Cyclic dinucleotides (CDNs) are second messengers that activate stimulator of interferon genes (STING). The cGAS-STING pathway plays a promising role in cancer immunotherapy. Here, we describe the synthesis of CDNs containing 7-substituted 7-deazapurine moiety. We used mouse cyclic GMP-AMP synthase and bacterial dinucleotide synthases for the enzymatic synthesis of CDNs. Alternatively, 7-(het)aryl 7-deazapurine CDNs were prepared by Suzuki-Miyaura cross-couplings. New CDNs were tested in biochemical and cell-based assays for their affinity to human STING. Eight CDNs showed better activity than 2'3'-cGAMP, the natural ligand of STING. The effect on cytokine and chemokine induction was also evaluated. The best activities were observed for CDNs bearing large aromatic substituents that point above the CDN molecule. We solved four X-ray structures of complexes of new CDNs with human STING. We observed π-π stacking interactions between the aromatic substituents and Tyr240 that are involved in the stabilization of CDN-STING complexes.
- MeSH
- cytokiny MeSH
- interferony MeSH
- lidé MeSH
- ligandy MeSH
- membránové proteiny * metabolismus MeSH
- myši MeSH
- nukleotidy cyklické * chemie MeSH
- nukleotidyltransferasy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Five 2'-deoxyribonucleoside triphosphates (dNTPs) derived from epigenetic pyrimidines (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil, and 5-formyluracil) were prepared and systematically studied as substrates for nine DNA polymerases in competition with natural dNTPs by primer extension experiments. The incorporation of these substrates was evaluated by a restriction endonucleases cleavage-based assay and by a kinetic study of single nucleotide extension. All of the modified pyrimidine dNTPs were good substrates for the studied DNA polymerases that incorporated a significant percentage of the modified nucleotides into DNA even in the presence of natural nucleotides. 5-Methylcytosine dNTP was an even better substrate for most polymerases than natural dCTP. On the other hand, 5-hydroxymethyl-2'-deoxyuridine triphosphate was not the best substrate for SPO1 DNA polymerase, which naturally synthesizes 5hmU-rich genomes of the SPO1 bacteriophage. The results shed light onto the possibility of gene silencing through recycling and random incorporation of epigenetic nucleotides and into the replication of modified bacteriophage genomes.
- MeSH
- 5-methylcytosin * MeSH
- deoxyribonukleosidy MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- DNA metabolismus MeSH
- epigeneze genetická MeSH
- nukleotidy metabolismus MeSH
- pyrimidinové nukleotidy * MeSH
- pyrimidiny MeSH
- restrikční enzymy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
5-(β-d-Glucopyranosyloxymethyl)-2'-deoxyuridine and -cytidine 5'-O-triphosphates were prepared and used for polymerase-mediated (primer extension or PCR) synthesis of DNA containing glucosylated 5-hydroxymethyluracil (5hmU) or 5-hydroxymethyluracil (5hmC). The presence of any glucosylated pyrimidines fully protected DNA from cleavage by type II restriction endonucleases. On the other hand, while the presence of glucosylated 5hmU completely inhibited transcription by bacterial (Escherichia coli) RNA polymerase, the DNA containing the corresponding glucosylated 5hmC allowed a similar level of transcription as natural DNA. This suggests different roles of these hypermodified bases in the epigenetic regulation of transcription in bacteriophages or kinetoplastid parasites. Consequently, enzymatic glucosylation of 5hmC-containing DNA can be used for tuning of transcription activity.
