New insights into the binding mode of pyridine-3-carboxamide inhibitors of E. coli DNA gyrase

Article abstract of DOI:10.1016/j.bmc.2019.06.015

Previously we have reported on a series of pyridine-3-carboxamide inhibitors of DNA gyrase and DNA topoisomerase IV that were designed using a computational de novo design approach and which showed promising antibacterial properties. Herein we describe th

Full text of DOI:10.1016/j.bmc.2019.06.015

Accepted Manuscript
New insights into the binding mode of pyridine-3-carboxamide inhibitors of E.
coli DNA gyrase
Sarah Narramore, Clare E.M. Stevenson, Anthony Maxwell, David M. Lawson,
Colin W.G. Fishwick
PII:
S0968-0896(19)30414-6
https://doi.org/10.1016/j.bmc.2019.06.015
BMC 14952
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 March 2019
24 May 2019
6 June 2019
Please cite this article as: Narramore, S., Stevenson, C.E.M., Maxwell, A., Lawson, D.M., Fishwick, C.W.G., New
insights into the binding mode of pyridine-3-carboxamide inhibitors of E. coli DNA gyrase, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.06.015
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New insights into the binding mode of pyridine-3-carboxamide inhibitors of E. coli DNA gyrase
Sarah Narramore,1 Clare E. M. Stevenson,2 Anthony Maxwell,2 David M. Lawson and Colin W.G.
Fishwick*1.
1. School of Chemistry and the Astbury Centre for Structural Molecular Biology, University of Leeds,
Leeds LS2 9JT, UK.
2. Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4
7UH, UK
Abstract
Previously we have reported on a series of pyridine-3-carboxamide inhibitors of DNA gyrase and
DNA topoisomerase IV that were designed using a computational de novo design approach and
which showed promising antibacterial properties. Herein we describe the synthesis of additional
examples from this series aimed specifically at DNA gyrase, along with crystal structures confirming
the predicted mode of binding and in vitro ADME data which describe the drug-likeness of these
compounds.
Introduction
Antibacterial resistance continues to be a growing threat to human health worldwide. The rate of
discovery of new antibacterials is far outstripped by the rate at which resistance is spreading, and
there remains an urgent need for the development of new antibacterial drugs1. Of particular concern
is the emergence of bacterial strains that appear to be totally resistant to all current antibacterials.
This was recently underlined by the CDC, who reported a case in which a patient in the US was
infected with a strain of Klebsiella pneumoniae that was resistant to all available antibiotics.2

DNA topoisomerases are an essential class of enzymes responsible for maintaining the topological
state of DNA (e.g. supercoiling)3. The ATP-dependent bacterial type II topoisomerases, DNA gyrase
and DNA topoisomerase IV, contribute to the maintenance of the correct level of supercoiling in
bacterial DNA. These enzymes are hetero-tetramers, e.g. gyrase consists of two subunits, GyrA and
GyrB, which form an A2B2 complex4. These enzymes have been well-validated as antibacterial targets
as the fluoroquinolone antibiotics act at the DNA-cleavage site of these enzymes. The
aminocoumarin antibiotics, including novobiocin, which was used clinically in the 1960’s, target the
ATP-binding site. Efforts to discover novel inhibitors of the ATP-binding site of these enzymes have
been ongoing for more than four decades. However, only two compounds have progressed into the
clinic5.
We have previously reported a series of pyridine-3-carboxamide inhibitors of DNA gyrase and
topoisomerase IV which displayed antibacterial activity6. These compounds, designed to bind to the
ATP-binding site within the gyrase using the de novo molecular design program SPROUT7, were
developed into a series possessing promising antibacterial activity. However, the computationally
predicted binding mode of these molecules has not been previously validated experimentally and
little assessment was made of the potential “drug-likeness” of compounds in this series. We now
report the detailed structural characterisation of the binding mode of these inhibitors within
bacterial GyrB as well as delineation of a number of key physicochemical parameters of importance
in considering their potential for progression into antibacterial drugs.
Results and discussion
Biological activity of novel pyridine-2-urea-3-carboxamide (PUC) inhibitors
As part of a program designed to expand upon our previously reported studies6, a small library of
novel PUC inhibitors was synthesised with a view to establishing the best candidates for subsequent
co-crystallisation studies with the target enzymes. As with previous examples6, the synthetic strategy

