Despite the eradication of smallpox four decades ago, poxviruses continue to be a threat to humans and animals. The arsenal of anti-poxvirus agents is very limited and understanding mechanisms of resistance to agents targeting viral DNA polymerases is fundamental for the development of antiviral therapies. We describe here the phenotypic and genotypic characterization of poxvirus DNA polymerase mutants isolated under selective pressure with different acyclic nucleoside phosphonates, including HPMPC (cidofovir), cHPMPC, HPMPA, cHPMPA, HPMPDAP, HPMPO-DAPy, and PMEO-DAPy, and the pyrophosphate analogue phosphonoacetic acid. Vaccinia virus (VACV) and cowpox virus drug-resistant viral clones emerging under drug pressure were characterized phenotypically (drug-susceptibility profile) and genotypically (DNA polymerase sequencing). Different amino acid changes in the polymerase domain and in the 3'-5' exonuclease domain were linked to drug resistance. Changes in the 3'-5' domain emerged earlier than in the polymerase domain when viruses acquired a combination of mutations. Our study highlights the importance of poxvirus DNA polymerase residues 314, 613, 684, 688, and 851, previously linked to drug resistance, and identified several novel mutations in the 3'-5' exonuclease domain (M313I, F354L, D480Y) and in the DNA polymerase domain (A632T, T831I, E856K, L924F) associated with different drug-susceptibility profiles. Furthermore, a combination of mutations resulted in complex patterns of cross-resistance. Modeling of the VACV DNA polymerase bearing the newly described mutations was performed to understand the effects of these mutations on the structure of the viral enzyme. We demonstrated the emergence of drug-resistant DNA polymerase mutations in complex patterns to be considered in case such mutations should eventually arise in the clinic.
- Publikační typ
- časopisecké články MeSH
Syntheses of α-branched alkyl and aryl substituted 9-[2-(phosphonomethoxy)ethyl]purines from substituted 1,3-dioxolanes have been developed. Key synthetic precursors, α-substituted dialkyl [(2-hydroxyethoxy)methyl]phosphonates were prepared via Lewis acid mediated cleavage of 1,3-dioxolanes followed by reaction with dialkyl or trialkyl phosphites. The best preparative yields were achieved under conditions utilizing tin tetrachloride as Lewis acid and triisopropyl phosphite. Attachment of purine bases to dialkyl [(2-hydroxyethoxy)methyl]phosphonates was performed by Mitsunobu reaction. Final α-branched 9-[2-(phosphonomethoxy)ethyl]purines were tested for antiviral, cytostatic and antiparasitic activity, the latter one determined as inhibitory activity towards Plasmodium falciparum enzyme hypoxanthine-guanine-xanthine phosphoribosyltransfesase. In most cases biological activity was only marginal.
- MeSH
- antiparazitární látky chemická syntéza chemie farmakologie MeSH
- antivirové látky chemická syntéza chemie farmakologie MeSH
- dioxolany chemie MeSH
- DNA viry účinky léků MeSH
- inhibitory enzymů chemická syntéza chemie farmakologie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- molekulární struktura MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- pentosyltransferasy antagonisté a inhibitory metabolismus MeSH
- Plasmodium falciparum účinky léků enzymologie MeSH
- puriny chemická syntéza chemie farmakologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
While noncanonic xanthine nucleotides XMP/dXMP play an important role in balancing and maintaining intracellular purine nucleotide pool as well as in potential mutagenesis, surprisingly, acyclic nucleoside phosphonates bearing a xanthine nucleobase have not been studied so far for their antiviral properties. Herein, we report the synthesis of a series of xanthine-based acyclic nucleoside phosphonates and evaluation of their activity against a wide range of DNA and RNA viruses. Two acyclic nucleoside phosphonates within the series, namely 9-[2-(phosphonomethoxy)ethyl]xanthine (PMEX) and 9-[3-hydroxy-2-(phosphonomethoxy)propyl]xanthine (HPMPX), were shown to possess activity against several human herpesviruses. The most potent compound was PMEX, a xanthine analogue of adefovir (PMEA). PMEX exhibited a single digit µM activity against VZV (EC50 = 2.6 µM, TK+ Oka strain) and HCMV (EC50 = 8.5 µM, Davis strain), while its hexadecyloxypropyl monoester derivative was active against HSV-1 and HSV-2 (EC50 values between 1.8 and 4.0 µM). In contrast to acyclovir, PMEX remained active against the TK- VZV 07-1 strain with EC50 = 4.58 µM. PMEX was suggested to act as an inhibitor of viral DNA polymerase and represents the first reported xanthine-based acyclic nucleoside phosphonate with potent antiviral properties.
