Structure-guided design of antibacterials that allosterically inhibit DNA gyrase

Article abstract of DOI:10.1016/j.bmcl.2019.03.029

A series of DNA gyrase inhibitors were designed based on the X-ray structure of a parent thiophene scaffold with the objective to improve biochemical and whole-cell antibacterial activity, while reducing cardiac ion channel activity. The binding mode and overall design hypothesis of one series was confirmed with a co-crystal structure with DNA gyrase. Although some analogs retained both biochemical activity and whole-cell antibacterial activity, we were unable to significantly improve the activity of the series and analogs retained activity against the cardiac ion channels, therefore we stopped optimization efforts.

Full text of DOI:10.1016/j.bmcl.2019.03.029

Accepted Manuscript
Structure-guided design of antibacterials that allosterically inhibit DNA gyrase
Reema K. Thalji, Kaushik Raha, Daniele Andreotti, Anna Checchia, Haifeng
Cui, Giovanni Meneghelli, Roberto Profeta, Federica Tonelli, Simona
Tommasi, Tania Bakshi, Brian T. Donovan, Alison Howells, Shruti Jain,
Christopher Nixon, Geoffrey Quinque, Lynn McCloskey, Benjamin D. Bax,
Margarete Neu, Pan F. Chan, Robert A. Stavenger
PII:
S0960-894X(19)30168-4
https://doi.org/10.1016/j.bmcl.2019.03.029
BMCL 26343
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
29 January 2019
15 March 2019
21 March 2019
Please cite this article as: Thalji, R.K., Raha, K., Andreotti, D., Checchia, A., Cui, H., Meneghelli, G., Profeta, R.,
Tonelli, F., Tommasi, S., Bakshi, T., Donovan, B.T., Howells, A., Jain, S., Nixon, C., Quinque, G., McCloskey, L.,
Bax, B.D., Neu, M., Chan, P.F., Stavenger, R.A., Structure-guided design of antibacterials that allosterically inhibit
DNA gyrase, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.03.029
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Structure-guided design of antibacterials
that allosterically inhibit DNA gyrase
Leave this area blank for abstract info.
Reema K. Thalji, Kaushik Raha, Daniele Andreotti, Anna Checchia, Haifeng Cui, Giovanni Meneghelli,
Roberto Profeta, Federica Tonelli, Simona Tommasi, Tania Bakshi, Brian T. Donovan, Alison Howells, Shruti
Jain, Christopher Nixon, Geoffrey Quinque, Lynn McCloskey, Benjamin D. Bax, Margarete Neu, Pan F. Chan,
and Robert A. Stavenger

Bioorganic & Medicinal Chemistry Letters
Structure-guided design of antibacterials that allosterically inhibit DNA gyrase
a
a
b
b
a
Reema K. Thalji , Kaushik Raha , Daniele Andreotti , Anna Checchia , Haifeng Cui , Giovanni
b,c
b,c
b
b,c
a
a
Meneghelli , Roberto Profeta , Federica Tonelli , Simona Tommasi , Tania Bakshi , Brian T. Donovan ,
Alison Howells , Shruti Jain , Christopher Nixon , Geoffrey Quinque , Lynn McCloskey , Benjamin D.
Bax , Margarete Neu , Pan F. Chan and Robert A. Stavenger
d
d
a
a
a
e,f
f
a,
a,*
a
b
c
GlaxoSmithKline, Collegeville, PA 19426, USA, EVOTEC Center of Drug Discovery and Development, 37135, Verona, Italy, Current address: Fabbrica
d
e
Italiana Sintetici (FIS), 36075, Montecchio Maggiore, Vicenza, Italy, Inspiralis Ltd. Innovation Centre, Norwich, NR4 7GJ, UK, Current address: Medicines
Discovery Institute, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, UK, GlaxoSmithKline, Stevenage, Hertfordshire, SG1 2NYH, UK.
f
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of DNA gyrase inhibitors were designed based on the X-ray structure of a parent
thiophene scaffold with the objective to improve biochemical and whole-cell antibacterial
activity, while reducing cardiac ion channel activity. The binding mode and overall design
hypothesis of one series was confirmed with a co-crystal structure with DNA gyrase. Although
some analogs retained both biochemical activity and whole-cell antibacterial activity, we were
unable to significantly improve the activity of the series and analogs retained activity against the
cardiac ion channels, therefore we stopped optimization efforts.
Keywords:
Antibacterial
Topoisomerase
2
009 Elsevier Ltd. All rights reserved.
Multi-drug resistant (MDR) bacteria, especially MDR Gram-
negative bacteria, are a rising public health threat. Without
effective antibiotic therapy, many mainstays of modern medicine
compound 1 only showed significant activity against DNA
gyrase with essentially no activity against the related enzyme
7
topoisomerase IV (IC50 >500 µM in a decatenation of
1
such as chemotherapy and major surgery would not be possible.
kinetoplast DNA inhibition assay). As part of an overall
medicinal chemistry program on this series, we sought to modify
the core of these inhibitors. Examination of the binding mode of
2
This high unmet medical need has been noted by societies,
3
4
governments, and even the United Nations. Due to this, the
need to discover and develop novel treatments for resistant
bacterial infections is higher than ever. To address this need, new
7
1 to Staphylococcus aureus (S. aureus) DNA gyrase (Figure
3A) suggested that two cyclization strategies to bicyclic
templates could be tolerated (Figure 1). Ring closure route a
retains the thiophene core and fuses a 6-ring heterocycle, while
ring closure route b changes the thiophene to a 6-membered
benzo- or heterocycle and fuses a 5- or 6-membered heterocycle.