was informed by a number of molecular modelling techniques. Putative compounds were initially
developed via de novo design utilising SPROUT. The most likely inhibitors of E. coli GyrB were then
identified via docking studies using Glide, with the highest-scoring compounds taken forward for
synthesis9. Following synthesis of the identified compounds, the inhibitory activity of these
compounds, together with their antibacterial activities, was established as described previously6. A
summary of the biological activities of these compounds are given below (Table 1).
Table 1. Biological activity of selected pyridine urea carboxamide compounds. (See Supporting
Information).
Compound
R
IC50 (µM)
Ec gyrase
MICs (µg/ mL)
SA
EC
1
2
0.41
>64
4
>64
0.019
>64
NH2
3
4
5
6
0.089
0.086
0.037
0.15
>64
64
>64
>64
>64
>64
N
16
64

7
0.20
n.d.
n.d.
8
9
0.082
0.12
>64
64
>64
>64
N
Ec gyrase, E. coli DNA gyrase ATPase; SA, S. aureus ATCC 29213; EC, E. coli ATCC 25922.
As shown in Table 1, several of these compounds were found to be potent inhibitors of E. coli DNA
gyrase, particularly compound 2. However, with regards to antibacterial activity, although
compound 2 shows some promise against S. aureus (MIC = 4 g/ml), the rest of the inhibitors were
less active and unfortunately, none of the compounds tested showed any activity against the Gram
negative organism E. coli. This was somewhat disappointing given the encouraging antibacterial
activity we had previously established for this class of compounds (e.g. compound 10, Figure 1)6.
Recent work by Hergenrother has correlated specific structural aspects of small molecules with their
ability to penetrate into bacterial cells8. This work has demonstrated that the presence of a primary
amine, along with a relatively flat molecular shape (corresponding to a ‘globularity’ of 0.25 or lower,
where flat molecules such as benzene have a globularity = 0, and spherical molecules like
adamantane have globularity = 1 ), together with a limited degree of molecular flexibility (five or
fewer rotatable bonds), maximises the likelihood of cell penetration into E. coli bacteria.
Furthermore, the work has strongly linked these structural features with penetration into the
bacteria cell via porins. Applying these analyses (using the online eNTRy properties calculator7) to
molecules 1 – 10 confirms that, although all molecules have favourably low globularity (values
ranging from 0.022 – 0.057), almost all are predicted to poorly enter E. coli bacteria, due to either
the lack of a primary amine function (molecules 1, 2, 4 – 10), and, in the case of 1, 2, and 10, also
having greater than five rotatable bonds. An interesting exception however, is the primary amine-
based system 3 which, despite conforming well to all three requirements, lacks inhibitory activity

towards E. coli (see Supporting Information). This may indicate that other factors such as the role of
bacterial efflux pumps, may be of prime importance in dictating the antibacterial activity for this
class of molecules in E. coli. Indeed, we had previously reported that, although devoid of
antibacterial activity in the presence of wild type E. coli, a number of similar pyridine-3-
carboxamides showed antibacterial activity (8 – 32 g/ml) towards E. coli lacking an efflux pump6.
Characterisation of the binding of PUC inhibitors using crystallisation and X-ray diffraction studies
In order to investigate the binding modes of the PUC inhibitors within GyrB, co-crystallisation of a
number of the inhibitors with a 24 kDa subunit of the GyrB unit (‘GyrB24’) of the DNA gyrase enzyme
was carried out9. The gene for the 24 kDa subunit of GyrB was transformed into a pNIC vector to give
a hexahistidine tagged version of the protein. The protein was expressed and purified using a nickel-
affinity chromotography and following purification the histidine tag was cleaved. The protein was
crystallised in the presence of compounds 2, 4, 5 and 10 respectively. We had previously reported
that compound 10 (Figure 1) had shown encouraging degrees of biological activity.
O
HN
N
N
O
H
N
N
H
H
10
E. coli GyrB IC50 (µM): 0.099
S. aureus MIC50 (µg/mL): 1.0
E. faecalis MIC50 (µg/mL): 0.5
Figure 1. Structure and biological activities of previously reported GyrB inhibitor 10
The crystal structures of GyrB24 with compounds 2, 4 and 5 bound respectively, were determined to
2.50-, 1.95-, and 1.90-Å resolution respectively (see Supporting Information). An additional crystal
structure involving our previously reported inhibitor 10 (Figure 1) was also determined at 2.35- Å
resolution. The models each consisted of one monomer of protein in the asymmetric unit with one

