Ivanka Nikolova

Development of resistance to disoxaril in Coxsackie B1 virus-infected newborn mice

Treatment with disoxaril (a WIN compound binding to the hydrophobic pocket within the enterovirus VP1 capsid protein) in newborn mice infected with Coxsackie B1 virus, for 10 days post virus inoculation at a daily subcutaneous dose of 25mg/kg decreased the virus titer in the mouse brain till day 7. Thereafter (on days 8 and 9) drug-resistant virus progeny in brain samples isolates was noted with disoxaril IC(50) values of >40 microM as compared to 0.59-1.37 microM in placebo. Study on phenotypic characteristics of the disoxaril-resistant mutants (as obtained in vivo or in cell culture experiments in parallel with the wild disoxaril-sensitive virus) showed: (i) larger size of virus plaques under agar overlay; (ii) more irregular shape of virus plaques; (iii) markedly less thermostability at 50 degrees C; (iv) slightly increased pathogenicity for newborn mice. The results obtained provide convincing evidence for the development of drug-resistant progeny of Coxsackie B1 virus in the brain of mice following treatment with disoxaril.

Anti-enteroviral triple combination of viral replication inhibitors: activity against coxsackievirus B1 neuroinfection in mice

Background Chemotherapy is an important tool for controlling enterovirus infections, but clinically effective anti-enterovirus drugs do not currently exist, mainly due to the development of drug resistance. We investigated the combination effects of enterovirus replication inhibitors in order to limit this process. In previous studies, we showed the efficacy of consecutive alternating administration of the triple combinations disoxaril/guanidine/oxoglaucine and pleconaril/guanidine/oxoglaucine against coxsackievirus B1 infection in newborn mice. Drug sensitivity tests of the viral brain isolates showed that these drug combinations prevented the development of drug resistance. Methods In the current study, we replaced guanidine-HCl with enteroviral RNA synthesis inhibitor MDL-860 to test the effect of a new triple combination—pleconaril/MDL-860/oxoglaucine—applied via consecutive alternating administration in newborn mice infected subcutaneously with 20 MLD50 of coxsackievirus B1. Results The pleconaril/MDL-860/oxoglaucine combination via consecutive alternating administration showed high activity at the 75 mg/kg MDL-860 dose: a protective effect of 50% and a pronounced suppression of brain virus titers. Moreover, along with prevention of drug resistance, a phenomenon of increased drug sensitivity was established. MDL-860 sensitivity in pleconaril/MDL-860/oxoglaucine increased 8.2 times vs. placebo (29 times vs. monotherapy) on day 7 and oxoglaucine sensitivity—4.9 times vs. placebo (by 6.8 times vs. monotherapy) on day 13. As concerns pleconaril, a demonstrable prevention of drug resistance was registered without increase of drug sensitivity. Daily, simultaneous administration of pleconaril/MDL-860/oxoglaucine showed no protective effects and led to a rapid development of drug resistance. Conclusions These results add new support for using consecutive alternating administration treatment courses to achieve clinically effective chemotherapy of enterovirus infections.

Effect of consecutive alternating administration (CAA) of a triple anti-enteroviral combination on Coxsackievirus B1 neuroinfection in mice

Currently, clinically effective antivirals for use in the treatment of enteroviral (EV) infections do not exist. The main reason is the development of drug resistance, the principle obstacle in the development of EV infection chemotherapy, based til now on monotherapy. The most important achievement of our previous studies was the development of a novel scheme for in vivo application of a triple combination of EV inhibitors with different modes of action against Coxsackievirus B (CVB) infections in mice. It consists of consecutive alternating administration (CAA) of the substances in the combination. Here, we tested the effect of the triple combination pleconaril, guanidine-HCl, and oxoglaucine (PGO) via CAA in newborn mice infected with a neurotropic strain of CVB1 (20 LD50 per mouse). This combination manifested a considerable protective effect with pleconaril doses of 25-200mg/kg: it decreased mortality rate (protection index, PI, between 31.3% and 67.7%) and increased mean survival time (MST) by 4-6days. Pleconaril monotherapy demonstrated activity similar to that of PGO via CAA, as measured by PI values, but MST values were slightly lower. However, it also greatly suppressed growth of infected suckling mice, especially at 200mg/kg. This toxic effect was avoided with CAA of PGO at pleconaril doses of 25-100mg/kg. Pleconaril monotherapy administered every 3days was ineffective. The PGO with CAA treatment course decreased infectious virus content, whereas pleconaril monotherapy did not. Analysis of drug-sensitivity in brain samples from CVB1 infected mice, based on IC50 (50% inhibitory concentration) values from cell culture experiments, showed that the CAA course counteracted the development of drug resistance to pleconaril and oxoglaucine in the triple PGO combination and increased drug sensitivity. In contrast, pleconaril and oxoglaucine monotherapies resulted in drug resistance. This data clearly proves the effectiveness of the proposed novel approach-the CAA treatment course-for combined application of EV replication inhibitors.

