MOLECULAR DESIGN OF INHIBITORS OF IN-VITRO ORIC DNA-REPLICATION BASEDON THE POTENTIAL TO BLOCK THE ATP BINDING OF DNAA PROTEIN

DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coil, is activated by binding to ATP. We earlier reported that 3-acetoxy-2,2'-bi-1H-indol inhibited the ATP binding to DnaA pro tein (Sasaki, S., Mizushima, T., Hashimoto, T., Maeda, M., and Sekimiz u, K. (1994) Bioorg. Med. Chem. Lett. 4, 1771-1774), In the present st udy, derivatives of 3-acetoxy-2,2'-bi-1H-indol with different lengths of aliphatic chains at the 3-O position were synthesized, and their po tential to inhibit the ATP binding to DnaA protein was examined. Elong ation of the aliphatic chain resulted in inhibition of the ATP binding to DnaA protein at lower concentrations. Among the derivatives, 3-[N- (11-carboxyundecyl)] carbamoylmethoxy-2,2'-bi-1H-indol (structure 7 (3 -CUCM-BI)) exhibited the most potent inhibition with an IC50 value of 7 mu M. The mode of the inhibition was competitive. We further demonst rated that structure 7 (3-CUCM-BI) inhibited DNA replication of the or iC plasmid in a system reconstituted from purified proteins. This inhi bition was specific for the initiation of DNA replication rather than for the elongation. The inhibition was overcome by preincubation of Dn aA protein with ATP. Furthermore, structure 7 (3-CUCM-BI) showed littl e inhibition on DNA synthesis in the ABC primosome system. We propose that structure 7 (3-CUCM-BI) functions in the in vitro oriC DNA replic ation by inhibiting the ATP binding to DnaA protein.