5
ways of working are required. One example of this is the
Innovative Medicines Initiative’s ENABLE project, whose aim is
to optimize hits and leads which target Gram-negative bacteria in
6
semi-open innovation, consortium format.
As part of the
1
0
ENABLE project, we recently reported a series of thiophene-
based antibacterials which target the bacterial topoisomerase
DNA gyrase via binding to a previously unidentified allosteric
We hypothesized that appropriately constraining the core might
improve biochemical activity at DNA gyrase; here we report our
results on the synthesis and evaluation of exemplars of both
cyclization approaches (Figure 2).
7
site on the enzyme. The bacterial topoisomerases DNA gyrase
and topoisomerase IV are essential enzymes which control DNA
topology and are the targets of the clinically validated
fluoroquinolone antibiotics, in addition to several other classes
The gyrase inhibitors 2 through 12 (Figure 2) were
synthesized as described in Schemes 1-7. The synthesis of
thieno[3,2-d]pyrimidin-4(3H)-one core inhibitor 2, derived from
ring closure route a (Figure 1), started with commercially
available thiophene 13 (Scheme 1). Installation of the 2-
chlorophenyl group via Suzuki coupling followed by
saponification provided the corresponding carboxylic acid.
8
9
reported in the literature or in clinical development.
The thiophene-based antibacterial 1 (Figure 1) is an inhibitor
of bacterial DNA gyrase with modest antibacterial activity
7
against a range of Gram-negative bacterial pathogens. Unlike
most other inhibitors of bacterial topoisomerases, the lead
PyBOP-mediated
coupling
with
the
protected
(S)-

phenylethylenediamine provided 14. Heating in the presence of
trimethoxymethane and acetic anhydride provided the cyclized
product which was deprotected with trifluoracetic acid to give
compound 2.
Scheme 2. Reagents and conditions: (a) 2-chlorobenzeneboronic acid,
CsF, Pd(PPh , DMF, H O, 100 ºC, 30%; (b) NH OH, dioxane, aq. NaOH,
9%.; (c) CDI, Et N, THF, 60 ºC, 79%; (d) alcohol, PPh , DIAD, THF, rt; (e)
Cl2,; (f) 1H-pyrazole-1-carboximidamide, iPr EtN, CH Cl , 49%
O; (h) 1) MeNO . Triton-B, MeOH, 0 ºC; 2) LiAlH
O, NaHCO , THF/H O (5% over 3 steps).
3
)
4
2
2
8
3
3
TFA, CH
2
2
2
2
Figure 1. Gyrase inhibitor 1 and new bicyclic designs.
over 2 steps; (g) HCl, Et
THF, 40 ºC, Boc
2
2
4
,
2
3
2
The synthesis of O-alkylated indazoles 4 and 5 proceeded with
a similar route and is shown in Scheme 3, starting with Suzuki
coupling and methyl ester formation to give 24. Displacement of
the aryl fluoride with hydrazine at 100 ºC, followed by
protection/activation with Boc-anhydride and treatment with a
strong base gave the indazolanone core 25. Mitsunobu reaction
with alcohol 18 followed by removal of the Boc group with
trifluoracetic acid and swapping to the HCl salt gave target
compound 4. Selective protection of the primary amine of 4 with
Boc-anhydride followed by methylation of the indazole nitrogen
with iodomethane, deprotection, and salt switching gave
compound 5.
Figure 2. DNA gyrase inhibitors disclosed in this paper.
Scheme 3. Reagents and conditions: (a) 2-chlorobenzeneboronic acid,
Cs
c) hydrazine, MeOH, 100 ºC, 75%; (d) Boc
aq. NaOH, 71% over 2 steps; (f) 19, PPh , DIAD, THF, rt; 73%; (g) TFA,
CH Cl ; (h) HCl, Et O, 67% over 2 steps; (i) Boc O, NaHCO , THF, 0 ºC; (j)
MeI, K CO , DMF; (k) TFA, CH Cl ; (l) HCl, Et O, 33% over 4 steps from 5.
2
CO
3
, Pd(PPh
3
)
4
, dioxane, H
2
O, 100 ºC, 91%; (b) SOCl
2
, tol, MeOH, 92%;
(
2
O, iPr NEt, DMAP, CH
2
2
2
Cl ; (e)
3
2
2
2
2
3
2
3
2
2
2
Scheme 1. Reagents and conditions: (a) 2-chlorobenzeneboronic acid,
K
2
CO
c) N-Boc-(S)-phenylethylenediamine, iPr
trimethoxymethane, Ac O 100 ºC, 25%; (e) TFA, CH
3
, Pd(PPh
3
)
4
, dioxane, H
2
O, 100 ºC, 98%; (b) LiOH, MeOH, H
NEt, PyBOP, MeOH, 67%; (d)
Cl , 61%.
