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MedChemComm
Page 4 of 6
DOI: 10.1039/C6MD00229C
COMMUNICATION
Journal Name
The efficacy of ciprofloxacin
9
(IC50 0.5
µ
M) was severely Compound
M with a faint band occurring at 3
piperazine ring, with complete supercoiling observable at 25 linear band occurs at approximately the same concentration as
M for compound 18. A restoration of activity is observed inhibition in the corresponding supercoiling assay (Fig. 4). This
when the coumarin is present with an IC50 of 3 M for is consistent with observations of ciprofloxacin, which exhibits
compound , comparable to SD8 (Figure 4). These results a correlation between supercoiling and cleavage stabilisation
demonstrate a favourable interaction when fragments and inhibitory concentrations.
4
displays a strong linear band from 100
µM to 10
attenuated with the introduction of the linker onto the
µ
µ
M (Fig. 5).The loss of the
µ
µ
4
7
18 are combined that is not seen with a simple aromatic ring. These results illustrate that the coumarin fragment contributes
This is in contrast to the poor inhibition displayed by each to the inhibitory efficacy of hybrid 4. The poor inhibition
fragment individually; indicating a synergistic effect is vital for displayed by each constituent fragment of
the observed activity. The coumarins and showed no synergistic effect is vital for the observed activity. Moreover, it
appreciable effect at 50 M. Furthermore, the free amine preserves the ability to stabilise the gyrase-DNA covalent
failed to cause inhibition at physiologically irrelevant complex. The unfunctionalised scaffold will act as a suitable
concentrations of 300 M (data not shown). Of the ester template from which future structure activity relationships can
protected analogues 10 11 15 and 17, only the esterified be explored. This proof of concept work serves as an example
ciprofloxacin fragment 10 retained inhibition, albeit with of a natural product-guided fragment-based approach to
4 shows a
6
7
µ
7
µ
,
,
attenuated efficacy. Supercoiling was inhibited from 3-50
with an IC50 of ~10 M (Supplementary Information Fig. 1S).
Compounds 11 15 and 17 showed no effect at maximal
µ
M
developing novel inhibitors of DNA gyrase.
µ
,
Acknowledgements
concentrations of 100
1S).
µM (supplementary information Figure
To study the mode of action, we investigated the effect of the
compounds 16 and 18 on the gyrase cleavage-religation
MJA was funded by a Dean’s studentship. SJH was funded by
BBSRC (Grant no. BB/I002049/1). Work in AM’s lab is
supported by grant BB/J004561/1 from BBSRC and the John
Innes Foundation. We thank the EPSRC Mass spectrometry
service (Swansea) for HRMS.
4,
equilibrium (Fig 5). This would demonstrate that the
compounds were able to inhibit gyrase through the same
mechanism as ciprofloxacin. If a reaction between gyrase and
DNA in the presence of ciprofloxacin is terminated by the
addition of SDS and proteinase K, cleaved DNA is revealed. This
is a manifestation of the covalent bonds formed between the
enzyme and the DNA, which are stabilised by intercalation of
the quinolone drug into strand breaks.25 Experimentally, when
supercoiled DNA is used as the substrate, this is represented
by the presence of a linear band. SD8 was used as a negative
control, no linear band being visible due to its ability to block
DNA binding and prevent strand passage cycle taking place.
Notes and references
1
H. W. Boucher, G.H. Talbot, J. S. Bradley, J. E. Edwards, D.
Gilbert, L. B. Rice, M. Scheld, B. Spellberg, and J. Bartlett, Clin
Infect Dis 2009, 48, 1; World Health Organization
Antimicrobial Resistance: Global Report on Surveillance;
Geneva, Switzerland, 2014; pp 1-256.
2
J. B. Cross, J. Zhang, M. F. Mesleh, J. A. Romero, B. Wang, D.
Bevan, K. M. Poutsiaka, F. Epie, T. Moy, A. Daniel, J. Shotwell,
B. Chamberlain, N. Carter, O. Andersen, J. Barker, M. D.
Ryan, C. A. Metcalf 3rd, J. Silverman, K. Nguyen, B. Lippa, R.
Ciprofloxacin
bands visible down to 0.3
100 M for 16 and from 100
9
was used as positive control with strong linear
M. Linear bands are visible only at
M to 50 M for 18.
µ
µ
µ
µ
E. Dolle, ACS Med. Chem. Lett. 2016, 7, 374.
R. C. Clark, S. Y. Lee, M. Searcey, D. L. Boger, Nat. Prod. Rep.
2009, 26, 465.
M. M.Cominetti, S. A. Goffin, E. Raffel, K. D. Turner, J. C.
Ramoutar, M .A. O'Connell, L. A. Howell, M. Searcey. Bioorg.
Med. Chem. Lett. 2015, 25, 4878.
3
4
5
6
H. B. Qiu, X. Y. Chen, Q. Li, W. J. Qian, S. M. Yu, G. L. Tang, Z.
J. Yao, Tetrahedron Lett., 2014, 55, 6055.
J. Schimana, H. P. Fiedler, I. Groth, R. Sussmuth, W. Beil, M.
Walker and A. Zeeck, J. Antibiot. 2000, 53, 779.
J. J. Champoux, Annu. Rev. Biochem. 2001, 70, 369.
M. Gellert, K. Mizuuchi, M. H. O'Dea, and H. A. Nash, P. Natl.
Acad. Sci. USA 1976, 73, 3872
7
8
9
F. Collin, S. Karkare and A. Maxwell, Appl. Microbiol. Biot.
2011, 92, 479.
10 A. Wohlkonig, P. F. Chan, A. P. Fosberry, P. Homes, J. Huang,
M. Kranz, V. R. Leydon, T. J. Miles, N. D. Pearson, R. L. Perera,
A. J. Shillings, M. N. Gwynn, B. D. Bax, Nat. Struct. Mol. Biol.
2010, 17, 1152; I. Laponogov, M. K. Sohi, D. A. Veselkov, X. S.
Pan, R. Sawhney, A. W. Thompson, K. E. McAuley, L. M.
Figure 5. Effects of compounds 4, 16 and 18 on cleavage-complex formation by wild
type E. coli gyrase in the absence of ATP. Supercoiled pBR322 plasmid (DNA) is used as
a negative control (-), and incubated in the presence of gyrase as a positive control (+).
SD8 is used as an additional negative control at a concentration of 1 µM. Ciprofloxacin
(9) is used as a comparator. NC, nicked circle; L, linear band; SC, supercoiled.
Fisher and M. R. Sanderson, Nat. Struct. Mol. Biol. 2009, 16
667; I. Laponogov, X.-S Pan, D. A. Veselkov, K. E. McAuley, L.
M. Fisher and M. R. Sanderson, PloS One 2010, , e11338.
,
5
4 | J. Name., 2012, 00, 1-3
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