Scavenging effects of metal complexes of water-soluble thiacalix[4]arenetetrasulfonate on superoxide anion radicals.

The scavenging effects of metal complexes of thiacalix[4]arenetetrasulfonate (Me-TCAS[4], Me=H2, Fe³(+), Mn³(+), Mn²(+), Cu²(+), and Zn²(+)) on superoxide anion radicals (O2⁻) generated from the xanthine-xanthine oxidase system were investigated by the nitroblue tetrazolium (NBT) method and electron spin resonance (ESR) spin-trapping method using 5,5-dimethyl-1-pyrroline-N-oxide as a trapping reagent. As a reference, calix[4]arenetetrasulfonate (H2)-CAS[4]), calix[6]arenehexasulfonate (H2-CAS[6]) and calix[8]areneoctasulfonate (H2-CAS[8]) were also examined. The results by the NBT method indicated that Fe³(+)- and Mn³(+)-TCAS[4] exhibited the highest O2⁻ scavenging activity among Me-TCAS[4] and H2-CAS[n] (n = 4, 6, 8) in this study. The IC50 values of Fe³(+)- and Mn³(+)-TCAS[4] for O2⁻ scavenging activity were estimated to be 5.3 and 7.8 µM, respectively, and were almost the same as those of tannin acid, catechin and their derivatives, which are known as very effective scavengers of O2⁻. Scavenging activities were in the order of Fe³(+)- and Mn³(+)-TCAS[4]>>Mn²(+)-, Cu²(+)-, and Zn(2+)-TCAS[4]>>H(2)-TCAS[4] and H2-CAS[n] (n=4, 6, 8). Each activity of Me-TCAS[4] (Me=Fe³(+), Mn³(+), Mn²(+), Cu²(+), and Zn²(+)) was higher than that of the corresponding metal ion, indicating that H2-TCAS[4] has the ability to raise the activity of the metal ion itself by forming a complex. Also, the ESR spin-trapping method revealed that Fe³(+)- and Mn³(+)-TCAS[4] showed high O2⁻ scavenging activities, similarly to the results by the NBT method.

A new screening method to identify inhibitors of the Lol (localization of lipoproteins) system, a novel antibacterial target.

As the Lol system, which is involved in localization of lipoproteins, is essential for Escherichia coli growth and widely conserved among gram-negative bacteria, it is considered to be a promising target for the development of anti-gram-negative bacterial agents. However, no high-throughput screening method has so far been developed to screen for Lol system inhibitors. By combining three assay systems (anucleate cell blue assay, Lpp assay, and LolA-dependent release inhibition assay) and a drug susceptibility test, we have successfully developed a new screening method for identification of compounds that inhibit the Lol system. Using this new screening method, we screened 23,600 in-house chemical compounds and found 2 Lol system inhibitors. We therefore conclude that our new screening method can efficiently identify new antibacterial agents that target the Lol system.

A 4-aminofurazan derivative-A189-inhibits assembly of bacterial cell division protein FtsZ in vitro and in vivo.

Out of 95,000 commercially available chemical compounds screened by the anucleate cell blue assay, 138 selected hit compounds were further screened. As a result, A189, a 4-aminofurazan derivative was found to inhibit FtsZ GTPase with an IC(50) of 80 mug/ml and to exhibit antibacterial activity against Staphylococcus aureus and Escherichia coli. Light scattering demonstrated that A189 inhibited FtsZ assembly in vitro, and microscopic observation of A189-treated E. coli indicated that A189 perturbed FtsZ ring formation and made bacterial cells filamentous. However, nucleoids staining with DAPI revealed that A189 did not affect DNA replication and chromosome segregation in bacterial filamentous cells. Furthermore, A189 made sulA-deleted E. coli cells filamentous. Taken together, these findings suggest that A189 inhibits FtsZ GTPase activity, resulting in perturbation of FtsZ ring formation, which leads to bacterial cell death.