Quantitative assessment of mRNA cap analogues as inhibitors of in vitro translation

Fifty-eight analogues of the 5'-terminal 7-methylguanosine-containing cap o f eukaryotic messenger RNA were synthesized and tested for their ability to inhibit in vitro protein synthesis. A new algorithm was developed for extr acting Kr, the dissociation constant for the cap analogue.eIF4E complex, fr om 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 dramatica lly. Aryl substitution at N7 improved the efficacy of guanine nucleoside mo nophosphate analogues. Substitution of the aromatic ring at the para positi on with methyl or NO2 groups abolished this effect, but substitution with C l or F enhanced it. By contrast, aryl substitution at N7 in nucleoside di- or triphosphate analogues produced only minor effects, both positive and ne gative. By far the strongest determinants of inhibitory activity for cap an alogues were phosphate residues. The beneficial effect of more phosphate re sidues was related more to anionic charge than to the number of phosphate g roups per se. The second nucleotide residue in analogues of the form m(7)Gp ppN 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'-positi on of this ribose had no effect. Non-nucleotide-based cap analogues contain ing benzimidazole derivatives were inhibitory, though less so than those co ntaining 7-methylguanine.