DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by binding to ATP. We earlier reported that 3-acetoxy-2,2 -bi-1H-indol inhibited the ATP binding to DnaA protein (Sasaki, S., Mizushima, T., Hashimoto, T., Maeda, M., and Sekimizu, K. (1994) Bioorg. Med. Chem. Lett. 4, 1771-1774). In the present study, derivatives of 3-acetoxy-2,2 -bi-1H-indol with different lengths of aliphatic chains at the 3-O position were synthesized, and their potential to inhibit the ATP binding to DnaA protein was examined. Elongation 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 IC 50 value of 7 M. The mode of the inhibition was competitive. We further demonstrated that structure 7 (3-CUCM-BI) inhibited DNA replication of the oriC plasmid in a system reconstituted from purified proteins. This inhibition was specific for the initiation of DNA replication rather than for the elongation. The inhibition was overcome by preincubation of DnaA protein with ATP. Furthermore, structure 7 (3-CUCM-BI) showed little inhibition on DNA synthesis in the ABC primosome system. We propose that structure 7 (3-CUCM-BI) functions in the in vitro oriC DNA replication by inhibiting the ATP binding to DnaA protein.Replication of chromosomal DNA in Escherichia coli is regulated at the step of initiation. DnaA protein is the initiation factor for chromosomal DNA replication (1-3); thus, DnaA protein has been considered to play an important role in regulating DNA replication. DnaA protein has a high affinity for ATP (K d ϭ 0.03 M) and for ADP (K d ϭ 0.1 M) (4). In the oriC DNA replication system reconstituted from purified proteins, the ATP binding form of DnaA protein is active in DNA replication, whereas the ADP binding form is inactive (4). These results suggest that the ATP binding to DnaA protein activates the protein; however, the possibilities that the ADP binding to the protein inhibits the activity of DnaA protein in the initiation of oriC DNA replication and that the ATP binding to the protein is not essential for the process would need to be excluded. Studies on DnaAcos protein, which loses the affinity for ATP and ADP but is active in the initiation of oriC DNA replication in vitro, imply this notion (5, 6). To better understand the requirement of the ATP binding for the initiation of oriC DNA replication, development of specific inhibitors for the ATP binding to DnaA protein and examination of their effects on oriC DNA replication in vitro are important. The availability of such inhibitors would be good tools to study the biological relevance of the ATP binding to DnaA protein.We reported that 3-acetoxy-2,2Ј-bi-1H-indol inhibited the ATP binding of DnaA protein (7). This indol is the first known synthetic organic compound to inhibit the ATP binding of DnaA protein. However, concentration of...

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Pharmacological treatment of hypercalcemia is essential for patients with parathyroid carcinoma and intractable primary hyperparathyroidism (PHPT). Use of the calcimimetic cinacalcet hydrochloride (cinacalcet) is an option to treat such patients. We investigated the efficacy and safety of cinacalcet in Japanese patients with parathyroid carcinoma and intractable PHPT. Five Japanese patients with parathyroid carcinoma and two with intractable PHPT were enrolled in an open-label, single-arm study consisting of titration and maintenance phases. Cinacalcet doses were titrated until the albumin-corrected serum calcium concentration decreased to 10.0 mg/dL or less or until dose escalation was considered not necessary or feasible. Serum calcium concentration at the baseline was 12.1 ± 1.3 mg/dL (mean ± standard deviation; range 10.4-14.6 mg/dL) and decreased to 10.1 ± 1.6 mg/dL (range 8.6-13.3 mg/dL) at the end of the titration phase with cinacalcet at a dosage of up to 75 mg three times a day. At the end of the titration phase, at least a 1 mg/dL reduction in serum calcium concentration from the baseline was observed in five patients (three with carcinoma and two with PHPT), and it decreased to the normocalcemic range in five patients (three with carcinoma and two with PHPT). Common adverse events were nausea and vomiting. One patient discontinued participation in the study because of an adverse event, liver disorder. Cinacalcet effectively relieved hypercalcemia in 60% of the Japanese patients with parathyroid carcinoma and might be effective in those with intractable PHPT. The drug might be tolerable and safe at a dosage of at most 75 mg three times a day.

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