Walkmycin B targets WalK (YycG), a histidine kinase essential for bacterial cell growth.

The WalK (a histidine kinase)/WalR (a response regulator, aka YycG/YycF) two-component system is indispensable in the signal transduction pathway for the cell-wall metabolism of Bacillus subtilis and Staphylococcus aureus. The inhibitors directed against WalK would be expected to have a bactericidal effect. After we screened 1368 culture broths of Streptomyces sp.

Inhibitors targeting two-component signal transduction.

A two-component signal transduction system (TCS) is an attractive target for antibacterial agents. In this chapter, we review the TCS inhibitors developed during the past decade and introduce novel drug discovery systems to isolate the inhibitors of the YycG/YycF system, an essential TCS for bacterial growth, in an effort to develop a new class of antibacterial agents.

Response regulator YycF essential for bacterial growth: X-ray crystal structure of the DNA-binding domain and its PhoB-like DNA recognition motif.

A response regulator YycF and its cognate sensor kinase YycG constitute the two-component signal transduction system essential for growth of Gram-positive bacteria with a low GC content. We have determined the X-ray crystal structure of the effector domain of Bacillus subtilis YycF involved in DNA binding. The structure, containing a winged helix-turn-helix motif, was found to be very similar to that of the response regulator PhoB from Escherichia coli.

Ascochytatin, a novel bioactive spirodioxynaphthalene metabolite produced by the marine-derived fungus, Ascochyta sp. NGB4.

Ascochytatin, a new spirodioxynaphthalene metabolite produced by a marine-derived fungus, was found from a screening program focused on the bacterial two-component regulatory system. The structure of ascochytatin was determined by spectroscopic methods, including NMR and MS. The relative stereochemistry was determined by an X-ray crystallographic analysis, and the absolute stereochemistry was determined by the modified Mosher's method.

B1500, a small membrane protein, connects the two-component systems EvgS/EvgA and PhoQ/PhoP in Escherichia coli.

Two-component signal-transduction systems (TCSs) of bacteria are considered to form an intricate signal network to cope with various environmental stresses. One example of such a network in Escherichia coli is the signal transduction cascade from the EvgS/EvgA system to the PhoQ/PhoP system, where activation of the EvgS/EvgA system promotes expression of PhoP-activated genes. As a factor connecting this signal transduction cascade, we have identified a small inner membrane protein (65 aa), B1500.

Targeting two-component signal transduction: a novel drug discovery system.

We have developed two screening systems for isolating inhibitors that target bacterial two-component signal transduction: (1) a differential growth assay using a temperature-sensitive yycF mutant (CNM2000) of Bacillus subtilis, which is supersensitive to histidine kinase inhibitors, and (2) a high-throughput genetic system for targeting the homodimerization of histidine kinases essential for the bacterial two-component signal transduction. By using these methods, we have been able to identify various types of inhibitors that block the autophosphorylation of histidine kinases with different modes of actions.