Linker-switch approach towards new ATP binding site inhibitors of DNA gyrase B

Article abstract of DOI:10.1016/j.ejmech.2016.09.040

Due to increasing emergence of bacterial resistance, compounds with new mechanisms of action are of paramount importance. One of modestly researched therapeutic targets in the field of antibacterial discovery is DNA gyrase B. In the present work we synthesized a focused library of potential DNA gyrase B inhibitors composed of two key pharmacophoric moieties linked by three types of sp³-rich linkers to obtain three structural classes of compounds. Using molecular docking, molecular dynamics and analysis of conserved waters in the binding site, we identified a favourable binding mode for piperidin-4-yl and 4-cyclohexyl pyrrole-2-carboxamides while predicting unfavourable interactions with the active site for piperazine pyrrole-2-carboxamides. Biological evaluation of prepared compounds on isolated enzyme DNA gyrase B confirmed our predictions and afforded multiple moderately potent inhibitors of DNA gyrase B. Namely trans-4-(4,5-dibromo-1H-pyrrole-2-carboxamide)cyclohexyl)glycine and 4-(4-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)piperidin-1-yl)-4-oxobutanoic acid with an IC₅₀value of 16 and 0.5?μM respectively.

Full text of DOI:10.1016/j.ejmech.2016.09.040

Accepted Manuscript
Linker-switch approach towards new ATP binding site inhibitors of DNA gyrase B
Marko Jukič, Janez Ilaš, Matjaž Brvar, Danijel Kikelj, Jožko Cesar, Marko Anderluh
PII:
S0223-5234(16)30770-X
10.1016/j.ejmech.2016.09.040
EJMECH 8906
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 May 2016
Revised Date: 18 August 2016
Accepted Date: 13 September 2016
Please cite this article as: M. Jukič, J. Ilaš, M. Brvar, D. Kikelj, J. Cesar, M. Anderluh, Linker-switch
approach towards new ATP binding site inhibitors of DNA gyrase B, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.040.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Linker-switch approach towards new ATP binding site inhibitors of DNA Gyrase B
1
1
2
1
1*
1*
Marko Jukič , Janez Ilaš , Matjaž Brvar , Danijel Kikelj , Jožko Cesar and Marko Anderluh
1
University of Ljubljana, Faculty of Pharmacy, Department of Medicinal Chemistry, Aškerčeva cesta 7, 1000
Ljubljana, Slovenia
2
National Institute of Chemistry, Laboratory for Biocomputing and Bioinformatics, Hajdrihova ulica 19, 1001
Ljubljana, Slovenia
*
Corresponding authors: Jožko Cesar and Marko Anderluh, University of Ljubljana, Faculty of Pharmacy,
Department of Pharmaceutical Chemistry, Aškerčeva 7, 1000 Ljubljana, Slovenia, Tel: +386-1-47-69-639, Fax:
+
386-1-425-80-31, E-mail: jozko.cesar@ffa.uni-lj.si; marko.anderluh@ffa.uni-lj.si.
Abstract: Due to increasing emergence of bacterial resistance, compounds with new mechanisms of action are
of paramount importance. One of modestly researched therapeutic targets in the field of antibacterial discovery
is DNA gyrase B. In the present work we synthesized a focused library of potential DNA gyrase B inhibitors
3
composed of two key pharmacophoric moieties linked by three types of sp -rich linkers to obtain three structural
classes of compounds. Using molecular docking, molecular dynamics and analysis of conserved waters in the
binding site, we identified a favourable binding mode for piperidin-4-yl and 4-cyclohexyl pyrrole-2-
carboxamides while predicting unfavourable interactions with the active site for piperazine pyrrole-2-
carboxamides. Biological evaluation of prepared compounds on isolated enzyme DNA gyrase B confirmed our
predictions and afforded multiple moderately potent inhibitors of DNA gyrase B. Namely trans-4-(4,5-dibromo-
1
H-pyrrole-2-carboxamide)cyclohexyl)glycine
and
4-(4-(3,4-dichloro-5-methyl-1H-pyrrole-2-
carboxamido)piperidin-1-yl)-4-oxobutanoic acid with an IC values of 16 and 0.5 ꢀM respectively.
5
0
Keywords: antibiotics, antibacterials, inhibitor, DNA Gyrase B, ATP binding site, pyrrole-2-carboxamides,
structure-based drug design, ligand-based drug design
1
.
Introduction
Continuous use of antibiotics from their discovery until now, calls for caution and critical examination
[
[1]]. Effectiveness and general availability of these active compounds is in disagreement with their expanding
and often erroneous utilisation [[2], [3]]. Careful use of antibacterials is imperative, especially in the light of
bacterial resistance in community and nosocomial infections, rendering established therapies ineffective [[4]].
Several mechanisms that confer bacteria with antibiotic resistance are reported. The most common are
enzymatic modification or degradation of the active compounds, physical removal from the cell, reduced uptake
and modification or overexpression of the target site [[5], [6]]. In the light of bacterial adaptation to modern
therapeutic approaches, there is an urgent need for new platforms and programmes on antibacterial discovery
[
[7]]. Among all currently reported potential targets in the field of antibacterials, only few have proceeded to
further studies, so new targets and active compounds with novel and synergistic pharmacodynamic mechanisms
are in critical demand [[8], [9]]. To challenge the demand for new type of antibacterial compounds overlooked
chemical spaces, such as marine natural products, may be used as a fruitful source of new hits & leads [[9]]. In
recent years we have completed FP7-funded integrated project MAREX (Exploring Marine Resources for
Bioactive Compounds), in which we sought for new bioactive compounds from marine sources and used them
as a starting point for hit-to-lead development (http://www.marex.fi/).
One of the validated but nevertheless, therapeutically less-explored targets is subunit B of DNA gyrase
(
DNA GyrB or GyrB). It is an essential bacterial enzyme and it belongs to type II topoisomerases. The enzyme
catalyses negative supercoiling of DNA [[11]]. Replication and transcription introduces increasing strain on
DNA chain ahead of replication bubble and in the process to relieve strain, topoisomerase enzymes bind to
DNA molecule and introduce transient breaks in DNA strands. Topoisomerase I enzymes cut one strand, while
topoisomerases II cut and anneal both strands. After the process is complete, DNA backbone is recoupled and
overall connectivity of DNA remains intact [[12]]. DNA gyrase possesses heterotetrameric structure consisting
of two GyrA subunits responsible for nucleotide binding and operation on DNA molecule, and two GyrB
subunits that hydrolyse adenosine triphosphate (ATP) providing free energy required for enzyme
conformational movements and introduction of negative supercoils [[13]].
GyrA is utilised as a therapeutic target in clinical practice with a well-known class of fluoroquinolone
antibacterials 1 (Figure 1). Their mechanism of action resides in stabilisation of DNA-GyrA complex, ultimately
stopping bacterial replication cycle [[14]]. On the other hand, GyrB subunit inhibitors are not present in materia