molecule of inhibitor bound in the ATP-binding site. The crystal structures confirmed that these
inhibitors bind in the ATPase site of the enzyme in an orientation closely resembling that originally
designed using SPROUT6, and also consistent with the binding pose predicted by using a
computational docking utilising the Glide programme10 (Figure 2).
Figure 2 Comparison of binding poses of inhibitors predicted using Glide docking in E. coli GyrB (bottom row)
with X-ray co-crystal structures where the key residues which form interactions with the inhibitors are shown
in green and the protein surface in grey (top row). Dashed lines indicate a hydrogen bond where the
interatomic distance is <3.5 Å. A) Compound 2 co-crystallised; B) Compound 4 co-crystallised; C) Compound 5
co-crystallised; D) Compound 10 co-crystallised; E) Compound 2 docked; F) Compound 4 docked; G) Compound
5 docked; H) Compound 10 docked.
Inspection of the crystal structures of GyrB containing these inhibitors reveals a number of aspects
common to all co-crystal structures. Specifically, as is the case with a number of other known
inhibitors which bind at the ATP-binding site of GyrB, a hydrogen bonding network involving Asp-73
and a conserved water molecule in the binding site can be seen. The hydrogen bond donation from
the urea moiety and hydrogen bond acceptance from the pyridine rings within the inhibitor
molecules closely mirror the poses predicted via the computational modelling studies.
The co-crystal structures obtained also compare favourably with previous series of GyrB inhibitors;
including ethyl urea series such as those generated by Panchaud et al in 2017 (Figure 3)11. The

binding mode of the compounds presented in this work is highly conserved with these previous
examples – featuring the same crucial interactions between the urea/pyridine moiety, Asp-73, and
the same conserved water molecule. This observation corresponds well with the similar GyrB IC50
values for these two example compounds of 0.125 µM and 0.03 µM respectively – with the slightly
higher potency of compound 2 explained via the introduction of hydrogen-bond interactions
between the terminal pyridine and Arg-136. The overall structure of the ATP binding site is highly
conserved between the Panchaud et al example and our newly generated structures, with the
residues occupying near-identical areas and orientations.
A)
B)
Figure 3 Comparison of a previous E. coli GyrB X-ray co-crystal structure (PDBID: 5MMN)11 with our newly-
generated X-ray co-crystal structures. A) The co-crystal structure of 1-ethyl-3-[8-methyl-5-(2-methyl-pyridin-4-
yl)-isoquinolin-3-yl]-urea with E. coli GyrB obtained by Panchaud et al in 201711. B) The co-crystal structure of
compound 2 with E. coli GyrB.
ADME Profiling of PUC inhibitors.
Compound 2, 3, 4 and 10 were profiled for their in vitro ADME properties; the results are shown in
Table 2.
Table 2. ADME properties of selected PUC inhibitors.