2
O, 86%;
The synthesis of the N-alkylated indazoles 6 and 7 started with
arylation of 5-bromo-indazole (26) followed by iodination in the
presence of a strong base to give compound 27 (Scheme 4).
Blocking the N1-H upon reaction with either Boc-anhydride or
iodomethane provided compounds 28 and 29, respectively, which
were each aminated using palladium catalysis and benzophenone
imine followed by treatment with hydroxylamine to give the 3-
amino analogs 30 and 31, respectively. Heating with nitrostyrene
provided the 1,4-addition products which were reduced with
nickel boride in cold THF/methanol. Trifluoracetic acid
deprotection of the N-Boc group of 30 and formation of the HCl
salt provided target compound 6. Target 7 was made similarly
from 31 by simple formation of the HCl salt following reduction
of the nitrostyrene adduct.
(
2
2
2
2
The synthesis of 3 and derivatives 11 and 12 is described in
Scheme 2. Arylation of bromide 15 provided 16 followed by
formation of the N-hydroxy amide under basic conditions
followed by cyclization with carbonyldiimidazole provided the
key benzisoxazolone intermediate 17. Treatment with alcohol 18
under Mitsunobu conditions afforded the desired O-alkylated
product 21. Formation of the HCl salt afforded target compound
3
.
Alternatively, reaction of 21 with 1H-pyrazole-1-
carboximidamide followed by HCl salt formation provided the
guanidine analog 12. In an analogous manner, intermediate 17
was coupled with Boc-protected 2-amino-1-(pyridin-2-yl)ethanol
(19), followed by deprotection and salt formation to give target
1
1.

Scheme 7. Reagents and conditions: (a) TMSCHN
, H O, 42 % over 2 steps; (c) 2-chlorobenzeneboronic acid, CsF,
Pd(PPh , DMF, 100 ºC, 16%; (d) NH OH, dioxane, aq. NaOH.; (e) CDI,
Et N, CH Cl , 23% over 2 steps; (f) 19, PPh , DIAD, THF, rt; (g) TFA,
CH Cl (h) HCl, Et O, 29% over 3 steps.
2 2 2
, CH Cl , MeOH; (b)
Scheme 4. Reagents and conditions: (a) 2-chlorobenzeneboronic acid,
CO , Pd(PPh , dioxane, H O, 160 ºC, 70%; (b) I , KOH, DMF; (c)
Boc O, Et N, DMAP, CH CN, 75% over 2 steps; (d) MeI, K CO , DMF,
0%; (e) benzophenone imine, Pd (dba) , xantphos, Cs CO , dioxane, 100 ºC,
OH, MeOH, 45-47% over 2 steps; (g) nitrostyrene, 60 ºC, 46-53%; (h)
Br
2
2
Cs
2
3
3
)
4
2
2
3
)
4
2
2
3
3
2
3
3
2
2
3
9
2
3
2
3
2
2
2
(f) NH
2
The cyclized analogs of compound 1 were initially tested in a
NiCl
2
4 2 2 2
, NaBH , MeOH, THF, -20 ºC; (i) TFA, CH Cl ; (j) HCl, Et O.
1
1
DNA gyrase biochemical assay and profiled against a panel of
1
2
The synthesis of isoquinoline 8 started with S Ar reaction of
N
Gram-negative organisms,
in addition to a mammalian
13
compound 32 with mono-protected diamine 33 (Scheme 5).
Suzuki coupling with 2-chlorobenzeneboronic acid provided
compound 34 followed by deprotection of the N-Boc group, and
formation of the HCl salt gave target compound 8.
cytotoxicity assay in mouse LY5178Y TK +/- cells (Table 1).
Inhibitor 2, representing ring closure strategy a (Figure 1),
retained modest inhibition of DNA gyrase and demonstrated an
improvement in cytotoxicity over the parent thiophene analog.
Unfortunately, no whole cell antibacterial activity was observed
across the range of wild-type (WT) Gram-negative pathogens
tested (MICs >128 µg/mL). Activity could be detected when
efflux was impaired by deletion of tolC in Escherichia coli (E.
coli ΔtolC MIC = 2-4 µg/mL) suggesting that efflux was a
1
4
contributor to the lack of WT antibacterial activity. On-target
activity at DNA gyrase was confirmed by an 8-fold shift in MIC
7
against an E. coli ΔtolC GyrB E793K target mutant (a mode-of-
Scheme 5. Reagents and conditions: (a) 34, Pyridine, 145 ºC microwave;
action tool strain) relative to the isogenic parental strain. Due to
the lack of activity against WT strains, additional analogs in this
series were not pursued.
(b) 2-chlorobenzeneboronic acid, Cs , Pd(PPh
2
CO
3
3 4 2
) , dioxane, H O, 130 ºC
microwave; (c) TFA, CH Cl ; (d) HCl, Et O, 12% over 4 steps.
2
2
2
Synthesis of the pyridylisoxazole 9 is shown in Scheme 6.
Treatment of 5-bromo-2-hydroxynicotinic acid (35) with thionyl
chloride and methanol with a catalytic amount of DMF provided
the corresponding methyl ester. Formation of the N-
hydroxyamide followed by sodium hydroxide-promoted
cyclization gave the fused isoxazolopyridinone 36. Successive
Mitsunobu reaction with alcohol 18, Suzuki coupling,
deprotection and HCl salt formation gave the target 9.