ACCEPTED MANUSCRIPT
medica. Two compounds: chlorobiocin and novobiocin 2 (Figure 1) proceeded through late clinical research,
however the whole class of aminocoumarin antibiotics was withdrawn from clinical practice due to in vivo
toxicity [[15]].
Figure 1. Ciprofloxcin 1 as a representative of fluoroquinolone class of antibiotics together with clorobiocin and novobiocin
2
as major representatives of aminocoumarin class of antibiotics. Key interactions with DNA Gyrase B binding site are
depicted by dashed lines (E. coli residue numbering) [[22]].
In spite of aminocoumarin class withdrawal, there remains a steady research activity on novel inhibitors
of DNA Gyr B and extensive reviews report on the subject, covering industrial programmes as well as academic
efforts [[16]-[19]]. Research can be rationalized by a number of different contributing factors: Firstly, the
binding mode of earliest reported compounds (2, Figure 1) has been well characterized and defined with high-
resolution crystallographic data available [[20]-[23]]. Secondly, the ATP binding pocket is positioned on a
distinct location away from DNA Gyr A, therefore novel and synergistic pharmacodynamics can be
accomplished without transfer of bacterial resistance. Despite the fact that selectivity on DNA Gyr B is not
trivial as ATP is an omnipresent compound in biological systems, selectivity on this particular target can
effectively be achieved [[24]]. Scaffolds of GyrB inhibitors encompass a broad chemical space, for example:
indolin-2-ones 3, bithiazoles 4, benzimidazole ureas 5, azaindole ureas 6, indazoles 7, aminopiperidines 8,
pyrrole-2-carboxamides 9 and cyclothialidines 10 (Figure 2), but nevertheless comply with key structural
requirements imposed by GyrB [[25]-[32]]. Namely, all compounds possess donor/acceptor pattern where N-H
is the most common H-bond donor group and acceptor is either amide carbonyl oxygen or heterocyclic nitrogen
atom on one side, and an acidic or polar functionality at the other terminal side separated by a more or less
sterically constricted or bulky linker moiety.
Figure 2. Reported small-molecule inhibitors of DNA Gyrase B.
Thirdly, GyrB target shares its structural similarity with the other member of type II topoisomerases, Topo IV.
Topo IV relaxes positive supercoils ahead of a translocating DNA polymerase, relieving topological strain.
Furthermore, it is also responsible for decatenation of interlinked newly replicated DNA strands [[33]].
Analogous enzymes in topoisomerase II class thus open the possibility of dual targeting. Such approach would
consequently lead to inhibition of bacterial replication on multiple enzymatic steps. Indeed, many of the
reported small molecule inhibitors display potency on both GyrB and ParE subunit on Topo IV [[34]-[36]].
Finally, there is a substantial body of selective GyrB or dual targeting GyrB/ParE inhibitors with nanomolar
potency on isolated enzymes and good antimicrobial activity described in the literature. GyrB inhibitors with

