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Organic & Biomolecular Chemistry
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Journal Name
COMMUNICATION
bromoacetamides using Cs2CO3 as a base led to the desired
products.13 Alterations in the thiophene section of the molecule
by exchange of the halogen substituted thiophene with a phenyl
Conclusions
DOI: 10.1039/C9OB01339C
constitutes a new class of ClpXP inhibitors that
In conclusion,
1
neither act on peptidase nor chaperone activities but requires
the whole proteolytic complex for impairing its substrate
turnover. The compound binds stereospecifically into a yet
unidentified binding pocket on ClpP, which according to our SAR
group (
protease assay (Figure 3C). Propylation
methylation ( ) of the nitrogen located in the linker almost
completely prevented inhibition. However, shortening of the
linker and removal of the nitrogen ( ) still retained some
2) resulted in a tenfold lower IC50 value in the ClpXP
(3)
instead of
4
data exhibits
a defined shape allowing only restricted
5
modifications. The elucidation of this pocket in future studies
may further unravel the precise mechanism of action and guide
the design of improved inhibitors also bearing in situ activities.
activity, although no full inhibition could be obtained.
Replacement of one phenyl group by a methyl substituent at the
hydantoin core structure (6) resulted in a 40-fold drop in
potency. This suggests that sterically demanding groups are
necessary for potent inhibition. Hence, the role of additional
methyl substituted phenyl groups was further elucidated.
Conflicts of interest
There are no conflicts to declare.
Disubstituted compound
compound , bearing two equally methyl substituted phenyl
moieties, showed complete loss of activity. This result indicates
that only one enantiomer of is binding to ClpP as the methyl
1 exhibited the highest potency while
7
Acknowledgements
1
This work was supported by the Deutsche Forschungsgemein-
schaft SI1096/8-1 (ClpP) and CIPSM.
substituent seems to clash with the binding site in one of the
two possible conformations. Consequently, enantiomers were
separated by chiral high-performance liquid chromatography.
Due to the small amount of isolated enantiomers the absolute
configuration could not be assigned by X-ray analysis.
Notes and references
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Individual testing of both enantiomers in the ClpXP protease
assay revealed that only
1-E1 showed inhibition, while the other
2.
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enantiomer ( -E2) was completely inactive (Figure 3C). This
1
suggests the existence of a defined, stereospecific and 3.
druggable binding pocket enabling further studies to improve
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4.
5.
6.
the in situ activity via parameters such as uptake and stability.
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and LC-MS/MS. Only a slight reduction of alpha-hemolysin, a
major virulence factor linked to ClpXP activity, was observed for
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the racemate and
(Supporting Figure 2). However, secretome analysis of
1-E2 but not for 1-E1 treated samples
1
enantiomers by LC-MS/MS showed no change of alpha-
hemolysin compared to DMSO treated samples (Supporting
Table 1). We thus conclude that physicochemical parameters
need to be optimized for more effective studies on whole cells.
A)
C)
O
HN
O
N
S
N
Cl
1
O
100
75
50
25
0
1- E1
1- E2
7.
8.
hydantoin
linker
thiophene
B)
3
4
4
4
5
6
6
6
7
7
7
-log c [c/M]
1
2
3
4
100
75
50
25
0
9.
2
3
5
-log c [c/M]
10.
11.
5
6
7
100
75
50
25
0
4
6
5
7
12.
13.
5
-log c [c/M]
Figure 3 Structure-activity relationship studies of 1 analogs in ClpXP-protease assay. A)
Dissection of 1 into 3 parts. B) Chemical structures of 1 analogs. C) Inhibition data of all
compounds in the ClpXP protease assay.
N. T. Tzvetkov, H. Euler and C. E. Müller, Beilstein J. Org.
Chem., 2012, 8, 1584–1593.
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