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Biochemical and biological properties of 5-bromotubercidin: Differential effects on cellular DNA-directed and viral RNA-directed RNA synthesis

We have shown that 5-bromotubercidin (BrTu), a cytotoxic pyrrolo[2, 3-d]pyrimidine analogue of adenosine can be used as a metabolic probe which possesses biological specificity and selectivity: 1) BrTu entered mammalian cell nucleotide pool by phosphorylation, and it was in unmodified form incorporated into RNA and perhaps DNA. As a consequence BrTu (5 µ g/ml ; 15 µ M) reversibly inhibited mammalian cell growth, RNA and DNA synthesis ; 2) BrTu inhibited the synthesis of heterogeneous nuclear RNA and the processing of the 45S RNA precursor into 28S and 18S ribosomal RNA ; 3) Since BrTu did not inhibit purine biosynthesis de novo, its inhibitory effects were probably the result of a primary action on polynucleotide synthesis. This conclusion was fortified by the pattern of BrTu nucleotide polymerization catalyzed by E. coli RNA polymerase with synthetic (e.g., poly d(A-T) and poly dT) and natural calf thymus DNA polynucleotides as templates, indicating that the presence of bulky C-Br group at the N-7 position of adenine does not interfere with the formation of polynucleotide helices ; 4) The growth and RNA sythesis of picornaviruses were completely resistant to concentrations of BrTu (300 µ M) much higher than those (15 µ M) which strongly inhibited the growth of the host cell, of DNA virus vaccinia, and of the DNA-dependent RNA synthesis of uninfected cells. BrTu thus discriminates between cellular DNA-directed and viral RNA-directed RNA synthesis, and can be used as a metabolic probe for studying the cell-virus relationship ; 5) BrTu is a substrate for adenosine kinase (Km=24± 4.3 µ M), but is not a substrate for all the kinase systems which are capable of utilizing adenine nucleotides. It is also a potent inhibitor of adenosine kinase and low concentrations of BrTu (1.5 µ M), which did not inhibit cell growth, blocked phosphorylation and the cellular uptake of other, highly cytotoxic pyrrolo-pyrimidine nucleoside analogues, such as tubercidin and deazanebularin. This block in cellular uptake of toxic analogues was associated with the protective effect of BrTu against cell killing by the analogues providing a means for controlling the rate of metabolism of these cytotoxic compounds in biologycal systems.

5-bromotubercidin ; pyrrolo-pyrimidines ; metabolic probe ; virus-directed RNA synthesis ; mouse fibroblasts

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