ACCEPTED MANUSCRIPT
activity on clinically-relevant strains and problematic bacteria from family of Mycobacteriaceae are also
reported [[37], [38]]. Nonetheless, complex scaffolds, polyaromatic planar structures, undesirable chemical
functionalities, improper physico-chemical properties, bacterial resistance by active transport, narrow spectrum
of activity and other complications prevent further clinical evaluation of majority of compounds with reported
potency on DNA Gyr B [[39]]. In this study, we investigate three structural classes of compounds as potential
inhibitors of DNA gyrase B.
2
.
Results and discussion
2
.1 Computational studies and design
In our previous research, we have evaluated analogues of marine natural products, specifically oroidin
and clathrodin 11 (Figure 3) secondary metabolites of Agelas marine sponges for their modulatory activity on
voltage-gated sodium channels [[40]]. However we were aware that marine alkaloids from Agelas sponges
possess notable antimicrobial activity [[41]-[44]]. Moreover, clathrodin and oroidin (
Figure 3, b) as well as our piperazine clathrodin (oroidin) analogues incorporate a pyrrole-2-
carboxamide moiety and are structurally similar to chlorobiocin and numerous small-molecule selective GyrB
inhibitors [[25]-[32]]. We thus postulated that marine alkaloid oroidin scaffold could be used as a basis for
design of synthetically accessible and simple novel GyrB inhibitors. Contrary to majority of published inhibitors
3
that employ a sterically rigid linker between pyrrole-2-carboxamide and acidic group, we focused on simple sp -
rich scaffolds that could favourably position the terminal functional groups and possibly mimic the L-noviose
moiety found in aminocoumarins clorobiocin and novobiocin (
Figure 3, a) [[45], [46]].
a)
b)
Figure 3. a) Fragment-based design approach and ReCore top ranking fragments from the ZINC library fragment database;
b) definition of core replacement strategy in LeadIT ReCore module.
We employed a fragment-based design in order to replace the native aminopropene flexible linker of
oroidin lead compound [[47]]. After construction of fragment library from ZINC Lead-Like subset, we
performed a screening using oroidin central 2-aminopropene linker as a query. We selected ten ReCore top
scoring fragments and identified short aliphatic chains and aminocyclohexanes (
Figure
3, a) as major structural motifs for linker design. Due to structural flexibility of
aminopropene(ane) chains, availability of piperazine oroidin analogues from our previous study and literature
reports on DNA GyrB inhibitors incorporating a piperdine central linker, we decided to design, prepare and
evaluate simple pyrrole-2-carboxamides and indole-2-carboxamides with central piperazine, aminopiperidine
and cyclohexane linker moieties (Figure 4, a).
Next, we examined ANP-GyrB crystal complex (ANP- phosphoaminophosphonic acid adenylate ester
mimics native ATP, PDB entry: 1EI1) together with binding modes of published small-molecule inhibitors and
identified highly conserved adenine binding pocket (Figure 4, b) [[49]]. Among all reported protein structures
with 95% sequence similarity, we focused on crystal structure with bithiazole small-molecule inhibitor 12
(
0
Figure 4, a, b) for further studies [[26]]. Docking protocol validation was successful with an RMSD value of
.16 Å. Our model could also replicate the experimental binding mode of previously reported DNA Gyrase B
inhibitors and was shown to be robust also in virtual screening scenarios. Bithiazole 12 shares a common
binding mode where terminal amide fits in highly defined (Val43, Val167) hydrophobic pocket and forms
hydrogen bonds towards Asp73 (E. coli GyrB numbering) with the assistance of water molecule [[21]]. The
same space is occupied by adenine moiety of ANP (Figure 4, b; depicted grey in background), yet where ANP
phosphate chain interacts with Lys103, small molecule inhibitors turn towards cavity entrance and form crucial

