Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive soft-tissue sarcomas that arise from neural tissues and carry a poor prognosis. Previously, we found that the glutamine amidotransferase inhibitor JHU395 partially impeded tumor growth in preclinical models of MPNST. JHU395 inhibits de novo purine synthesis in human MPNST cells and murine tumors with partial decreases in purine monophosphates. On the basis of prior studies showing enhanced efficacy when glutamine amidotransferase inhibition was combined with the antimetabolite 6-mercaptopurine (6-MP), we hypothesized that such a combination would be efficacious in MPNST. Given the known toxicity associated with 6-MP, we set out to develop a more efficient and well-tolerated drug that targets the purine salvage pathway. Here, we report the discovery of Pro-905, a phosphoramidate protide that delivered the active nucleotide antimetabolite thioguanosine monophosphate (TGMP) to tumors over 2.5 times better than equimolar 6-MP. Pro-905 effectively prevented the incorporation of purine salvage substrates into nucleic acids and inhibited colony formation of human MPNST cells in a dose-dependent manner. In addition, Pro-905 inhibited MPNST growth and was well-tolerated in both human patient-derived xenograft (PDX) and murine flank MPNST models. When combined with JHU395, Pro-905 enhanced the colony formation inhibitory potency of JHU395 in human MPNST cells and augmented the antitumor efficacy of JHU395 in mice. In summary, the dual inhibition of the de novo and purine salvage pathways in preclinical models may safely be used to enhance therapeutic efficacy against MPNST.
- MeSH
- antimetabolity terapeutické užití MeSH
- glutamin MeSH
- lidé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory nervové pochvy * farmakoterapie MeSH
- neurofibrosarkom * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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
Polyomavirus infections occur commonly in humans and are normally nonfatal. However, in immunocompromised individuals, they are intractable and frequently fatal. Due to a lack of approved drugs to treat polyomavirus infections, cidofovir, a phosphonate nucleotide analog approved to treat cytomegalovirus infections, has been repurposed as an antipolyomavirus agent. Cidofovir has been modified in various ways to improve its efficacies as a broad-spectrum antiviral agent. However, the actual mechanisms and targets of cidofovir and its modified derivatives as antipolyomavirus agents are still under research. Here, polyomavirus large tumor antigen (Tag) activities were identified as the viral target of cidofovir derivatives. The alkoxyalkyl ester derivatives of cidofovir efficiently inhibit polyomavirus DNA replication in cell-free human extracts and a viral in vitro replication system utilizing only purified proteins. We present evidence that DNA helicase and DNA binding activities of polyomavirus Tags are diminished in the presence of low concentrations of alkoxyalkyl ester derivatives of cidofovir, suggesting that the inhibition of viral DNA replication is at least in part mediated by inhibiting single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) binding activities of Tags. These findings show that the alkoxyalkyl ester derivatives of cidofovir are effective in vitro without undergoing further conversions, and we conclude that the inhibitory mechanisms of nucleotide analog-based drugs are more complex than previously believed.
- MeSH
- antigeny virové nádorové * MeSH
- cytosin MeSH
- DNA virů genetika MeSH
- estery farmakologie MeSH
- lidé MeSH
- nukleotidy MeSH
- Polyomavirus * genetika MeSH
- replikace DNA MeSH
- replikace viru MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.
