Walkmycin B targets WalK (YycG), a histidine kinase
essential for bacterial cell growth
Ario Okada1,5, Masayuki Igarashi2,5, Toshihide Okajima3, Naoko Kinoshita2, Maya Umekita2, Ryuichi Sawa2,
Kunio Inoue2, Takafumi Watanabe1, Akihiro Doi1, Aaron Martin4, John Quinn4, Yoshio Nishimura2
and Ryutaro Utsumi1
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. by a
differential growth assay, walkmycin A, B and C, which were produced by strain MK632-100F11, were purified using silica-gel
column chromatography and HPLC. In this paper, the chemical structure of the major product (walkmycin B) was determined
to be di-anthracenone (C44H44Cl2O14), which was very similar to BE40665A. MICs of walkmycin B against B. subtilis and
S. aureus were 0.39 and 0.20lgml?1, and IC50measurements against WalK were 1.6 and 5.7lM, respectively. To clarify the
affinity between WalK and walkmycin B, surface plasmon resonance was measured to obtain the equilibrium dissociation
constant, KD1, of 7.63lM at the higher affinity site of B. subtilis WalK. These results suggest that walkmycin B inhibits WalK
autophosphorylation by binding to the WalK cytoplasmic domain.
The Journal of Antibiotics (2010) 63, 89–94; doi:10.1038/ja.2009.128; published online 8 January 2010
Keywords: antibiotic; histidine kinase inhibitor; MRSA; two-component signal transduction; WalK
In the past decade, development of bacterial genomics, bioinformatics
and gene manipulation have led to the discovery of many novel
protein targets for antibacterial agents.1For instance, the two-
component signal transduction systems of bacteria, which consist of
two proteins, histidine kinase (HK) and response regulators, have
received increasing attention on account of their potential as novel
antibacterial drug targets.2–4
Earlier work has reported on five chemotypes (cyclohexene, closan-
tel, benzimidazole, trityl and bisphenol) that possess inhibitory activity
against KinA (an HKwith an IC50value ranging from 2 to 20mM) and
that result in antibacterial activity.5These compounds also had an
appreciable effect on the cell membrane integrity or caused hemolysis
of equine erythrocytes. Although they were inhibitors against non-
essential HKs, no absolute proof has been presented showing that
growth inhibition was the direct consequence of HK inhibition.
The WalK/WalR (aka YycG/YycF) two-component signal transduc-
tion system has appeared as a promising candidate, as it is essential for
bacterial survival and is specific to low G+C% Gram-positive bacteria,
pneumoniae, Enterococcus faecalis and S. pyogenes.6–10Recent studies
have unveiled a conserved function for this system in different
bacteria, defining this signal transduction pathway as a master
regulatory system for cell-wall metabolism, owing to which YycG/
YycF has been accordingly renamed as WalK/WalR.11,12Thus,
inhibitors directed against WalK/WalR may have a bactericidal effect
and may be active against an array of clinically important
WalK is a membrane-linked kinase, with a conserved C-terminal
cytoplasmic HK region, including ATP binding and phosphoacceptor
domains. Sequence similarities indicate that WalK phosphorylation
occurs on conserved residue His387(all amino acid sequence coordi-
nates are those of the prototypical B. subtilis proteins), although the
aminoterminal domain of the WalK HK shows considerable variation
among species. The conserved C-terminal cytoplasmic HK region can
be used for screening WalK inhibitors.
First, inhibitors targeting WalK (C-terminal cytoplasmic HK region)
of B. subtilis were analyzed to obtain imidazole derivatives (NH125).13
Imidazole derivatives had antibacterial activity in drug-resistant
S. aureus, E. faecalis and S. pneumoniae, as well as in B. subtilis, with
MICs of 0.39–6.25mgml?1. However, the inhibitors have caused
structural alteration of HK leading non-specifically to aggregation.
The specific hypersensitivities shown by temperature-sensitive
mutants indicate that use of these mutants in whole-cell screening
Received 26 October 2009; revised 21 November 2009; accepted 24 November 2009; published online 8 January 2010
1Department of Bioscience, Graduate School of Agriculture, Kinki University, Nara, Japan;2Microbial Chemistry Research Center, Tokyo, Japan;3Institute of Science
and Industrial Research, Osaka University, Osaka, Japan and4ICx Nomadics, Oklahoma City, OK, USA
5These authors contributed equally to this work.
Correspondence: Dr R Utsumi, Department of Bioscience, Graduate School of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan.
The Journal of Antibiotics (2010) 63, 89–94
& 2010 Japan Antibiotics Research Association All rights reserved 0021-8820/10 $32.00
the Research and Development Program for New Bio-Industry Initiatives
(2006–2010) of the Bio-Oriented Technology Research Advancement
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Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
An antibacterial agent targeting WalK, a histidine kinase
A Okada et al
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