ACCEPTED MANUSCRIPT
contacts with positively charged Arg136 and Arg76 residues [[20]]. Inhibitor-Arg136 interaction was shown to
be important by mutation studies leading to aminocoumarin loss of activity [[23]].
a)
b)
Figure 4. a) Design of piperazin-1-yl, piperidin-4-yl and 4-cyclohexyl pyrrole-2-carboxamide (indole-2-carboxamide)
compound libraries; b) 2D projection alignment of ANP (PDB entry: 1EI1, depicted grey in background) and bithiazole 12-
GyrB crystal complex (PDP: 4DUH, depicted blue in foreground) revealing highly conserved ATP binding site and key
interactions of bithiazole 12 (highlighted residues are conserved in both aligned crystal complexes). [[48]].
a)
b)
Figure 5. Identification of reported water locations in locally aligned crystal-complexes similar to 4DUH chain B query
depicted green with calculated solvent accessible surface in grey colour); a) random bulk water clusters, b) conserved water
(
cluster in vicinity of Asp73 (E. coli numbering, water location data clusters depicted as blue spheres, r = 1 Å, red arrow
conserved water cluster) [[52]].
With essential small-molecule inhibitor-protein interactions identified, we turned our attention towards
conserved water molecules [[50]]. Due to their importance in inhibitor binding to ATP site of GyrB, we tried to
assert their location in order to validate applicability of conserved water-mediated hydrogen bond interactions in
our computational studies. Using ProBiS (Protein Binding Sites Detection) database of non-redundant PDB

ACCEPTED MANUSCRIPT
entries, we identified most similar locally-aligned protein structures to bithiazole-GyrB complex query (PDB
entry: 4DUH) [[51]]. We compiled crystallographic water location data and successfully enumerated the random
bulk water molecules (Figure 5, a; depicted blue) surrounding our protein, as well as identified small spatial
clusters where water molecules are reported across multiple similar protein structures (Figure 5, b; blue sphere).
This observation substantiated the conserved water-mediated hydrogen bond formation in the vicinity of key
Asp73 (E. coli numbering) residue.
Consecutively, we designed three compounds with central piperazine 13, aminopiperidine 14 and
cyclohexane 15 linker moieties for binding mode studies (Figure 6). Piperazine and aminopiperidine linkers
were derivatised with malonate fragment on one terminal side while cyclohexane linker was derivatised with an
analogous glycine fragment. All three linkers were derivatised with key pyrrole-2-carboxamide scaffold on the
other terminal side of the molecules. Designed compounds were evaluated by FILTER 2.5.1.4 software
(
OpenEye Scientific Software, Inc., Santa Fe, NM, USA; www.eyesopen.com) for possible aggregation
properties, solubility problems as well as checked against known PAINS (pan-assay interference compounds)
substructures [[53]]. For docking studies E. coli DNA Gyrase B crystal complex (PDB entry: 4DUH) was
selected and docking performed using OpenEye OEDocking 3.0.1 software package (OpenEye Scientific
Software, Inc., Santa Fe, NM, USA; www.eyesopen.com) [[54], [55], [56]].
Figure 6. Model compounds with central piperazine, aminopiperidine and cyclohexane linker moieties designed for further
binding mode studies.
Inspection of calculated docked poses (Error! Reference source not found. compounds in a, b, c analogous to
compounds 13, 14, 15 in Figure 6) revealed the common binding mode as reported beforehand (PDB entry
4
DUH) [[26]]. Indeed brominated pyrrole perfectly accommodated in tightly defined adenine pocket of GyrB
and interacted with Asp73 (Error! Reference source not found., E. coli numbering). Pyrrole-2-carboxamide
moiety was in favourable location to complement hydrogen bonding towards Asp73 via vicinal conserved water
molecule. Binding modes of designed aminopiperidine 14 (Error! Reference source not found., b) and
cyclohexane 15 (Error! Reference source not found., c) with respective terminal malonate and glycine
moieties are analogous and enable hydrogen bonding or ionic interactions towards Arg136 and Arg76 residues.
Contrary, piperazine 13 (Error! Reference source not found., a) only partially adopts the observed common
binding mode as it kinks towards Gly101 and cannot form a meaningful contact towards Arg76. Arg76 allows
favourable interactions with aromatic residues adjacent to the terminal carboxylate, while offering repulsion in
the case of positively charged moieties in the linker region. As a consequence, in the case of piperazine 13 and
piperidine 14, the ring nitrogen was incorporated into an amide bond to lower its basicity (Figure 6). Due to
piperazine linker conformation, the terminal polar moiety protrudes into phosphate binding pocket (Error!
Reference source not found., Lys103). Crucial interactions at the ATP entrance could not be observed and we
postulate that compounds with this type of scaffold do not effectively interact with GyrB active site. Docking
studies of pyrrole-2-carboxamides were not focused on relative compound ranking, nevertheless we observed
that compound with 1,4-disubstituted cyclohexane linker 15 scored more favourably compared to other studied
scaffolds.