- Publikační typ
- časopisecké články MeSH
New 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidine (PMEO-DAPy) and 1-[2-(phosphonomethoxy)ethyl]-5-azacytosine (PME-5-azaC) prodrugs were prepared with a pro-moiety consisting of carbonyloxymethyl esters (POM, POC), alkoxyalkyl esters, amino acid phosphoramidates and/or tyrosine. The activity of the prodrugs was evaluated in vitro against different virus families. None of the synthesized prodrugs demonstrated activity against RNA viruses but some of them proved active against herpesviruses [including herpes simplex virus (HSV), varicella-zoster virus (VZV), and human cytomegalovirus (HCMV)]. The bis(POC) and the bis(amino acid) phosphoramidate prodrugs of PMEO-DAPy inhibited herpesvirus replication at lower doses than the parent compound although the selectivity against HSV and VZV was only slightly improved compared to PMEO-DAPy. The mono-octadecyl ester of PME-5-azaC emerged as the most potent and selective PME-5-azaC prodrug against HSV, VZV and HCMV with EC50's of 0.15-1.12µM while PME-5-azaC only had marginal anti-herpesvirus activity. Although the bis(hexadecylamido-l-tyrosyl) and the bis(POM) esters of PME-5-azaC were also very potent anti-herpesvirus drugs, these were less selective than the mono-octadecyl ester prodrug.
- MeSH
- antivirové látky chemická syntéza chemie farmakologie MeSH
- buněčné linie MeSH
- Cytomegalovirus účinky léků MeSH
- lidé MeSH
- organofosfonáty chemická syntéza chemie farmakologie MeSH
- prekurzory léčiv chemická syntéza chemie farmakologie MeSH
- pyrimidinové nukleosidy chemie MeSH
- Simplexvirus účinky léků MeSH
- virus varicella zoster účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Human papillomavirus (HPV) is responsible for cervical cancer, and its role in head and neck carcinoma has been reported. No drug is approved for the treatment of HPV-related diseases but cidofovir (CDV) exhibits selective antiproliferative activity. In this study, we analyzed the effects of CDV-resistance (CDVR) in two HPV(+) (SiHaCDV and HeLaCDV) and one HPV(-) (HaCaTCDV) tumor cell lines. Quantification of CDV metabolites and analysis of the sensitivity profile to chemotherapeutics was performed. Transporters expression related to multidrug-resistance (MRP2, P-gp, BCRP) was also investigated. Alterations of CDV metabolism in SiHaCDV and HeLaCDV, but not in HaCaTCDV, emerged via impairment of UMP/CMPK1 activity. Mutations (P64T and R134M) as well as down-regulation of UMP/CMPK1 expression were observed in SiHaCDV and HeLaCDV, respectively. Altered transporters expression in SiHaCDV and/or HeLaCDV, but not in HaCaTCDV, was also noted. Taken together, these results indicate that CDVR in HPV(+) tumor cells is a multifactorial process.