Anti-Herpes Simplex Virus Type 1 Activity of Specially Selected Groups of Tannins

Anti-herpes simplex virus (HSV-1) activity of 9 ellagitannins, including 6 natural compounds (castalin, vescalin, acutissimin A, epiacutissimins A and B, mongolicain) and 3 vescalagin synthetic derivatives (VgSBuSH, VgSOctSH, VgOMe), and 13 gallotannin-type compounds [Gal-01A, Gal-01B, Gal-02A, Gal-02B, Gal-03M, Gal-04A, Gal-04B, Gal-05M, Gal-07, Gal-08, Gal-09, Gal-11M (tannic acid), as well as Gal-12 (gallic acid), Gal-13 and Gal-14 (ellagic acid)] were examined in MDBK monolayer cell culture. Their antiviral activity was determined by the cytopathic effect (CPE) inhibition test and their cytotoxicity was evaluated through the neutral red uptake assay. In general, the series of ellagitannins showed a significantly stronger activity against HSV-1 replication than that of the gallotannins. Six of the tested ellagitannins manifested a well-pronounced activity: epiacutissimin B (selectivity index, SI>60.6), epiacutissimin A (SI>55.5), acutissimin A (SI>34.8), mongolicain (SI>32.5), VgSBuSH (SI>24.6) and VgOMe (SI>22.0). Four gallotannin-type compounds inhibited the replication of HSV-1 at a lower but still significant extent: Gal-04B (SI>35.7), Gal-04A (SI>28.5), Gal-11M (tannic acid) (SI>25) and Gal-05M (SI=15.6).

Synthesis and anti-enterovirus activity of new analogues of MDL-860

A series of twelve novel compounds, analogues of antiviral agent MDL-860 were synthesized and their antiviral activity was evaluated in vitro against enteroviruses poliovirus 1 (PV1), Coxsackieviruses B1 (CVB1) and Coxsackieviruses B3 (CVB3). Compounds 14, 24 and 25 manifested strong antiviral effects against CVB1 and PV1 (SI values of 405 and 118 for CVB1 and PV1 respectively). In contrast to the wide anti-enteroviral activity of MDL-860, these three compounds were inactive against CVB3. Compounds 14, 24 and 25 along with MDL-860 were tested in vivo in mice infected with CVB1. Marked protective effects of compounds 14 and 24 were established, PI values of 50% and 33.3%, respectively. In addition, almost all of the tested compounds manifested very low toxicity.

Antiviral Combination Approach as a Perspective to Combat Enterovirus Infections.