ACCEPTED MANUSCRIPT
a)
b)
c)
Figure 7. a) docked pose of piperazin-1-yl pyrrole-2-carboxamide 13 (depicted green) with superimposed bithiazole in
experimental binding mode (PDB entry: 4DUH, depicted orange) and emphasized binding pocket colored gray. b) docked
pose of piperidin-4-yl pyrrole-2-carboxamide 14 (depicted light red) with re-docked validation experiment (PDB entry:
4
DUH; bithiazole inhibitor, depicted orange) , c) docked pose of 4-cyclohexyl pyrrole-2-carboxamide 15 (depicted
magenta); Only key amino acid residues are rendered as sticks coloured by atom; Conserved water molecule is presented as
blue sphere [[52]].
2
.2 Molecular dynamic simulations
In order to evaluate the calculated binding modes of designed linker pyrrole-2-carboxamide scaffold
compounds in time domain, we performed an MD study under simulated solvated system conditions. Crystal
structure complex with bithiazole inhibitor (PDB entry: 4DUH) chain A was selected. A comparative MD study
was conducted for piperazine 13, aminopiperidine 14 and cyclohexane 15, and GyrB model complexes and
trajectories were analysed (Figure 8).
a)
b)

ACCEPTED MANUSCRIPT
c)
Figure 8. RMSD plots during MD production run. Protein backbone RMSD depicted in blue, ligand atom RMSD depicted
in red. a) 13; b) 14; c) 15 (Figure 6).
•
•
•
3-(4-(4,5-dibromo-1H-pyrrole-2-carbonyl)piperazin-1-yl)-3-oxopropanoic acid 13: In the first case of
the ligand with piperazine central linker we can observe elevated movement of small molecule (RMSD
increasing from 1.2 to 2.7 Å). The protein backbone RMSD settles immediately after 1 ns of simulation
time at around 2.5 Å We can conclude that this scaffold is the most flexible and offers the least protein
structure stabilisation compared to other two compounds. (Figure 8, a).
3-(4-(4,5-dibromo-1H-pyrrole-2-carboxamido)piperidin-1-yl)-3-oxopropanoic acid 14: Examination of
modelled ligand bearing aminopiperidine central linker revealed relatively stable RMSD for the ligand
fluctuating around 1.2 Å with occasional small movements and consistent RMSD of the protein
backbone around 1.8 Å. Comparison of all three structures points that this compound moderates protein
movement and stabilises its structure through simulation time. (Figure 8, b).
(4-(4,5-dibromo-1H-pyrrole-2-carboxamido)cyclohexane-1-carbonyl)glycine 15: Third modelled
ligand with 1,4-substituted cyclohexane linker sits compactly in the binding site with consistent RMSD
between 1.2 and 1.5 Å. It binds to the active site tightly without major conformational movements but
nonetheless, provides less protein stabilisation as demonstrated by RMSD of protein backbone atoms
that gradually increases through simulation time towards stable 2.5 Å (Figure 8, c).
Ligand-protein interactions were evaluated all along production run in 1.2 ps intervals and plotted for
examination (Figure 9).
a)
b)
c)
Figure 9. Abscissa: interacting amino acid residues around modelled ligand; Ordinate: interaction fraction where 1
represents 100 % occupation throughout simulation time of 10 ns. Values greater than 1 indicate multiple interactions with
the amino acid residues. a) piperazine 13; (b) piperidine 14; c) cyclohexane 15; (light blue bars represent hydrophobic
interactions; green bars represent hydrogen bonds, magenta bars indicate ionic interactions and dark blue bars indicate water
bridges).

