Novenamines as inhibitors of two independent enzymes during DNA replication in a toluenized Escherichia coli cell system

Abstract

The amphiphilic novenamines described in this report have been shown previously to be specific inhibitors of human immunodeficiency virus type 1 reverse transcriptase-associated ribonuclease, which they inhibit when they are in the micellar state but not when they are monomeric. These compounds also inhibit the bacterial enzyme DNA gyrase, which is essential for DNA replication. Hence, the present studies were initiated to determine whether the molecular species inhibiting the gyrase reaction was the monomeric or the micellar form. For this purpose, the rate of DNA replication was measured in a toluenized Escherichia coli cell system in the presence of increasing concentrations of novenamines. The resulting concentration-response curves proved anomalous, suggesting the involvement of micelles or some other, noncovalently aggregated forms of the inhibitors. The results were analyzed in terms of a variety of kinetic schemes and were found to be most consistent with the model where novenamines inhibit replicative DNA synthesis predominantly as cooperative dimers and, to a lesser extent, as monomers, but not as highly aggregated micelles. Based on this analysis and the knowledge that novobiocin and all novenamine-containing analogs are powerful gyrase inhibitors, we conclude that the target of the cooperative, dimeric inhibition is the gyrase, whereas the monomers of the novenamines inhibit another enzyme species involved in the bacterial DNA replication process.