Abstract Human enteroviruses distributed worldwide are causative agents of a broad spectrum of diseases with extremely high morbidity, including a series of severe illnesses of the central nervous system, heart, endocrine pancreas, skeleton muscles, etc., as well as the common cold contributing to the development of chronic respiratory diseases, including the chronic obstructive pulmonary disease. The above mentioned diseases along with the significantly high morbidity and mortality in children, as well as in the high-risk populations (immunodeficiencies, neonates) definitely formulate the chemotherapy as the main tool for the control of enterovirus infections. At present, clinically effective antivirals for use in the treatment of enteroviral infection do not exist, in spite of the large amount of work carried out in this field. The main reason for this is the development of drug resistance. We studied the process of development of resistance to the strongest inhibitors of enteroviruses, WIN compounds (VP1 protein hydrophobic pocket blockers), especially in the models in vivo, Coxsackievirus B (CV-B) infections in mice. We introduced the tracing of a panel of phenotypic markers (MIC50 value, plaque shape and size, stability at 50℃, pathogenicity in mice) for characterization of the drug-mutants (resistant and dependent) as a very important stage in the study of enterovirus inhibitors. Moreover, as a result of VP1 RNA sequence analysis performed on the model of disoxaril mutants of CVB1, we determined the molecular basis of the drug-resistance. The monotherapy courses were the only approach used till now. For the first time in the research for anti-enterovirus antivirals our team introduced the testing of combination effect of the selective inhibitors of enterovirus replication with different mode of action. This study resulted in the selection of a number of very effective in vitro double combinations with synergistic effect and a broad spectrum of sensitive enteroviruses. The most prospective attainment in our examinations in this field was the development of a novel scheme for the combined application of anti-enteroviral substances in coxsackievirus B1 neuroinfection in newborn mice. It consisted of a consecutive, alternating and non simultaneous administration of the substances in the combination. The triple combination - disoxaril- guanidine. HCl-oxoglaucine (DGO) showed a high effectiveness expressed in the marked reduction of the mortality rate in infected mice as compared both to the placebo group, and to the partner compounds used alone every day, and to the same combination applied simultaneously every day. The studies of the drug sensitivity of viral brain isolates from mice treated with DGO combination showed not only preserved, but even increased sensitivity to the drugs included in the combination. Obviously, the consecutive alternating administration of anti-enteroviral substances hinders the occurrence of drug-resistance in the course of the experimental enteroviral infections in mice. Abstract Хуманите ентеровируси дистрибуирани низ целиот свет се предизвикувачки агенси на широк спектар на болести со екстремно висок морбидитет, вклучително и серија тешки бо- лести на централниот нервен систем, срцето, ен- докриниот панкреас, мускулите на скелетот, итн., како и обичната настинка што придонесува за развој на хроничните респираторни болести, вклучувајќи го хроничното опструктивно бело- дробно заболување. Горенаведените болести, заедно со значително високиот морбидитет и морталитет кај децата, како и кај популациите со висок ризик (имунодефициенција, новороде- ни), дефинитивно, ја формулираат хемотерапи- јата како главна алатка за контрола на ентерови- русните инфекции. Во моментов не постојат клинички ефективни антивирусни лекови за употреба во лекувањето на ентеровирусната ин- фекција, и покрај големиот труд вложен на ова поле. Главна причина за ова е развојот на от- порноста на лекови. Ние го студиравме проце- сот на развој на отпорноста на најсилните инхи- битори на ентеровируси, WIN-соединенија (VP1 protein hydrophobic pocket blockers), особено кај моделите ин виво, Coxsackievius B (CV-B) ин- фекциите кај глувците. Воведовме следење на панел на фенотипските маркери (MIC50 вред- ност, обликот и големина на плочката, стабил- ност на 50oC, патогеноста кај глувци) за карак- теризација на лековите-мутанти (отпорни и за- висни), како многу важна фаза во студијата на ентеровирусните инхибитори. Згора на тоа, како резултат на VP1 РНК секвентната анализа на- правена врз модел на disoxaril мутанти на CVB1, ја одредивме молекуларната основа на отпор- носта на лекови. Монотерапевтските начини беа единстве- ниот пристап што се користеше досега. Прв пат во истражувањето на антиентеровирусните ан- тивируси нашиот тим воведе тестирање на ком- биниран ефект на селективни инхибитори на ентеровирусна репликација со различен начин на дејство. Ова истражување резултираше со избор на голем број многу ефикасни ин витро двојни комбинации со синергистичко дејство и широк спектар на чувствителни ентеровируси. Најпроспективното постигнување во нашите ис- тражувања од оваа област беше развојот на нова шема за комбинираната примена на антиенте- ровирусните супстанции во coxsackievirus B1 невроинфекцијата кај новородени глувци. Тоа се состоеше од последователно, наизменично и несимултано давање на супстанциите во комби- нација. Тројната комбинација - disoxaril-guanidine. HCl-oxoglaucine (DGO) покажа висока ефи- касност изразена во означено намалување на стапката на смртност кај заразените глувци во споредба со плацебо-групата, и со партнерските соединенија што се користат сами секој ден, а на истата комбинација што се применува исто- времено секој ден. Студиите за чувствителноста на изолатите на вирус од мозок од глувци, тре- тирани со DGO-комбинација, покажаа не само зачувана, но дури и зголемена чувствителност на лековите вклучени во комбинацијата. Очи- гледно, последователното наизменично давање антиентеровирусни супстанции го спречува јаву- вањето на отпорност на лекови во текот на експе- рименталната ентеровирусна инфекција кај глув- ците.

Disoxaril Mutants of Coxsackievirus B1: Phenotypic Characteristics and Analysis of the Target VP1 Gene

Disoxaril inhibits enterovirus replication by binding to the hydrophobic pocket within the VP1 coat protein, thus stabilizing the virion and blocking its uncoating. Disoxaril-resistant (RES) mutants of the Coxsackie virus B1 (CVB1/RES) were derived from the wild disoxaril- sensitive (SOF) strain (CVB1/SOF) using a selection approach. A disoxaril-dependent (DEP) mutant (CVB1/DEP) was obtained following nine consecutive passages of the disoxaril- resistant mutant in the presence of disoxaril. Phenotypic characteristics of the disoxaril mutants were investigated. A timing-of-addition study of the CVB1/DEP replication demonstrated that in the absence of disoxaril the virus particle assembly stopped. VP1 RNA sequences of disoxaril mutants were compared with the existing Gen Bank CVB1 reference structure. The amino acid sequence of a large VP1 196 - 258 peptide (disoxaril-binding region) of CVB1/RES was significantly different from that of the CVB1/SOF. Crucially important changes in CVB1/RES were two point mutations, M213H and F237L, both in the ligand-binding pocket. The sequence analysis of the CVB1/DEP showed some reversion to CVB1/SOF. The amino acid sequences of the three VP1 proteins are presented