We designed and synthesized nucleosides bearing aminophenyl- or aminonaphthyl-3-methoxychromone fluorophores attached at position 5 of cytosine or thymine and converted them to nucleoside triphosphates. The fluorophores showed solvatochromic fluorescence with strong fluorescence at 433-457 nm in non-polar solvents and very weak fluorescence at 567 nm in alcohols. The nucleosides and nucleotides also showed only negligible fluorescence in alcohols or water. The triphosphates were substrates for DNA polymerase in the enzymatic synthesis of modified DNA probes that showed only very weak fluorescence in aqueous buffer but a significant light-up and blue shift were observed when they interacted with proteins (histone H3.1 or p53 for double-stranded DNA probes or single-strand binding protein for single-stranded oligonucleotide probes). Hence, nucleotides have good potential in the construction of DNA sensors for studying protein-DNA interactions. The modified dNTPs were also transported into cells using a cyclodextrin-based transporter but they were not incorporated into the genomic DNA.
Synthesis of base-modified oligonucleotides and DNA by polymerase incorporations of modified nucleoside triphosphates (dNTPs) is summarized. The crosscoupling synthesis of modified dNTPs, methods of their enzymatic incorporations, as well as their applications in bioanalysis and chemical biology are discussed.
- MeSH
- biochemie metody trendy MeSH
- diagnostické techniky molekulární MeSH
- DNA-dependentní DNA-polymerasy * MeSH
- nukleové kyseliny, nukleosidy a nukleotidy * chemická syntéza MeSH
- pojmy organické chemie MeSH
- polymerázová řetězová reakce MeSH
- techniky syntetické chemie MeSH
- techniky syntézy na pevné fázi * MeSH
- Publikační typ
- práce podpořená grantem MeSH
A series of O-phenyl methyl-, ethyl- and benzylalanyl phosphoramidate pronucleotides derived from cytostatic 6-aryl-7-deazapurine ribonucleosides were prepared by the cross-coupling reactions of the 2',3'-isopropylidene protected 6-chloro-7-deazapurine ribonucleoside phosphoramidates with (het)arylboronic acids or -stannanes followed by deprotection. Most of the prepared prodrugs exerted in vitro cytostatic effects against both solid tumor and lymphoid cancer cells within low micromolar range of concentrations. These activities were in general weaker or comparable to the activities of the parent nucleosides. Additional testing of selected prodrugs suggests that the lack of activity improvement over parent nucleosides is not due to the lack of permeability or inefficient catabolism of alanyl-ester by intracellular hydrolases. More likely, active efflux of prodrugs may play a role in their weak cytotoxic activity.
- MeSH
- antitumorózní látky chemie farmakologie MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie MeSH
- nádorové buněčné linie MeSH
- purinové nukleosidy chemie farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A series of novel sugar-modified derivatives of cytostatic 6-hetaryl-7-deazapurine ribonucleosides: 2′-C-methylribonucleosides, arabinonucleosides and 2′-deoxy-2′-fluoroarabinonucleosides bearing an alkyl, aryl and hetaryl group in position 6 were prepared by palladium catalyzed cross-coupling reactions of corresponding (protected) 6-chloro-(7-fluoro)-7-deazapurine nucleosides with (het)arylboronic, hetarylstannanes and trimethylaluminium eventually followed by deprotection. Key intermediate 6-chloro-7-deazapurine 2′-C-methyl-β-D-ribofuranoside was prepared via a stereoselective nucleobase anion glycosylation with toluoyl-protected 1,2-anhydro-2-C-methylribofuranose. The 1,2-anhydro sugar was synthesized in 3 steps starting from readily available 2-C-methylribonolactone. The 6-chloro-7-deazapurine arabinofuranoside intermediate was obtained by epimerization from 3′,5′-protected 6-chloro-7-deazapurine ribofuranoside via 2′-hydroxyl oxidation followed by reduction. None of the prepared compounds showed any considerable cytostatic or antiviral activity.
- MeSH
- inhibitory enzymů chemická syntéza chemie farmakologie MeSH
- molekulární struktura MeSH
- organické sloučeniny křemíku chemická syntéza chemie farmakologie MeSH
- restrikční endonukleasy typu II antagonisté a inhibitory metabolismus MeSH
- štěpení DNA účinky léků MeSH
- stereoizomerie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