Initial investigation of the scope of ring closure strategy b
Figure 1) resulted in compounds 3-8, representing 3-O-alkyated
benzoisoxazoles, 3-O- and 3-N-alkyated indazoles, and 1-N-
alkylated isoquinolines. The benzoisoxazole 3 demonstrated
promising DNA gyrase biochemical activity and modest (8-16
µg/mL) activity against WT E. coli and Acinetobacter baumannii
(
(
A. baumannii), with activity against Klebsiella pneumoniae (K.
pneumoniae) and Pseudomonas aeruginosa (P. aeruginosa)
being considerably weaker (MICs ≥128 µg/mL, not shown). Very
potent activity was observed in the efflux knock-out stains and
the ≥16-fold shift in MIC in the GyrB E793K mutant
demonstrated on-target activty (Table 1).
To confirm that the binding mode of compound 3, we initially
solved a 2.7Å co-crystal structure with S. aureus DNA gyrase
1
5
and 20-12p-8 double nicked DNA. This showed that compound
indeed bound as expected (Figure 3B) to the same allosteric
Scheme 6. Reagents and conditions: (a) SOCl
8%; (b) NH OH, dioxane, aq. NaOH, 94%; (c) NaOH, 70 ºC, 59%; (d) PPh
DIAD, THF, rt, 68%; (e) 2-chlorobenzeneboronic acid, CsF, Pd(PPh
DMF, H O, 100 ºC, (f) TFA, CH Cl ; (g) HCl, Et O, 18% over 3 steps.
2
, DMF, MeOH, 65 ºC,
3
9
2
3
,
7
site as compound 1. The DNA used for this structure contained
artificial nicks at each binding site, and the central four base-pairs
of the DNA were not well defined in electron density maps. To
try to obtain a clear view of a DNA-cleavage complex with
compound 3 (see next paragraph), we repeated the structural
studies using a purified DNA-cleavage complex with 20-447T
3 4
) ,
2
2
2
2
Scheme 7 shows the synthesis of inhibitor 10. Conversion of
8 to the corresponding methyl ester followed by bromination
3
gave compound 39. Suzuki coupling followed by treatment with
hydroxylamine under basic conditions gave compound 40.
Cyclization with carbonyldi-imidazole gave intermediate 41
which was treated with alcohol 18 under Mitsunobu conditions,
followed by deprotection and formation of the HCl salt to give
1
5
DNA. We obtained a 3.4Å crystal structure of compound 3 co-
crystallized with a purified DNA-cleavage complex of S. aureus
gyrase. This 3.4Å structure showed compound 3 bound as seen in
the 2.7Å structure, as expected (data not shown).
1
0.
We have previously reported that compound 1 gave both
single- and double-stranded DNA cleavage with E. coli DNA
gyrase. Using a fusion truncate of DNA gyrase from K.
7
pneumoniae, we showed that compound 3 also enhanced single-
stranded and double-stranded DNA cleavage products (data not
shown) indicating the mechanism of action of compound 3 is via
stabilization of ternary cleaved complexes. In addition to

bonds from R630 to 3 is similar to that in panel A from R630 to 1; but no H-
bonds are observed to E634 with 3 (maybe because of a small movement of
the end of the side-chain of R630). C) Superposition of crystal structures in A
and B. Figure drawn with pymol (The PyMOL Molecular Graphics System,
Schrödinger, LLC).
inhibition of DNA gyrase (IC50 = 0.16 µM, Table 1), compound 3
weakly inhibited E. coli TopoIV activity (IC50 was ~ 90 µM in a
decatenation of kinetoplast DNA inhibition assay ) and induced
TopoIV-mediated DNA cleavage breaks (data not shown), and
was also weakly active against the human enzyme (IC50 was
1
6
~
210 µM against human topoisomerase II in a decatanation of
As analogs 4-8 displayed disappointing antibacterial activities,
we returned to the benzisoxazole scaffold of compound 3 and
synthesized the three aza-analogs 9-11 and the guanidine 12.
Although all analogs retained modest to good levels of activity
against DNA gyrase, none showed an improvement in
antibacterial activity in either WT or efflux strains of E. coli (12
gave a 2-fold improvement against A. baumannii, Table 2). No
activity (MIC >128 µg/mL) was observed against K. pneumoniae
and P. aeruginosa, except for compound 12 which gave an MIC
of 16 µg/mL against both K. pneumoniae and P. aeruginosa (data
not shown). On-target activity was maintained for some (e.g. 9
and 11) but was less clear for analogs 10 and 12. Indeed, the
broader spectrum activity of compound 12 could be due to an
additional non-specific mechanism of action, potentially related
to the guanidine substituent which is installed in an effort to alter
the polarity of the series.
16
kDNA inhibition assay )...
