5762
T. Mendgen et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5757–5762
Figure 4. Formation of the adduct between tulipaline derivatives and UNAG via a covalent tulipaline–MurA intermediate.
Since the reactivity of the inhibitors towards sulfhydryl nucleo-
References and notes
philes is probably the decisive factor for inactivation in the full
medium and may also be related to MurA binding, we studied
the reaction of a sub-group of compounds with glutathione
(cf. Table 2). Fosfomycin is inert against glutathione. For the other
compounds, there is a clear relationship between glutathione reac-
tivity and inhibition of MurA. This indicates—along with the inac-
tivity of the compounds against the C115D mutant—that the
formation of a covalent intermediate between the cysteine sulfur
and the inhibitors is probably required to form the non-covalent
suicide inhibitor that was observed in the cnicin–UNAG–MurA
crystal structure. This is illustrated in Figure 4.
To further elucidate whether MurA is the relevant molecular
target of the tulipaline derivatives and cnicin, we studied the activ-
ity of the compounds against E. coli cells harboring the C115D mu-
tant MurA. Details of these experiments are given in the
Supplementary data. The experiments clearly show, however, that
expression of the C115D mutant MurA makes E. coli resistant to-
wards fosfomycin but not towards cnicin or the tulipaline deriva-
tives. Therefore, MurA is not the only target addressed by cnicin
and the tulipalines.
In conclusion, our data demonstrates that the natural products
1-tuliposide B and the lactonized aglycon ( )-tulipaline B are po-
tent inhibitors of MurA, which may partly explain the known anti-
bacterial activity of these compounds. In the context of drug
discovery, the ‘desired’ endpoints MurA inhibition and antibiotic
activity can not be sufficiently separated from ‘undesired’ non-spe-
cific binding to sulfhydryl nucleophiles. Further steps in this direc-
tion must be focused on structural modifications that allow the
separation of the desired and undesired endpoints, as realized in
fosfomycin.
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lM
UNAG, 0.125% BSA and inhibitor (10
ll, aqueous solution
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10 min at 37 °C. To determine the influence of the substrate UNAG on the
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the following concentrations: E. coli WT and mutant MurA 12 nM, BSA 0.1%,
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stopped after 60 min at 37 °C by adding 100 of Lanzetta reagent. The
ll, 625 lM) resulting in a total volume of 100 ll with
l
l
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20. Screening for antimicrobial activities was performed by monitoring the
optical density (OD) at 590 nm using 96-well U-bottom polystyrene
microtiter plates. Bacterial cells from an overnight culture of E. coli
BL21(DE3) grown in LB- or minimal medium were diluted 50-fold with
Acknowledgment
the respective medium, resulting in an OD (590 nm) of 0.03. 245
dilution were dispensed into the wells containing of test solutions
prepared in DMSO, giving a final concentration of the test compounds of
200 M. Control wells were prepared with fosfomycin. Plates were
ll of the
This work was supported by the Deutsche Forschungsgemeins-
chaft (KL 1356).
5 ll
l
incubated at 37 °C for 7 h. Bacterial growth was measured as increase in
OD 590 nm. Antimicrobial activity (growth inhibition, GI) was calculated as
the OD percentage relative to an uninhibited control.
Supplementary data
Supplementary data associated with this article can be found, in