Human
Plasma
Caco-2
Caco-2 efflux
ratioa
Aqueous
Compound solubilitya
(μM)
Human liver Mouse liver
microsome microsome
t1/2a (mins) t1/2a (mins)
permeability
protein
A-Ba (Papp×106/ Papp(B-A)/
bindinga (%)
cm·s-1)
Papp(A-B)
2
3
2.4
97.2
50.2
90.5
99.9
11.83
2.82
20
290
128
46
n/d
n/d
24
88.5
32.6
4.26
4.02
1.23
2.78
30.8
n/a
4
10
<2.67b
58
a) assays carried out by Shanghai ChemPartner Ltd. b) Papp values were expressed as “<” than the values that
were calculated using the minimum concentration of the standards for receiver sides as the concentration in
the receivers was below the quantitation limit.
A review of the data in Table 2 reveals some interesting trends in physicochemical properties within
the PUC series of inhibitors. The aqueous solubility of compounds 2, 4 and 10 was found to be rather
low suggesting that these specific structures within the PUC series would be unattractive in terms of
development as orally bioavailable drug leads. This is mirrored by the high levels of plasma protein
binding seen for these compounds. However, compound 3, containing an aminopyrolidine
substituent, showed a much higher aqueous solubility and correspondingly lower plasma protein
binding.
An estimate of the ability of these compounds to cross biological membranes was also carried out
using the Caco-2 permeability assay, which uses a human intestinal cell line to model absorption of a
compound from the gut12. The rate of transfer of compound is measured in both directions across
the cell membrane. The rate of transfer in the A-B direction (mimicking transfer from the gut to the
blood) and the efflux ratio (an indicator that a compound will fail to be absorbed due to efflux) are
given in Table 2. Compounds 2 and 3 performed the best in this assay showing a moderate
permeability and lower efflux ratios than the other compounds. Compound 4 displayed low

permeability and a high efflux ratio. Full data could not be obtained for compound 10, due to
solubility preventing reliable measurements to be made.
Microsomal stability assays were also carried out in mice liver microsomes and human liver
microsomes respectively. In the human liver microsome assay, the most stable compound tested
was compound 3, which had a half-life (t1/2) of 290 minutes. Compounds 2 and 10 were the least
metabolically stable. This suggests that the aryl amine groups are more sensitive metabolically than
the pyrazole-based moieties. Addition of functional groups to the aniline rings which are known to
slow metabolism, such as CF3 could increase the metabolic half-life of these compounds13.
Finally, we also investigated the selectivity of this class of inhibitor for bacterial gyrase versus
inhibition of human topoisomerase II. Four representative compounds (2, 3, 4, and 5) were tested
for activity against human topoisomerase IIα. In all cases, we found little or no inhibition of the
human enzyme with compound concentrations up to 100 µM, suggesting that the human enzyme is
at least 1000-fold less sensitive than bacterial gyrase (data not shown).
Conclusions
In this study, protein X-ray crystallography has been used to explore the detailed mode of binding of
the pyridine carboxamide class of antibacterial agents, to their bacterial DNA gyrase target. These
studies reveal that the binding mode of these inhibitors closely corresponds to that predicted via the
structure-based design approach used in their conception, in particular, confirming the importance
of an exquisite network of H-bonds involving all three nitrogen atoms shared between the urea and
pyridine units within the inhibitors, a bound water molecule, and serine and aspartate amino acids
derived from the protein.
A preliminary analysis of the development potential of these compounds for use as antibacterials via
measurement of a range of in vitro ADMET parameters reveals that, whilst some of the compounds
possess a rather low degree of aqueous solubility, with a corresponding increase in plasma-protein

binding, solubility can be increased to encouraging levels via suitable structural alteration, as
underlined by compound 3, which also shows promising levels of metabolic stability. However, the
weak antimicrobial activity measured for this compound, despite the good affinity it displays for
GyrB, underlines one of the key challenges of target-based antimicrobial drug design in terms of
producing compounds with high target affinity and selectivity, but which also possess the right blend
of physicochemical properties to allow passage into- and retention within- the bacterial cell, a
requirement that is particularly challenging for entry into Gram negative organisms.
Experimental Section
The Supporting Information contains a complete general Experimental Section, including all
procedures and equipment used. Chemicals were from commonly used suppliers (Aldrich, Acros, and
Alfa Aesar) and used without purification. The purities of compounds submitted for screening were
≥95% as determined by UV analysis of liquid chromatography (HPLC) chromatograms at 254 nM.
Representative Synthesis for Compound 1. Synthesis of 4-chloro-6-[(ethylcarbamoyl)amino]-N-
(pyridin-3-yl)pyridine-3-carboxamide. Ethyl 4-chloro-6-(3-ethylureido)nicotinate6 (700 mg, 2.57
mmol) was suspended in NaOH (2 M, 30 ml) and heated to 80°C for 16 hours. The reaction mixture
was then cooled to 0°C and acidified to pH 4 by dropwise addition of cHCl. The resulting precipitate
was collected using filtration, washed with water and dried under air to afford the carboxylic acid
intermediate (516 mg, 2.12 mmol, 83%) as pale-yellow microcrystals. The carboxylic acid (516 mg,
1.12 mmol) and 3-amino pyridine (439 mg, 4.70 mmol) were dissolved in EtOAc (5 ml), and pyridine
(0.5 ml) and T3P (5 ml, 58.2 mmol, 50% solution in EtOAc) were added. The reaction mixture was
stirred at room temperature for 18 hours and then basified to pH 10 by addition of aqueous NaOH (2
M). The mixture was then stirred for 20 minutes and the product collected by filtration to afford the
product (230 mg, 0.72 mmol, 34%) as pale orange microcrystals, m.p. 210.4-215.6 °C; Rf 0.24 (1:1
EtOAcPet); H (300 MHz, DMSO-d6) 10.74 (s, 1 H, 4-NH), 9.57 (s, 1 H, 6-NHCONHCH2CH3), 8.84 (d, J
2.2 Hz, 1 H, 3-pyridyl 2-H), 8.47 (s, 1 H, 2-H), 8.33 (d, 1 H, J 3.8 Hz, 3-pyridyl 6-H), 8.15 (d, J 8.8 Hz, 1