Encouraged by the initial data of compound 3 and validation
of the ring closure b hypothesis, several related 5,5 and 5,6 fused
heterocycles were prepared (4-8). Relative to compound 3, these
analogs had reduced DNA gyrase activity and weaker
antimicrobial activity in WT and efflux knock-out strains of E.
coli and WT A. baumannii (Table 1). Interestingly, slight
improvements in activity against K. pneumoniae relative to
compound 3 were observed for some analogs. Like compound 3,
none of the newer analogs possessed activity against WT P.
aeruginosa (MIC >128 µg/mL). Unfortunately, whole cell on-
target activity was not demonstrated for these analogs as no MIC
shift was observed in the E. coli GyrB E793K mutant, perhaps
suggesting that some of the activity observed is not solely due to
inhibition of DNA gyrase.
Despite the disappointing antibacterial activity, we tested
several analogs against cardiac ion channels to determine if
rigidifying the core would impact these activities (Table 2).
1
7
Although 3 did have attenuated inhibition of both the hERG and
1
8
Na 1.5 channels compared to compound 1 (Table 2), the effect
V
was not dramatic. Compounds 9, 10, and 12 also retained
relatively potent inhibition against one or both of the cardiac ion
channels tested, highlighting the difficulty in optimizing safety
parameters in addition to activity in antibacterial research and
development.
In summary, we have synthesized and tested several 5,6 and
6
,6 fused core analogs of thiophene antibacterial 1 . Although
both initial design hypotheses were successful in delivering
analogs with in vitro activity against DNA gyrase, none of the
analogs had improved whole cell antibacterial activity relative to
the parent compound 1. Moreover, several liabilities of the series,
specifically cytotoxicity and cardiac ion channel activity, were
not mitigated with this ring-fusion strategy. As such, we stopped
work on the present ring-fusion approach. A complete account of
our efforts on monocyclic series, including liabilities and reasons
7
for termination, will be reported separately.
Figure 3. A) 2.22 Å crystal structure (pdb code: 5NPP) of 1 (yellow carbons)
in complex with S. aureus DNA gyrase (GyrA blue carbons, GyrB magenta
7
carbons) and DNA (green atom, lower left) . Nitrogens blue, oxygens red,
phosphorous orange chlorine green. Semi-transparent surface is shown on
GyrA but not GyrB. Semi-transparent surface is shown on GyrA but not
GyrB. Hydrogen bonds (dotted yellow lines) from E634 (=E793 in E.coli) go
to 1 and R630. R630 also forms H-bonds to 1. B) 2.7 Å crystal structure of 3
(white carbons) in complex with S. aureus DNA gyrase and DNA (pdb code
6
QX1). Hydrogen bond is shown as black dotted line. Note, the hydrogen

Table 1. Biochemical and bacterial susceptibility data for bicyclic gyrase inhibitors 1-8.
DNA
Gyrase
IC50
E.coli
TOP10
ΔtolC
E.coli
TOP10 ΔtolC
GyrB E793K
E.coli
7623
ΔtolC
E.coli
K. pneumoniae
1161486
A. baumannii Cytotoxicity
Cmpd
7
623
BM4454
TC50
(µM)
(µg/mL)
1
2
3
4
5
6
7
8
0.04
2.0
0.16
1.6
5.0
3.2
7.9
10
≤0.125
2
≤0.125
2
2
4
4
2
4
16
2
2
2
4
4
2
8
≤0.125
2-4
≤0.125
2
2
4
4
2
32
>128
≥128
32
>128
64
4
>128
8
32
64
64
128
32
5
16
10
3
nd
5
>128
8-16
32
>128
128
>128
64
>128
32
5
3
Table 2. Biochemical, bacterial susceptibility and cardiac ion channel data for 5,6-fused gyrase inhibitors 3 and 9-12.
DNA
Gyrase
IC50
E.coli
TOP10
ΔtolC
E.coli
TOP10 ΔtolC
GyrB E793K
E.coli
7623
ΔtolC
A.
V
hERG Na 1.5
17 18
E.coli
Cytotoxicity
IC50
IC50
Cmpd
baumannii
BM4454
7
623
TC50
(µM)
(µM)
(µM)
(µg/mL)
1
3
9
0.04
0.16
0.5
1.6
0.5
≤0.125
≤0.125
1
8
0.25
0.25
4
2
16
16
8
8
8-16
64
>128
64
8
≤0.125
≤0.125
1
4
0.25
0.25
4
8
5
10
20
5
3
11
6.7
23
nd
3.6
nd
9.5
13
22
24
16
nd
nd
64
>128
32
4
1
1
1
0
1
2
0.16
0.5
Resistance, 2014. Available at https://obamawhitehouse.
archives.gov/sites/default/files/microsites/ostp/PCAST/pcast_amr
_
jan2015.pdf. Accessed February 1, 2017,
Acknowledgments
4
.
See https://www.un.org/pga/71/event-latest/high-level-meeting-
on-antimicrobial-resistance/.
Some of the research leading to these results received funding
from the Innovative Medicines Initiative Joint Undertaking under
Grant Agreement 115583, resources of which are composed of
financial contribution from the European Union’s Seventh
Framework Programme (FP7/2007-2013) and the in-kind
contributions of European Federation of Pharmaceutical
Industries and Associations (EFPIA) companies. The ENABLE
project is also financially supported by contributions from
academic and small-medium enterprise (SME) partners. We
thank Gaochao Tian, Mehul Patel, Jeffrey Carson and Tony
Jurewicz (GSK) for development of the high throughput gyrase
assay, Hong-Wei Sun (GSK) for synthetic assistance, Luz Helena
Kryn for the cytotoxicity data, Pierre Despeyroux and Frédéric
Jeannot (Evotec) for assistance with re-preparing some analogs
and Eric ꢀaꢁꢂuꢃ (Evotec) for critical reading of the manuscript.