ACCEPTED MANUSCRIPT
Visual inspection of MD trajectories corroborates binding mode predicted by docking studies where pyrrole-2-
carboxamide sits firmly in the adenine binding pocket without excessive movements. Aminopiperidine 14
(
Figure 9, b) and cyclohexyl pyrrole-2-carboxamide 15 (Figure 9, c) favourably present the terminal flexible
fragments towards arginine residues at ATP binding site entrance. Piperazine pyrrole-2-carboxamide 13 (Figure
, a) adopts a conformation that is kinked and interacts with diphosphate binding site. Interaction diagrams
9
support previous observations and substantiate presence of water-mediated hydrogen bonds in adenine pocket
near Asp73 where water bridge interaction is present. These postulated crucial contacts are preserved constantly
throughout simulation time by aminopiperidine 14 (Figure 9, b) and cyclohexyl pyrrole-2-carboxamide 15
(
Figure 9, c). Furthermore, aminopiperidine 14 (Figure 9, b) enables additional hydrogen bond towards Gly101,
an interaction already reported beforehand by Brvar et al. [[26]]. In contrast to other two compounds, piperazine
3 (Figure 9, a) buries halogenated pyrrole in adenine pocket too, but is kinked towards diphosphate binding
1
site. Its terminal acidic moiety interacts with Lys103 and Lys110 almost entire simulation time. The MD study
thus supports favourable binding modes of model compounds with aminopiperidine (14) and cyclohexane (15)
central linker, but also reveals flexibility and additional space at the binding site opening towards bulk solvent.
Further experimental studies on terminal fragment incorporating a carboxylate or ester group should adequately
complement this data set and shed light into favourable conformational requirements.
2
.3 Chemistry
Synthesis of compounds with piperazin-1-yl pyrrole-2-carboxamide scaffolds 20, 21 is outlined in the
Scheme 1, while synthesis of piperazin-1-yl compounds with terminal aminoimidazole fragment 22a-e (Table 1)
was reported before [40]. In the first step N-benzyloxycarbonyl (CBz) protected piperazine 16 was reacted with
4
,5-dibromo-pyrrole-2-carboxylic acid 17 using classic amide coupling protocol with EDC (1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide) and HOBt (1-hydroxybenzotriazole) with N-methylmorpholine (NMM) as
a base in N,N’-dimethylformamide (DMF) as solvent to afford protected key intermediate 18. After Cbz
deprotection by catalytic reduction using 10% palladium on activated charcoal under hydrogen atmosphere in
THF/methanol solvent system, resulting free amine 19 was used for final acylation. Methyl malonylchloride was
employed in dichloromethane to afford methyl ester 20. The target compound 21 was obtained after hydrolysis
of the methyl esters with aqueous lithium hydroxide in THF.
Scheme 1. Reagents and conditions: (a) EDC, HOBt, NMM, DMF, 0 °C → r.t., 16 h; (b) H , Pd/C, AcOH , MeOH, THF,
2
gl
r.t., 4h; (c) methyl malonyl chloride, DIPEA, CH Cl , 0 °C → r.t., 3 h; (d) LiOH, THF, H O, r.t., 4h.
2
2
2
Compounds bearing piperidin-1-yl pyrrole-2-carboxamide and indole-2-carboxamide scaffolds 28, 29, 30 and
1 were synthesized according to the Scheme 2. N1-BOC protected 4-aminopiperidine 23 was alkylated via
3
reductive amination using sodium triacetoxyborohydride in dichloromethane towards 24. In the case of allyl
substituent, classic N-alkylation with allyl bromide was performed. Alkylated or 4-amino nonsubstituted
aminopiperidine 24 was consecutively subjected to amide coupling protocol with EDC carbodiimide and
hydroxybenzotriazole (HOBt) with N-methylmorpholine (NMM) as a base in dimethylformamide (DMF)
solvent to afford protected key intermediates 25 and 26 (Scheme 2). Tert-butyloxycarbonyl (BOC) protected
pyrrole-2-carboxamide and indole-2-carboxamides were deprotected to the free cyclic secondary amines 27 with
trifluoroacetic acid (TFA) in dichloromethane. Final alkylation/acylation step was conducted according to three
general routes; with acyl chlorides, via TBTU-promoted coupling in dichloromethane/DMF, or with alkyl
bromides performed in acetonitrile solvent to afford methyl esters. Hydrolysis of methyl/ethyl esters 28 and 29
in the ultimate step afforded final free acids 30 and 31.

ACCEPTED MANUSCRIPT
Scheme 2. Reagents and conditions: (a) benzaldehyde, sodium triacetoxyborohydride (Na(Ac) BH), CH Cl , 0 °C → r.t., 1 h
3
2
2
(
b) allyl bromide, K CO , CH Cl , 0 °C → r.t., 15 h; (c) EDC, HOBt, NMM, DMF, 0 °C → r.t., 16 h; (d) Trifluoroacetic
2 3 2 2
acid (TFA), CH2Cl2, 50 °C, 3 h; (e) acid fragment, TBTU, NMM, CH Cl , DMF, r.t. → 40 °C , 16 h; (f) acyl chloride,
2
2
DIPEA or Et N, CH Cl , 0 °C → r.t., 1 h; (g) alkyl halogenide, Cs CO , Acetonitrile (CH CN), r.t., 5 h; (h) LiOH, THF,
3
2
2
2
3
3
H O, r.t., 2-8 h.
2
Synthetic route to the third compound series incorporating a stereoisomerically pure 1,4-disubstituted
cyclohexane linker is depicted in the Scheme 3. Stereoisomerically pure 1,4-disubstituted cyclohexanes amino
acids were protected as methyl esters 32, and then coupled to 4,5-dibromo-pyrrole-2-carboxylic acid 17 using
EDC/HOBt procedure to afford protected key intermediates 33. Methyl ester deprotection with LiOH in
THF/water to free acids 34 was followed by second amide coupling using either EDC/HOBt or TBTU
procedure towards methy ester protected compounds 35. Final compounds 36 were afforded by methyl ester
hydrolysis using aforementioned protocol.
Scheme 3. Reagents and conditions: (a) Thionyl chloride (SOCl ), MeOH, 40 °C, 2h; (b) EDC, HOBt, NMM, DMF, 0 °C →
2
r.t., 16 h; (c) LiOH, THF, H O, r.t., 2-8 h; (d) amino acid methyl ester, TBTU, NMM, CH Cl , DMF, r.t. → 40 °C , 16 h.
2
2
2
2
.4 DNA gyrase B inhibition assays

