Unmetabolized 5,6-dichlororibofuranosylbenzimidazole rather than its monophosphate metabolites is probably the active transcription inhibitor.

The question of whether or not the monophosphate metabolites DRB2′P and DRB5′P of the transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) constitute the active form of the inhibitor was investigated in intact salivary gland cells of Chironomus tentants. The intracellular phosphorylation of DRB to DRB2′P as well as to DRB5′P could be lowered to a considerable extent by addition of a 15-fold excess of adenosine or 3′-deoxyadenosine without lowering the intracellular concentration of unmetabolized [3H]DRB. Nevertheless, the inhibition of hnRNA synthesis remained essentially unaffected. The addition of excess guanosine interfered only with the formation of 3H-labelled DRB5′P isomer while uridine was without any detectable effect on the metabolism of DRB to any of the DRB monophosphate isomers. When in another type of experiment with DRB-inhibited cells the unmetabolized DRB was washed out, whereas the monophosphate isomers, formed before removal of DRB, remained trapped intracellularly, it was found that the inhibitory activity was abolished and the initiation of RNA synthesis was resumed. The experimental data taken together strongly indicate that the DRB metabolites DRB2′P and DRB5′P are not involved in the inhibitory activity, and that it is the unmetabolized compound that acts as active transcription inhibitor.