5. Simpkin, V.I.; Tenwick, M. J.; Kelly, R. Mossialos, E.
Incentivizing innovation in antibiotic drug discovery and
development: progress, challenges and next steps. J. Antibiotics.
2
017, 170, 1087-1096.
6
7
.
.
See: http://nd4bb-enable.eu/
Chan, P. F.; Germe, T. Bax, B. D.; Huang, J.; Thalji, R. K.;
Bacqué, E.; Checchia, A.; Chen, D.; Cui, H.; Ding, X. Ingraham,
K.; McCloskey, L.; Raha, K; Srikannathasanc, V.; Maxwell, A.;
Stavenger R. A. Thiophene antibacterials that allosterically
stabilize DNA-cleavage complexes with DNA gyrase. Proc. Natl.
Acad. Sci. USA. 2017, 114, E4492-E4500.
8
9
.
.
Chan, P.F.; Huang J.; Bax B.D.; Gwynn, M.N. (2013) Recent
developments in inhibitors of bacterial type IIA topoisomerases.
Antibiotics: Targets, Mechanisms and Resistance, eds Gualerzi
CO, Brandi L, Fabbretti A, Pon CL (Wiley, Weinheim, Germany),
pp 263–297.
a) Taylor, S.N.; Morris, D.H.; Avery, A.K.; Workowski, K.A.;
Batteiger, B.E.; Tiffany, C.A.; Perry, C.R.; Raychaudhuri, A;,
Scangarella-Oman, N.E.; Hossain, M.; Dumont, E.F. Gepotidacin
for the Treatment of Uncomplicated Urogenital Gonorrhea: A
Phase 2, Randomized, Dose-Ranging, Single-Oral Dose
Evaluation. Clin. Infect. Dis. 2018, 67, 504-512. b) Basarab, G. S.;
Kern, G. H.; McNulty, J. Mueller, J. P.; Lawrence, K;
Vishwanathan, K.; Alm, R. A.; Barvian, K.; Doig, P.; Galullo, V.;
Gardner, H.; Gowravaram, M.; Huband, M; Galullo, V.; Gardner,
H.; Gowravaram, M.; Huband, M.; Kimzey, A.; Morningstar, M;
Kutschke, A.; Lahiri, S. D.; Perros, M.; Singh, R.; Schuck, V. J.
A.; Tommasi, R.; Walkup, G.; Newman, J.V. Responding to the
challenge of untreatable gonorrhea: ETX0914, a first-in-class
agent with a distinct mechanism-of-action against bacterial Type
II topoisomerases. Sci. Rep. 2015, 5, 11827.
References and notes
1
2
.
.
Piddock, L.J.V. The crisis of no new antibiotics—what is the way
forward? Lancet Infect. Dis. 2012, 12, 249-253.
Spellberg, B.; Guidos, R.; Gilbert, R.; Bradley, J.; Boucher, H.
W.; Scheld, W. M.; Bartlett, J. G.; Edwards, J. The Epidemic of
Antibiotic-Resistant Infections: A Call to Action for the Medical
Community from the Infectious Diseases Society of America.
Clin. Infect. Dis. 2008, 46, 155-164.
3
.
President’s Counꢁil of Advisors on Sꢁienꢁe and Teꢁhnology
(2014) Report to the President on Combating Antibiotic

1
1
0. a) Zheng, Y.; Tice, C.M.; Singh, S.B. Conformational control in
the following conditions: 50 mM HEPES-KOH (pH 7.6), 100 mM
potassium glutamate, 10 mM magnesium acetate, 10 mM
dithiothreitol, 1 mM ATP and 50 µg/ml BSA.
structure-based drug design. Bioorg. Med. Chem. Lett. 2017, 27,
2
825-2837. b) Fang, Z.; Song, Y.: Zhan, P.; Zhang, Q.; Liu, X.
Conformational restriꢁtion: an effeꢁtive taꢁtiꢁ in ‘follow-on’-based
drug discovery. Future Med. Chem. 2014, 6, 885-901.
Human topoisomerase II alpha assays were performed as follows:
1 U of human topo IIα (final assay ꢁonꢁentration 1.9 nM) was
inꢁubated with 200 ng kDNA in a 30 μl reaꢁtion at 37°C for 30
minutes under the following conditions: 50 mM Tris HCl (pH
7.5), 125 mM NaCl, 10 mM MgCl2, 5 mM DTT, 0.5 mM
EDTA, 0.1 mg/ml bovine serum albumin (BSA) and 1 mM ATP.
All reaꢁtions were stopped by the addition of 30 μl
chloroform/iso-amyl alꢁohol (24:1) and 20 μl Stop Dye (40%
sucrose, 100 mM Tris.HCl ( pH 7.5), 10 mM EDTA, 0.5 μg/ml
1. High-throughput DNA gyrase biochemical assay was developed
based on a preferential enhancement of fluorescence of a H19 dye
following binding to supercoiled DNA compared to relaxed DNA.