ACCEPTED MANUSCRIPT
All prepared compounds were tested for inhibitory activity on DNA gyrase B from E. coli using
commercial assay kit (Inspiralis Ltd, England). Several compounds that displayed notable potency against DNA
gyrase B from E. coli were also tested on DNA gyrase from S. aureus bacterial strain. Results are presented as
either residual activities of the enzyme in percent (RA = 0
– 100 %) at the 100 µM or as IC50 values for
compounds with IC50 predicted below 100 µM. In the Table 1, results for the first series of compounds with
piperazin-1-yl pyrrole-2-carboxamide scaffold 20, 21, 22 are presented. As expected, these compounds do not
possess any notable inhibition of isolated enzyme as they are not capable of forming key interactions at the ATP
binding site entrance. To explore this postulated key interaction, 20 with terminal methyl-protected malonate
was prepared along with its deporotected free acid 21. Free acid was modestly potent with the value of IC50 in
the low milimolar range, while the ester moiety remained inactive.
Table 1
Biological evaluation of compounds with piperazin-1-yl and piperazin-1-yl pyrrole-2-carboxamide scaffold
a
b
IC50 or (Kd) [µM] or RA [%]
compound
R1
R2
DNA Gyrase E. coli
100 %
0.17
DNA Gyrase S. aureus
100 %
0.041
c
negd
NB
2
2
0
1
78 %
n.t.
1080 µM
n.a.
2
2
2a
2b
100 %
100 %
n.t.
n.t.
2
2
2c
2d
2e
100 %
100 %
100%
n.t.
n.t.
n.t.
2
a
b
K determined by surface plasmon resonance; Residual activity of the enzyme at 100 µM of the tested
d
compound. Novobiocin was used as a positive control (IC50 = 0.17 µM on GyrB from E. coli and IC50 = 0.04
c
d
µM on GyrB from S. aureus); neg – control 1% DMSO; NB – novobiocin as a positive control. n.a. – non
active; n.t. – not tested.
With in silico studies in hand, we turned our attention towards piperidin-4-yl pyrrole-2-carboxamide
scaffold collected in the Table 2. At first, we performed a study on optimal length of terminal fragment
incorporating a methyl ester or acid functionality and identified a succinic acid 30c as optimal with an IC50
=
1
.55 µM for most potent free acid and 21 µM for methyl ester 28c. If we increased the chain length towards
glutaric acid analogues 28d, 30d we produced similarly potent compounds, but the same cannot be observed for
shorter fragments. Oxalic acid 30a is evidently too short for achieving any meaningful contacts in active site,
although interestingly its ethyl ester 28a displayed surprising potency with an IC in the low micromolar range.
50
Compounds incorporating a malonic fragment 28b and 30b displayed a diminished potency in the range of
factor 2 compared to most potent succinic acid analogues. Latter were also confirmed as selective inhibitors of
DNA gyrase B by surface plasmon resonance experiments and were measured with a K value of 12.6 µM.
d
Exchange of aliphatic terminal fragments with sterically rigid methyl 4-methylbenzoate 29b, 31b or methyl 4-
carbonylbenzoate moieties 29a, 31a afforded free acids that displayed potency in the low micromolar range.

