosphorylation state and enzymatic activities of 26S proteasomes in cells undergoing the programmed death. Here we have shown for the first time that proteasomes isolated from the nuclei of control and induced K562 cells differ in their subunit patterns, as well as in the phosphorylation state of subunits on threonine and tyrosine residues. It has been shown for the first time that trypsin- and chymotrypsin-like, and the endoribonuclease activities of nuclear 26S proteasomes are affected under influence of DR on K562 cells. Treatment of K562 cells with DR leads to modification of zeta/alpha5 and iota/alpha6 proteasomal subunits associated with RNase activity of proteasomes. These findings confirm our hypothesis about so-called reprogramming of nuclear proteasomes population in undergoing apoptosis K562 cells which is manifested by changes in proteasomal composition, phosphorylation state, and enzymatic activities during the programmed cell death.

[Reprogramming of nuclear proteasomes in K562 cells undergoing apoptosis. I. Effect of glutathione-depleting agent, diethylmaleate].

Here we have studied changes in the subunit composition, phosphorylation state and enzymatic activities of 26S proteasomes in cells undergoing the programmed cell death. Apoptosis in proerythroleukemic K562 cells was induced by glutathione-depleting agent, diethylmaleate (DEM). We have shown for the first time that proteasomes isolated from the nuclei of control and induces K562 cells differ in their subunit patterns, as well as in the phosphorylation state of subunits on threonine and tyrosine residues. We observed trypsin- and chymotrypsin-like activities on nuclear proteasomes and the specificity of proteasomal nucleolysis of several individual messenger RNAs (c-fos and c-myc) to be changed under effect of DEM on K562 cells. Treatment of K562 cells with DEM leads to modification of zeta/alpha5 and iota/alpha6 proteasomal subunits associated with RNAse activity of proteasomes. These findings confirm our hypothesis about so-called reprogramming of nuclear proteasome population in undergoing apoptosis K562 cells which is manifested by the changes in proteasomal composition, phosphorylation state, and enzymatic activities during the programmed cell death.

[A focal infiltration in the upper lobe of the right lung as a manifestation of the initial stage of secondary syphilis].

Among sexually transmitted diseases, the incidence of primary and secondary syphilis has substantially increased in Russian Federation within the recent years. Modern clinicians, acquainted only with conventional dermatovenerologic symptoms of the disease, are less aware of rare manifestations of syphilis as a generalized infection. A rare case of syphilis with infiltrative pulmonary lesion is discussed in the article; the attention of practitioners is drawn to "unusual" manifestations of syphilis.

[Regulation of the 26S proteasomes' endoribonuclease activity specificity in k562 cells under effect of differentiation and apoptosis inductors].

The specificity of 26S proteasomes' endoribonuclease activity has been shown to be changed under effect of erythroid differentiation (hemin) and programmed cell death (diethylmaleate) inductors in proerythroleukemic K562 cells. Treatment of K562 cells with apoptosis and differentiation inductors leads to the specific stimulation of RNase activity towards certain mRNA and to reduction of proteasome RNase activity towards other mRNA. The enzymatic activity under study has been demonstrated to be specifically and selectively dependent on phosphorylation of 26S proteasome subunits as well as on Mg and Ca ions. The conclusion is drawn that the specificity of the proteasomes' RNAse activity is regulated during differentiation and apoptosis, and selective regulation of the activity of different nuclease centers is suggested, the mechanism involving changes in phosphorylation of proteasome subunits and cation homeostasis.

[Specificity of changes in proteasome properties in diethylmaleate-induced apoptosis of K562 cells].

The participation of proteasome in the programmed cells death is now extensively investigated. Studies using selective inhibitors of proteasomes have provided a direct evidence of both pro- and anti-apoptotic functions of proteasomes. Such opposite roles of 26S proteasomes in regulation of apoptosis may be defined by the proliferative state of cell. The induction of apoptosis in K562 cells by diethylmaleate was used as a model to investigate changes in the subunit composition, phosphorylation state and enzymatic activities of 26S proteasomes undergoing the programmed cell death. Here we have shown that proteasomes isolated from the cytoplasm of control and diethylmaleate treated K562 cells differ in their subunit patterns, as well as in the phosphorylation state of subunits on threonine and tyrosine residues. It has been shown for the first time that proteolytic activity of 26S proteasomes is decreased, and endoribonuclease activity of 26S proteasomes is affected under diethylmaleate action on K562 cells. Treatment of K562 cells with an inductor of apoptosis--diethylmaleate--leads to modification of a proteasomal subunit (zeta/alpha5) associated with RNase activity of proteasomes. These data suggest the subunit composition and enzymatic activities of 26S proteasomes to be changed in K562 cells undergoing apoptosis, and that specific subtypes of 26S proteasomes participate in execution of programmed death of these cells.

[Phosphorylation of proteasomes and alpha-RNP from rat liver cells].