H, 3-pyridyl 4-H), 7.80 (s, 1 H, 5-H), 7.31-7.50 (m, 2 H, 3-pyridyl 5-H and 6-NHCONHCH2CH3), 3.18 (qd,
J 7.2 and 5.5 Hz, 2 H, 6-NHCONHCH2CH3), 1.09 (t, J 7.2 Hz, 3 H, 6-NHCONHCH2CH3); C (125 MHz
DMSO-d6) 163.1 (3-CONH), 155.0 (6-C), 154.0 (6-NHCONHCH2CH3), 148.0 (2-C), 144.8 (3-pyridyl 6-C),
141.6 (3-pyridyl 2-C), 141.2 (3-pyridyl 3-C), 135.5 (4-C), 126.5 (3-pyridyl 4-C), 124.8 (3-C), 123.7 (3-
pyridyl 5-C), 111.2 (5-C), 33.9 (6-NHCONHCH2CH3), 15.2 (6-NHCONHCH2CH3); νmax / cm-1 (solid) 3179,
1669, 1548; m/z (ES+) found MH+ 320.0912 C14H14ClN5O2 MH requires 320.0909; HPLC RT = 0.99 mins.
Synthesis of 6-[(ethylcarbamoyl)amino]-4-[(2-hydroxyphenyl)amino]-N-(pyridin-3-yl)pyridine-3-
carboxamide. 4-Chloro-6-[(ethylcarbamoyl)amino]-N-(pyridin-3-yl)pyridine-3-carboxamide (30 mg,
0.09 mmol) and 2-hydroxyaniline (30 mg, 0.09 mmol) were dissolved in ethanol (2 ml) and NEt3 (5
drops) was added. The reaction was heated to 80°C for 3 hours. The reaction was then allowed to
cool to room temperature and the solvent removed in vacuo. The resulting off white residue was
taken up in EtOAc (10 ml), washed with water (3 × 10 ml) and brine (10 ml), the volume reduced in
vacuo and the product collected by filtration to afford the product (7.5 mg, 0.191 mmol, 21%) as
pale red microcrystals, Rf 0.03 (EtOAc); H (500 MHz, DMSO-d6) 10.43 (br. s., 1 H, 3-CONH), 9.85 (s, 1
H, 6-NHCONHCH2CH3), 9.77 (s, 1 H, 4-NH), 9.14 (s, 1 H, 4-phenyl 6-H), 8.88 (s, 1 H, 3-pyridyl 2-H),
8.61 (s, 1 H, 2-H), 8.33 (d, 1 H, J 4.6 Hz, 3-pyridyl 6-H), 8.14 (d, J 7.8 Hz, 1 H, 3-pyridyl 4-H), 8.00 (br.
s., 1 H, 4-phenyl OH), 7.41 (dd, J 8.5, 4.8 Hz, 1 H, 3-pyridyl 5-H), 7.32 (d, J 7.8 Hz, 1 H, 4-phenyl 3-H),
7.23 (s, 1 H, 5-H), 6.91 - 7.03 (m overlapping, 2 H, 4-phenyl 4-H and 5-H), 6.86 (t, J 6.9 Hz, 1 H, 6-
NHCONHCH2CH3), 3.17 (qd, J 7.1, 6.9 Hz, 2 H, 6-NHCONHCH2CH3), 1.09 (t, J 7.1 Hz, 3 H, 6-
NHCONHCH2CH3); C (500 MHz, DMSO-d6) 166.9 (3-CONH), 155.9 (4-phenyl 2-C), 154.4 (6-C), 152.1
(4-C), 149.6 (6-NHCONHCH2CH3), 149.0 (5-C), 144.6 (3-pyridyl 2-C), 142.2 (3-pyridyl 6-C), 127.7 (3-
pyridyl 4-C), 126.5 (4-phenyl 1-C), 124.7 (4-phenyl 4-C), 123.5 (3-pyridyl 5-C), 122.1 (4-phenyl 5-C),
119.2 (4-phenyl 6-C), 115.8 (4-phenyl 3-C), 108.0 (3-C), 92.0 (2-C), 33.8 (6-NHCONHCH2CH3), 15.3 (6-
NHCONHCH2CH3); νmax / cm-1 (solid) 2969, 2811, 1698, 1642, 1543, 1517; m/z (ESI+) found MH+
393.1673, C20H20N6O3 MH requires 393.1670; HPLC RT = 1.38 mins.