- MeSH
- adenosindifosfát metabolismus farmakologie MeSH
- adenosintrifosfát metabolismus farmakologie MeSH
- alosterická regulace MeSH
- bakteriální proteiny antagonisté a inhibitory metabolismus MeSH
- enzymová indukce MeSH
- fosfofruktokinasy antagonisté a inhibitory metabolismus MeSH
- fruktosadifosfáty biosyntéza farmakologie MeSH
- fruktosafosfáty metabolismus farmakologie MeSH
- glukoneogeneze MeSH
- glykolýza MeSH
- hexosafosfáty metabolismus MeSH
- izoenzymy antagonisté a inhibitory metabolismus MeSH
- katalýza MeSH
- kinetika MeSH
- kyslík farmakologie MeSH
- L-laktátdehydrogenasa metabolismus MeSH
- Mycobacterium tuberculosis účinky léků enzymologie MeSH
- pyruvátkinasa metabolismus MeSH
- rekombinantní proteiny metabolismus MeSH
- substrátová specifita MeSH
- zpětná vazba fyziologická MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
In analogy to antiviral acyclic nucleoside phosphonates, a series of 5-amino-3-oxo-1,2,4-thiadiazol-3(2H)-ones bearing a 2-phosphonomethoxyethyl (PME) or 3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) group at the position 2 of the heterocyclic moiety has been synthesized. Diisopropyl esters of PME- and HPMP-amines have been converted to the N-substituted ureas and then reacted with benzoyl, ethoxycarbonyl, and Fmoc isothiocyanates to give the corresponding thiobiurets, which were oxidatively cyclized to diisopropyl esters of 5-amino-3-oxo-2-PME- or 2-HPMP- 1,2,4-thiadiazol-3(2H)-ones. The phosphonate ester groups were cleaved with bromotrimethylsilane, yielding N5-protected phosphonic acids. The subsequent attempts to remove the protecting group from N5 under alkaline conditions resulted in the cleavage of the 1,2,4-thiadiazole ring. Similarly, compounds with a previously unprotected 5-amino-1,2,4-thiadiazolone base moiety were stable only in the form of phosphonate esters. The series of twenty-one newly prepared 1,2,4-thiadiazol-3(2H)-ones were explored as potential inhibitors of cysteine-dependent enzymes - human cathepsin K (CatK) and glycogen synthase kinase 3β (GSK-3β). Several compounds exhibited an inhibitory activity toward both enzymes in the low micromolar range. The inhibitory potency of some of them toward GSK-3β was similar to that of the thiadiazole GSK-3β inhibitor tideglusib, whereas others exhibited more favorable toxicity profile while retaining good inhibitory activity.
- MeSH
- antitumorózní látky chemická syntéza chemie farmakologie MeSH
- buněčné linie MeSH
- GSK3B antagonisté a inhibitory metabolismus MeSH
- inhibitory enzymů chemická syntéza chemie farmakologie MeSH
- kathepsin K antagonisté a inhibitory metabolismus MeSH
- léky antitumorózní - screeningové testy MeSH
- lidé MeSH
- molekulární struktura MeSH
- nukleosidy chemická syntéza chemie farmakologie MeSH
- organofosfonáty chemická syntéza chemie farmakologie MeSH
- proliferace buněk účinky léků MeSH
- thiadiazoly chemická syntéza chemie farmakologie MeSH
- viabilita buněk účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
An enzymatic alternative to the chemical synthesis of chiral gem-difluorinated alcohols has been developed. The method is highly effective and stereoselective, feasible at laboratory temperature, avoiding the use of toxic heavy metal catalysts which is an important benefit in medicinal chemistry including the synthesis of drugs and drug precursors. Candida antarctica lipases A and B were applied for the enantioselective resolution of side-chain modified gem-difluorinated alcohols, (R)- and (S)-3-benzyloxy-1,1-difluoropropan-2-ols (1a and 1b), compounds serving as chiral building blocks in the synthesis of various bioactive molecules bearing a gem-difluorinated grouping. The catalytic activity of these lipases was investigated for the chiral acetylation of 1a and 1b in non-polar solvents using vinyl acetate as an acetyl donor. The dependence of the reaction course on various substrate and enzyme concentrations, reaction time, and temperature was monitored by chiral capillary electrophoresis (CE) using sulfobutyl ether β-cyclodextrin as a stereoselective additive of the aqueous background electrolyte. The application of CE, NMR, and MS methods has proved that the complex enzyme effect of Candida antarctica lipase B leads to the thermodynamically stable (S)-enantiomer 1b instead of the expected acetylated derivatives. In contrast, the enantioselective acetylation of racemic alcohol 1 was observed as a kinetically controlled process, where (R)-enantiomer 1a was formed as the main product. This process was followed by enzymatic hydrolysis and chiral isomerisation. Finally, single pure enantiomers 1a and 1b were isolated and their absolute configurations were assigned from NMR analysis after esterification with Mosher's acids.
Aberrant DNA methylation that results in silencing of genes has remained a significant interest in cancer research. Despite major advances, the success of epigenetic therapy is elusive due to narrow therapeutic window. A wide variety of naturally occurring epigenetic agents and synthetic molecules that can alter methylation patterns exist, however, their usefulness in epigenetic therapy remains unknown. This underlines the need for effective tumor models for large-scale screening of drug candidates with potent hypomethylation activity. In this study, we present the development of a cell-based DNA demethylation detection system, which is amenable for high content screening of epigenetic drugs in two-dimensional and three-dimensional cell culture models. Additionally, the detection system also supports the in vivo monitoring of demethylation efficacy of potential lead compounds from in vitro screens in tumor xenografts. The described detection system not only permits the continuous monitoring of demethylation but also of the induced cytostatic/cytotoxic drug effects in live cells, as a function of time. The detection system is fluorescence based and exploits the dominant ability of DNA methylation to inhibit gene transcription, and utilizes FLJ32130 gene, which is silenced on account of promoter hypermethylation in human colorectal cancer. The described work will provide the researchers with an efficient tool for epigenetic drug screens on a high throughput platform and would therefore benefit academic and industrial drug discovery. © 2016 International Society for Advancement of Cytometry.