- MeSH
- ABC transportéry biosyntéza MeSH
- chemorezistence genetika MeSH
- cytidintrifosfát biosyntéza MeSH
- cytosin analogy a deriváty farmakologie MeSH
- fosforylace MeSH
- HeLa buňky MeSH
- infekce papilomavirem farmakoterapie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- nádorové buněčné linie MeSH
- nádory děložního čípku farmakoterapie patologie virologie MeSH
- nukleosidmonofosfátkinasa biosyntéza metabolismus MeSH
- organofosfonáty farmakologie MeSH
- Papillomaviridae MeSH
- SLC transportéry biosyntéza MeSH
- uridintrifosfát biosyntéza MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Even number fatty acid residues-docosanoyl (behenoyl) and stearoyl were selected for introduction to the N(4)-position of (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine) (HPMPC, cidofovir), and its 5-azacytosine counterpart, (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine) (HPMP-5-azaC) with the aim to prepare a new type of lipophilic prodrugs. The study on the influence of these modifications to the stability and biological activity of both antivirals was performed. Different reactivity of both systems towards acylation reactions was also found: the 4-NH2 group of cidofovir was more reactive compared to that of HPMP-5-azaC. In 5-azacytosine derivatives, we found mostly a destabilizing effect of the N(4)-acylation but this could be compensated by a positive influence of the esterification of the phosphonate group. Chemical stability of the 5-azacytosine moiety in the HPMP series is increasing in the following order: HPMP-5-azaC
- MeSH
- antivirové látky chemická syntéza chemie farmakologie MeSH
- cytosin analogy a deriváty chemická syntéza chemie farmakologie MeSH
- Herpesviridae účinky léků MeSH
- hydrofobní a hydrofilní interakce * MeSH
- mikrobiální testy citlivosti MeSH
- molekulární struktura MeSH
- organofosfonáty chemická syntéza chemie farmakologie MeSH
- prekurzory léčiv chemická syntéza chemie farmakologie MeSH
- replikace viru účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Hexadecyloxypropyl esters of acyclic nucleoside phosphonates containing guanine (G) or hypoxanthine (Hx) and a (S)-[3-hydroxy-2-(phosphonomethoxy)propyl] [(S)-HPMP] or 2-(2-phosphonoethoxy)ethyl (PEE) acyclic moiety have been prepared. The activity of the prodrugs was evaluated in vitro against different virus families. Whereas ester derivatives of PEEHx and (S)-HPMPHx were antivirally inactive, monoesters of PEEG, and mono- and diesters of (S)-HPMPG showed pronounced antiviral activity against vaccinia virus and/or herpesviruses. Monoesters of (S)-HPMPG emerged as the most potent and selective derivatives against these DNA viruses. None of the compounds were inhibitory against RNA viruses and retroviruses.
- MeSH
- antivirové látky chemická syntéza chemie metabolismus farmakologie MeSH
- buněčné linie MeSH
- DNA viry účinky léků MeSH
- guanin analogy a deriváty chemie MeSH
- hypoxanthin chemie MeSH
- hypoxanthiny chemie MeSH
- kyseliny fosforité chemie MeSH
- lidé MeSH
- nukleosidy chemie MeSH
- organofosfonáty chemická syntéza chemie metabolismus farmakologie MeSH
- prekurzory léčiv metabolismus MeSH
- techniky syntetické chemie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Series of novel acyclic nucleoside phosphonates (ANPs) with various nucleobases and 2-(2-phosphonoethoxy)ethyl (PEE) chain bearing various substituents in β-position to the phosphonate moiety were prepared. The influence of structural alternations on antiviral activity was studied. Several derivatives exhibit antiviral activity against HIV and vaccinia virus (middle micromolar range), HSV-1 and HSV-2 (lower micromolar range) and VZV and CMV (nanomolar range), although the parent unbranched PEE-ANPs are inactive. Adenine as a nucleobase and the methyl group attached to the PEE chain proved to be a prerequisite to afford pronounced antiviral activity.
