Inhibition of eukaryotic translation by analogs of messenger RNA 5'-cap: chemical and biological consequences of 5'-phosphate modifications of 7-methylguanosine 5'-monophosphate (original) (raw)
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Biochemistry, 1989
New analogues of 7-methylguanosine 5'-monophosphate (m7GMP) were synthesized with modified 5'-phosphate moieties by replacement of -0 with -H, -CH3, or -NH2. Additional analogues were synthesized with &methyl-or 8-aminoguanine base substitutions or ring-opened ribose (2',3'-diol). These compounds were analyzed by 'H and 31P N M R for solution conformation. In addition, they were also analyzed for biological activity as analogues of mRNA 5'-caps by competition as inhibitors of translation in reticulocyte lysate. Substitution of oxygen on the 5'-monophosphate moiety by -H and -CH3 diminished the activity of the cap analogue as a competitive inhibitor; however, replacement by -NH2 did not diminish the activity of the analogue as an inhibitor. It was inferred from this result that cap binding proteins require a hydrogen bond acceptor as opposed to having an exclusive requirement for a second anionic group on the a-phosphate moiety. Inhibition results obtained with C8-substituted m7GMP analogues indicated that the 8-amino derivative was a better inhibitor than the 8-methyl derivative of m7GMP. The former is primarily anti whereas the latter is primarily syn with respect to glycosidic bond conformation. This result further supports the model that the anti conformation is the preferred form of the cap structure for interaction with cap binding proteins. The 2',3'-diol derivative of m7GMP was inactive as an inhibitor of translation.
Phosphorothioate analogs of m7GTP are enzymatically stable inhibitors of cap-dependent translation
Bioorganic & Medicinal Chemistry Letters, 2009
We report synthesis and properties of a pair of new potent inhibitors of translation, namely two diastereomers of 7-methylguanosine 5 0 -(1-thiotriphosphate). These new analogs of mRNA 5 0 cap (referred to as m 7 GTPaS (D1) and (D2)) are recognized by translational factor eIF4E with high affinity and are not susceptible to hydrolysis by Decapping Scavenger pyrophosphatase (DcpS). The more potent of diastereomers, m 7 GTPaS (D1), inhibited cap-dependent translation in rabbit reticulocyte lysate 8−foldand8-fold and 8−foldand15-fold more efficiently than m 7 GTP and m 7 GpppG, respectively. Both analogs were also significantly more stable in RRL than unmodified ones.
Quantitative Assessment of mRNA Cap Analogues as Inhibitors of in Vitro Translation †
Biochemistry, 1999
Fifty-eight analogues of the 5′-terminal 7-methylguanosine-containing cap of eukaryotic messenger RNA were synthesized and tested for their ability to inhibit in vitro protein synthesis. A new algorithm was developed for extracting K I , the dissociation constant for the cap analogue‚eIF4E complex, from protein synthesis data. The results indicated that addition of a methyl group to the N2 of guanine produced more inhibitory compounds, but addition of a second methyl group to N2 decreased the level of inhibition dramatically. Aryl substitution at N7 improved the efficacy of guanine nucleoside monophosphate analogues. Substitution of the aromatic ring at the para position with methyl or NO 2 groups abolished this effect, but substitution with Cl or F enhanced it. By contrast, aryl substitution at N7 in nucleoside di-or triphosphate analogues produced only minor effects, both positive and negative. By far the strongest determinants of inhibitory activity for cap analogues were phosphate residues. The beneficial effect of more phosphate residues was related more to anionic charge than to the number of phosphate groups per se. The second nucleotide residue in analogues of the form m 7 GpppN affected inhibitory activity in the order G > C > U > A, but there was no effect of 2′-O-modification. Opening the first ribose ring of m 7 GpppG analogues dramatically decreased activity, but alterations at the 2′position of this ribose had no effect. Non-nucleotide-based cap analogues containing benzimidazole derivatives were inhibitory, though less so than those containing 7-methylguanine.