Test compounds were incubated at room temperature for 1 hour
with 15 μg/ml relaxed pBR322 DNA (Topogen or Inspiralis), 60
nM of His-tagged E.coli DNA gyrase and 2.5 mM ATP in an
4
assay buffer of 20 mM Tris-HCl, 35 mM NH OAc (pH 8), 8 mM
MgCl , 4.6% glycerol, 0.005% Brij35 and 1 mM DTT. Relaxed
2
bromophenol blue), before being loaded on a 1.0% TAE
and supercoiled DNA forms were detected following incubation
with a 1 in 400 dilution of H19 dye (ProFoldin) and fluorescence
intensity reading with Ex: 485 nm and Em: 530 nm. See also:
Asha, M. K.; Debraj, D.; Prashanth, D; Edwin, J. R.; Srikanth, H.
S.; Muruganantham, N.; Dethe, S. M.; Anirban, B.; Jaya, B.;
Deepak, M.; Agarwal, A. In vitro anti-Helicobacter pylori activity
ofaflavonoidrichextract of Glycyrrhizaglabra and its probable
mechanisms of action. J. Ethnopharmacol. 2013, 145, 581-586.
2. Antibacterial MIC assays were determined according to Clinical
and Laboratory Standards Institute guidelines: Clinical and
Laboratory Standards Institute (2009) Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria That Grow
Aerobically: Approved Standard. (CLSI, Wayne, PA), 8th Ed,
Publication M07-A8.
3. Concha, N. O.; Huang, J.; Bai, X.; Benowitz, A. B.; Brady, P. G.;
LaShadric, C. G.; Kryn, L. H.; Holmes, D.; Ingraham, K.; Jin, Q.;
Kaushanaky, L. P.; MꢁCloskey, L.; Messer, J. A.; O’Keefe, H.;
Patel, A,; Satz, A. L.; Sinnamon, R. H.; Schneck, J. L.; Skinner, S.
S.; Summerfield, J.; Taylor, A. N.; Taylor, J. D.; Evindar, G.;
Stavenger, R. A. Discovery and characterization of a class of
pyrazole inhibitors of bacterial undecaprenyl phyrophosphate
synthase. J. Med. Chem. 2016, 59, 7299-7304.
(Tris.acetate 0.04 mM, EDTA 0.002 mM) agarose gel. Gels were
run at 90V for 2 hours. Bands were visualised by ethidium
bromide staining for 20 minutes. Gels were scanned using gel
documentation equipment (GeneGenius, Syngene, Cambridge,
UK) and IC50s were obtained with gel scanning software.
(GeneTools, Syngene, Cambridge,UK) Raw gel data (fluorescent
band volumes) collected from Syngene, GeneTools gel analysis
software were converted to a % of the 100% control (the fully
decatenated substrate) These were analysed using SigmaPlot
Version 13.0 (2015). (Enzymes and substrates provided by
Inspiralis Ltd)
1
1
17. hERG Qpatch assay as previously described in: Haile. P.A.;
Casillas, L.N.; Bury, M. J.; Mehlmann, J.F.; Singhaus, Jr., R.;
Charnley, A.K.; Hughes, T.V.; DeMartino, M.P.; Wang, G.Z.;
Romano, J.J.; Dong, X.; Plotnikov, N.V.; Lakdawala, A.S.;
Convery, M.A.; Votta, B.J.; Lipshutz, D.B.; Desai, B.M.; Swift,
B.; Capriotti, C.A.; Berger, S.B.; Mahajan, M.K.; Reilly, M.A.;
Rivera, E.J.; Sun, H.H.; Nagilla, R.; LePage, C.; Ouellette, M.T.;
Totoritis, R.D.; Donovan, B.T.; Brown, B.S.; Chaudhary, K.W.;
Gough, P.J.; Bertin, J.; Marquis, R.W. Identification of Quinoline-
Based RIP2 Kinase Inhibitors with an Improved Therapeutic
Index to the hERG Ion Channel. ACS Med. Chem. Lett. 2018, 26,
1039-1044..
1
1
4. Du, D.; Wang-Kan, X.; Neuberger, A.; van Veen, H.W.; Pos,
K.M.; Piddock, L.J.V.; Luisi, B.F. Multidrug efflux pumps:
structure, function and regulation. Nat. Rev. Microbiol. 2018, 16,
V
18. Na 1.5 Qpatch assay: Cell culture, assay solutions and growth
medium are identical to that described in Donovan, B.T.; Bakshi,
T.; Galbraith, S.; Nixon, C.J.; Payne, L.A.; Martens, S.F. Utility
of frozen cell lines in medium throughput electrophysiology
5
23-539.