Associated Content
Supporting Information
The Supporting Information is available free of charge on the ScienceDirect Publications website at
DOI:….
Experimental procedures, characterization of intermediates and target compounds,
description of protein production and purification, molecular modelling, biological
assays, x-ray crystallography.
Author information
Corresponding Author
*Phone: +44 (0) 1133 436510; e-mail: c.w.g.fishwick@leeds.ac.uk.
ORCID
Colin W. G. Fishwick: 0000-0003-1283-2181
Anthony Maxwell: 0000-0002-5756-6430
Notes and acknowledgements
The authors declare no competing financial interest.
The authors declare no competing financial interest.
Crystal data for compounds 2, 4, 5, and 10 have been deposited in the Protein Data Bank with
accession codes 6F86, 6F8J, 6F94 and 6F96
The authors thank Alison Howells (Inspiralis Ltd.) for carrying out supercoiling and relaxation assays.
Abbreviations used

ADME, absorption distribution metabolism excretion; ATP, adenosine triphosphate; BSA, bovine
serum albumin; CaCo-2, human carcinoma colon cell line; CDC, Centers for Disease Control and
Prevention; cHCl, concentrated hydrochloric acid; CHCl3, chloroform; DCM, dichloromethane; dCTP,
deoxycytosine triphosphate; dGTP, deoxyguanosine triphosphate; DMSO, dimethyl sulphoxide; E.
coli, Escherichia coli; EDTA, ethylenediaminetetraacetic acid; GyrA, DNA gyrase subunit A; GyrB, DNA
gyrase subunit B; DTT, dithiothreitol; EtOAc, ethyl acetate; HEPES, 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid; HPLC, high performance liquid chromatography; IC50, inhibitory
concentration at half maximal; KCl, potassium chloride; MeOH, methanol; MgCl2, magnesium
chloride; MgSO4 ,magnesium sulphate; MIC, minimum inhibitory concentration; m.p., melting point;
NAD+, nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide reduced form;
NaOH, sodium hydroxide; NEB, New England biolabs; NEt3, trimethylamine; NiSO4, nickel sulphate;
PCR, polymerase chain reaction; PEG, polyethylene glycol; Pet, petroleum ether; RT, room
temperature; S. aureus, Staphylococcus aureas; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide
gel electrophoresis; SOC, super optimal broth with catabolite suppression; T3P, propyl phosphonic
anhydride; Tris, tris(hydroxymethyl)aminomethane.
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Table of Contents graphic
O
Pro79
OMe
N
O
H2N
O
HN
N
N
H
O
Asn46
H2N
HN
N
H
N
N
NH2
H
H
O
O
H
O
Arg136
Asp73
Gly77
O