Nucleos Nucleot Nucleic Acids, 2003
A series of new mRNA anti reverse cap analogues (ARCA) was designed to obtain a tool for studying the mechanism of protein translation. Dinucleoside P 1 , P 3-triP P 1 , P 4-tetra-and P 1 , P 5-pentaphosphates, linked by a 5 0-to-5 0 phosphate bridge and composed of modified 7-methylguanosine and guanosine, have been synthesized. The hydroxyl group (2 0 OH or 3 0 OH) in 7-metylguanosine moiety was replaced by-OCH 3 or-H in order to obtain the cap analogues capable to be correctly incorporated into synthetic mRNA transcripts. Tri-, tetra-, and pentaphosphates were prepared by ZnCl 2 catalyzed condensation in DMF of derivatives of the 7-methylguanosine diphosphates with the guanosine mono-, di-and triphosphate P-imidazolides, respectively. The structures of the novel compounds were established by means of 1 H and 31 P NMR spectra.
Nucleosides, Nucleotides and Nucleic Acids, 2003
A series of new mRNA anti reverse cap analogues (ARCA) was designed to obtain a tool for studying the mechanism of protein translation. Dinucleoside P 1 , P 3 -tri-, P 1 , P 4 -tetra-and P 1 , P 5 -pentaphosphates, linked by a 5 0 -to-5 0 phosphate bridge and composed of modified 7-methylguanosine and guanosine, have been synthesized. The hydroxyl group (2 0 OH or 3 0 OH) in 7-metylguanosine moiety was replaced by -OCH 3 or -H in order to obtain the cap analogues capable to be correctly incorporated into synthetic mRNA transcripts. Tri-, tetra-, and pentaphosphates were prepared by ZnCl 2 catalyzed condensation in DMF of derivatives of the 7-methylguanosine diphosphates with the guanosine mono-, di-and triphosphate P-imidazolides, respectively. The structures of the novel compounds were established by means of 1 H and 31 P NMR spectra.
Bioorganic & Medicinal Chemistry Letters, 2013
Synthetic mRNA cap analogs are valuable tools in the preparation of modified mRNA transcripts with improved translational activity and increased cellular stability, and have recently attracted more attention because of their great potential in therapeutic applications. We have synthesized and tested isopropylidene dinucleotide cap analogs bearing a phosphorothioate group at the b position of the 5 0 ,5 0 -triphosphate bridge (two diastereomers of 2 0 ,3 0 -iPr-m 7 Gpp S pG), as synthetically simpler alternatives to previously obtained phosphorothioate cap analogs. To evaluate the utility of the new compounds in biological systems we determined their affinity to translation initiation factor 4E (eIF4E), and tested their translational properties in rabbit reticulocyte lysates (RRL) and in human immature dendritic cells (hiDCs). In order to explain the properties of isopropylidene analogs we performed 1 H NMR conformational analysis and correlated the absolute configuration at the b-phosphorous atom with previously synthesized m 7 Gpp S pG.
Novel cap analogs for in vitro synthesis of mRNAs with high translational efficiency
RNA, 2004
Synthetic analogs of the N7-methylated guanosine triphosphate cap at the 5 end of eukaryotic mRNAs and snRNAs have played an important role in understanding their splicing, intracellular transport, translation, and turnover. We report here a new series of N7-benzylated dinucleoside tetraphosphate analogs, b 7 Gp 4 G, b 7 m 3-O Gp 4 G, and b 7 m 2 Gp 4 G, that extend our knowledge of the role of the cap in translation. We used these novel analogs, along with 10 previously synthesized analogs, to explore five parameters: binding affinity to eIF4E, inhibition of cap-dependent translation in a rabbit reticulocyte lysate system, efficiency of incorporation into RNAs during in vitro transcription (% capping), orientation of the analog in the synthetic mRNA (% correct orientation), and in vitro translational efficiency of mRNAs capped with the analog. The 13 cap analogs differed in modifications of the first (distal) and second (proximal) guanine moieties, the first and second ribose moieties, and the number of phosphate residues. Among these were analogs of the naturally occurring cap m 3 2,2,7 Gp 3 G. These compounds varied by 61-fold in affinity for eIF4E, 146-fold in inhibition of cap-dependent translation, 1.4-fold in % capping, and 5.6-fold in % correct orientation. The most stimulatory analog enhanced translation 44-fold compared with uncapped RNA. mRNAs capped with b 7 m 2 Gp 4 G, m 7 Gp 3 m 7 G, b 7 m 3-O Gp 4 G, and m 7 Gp 4 m 7 G were translated 2.5-, 2.6-, 2.8-, and 3.1-fold more efficiently than mRNAs capped with m 7 Gp 3 G, respectively. Relative translational efficiencies could generally be explained in terms of cap affinity for eIF4E, % capping, and % correct orientation. The measurement of all five parameters provides insight into factors that contribute to translational efficiency.