- MeSH
- antivirové látky chemická syntéza chemie farmakologie MeSH
- buněčné linie MeSH
- herpes simplex farmakoterapie MeSH
- HIV infekce farmakoterapie MeSH
- HIV účinky léků MeSH
- kyseliny fosforité chemická syntéza chemie farmakologie MeSH
- lidé MeSH
- myši MeSH
- nukleosidy chemická syntéza chemie farmakologie MeSH
- Simplexvirus účinky léků MeSH
- vakcínie farmakoterapie MeSH
- viabilita buněk účinky léků MeSH
- virové nemoci farmakoterapie MeSH
- virus vakcinie účinky léků MeSH
- viry účinky léků MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
New Adefovir (PMEA) prodrugs with a pro-moiety consisting of decyl or decyloxyethyl chain bearing hydroxyl function(s), hexaethyleneglycol or a (5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl unit were prepared starting from the tetrabutylammonium salt of the phosphonate drug and an appropriate alkyl bromide or tosylate. Analogously, two esters of Cidofovir [(S)-HPMPC] bearing a hexaethyleneglycol moiety were prepared. The activity of the prodrugs was evaluated in vitro against different virus families. A loss in the antiviral activities of the hydroxylated decyl or decyloxyethyl esters and hexaethyleneglycol esters of PMEA against human immunodeficiency virus (HIV) and herpesviruses [including herpes simplex virus (HSV), varicella-zoster virus (VZV), and human cytomegalovirus (CMV)] occurred in comparison with the parent compound. On the other hand, the (5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl ester of PMEA showed significant activities against HIV and herpesviruses. (S)-HPMPC prodrugs exhibited anti-cytomegalovirus activities in the same range as the parent drug, whereas the anti-HSV and anti-VZV activities were one- to seven-fold lower than that of Cidofovir.
- MeSH
- adenin analogy a deriváty chemie MeSH
- antivirové látky chemická syntéza chemie toxicita MeSH
- Cytomegalovirus účinky léků MeSH
- cytosin analogy a deriváty chemie MeSH
- HIV účinky léků MeSH
- kyseliny fosforité chemie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- prekurzory léčiv chemická syntéza chemie toxicita MeSH
- Simplexvirus účinky léků MeSH
- virus varicella zoster účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
9-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) and its cyclic form were selected for further evaluation as potential drug candidates against poxvirus infections. To increase bioavailability of these compounds, synthesis of their structurally diverse ester prodrugs was carried out: alkoxyalkyl (hexadecyloxypropyl, octadecyloxyethyl, hexadecyloxyethyl), pivaloyloxymethyl (POM), 2,2,2-trifluoroethyl, butylsalicylyl, and prodrugs based on peptidomimetics. Most HPMPDAP prodrugs were synthesized in the form of monoesters as well as the corresponding cyclic phosphonate esters. The activity was evaluated not only against vaccinia virus but also against different herpes viruses. The most potent and active prodrugs against vaccinia virus were the alkoxyalkyl ester derivatives of HPMPDAP, with 50% effective concentrations 400-600-fold lower than those of the parent compound. Prodrugs based on peptidomimetics, the 2,2,2-trifluoroethyl, the POM, and the butylsalicylyl derivatives, were able to inhibit vaccinia virus replication at 50% effective concentrations that were equivalent or ∼10-fold lower than those observed for the parent compounds.
- MeSH
- adenin analogy a deriváty chemická syntéza chemie farmakologie MeSH
- antivirové látky chemická syntéza chemie farmakologie MeSH
- estery MeSH
- Herpesviridae účinky léků MeSH
- kultivované buňky MeSH
- lidé MeSH
- organofosforové sloučeniny chemická syntéza chemie farmakologie MeSH
- Poxviridae účinky léků MeSH
- prekurzory léčiv chemická syntéza chemie farmakologie MeSH
- proliferace buněk účinky léků MeSH
- RNA-viry účinky léků MeSH
- stereoizomerie MeSH
- virologie metody MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