- MeSH
- antitumorózní látky terapeutické užití MeSH
- epigeneze genetická * MeSH
- kolorektální nádory farmakoterapie genetika MeSH
- lidé MeSH
- metylace DNA účinky léků MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- proliferace buněk účinky léků MeSH
- promotorové oblasti (genetika) MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- xenogenní modely - testy antitumorózní aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Capillary electrophoresis (CE) and quantum mechanical density functional theory (DFT) were applied to the investigation of the acid-base and electromigration properties of important compounds: newly synthesized derivatives of 5-azacytosine - analogs of efficient antiviral drug cidofovir. These compounds exhibit a strong antiviral activity and they are considered as potential new antiviral agents. For their characterization and application, it is necessary to know their acid-base properties, particularly the acidity constants (pKa) of their ionogenic groups (the basic N(3) atom of the triazine ring and the acidic phosphonic acid group in the alkyl chain). First, the mixed acidity constants (pKa(mix)) of these ionogenic groups and the ionic mobilities of these compounds were determined by nonlinear regression analysis of the pH dependence of their effective electrophoretic mobilities. Effective mobilities were measured by CE in a series of background electrolytes in a wide pH range (2.0-10.5), at constant ionic strength (25mM) and constant temperature (25°C). Subsequently, the pKa(mix) values were recalculated to thermodynamic pKa values using the Debye-Hückel theory. The thermodynamic pKa value of the NH(+) moiety at the N(3) atom of the triazine ring was found to be in the range 2.82-3.30, whereas the pKa of the hydrogenphosphonate group reached values from 7.19 to 7.47, depending on the structure of the analyzed compounds. These experimentally determined pKa values were in good agreement with those calculated by quantum mechanical DFT. In addition, DFT calculations revealed that from the four nitrogen atoms in the 5-azacytosine moiety, the N(3) atom of the triazine ring is preferentially protonated. Effective charges of analyzed compounds ranged from zero or close-to-zero values at pH 2 to -2 elementary charges at pH≥9. Ionic mobilities were in the range (-16.7 to -19.1)×10(-9)m(2)V(-1)s(-1) for univalent anions and in the interval (-26.9 to -30.3)×10(-9)m(2)V(-1)s(-1) for divalent anions.
OBJECTIVE: The review covers basic principles of the prodrug strategy applied to antiviral nucleoside drugs or drug candidates. Specific role of amino acids as promoieties is explained with respect to transport mechanisms, pharmacokinetics and a low toxicity of compounds. Synthetic approaches to the most important representatives (compounds under clinical investigations or available on the market) are described, including valacyclovir, valganciclovir, valomaciclovir stearate, valcyclopropavir, valtorcitabine, valopicitabine and several attempts to amino acid modifications of antiretroviral nucleosides. METHOD: A special attention is paid to acyclic nucleoside phosphonates, where the phosphonic acid residue is esterified with a side-chain hydroxyl group of appropriate amino acid (serine, tyrosine) which can be used as single amino acid or as a part of dipeptides further modified on the terminal carboxyl function. The most advantageous pharmacokinetic profile and the best oral bioavailability were found in tyrosinebased prodrugs. RESULTS & CONCLUSION: Studies were performed successfully on 1-(S)-[3-hydroxy-2-(phosphonomethoxy) propyl]cytosine (cidofovir), 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine and some (R)-2- (phosphonomethoxy)propyl and 2-(phosphonomethoxy)ethyl derivatives including adefovir.
- MeSH
- adenin analogy a deriváty chemie farmakologie MeSH
- antivirové látky chemie farmakologie MeSH
- Cytomegalovirus účinky léků MeSH
- cytosin analogy a deriváty chemie farmakologie MeSH
- lidé MeSH
- nukleosidy chemie farmakologie MeSH
- nukleotidy chemie farmakologie MeSH
- organofosfonáty chemie farmakologie MeSH
- prekurzory léčiv chemie farmakologie MeSH
- virus varicella zoster účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH