The oligoadenylate synthetase-ribonuclease L pathway is a major player in the interferon-induced antiviral defense mechanism of cells. Upon sensing viral dsRNA, 5'-phosphorylated 2',5'-oligoadenylates are synthesized, and subsequently activate latent RNase L. To determine the influence of 5'-phosphate end on the activation of human RNase L, four sets of 5'-phosphonate modified oligoadenylates were prepared on solid-phase. The ability of these 5'-modified oligoadenylates bearing shortened, isosteric and prolonged phosphonate linkages to activate RNase L was explored. We found that isosteric linkages and linkages prolonged by one atom were in general well tolerated by the enzyme with the EC50 values comparable to that of the natural activator. In contrast, linkages shortened by one atom or prolonged by two atoms exhibited decrease in the activity.
- MeSH
- adeninnukleotidy chemická syntéza chemie farmakologie MeSH
- endoribonukleasy metabolismus MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- oligoribonukleotidy chemická syntéza chemie farmakologie MeSH
- organofosfonáty chemická syntéza chemie farmakologie 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
Cyclic dinucleotides (CDNs) are second messengers that bind to the stimulator of interferon genes (STING) and trigger the expression of type I interferons and proinflammatory cytokines. Here we evaluate the activity of 3',3'-c-di(2'F,2'dAMP) and its phosphorothioate analogues against five STING allelic forms in reporter-cell-based assays and rationalize our findings with X-ray crystallography and quantum mechanics/molecular mechanics calculations. We show that the presence of fluorine in the 2' position of 3',3'-c-di(2'F,2'dAMP) improves its activity not only against the wild type (WT) but also against REF and Q STING. Additionally, we describe the synthesis of the acyloxymethyl and isopropyloxycarbonyl phosphoester prodrugs of CDNs. Masking the negative charges of the CDNs results in an up to a 1000-fold improvement of the activities of the prodrugs relative to those of their parent CDNs. Finally, the uptake and intracellular cleavage of pivaloyloxymethyl prodrugs to the parent CDN is rapid, reaching a peak intracellular concentration within 2 h.
- MeSH
- estery chemie farmakologie terapeutické užití MeSH
- fosfáty chemie metabolismus farmakologie terapeutické užití MeSH
- HEK293 buňky MeSH
- interferon gama metabolismus MeSH
- krystalografie rentgenová MeSH
- leukocyty mononukleární cytologie účinky léků metabolismus MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie MeSH
- membránové proteiny agonisté metabolismus MeSH
- prekurzory léčiv chemická syntéza chemie metabolismus farmakologie MeSH
- teorie funkcionálu hustoty MeSH
- TNF-alfa metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cyclic dinucleotides are second messengers in the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, which plays an important role in recognizing tumor cells and viral or bacterial infections. They bind to the STING adaptor protein and trigger expression of cytokines via TANK binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) and inhibitor of nuclear factor-κB (IκB) kinase (IKK)/nuclear factor-κB (NFκB) signaling cascades. In this work, we describe an enzymatic preparation of 2'-5',3'-5'-cyclic dinucleotides (2'3'CDNs) with use of cyclic GMP-AMP synthases (cGAS) from human, mouse, and chicken. We profile substrate specificity of these enzymes by employing a small library of nucleotide-5'-triphosphate (NTP) analogues and use them to prepare 33 2'3'CDNs. We also determine affinity of these CDNs to five different STING haplotypes in cell-based and biochemical assays and describe properties needed for their optimal activity toward all STING haplotypes. Next, we study their effect on cytokine and chemokine induction by human peripheral blood mononuclear cells (PBMCs) and evaluate their cytotoxic effect on monocytes. Additionally, we report X-ray crystal structures of two new CDNs bound to STING protein and discuss structure-activity relationship by using quantum and molecular mechanical (QM/MM) computational modeling.
- MeSH
- biotest MeSH
- cytokiny metabolismus MeSH
- HEK293 buňky MeSH
- konformace proteinů MeSH
- leukocyty mononukleární účinky léků MeSH
- lidé MeSH
- membránové proteiny chemie metabolismus MeSH
- nukleotidy cyklické chemická syntéza farmakologie MeSH
- počítačová simulace MeSH
- regulace genové exprese účinky léků MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We have developed a robust solid-phase protocol which allowed the synthesis of chimeric oligonucleotides modified with phosphodiester and O-methylphosphonate linkages as well as their P-S and P-N variants. The novel O-methylphosphonate-derived modifications were obtained by oxidation, sulfurization, and amidation of the O-methyl-(H)-phosphinate internucleotide linkage introduced into the oligonucleotide chain by H-phosphonate chemistry using nucleoside-O-methyl-(H)-phosphinates as monomers. The H-phosphonate coupling followed by oxidation after each cycle enabled us to successfully combine H-phosphonate and phosphoramidite chemistries to synthesize diversely modified oligonucleotide strands.
This unit comprises the straightforward synthesis of protected 2'-deoxyribonucleoside-O-methyl-(H)-phosphinates in both 3'- and 5'-series. These compounds represent a new class of monomers compatible with the solid-phase synthesis of oligonucleotides using H-phosphonate chemistry and are suitable for the preparation of both 3'- and 5'-O-methylphosphonate oligonucleotides. The synthesis of 4-toluenesulfonyloxymethyl-(H)-phosphinic acid as a new reagent for the preparation of O-methyl-(H)-phosphinic acid derivatives is described. © 2017 by John Wiley & Sons, Inc.
- MeSH
- oligonukleotidy chemická syntéza MeSH
- organofosfonáty MeSH
- organofosforové sloučeniny chemie MeSH
- Publikační typ
- časopisecké články MeSH
The concept of conformational restriction leading to the preorganization of modified strands has proven to be successful and has afforded nucleic acid analogues with many interesting properties suitable for various biochemical applications. We utilized this concept to prepare a set of constrained oligonucleotides derived from 1,4-dioxane and 1,3-dioxolane-locked nucleoside phosphonates and evaluated their hybridization affinities towards their complementary RNA strands. With an increase of ΔTmper modification up to +5.2 °C, the hybridization experiments revealed the (S)-2',3'-O-phosphonomethylidene internucleotide linkage as one of the most Tm-increasing modifications reported to date. Moreover, we introduced a novel prediction tool for the pre-selection of potentially interesting chemical modifications of oligonucleotides.
The straightforward synthesis of sodium 4-toluenesulfonyloxymethyl-(H)-phosphinate and (H)-phosphinomethylisothiouronium tosylate as new reagents for the preparation of O- and S-methyl-(H)-phosphinic acid derivatives, respectively, is described. The reactivity of both reagents was demonstrated by the preparation of protected 2'-deoxyribonucleoside-O-methyl-(H)-phosphinates in the 5'- and 3'-series and 2',5'-dideoxyribonucleoside-5'-S-methyl-(H)-phosphinates. These compounds represent a new class of monomers compatible with the solid phase synthesis of oligonucleotides by H-phosphonate chemistry, as it was proved with the synthesis of a fully phosphonate heptamer.
Mycobacterium tuberculosis O(6)-methylguanine-DNA methyltransferase (MtOGT) contributes to protect the bacterial GC-rich genome against the pro-mutagenic potential of O(6)-methylated guanine in DNA. Several strains of M. tuberculosis found worldwide encode a point-mutated O(6)-methylguanine-DNA methyltransferase (OGT) variant (MtOGT-R37L), which displays an arginine-to-leucine substitution at position 37 of the poorly functionally characterized N-terminal domain of the protein. Although the impact of this mutation on the MtOGT activity has not yet been proved in vivo, we previously demonstrated that a recombinant MtOGT-R37L variant performs a suboptimal alkylated-DNA repair in vitro, suggesting a direct role for the Arg(37)-bearing region in catalysis. The crystal structure of MtOGT complexed with modified DNA solved in the present study reveals details of the protein-protein and protein-DNA interactions occurring during alkylated-DNA binding, and the protein capability also to host unmodified bases inside the active site, in a fully extrahelical conformation. Our data provide the first experimental picture at the atomic level of a possible mode of assembling three adjacent MtOGT monomers on the same monoalkylated dsDNA molecule, and disclose the conformational flexibility of discrete regions of MtOGT, including the Arg(37)-bearing random coil. This peculiar structural plasticity of MtOGT could be instrumental to proper protein clustering at damaged DNA sites, as well as to protein-DNA complexes disassembling on repair.
- MeSH
- bakteriální proteiny chemie genetika MeSH
- bodová mutace genetika MeSH
- krystalografie MeSH
- Mycobacterium tuberculosis genetika MeSH
- O(6)-methylguanin-DNA-methyltransferasa chemie genetika MeSH
- poškození DNA genetika MeSH
- sekundární struktura proteinů MeSH
- terciární struktura proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Protected N-branched nucleoside phosphonates containing adenine and thymine bases were prepared as the monomers for the introduction of aza-acyclic nucleotide units into modified oligonucleotides. The phosphotriester and phosphoramidite methods were used for the incorporation of modified and natural units, respectively. The solid phase synthesis of a series of nonamers containing one central modified unit was successfully performed in both 3'→5' and 5'→3' directions. Hybridization properties of the prepared oligoribonucleotides and oligodeoxyribonucleotides were evaluated. The measurement of thermal characteristics of the complexes of modified nonamers with the complementary strand revealed a considerable destabilizing effect of the introduced units. We also examined the substrate/inhibitory properties of aza-acyclic nucleoside phosphono-diphosphate derivatives (analogues of nucleoside triphosphates) but neither inhibition of human and bacterial DNA polymerases nor polymerase-mediated incorporation of these triphosphate analogues into short DNA was observed.
- MeSH
- adenin chemická syntéza chemie MeSH
- DNA-dependentní DNA-polymerasy metabolismus MeSH
- inhibitory syntézy nukleových kyselin chemická syntéza chemie farmakologie MeSH
- lidé MeSH
- nukleosidy chemická syntéza chemie farmakologie MeSH
- oligonukleotidy chemická syntéza chemie farmakologie MeSH
- organofosfonáty chemická syntéza chemie farmakologie MeSH
- sekvence nukleotidů MeSH
- thymin chemická syntéza chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