5. The 2.7Å complex of compound 3 was obtained using the double-
nicked 20-12p-8 DNA (which has an artificial nick at each
cleavage site) and was deposited in the PDB with code: 6qx1; its
structure was determined as described for some other structures
with the same DNA - see Srikannathasan, V.; Wohlkonig, A.;
Shillings, A.; Singh, O.; Chan, P.F.; Huang, J.; Gwynn, M.N.;
Fosberry, A.P.; Homes, P.; Hibbs, M.; Theobald, A.J.; Spitzfaden,
C.; Bax, B.D. Crystallization and initial crystallographic analysis
of covalent DNA-cleavage complexes of Staphyloccocus aureus
DNA gyrase with QPT-1, moxifloxaꢁin and etoposide’ Acta
Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2015, 71, 1242-
screening of hERG and Na
Methods, 2011, 64, 269–276, with the following exceptions: Cell
line: The HEK293-based Na 1.5 expressing cell line was derived
V
1.5 blockade. J. Pharmacol. Toxicol.
V
in-house by GSK Biological Reagents and Assay Design group.
The parental cell line, the ATCC line HEK293 cell line CRL-1573
was transfected with the Homo sapiens cardiac sodium voltage-
gated channel, hNa
V
1.5 (type V, α subunit, SCN5A, Entrez gene
id: 6331). For Na 1.5 experiments, all protocols were sampled at
V
20 kHz and filtered with a 4th order Bessel filter with a 200 Hz
cutoff. Series resistance was compensated at 50%. The holding
potential was -120 mV. The stimulus protocol consisted of
holding eaꢁh ꢁell at −120 mV for 20 ms, stepping to −30 mV for
50 ms and returning to the −120 mV holding potential. The
command protocol was delivered every 2 s (0.5 Hz). A separate
assay was run with the command protocol delivered every 500 ms
(2 Hz) to check for signs of frequency dependent effects on
potency. Data reported in Table 2 correspond to the 0.5 Hz assay.
Signal was measured at each sweep was the difference between
the maximum inward current seen in the first 10ms of the −30 mV
depolarizing step minus the mean current seen in the last 5 ms of
the same step. Currents were recorded at room temperature on the
Sophion Qpatch 48 HTX (Sophion Biosciences). Cells which had
1
3
246 for details. Then to try and obtain a clear view of compound
with cleaved DNA, a DNA-cleavage complex was purified and
co-crystallized with compound 3: A 1.4 fold excess of uncleaved
DNA 20-447T (dimer, 2 mM DNA, sequence in Srikanathasan et
al., 2015) and a 50-fold excess of moxifloxacin was added to S.
aureus GyrB27-A56 (GKdel) in 20 mM HEPES, 100 mM
Na
S200 10/300 column (GE Healthcare 17517501) in a running
buffer of 20 mM HEPES pH 7.0, 100 mM Na SO4 and 20 mM
2 4 2
SO , 5 mM MnCl , pH 7.0 and then applied to a Superdex
2
EDTA to remove the moxifloxacin, Mn ions and excess DNA.
The fractions containing the gyrase/DNA complex (with no drug)
were pooled and concentrated to approximately 15 mg/ml.
Crystallization: The DNA/protein complex was diluted 2:1 with
V
Na 1.5 currents greater than 0.25 nA were used for experiments,
2
0 mM HEPES pH 7.0 to reduce the Na
compound 3 was added to an end concentration of approximately
00 µM compound and 5% DMSO. Crystallization under paraffin
2
SO
4
concentration and
although the amplitude was usually much higher (~2.5 nA). For
all concentration response curve experiments, a negative control
consisting of external solution with 0.3% DMSO bracketed the
concentration–response dilution series. After the second negative
ꢁontrol was added, a positive ꢁontrol of 1000 μM ꢂuinidine was
added to fully block all current. Inhibition data were normalized
between the negative and positive controls. All solutions on each
plate were matched for vehicle concentration, 0.3% DMSO.
Above that concentration peak current amplitude dropped
significantly. In all experiments, each test solution concentration
was added three times (10 μl eaꢁh addition for 30 μl total, 3 s
6
oil was set up in Terazaki plates with 0.7 µl protein and 2.8 µl
well buffer (7.5-10% PEG 5000 MME, 130-190 mM BisTris pH
6
.2). The crystallizations were set up in the presence and absence
of 25mM MgCl . Before adding the oil, the drops were streak-
2
seeded. The crystals were cryo-protected in 15% Glycerol, 20%
PEG 5000 MME, 150 mM BisTris pH6.2, 2 mM EDTA, 2 mM
compound before flash freezing in liquid nitrogen for data
collection at the synchrotron. This complex of compound 3 with
cleaved DNA gave a 3.4Å structure with six complexes in the
asymmetric unit deposited with PDB code: 6qx2.
apart) and given
5
min to reach steady-state.
Rundown
compensation, where necessary, was done with a linear fit
anchored to the end of the control state. Curve fitting: all signals
were analyzed using Sophion Qpatch software version 5.0. Data
was collated using Microsoft Excel and all subsequent analyses
1
6. E.coli topoisomerase IV assays were performed as follows: 1 U of
topo IV (final assay concentration 15 nM) was incubated with 200
ng kDNA DNA in a 30 μl reaꢁtion at 37ºC for 30 minutes under

were done using GraphPad Prism version 6.05 for Windows
GraphPad Software, La Jolla, California USA,
(
www.graphpad.com). Measurements were fit to a four-parameter
logistic fit for dose-response curves: y = bottom + (top-bottom) /
Click here to remove instruction text...
(1+10^((logEC50-x) * Hill slope)).