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3539
compounds for chaperone binding10,18,19 and this technique was
now used also for the cyclopropyl-series of isosteres (Table 1).
Binding data clearly showed that the affinity of the lead com-
pound 2 for the chaperone PapD could be retained or even
improved after isosteric replacement of the carboxylic acid. The
benzene sulfonamide 12 was the best binder (entry 4, 100% rela-
tive PapD affinity) resulting in a completely abolished signal from
the compound. The methyl sulfonamide 10 and the tetrazole 16
displayed comparable affinities to 2 (entries 6 and 8 vs entry 2).
Decreased affinities were seen for the hydroxamic acid 6, amide
14 and also for the ring-opened analogue 20 (entries 10, 12 and
14). In addition, the reduced binding of 20 compared to the parent,
bicyclic lead 2 was central since it demonstrated the importance of
having the carboxylate positioned on the rigid scaffold. Interest-
ingly, an increased line-width was observed for one of the naphthyl
protons on compounds 10 and 20. This could indicate a different
binding mode or site for these compounds. However, considering
the potency of 10 (see below), a different binding site seems unli-
kely for this compound. Altogether, although a reduced binding
affinity was observed for some isosteres, all compounds bound to
PapD in a range that from previous studies was known to be ade-
quate for inhibitory activity on pilus biogenesis.
Next, all compounds were evaluated in a hemagglutination
(HA)-assay for their ability to inhibit P pilus biogenesis in E. coli
(Table 1, HA-titer).11 P pili expressing E. coli (HB101/pPAP5) were
cultured in the presence of 1.8 mM compound and the degree of
piliation could subsequently be assessed by the ability of pili-
cide-treated bacteria to agglutinate erythrocytes (Table 1, HA-ti-
ter). The reported HA-titer is the highest dilution of bacteria that
still provides hemagglutination and, thus, a high HA-titer denotes
a high abundance of pili on the bacterial surface. Rewardingly,
the HA-data clearly demonstrated an improved ability to block P
pili formation for the tetrazoles 15 and 16 and the acyl sulfona-
mides 9–12 (entries 3–8 vs entry 1 and 2, HA-titer). Neither the
hydroxamic acids 5 and 6 nor the primary amides 13 or 14 were
active (entries 9–12). A slight effect was seen for the ring-opened
derivatives 19 and 20 (entries 13 and 14), but there was an evident
loss in activity compared to the bicyclic lead 2 (entry 2).
Figure 2. Compounds 10 and 16 were evaluated in a biofilm assay where they
showed a dose-dependent pattern with an estimated IC50 around 30–40 lM for
compound 10 and 65–75 lM for 16. The corresponding carboxylic acid substituted
pilicide 2 displayed a 45% inhibition at 400 lM. The figure shows the average in-
hibition of duplicate runs and the standard deviations are shown in error bars.
inhibit pili-dependent biofilm formation. The increased potencies,
particularly for the sterically more demanding isosteres, open up
possibilities to further investigate this position from a medicinal
chemistry perspective.
Acknowledgments
We thank the Swedish Natural Science Research Council and
the Knut and Alice Wallenberg foundation for financial support.
Supplementary data
Supplementary data associated with this article can be found, in
Finally, the best compounds from the HA-titers and NMR-stud-
ies were further evaluated in a biofilm assay11 not only to confirm
their biological effect but also to be able to see if they behave in a
dose-dependent manner (Fig. 2). The results show that the com-
pounds could be suspended to a concentration as low as 22 lM,
with a dose-dependent behavior, and still displaying significant
biofilm inhibition.
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