In eukaryotic cells the population of proteasomes is heterogeneous. Here we have shown that proteasomes from nuclei and cytoplasm of rat liver cells differ in their subunit patterns. The subunit pattern of alpha-RNP differs from that of proteasomes, however, alpha-RNP particles contain the number of 26S proteasome subunits. Moreover, the proteasomes contain subunits of alpha-RNP. We have shown for the first time that nuclear proteasomes and alpha-RNP are hyperphosphorylated on threonine residues. Differences in phosphorylation state of subunits of nuclear and cytoplasmic proteasomes and alpha-RNP on threonine and tyrosine residues have been revealed. A suggestion is put forward that hyperphosphorylation of subunits may determine nuclear localization of these complexes in rat liver cells. The results obtained suggest that a highly specialized system of protein kinases and phosphatases may be involved in the regulation of phosphorylation state of different populations of proteasomes and alpha-RNP in rat liver cells.

[Effect of EGF on the activity of nuclear and cytoplasmic 26S proteasomes in A431 cells].

It has been first shown that EGF regulates a proteolytic activity of proteasomes. Following a 15 min action with 100 ng/ml EGF, three types of peptidase activity of both cytoplasmic and nuclear proteasomes were induced in A431 cells, although, this effect on different populations of proteasomes was selective. EGF preferentially stimulates chymotrypsin-like activity of cytoplasmic proteasomes, and induces a similar increase of chymotrypsin-like, trypsin-like and peptydylglutamyl peptide hydrolase activities of nuclear particles. Tyrphostin, an inhibitor of tyrosine kinase activity of EGF receptor, prevents the EGF effect on both proteolytic and RNase activity of nuclear and cytoplasmic proteasomes. It is concluded that EGF may rapidly and selectively stimulate enzymatic activity of EGF receptor.

[Biotechnological synthesis of ribavirin. Effect of ribavirin and its various combinations on the reproduction of Vaccinia virus]. / Biotekhnologicheskii sposob polucheniia ribavirina. Deistvie ribavirina i nekotorykh ego kombinatsii na reproduktsiiu virusa ospovaktsiny (Vaccinia virus).

The biotechnological method of synthesis of ribavirin, vidarabin, and 6-azauridine by the use of immobilized recombinant enzymatic preparations of nucleoside phosphorylase was improved. The effect of ribavirin and its combinations with the other synthesized nucleosides on the reproduction of Vaccinia virus was studied using cultures of Vero cells. The combination of ribavirin and vidarabin was shown to provide an antiviral effect at lesser concentrations than when these compounds were taken separately. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

[Selective effect of epidermal growth factor on the endoribonuclease activity of different subpopulations of proteasomes from A431 cell line. Specificity of extracellular proteasome population]. / Selektivnoe vliianie épidermal'nogo faktora rosta na éndoribonukleaznuiu aktivnost' razlichnykh subpopuliatsii proteasom v kletkakh linii A431. Spetsifichnost' populiatsii proteasom, éksportiruemykh iz kletok.

For the first time, it has been shown that population of proteasomes is heterogeneous in their RNAse activity. EGF exerts selective effect on different subpopulations of proteasomes. The RNAse activity of cytoplasmic proteasomes is induced under the influence of EGF on epidermoid carcinoma cell line A431. However, the activity of proteasomes isolated from culture medium and of nuclear proteasomes is inhibited by EGF. The above enzymatic activity has been shown to be specifically and selectively dependent on phosphorylation of proteasomal subunits in different subpopulations of proteasomes. Proteasome involvement in the coordinated control of specific messenger RNA molecules stability is suggested, and one of the mechanisms of this control might be an export of specific subpopulation of proteasomes from the cell.

[Selective effect of inductors of apoptosis on the endoribonuclease activity of 26S proteasomes and alpha-RNP particles in K562 cells: possible involvement of 26S proteasomes and alpha-RNP in the regulation of RNA stability]. / Differentsial'naia reguliatsiia éndoribonukleaznoi aktivnosti 26S-proteasom i alpha-RNP-chastits pri deistvii induktorov apoptoza na kletki linii K562: vozmozhnoe uchastie alpha-RNP-chastits i proteasom v kontrole nad stabil'nost'iu RNK pri programmirovannoi kletochnoi gibeli.

It has been shown that endoribonuclease activity of alpha-RNP particles and 26S proteasomes are changed under the action of inductors of programmed cell death. Treatment of K562 cells with inductors of apoptosis--doxorubicin (adriamycin) and diethylmaleate--lead to a significant stimulation of RNAse activity of alpha-RNP and to reduction of proteasome RNase activity. The enzymatic activity under study has been shown to be specifically and selectively dependent on phosphorylation of subunits of alpha-RNP particles and 26S proteasomes. The characteristics of RNAse activity of different subpopulations of proteasomes differ. The specificity of a subpopulation of proteasomes exported from the cell has been demonstrated. Proteasome and alpha-RNP involvement in the coordinated control of stability of various specific messenger RNA molecules is suggested, and one of the mechanisms of this control might be the export of specific subpopulation of proteasomes from the cell.