ACCEPTED MANUSCRIPT
These fragments were not pursued further due to results of SPR experiments that indicated nonspecific binding
to the G24 protein. Interesting compounds were also prepared with dihydroxyphenyl terminal fragment 27b that
were active with the IC s in low micromolar range. Next, we examined dibromopyrrole exchange and noticed a
5
0
marked increase in potency by using previously reported methyldichloropyrrole fragment while nonsubstituted
pyrrole 30f was completely devoid of activity. The most potent compound 4-(4-(((3,4-dichloro-5-methyl-1H-
pyrrol-2-yl)methyl)amino)piperidin-1-yl)-4-oxobutanoic acid 30e displayed an IC50 of 490 nM. Replacement of
dibromopyrrole with indole moiety afforded completely inactive compounds 29d-h, 31d-h as did derivatisation
of pyrrolo/indolo-amide nitrogen with bulky aliphatic or aromatic fragments 29g-j, 31g-j. This observation is in
accordance with strictly defined adenine binding pocket as observed in our in silico studies. Crucial interactions
of pyrrole-2-carboxamide hydrogen bond donor-acceptor motif towards Asp73 (E. coli numbering) via direct or
conserved water-mediated hydrogen bonds is also exemplified by compounds 29d-j and 31d-j (Table 3).
Table 2
Biological evaluation of compounds with piperidin-4-yl pyrrole-2-carboxamide scaffold
a
b
IC50 or (Kd) [µM] or RA [%]
compound
R1
R2
DNA Gyrase E. coli
100 %
0.17
DNA Gyrase S. aureus
100 %
0.041
c
negd
NB
2
2
3
2
3
2
3
2
3
5
>50 µM
4.35 µM
49.2 µM
13.7 µM
10.8 µM
20.9 µM
n.t.
8a
0a
8b
0b
8c
0c
8d
0d
74 %
n.t.
e
n.t.
80 %
n.t.
1.55 µM (SPR 12.6 µM)
11.2 µM
93.8 µM
n.t.
3.18 µM
71 %
2
2
7a
9a
100 %
n.t.
n.t.
f
480 µM
3
2
1a
9b
14.9 µM (SPR 256 µM)
100 %
84 %
n.t.

ACCEPTED MANUSCRIPT
63.1 µM (SPR *)
3
2
3
1b
9c
1c
100 %
n.t.
100 %
49.5 µM (SPR 11.2 µM)
100 %
2
2
2
7b
7c
8e
2.84 µM
8.08 µM
20.0 µM
61.4 µM
n.t.
n.t.
3
0e
0.48 µM
n.t.
2
3
a
8f
0f
100 %
100 %
b
n.t.
n.t.
K determined by surface plasmon resonance; Residual activity of the enzyme at 100 µM of the tested
d
compound. Novobiocin was used as a positive control (IC = 0.17 µM on GyrB from E. coli and IC = 0.04
5
0
50
c
d
e
µM on GyrB from S. aureus); neg – control 1% DMSO; NB – novobiocin as a positive control; Primary test
result against E. coli (ATCC 25922) is 10,2 % inhibition after 24h (50 µM); Primary test result against E. coli
f
(
ATCC 25922) is 11,5 % inhibition after 24h (50 µM). n.a. – non active; n.t. – not tested; * non-specific
binding.
Table 3
Biological evaluation of compounds with piperidin-4-yl indoloamide scaffold and bulky pyrrole-2-carboxamides
a
b
IC50 or (Kd) [µM] or RA [%]
compound
R1
R2
DNA Gyrase E. coli
100 %
0.17
DNA Gyrase S. aureus
100 %
0.041
c
negd
NB
2
2
6
100 %
n.t.
9d
88 %
n.t.
3
1d
100 %
n.t.
2
3
9e
1e
100 %
n.t.
n.t.
100 µM

ACCEPTED MANUSCRIPT
2
3
9f
1f
100 %
100 %
n.t.
n.t.
2
2
7d
9g
150 µM
100 %
n.t.
n.t.
3
2
3
2
3
2
1g
9h
1h
9i
100 %
100 %
100 %
36 %
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
1i
29 %
9j
100 %
3
1j
49.5 µM (SPR 11.2 µM) n.t.
a
b
K determined by surface plasmon resonance; Residual activity of the enzyme at 100 µM of the tested
d
compound. Novobiocin was used as a positive control (IC50 = 0.17 µM on GyrB from E. coli and IC50 = 0.04
c
d
µM on GyrB from S. aureus); neg – control 1% DMSO; NB – novobiocin as a positive control. n.a. – non
active; n.t. – not tested; * non-specific binding.
Third studied 4-cyclohexyl pyrrole-2-carboxamide scaffold presented in Table 4 confirmed our in silico studies
as well as our observations on piperidin-4-yl compounds. Only trans-cyclohexanes can present terminal
moieties in favourable positions. All analogues 35 and 36 were devoid of activity except trans-cyclohexane 36b
with terminal glycine that displayed an IC of 15.1 µM.
5
0
Table 4
Biological evaluation of 4-cyclohexyl pyrrole-2-carboxamides
a
IC50 [µM] or RA [%]
compound
scaffold
R2
DNA Gyrase E. coli
DNA Gyrase S. aureus
b
Neg
100 %
0.17
100 %
0.041
c
NB
3
5a
cis
100 %
n.t.

ACCEPTED MANUSCRIPT
3
3
3
6a
5b
6b
cis
100 %
n.t.
n.t.
n.t.
trans
trans
100 %
15.1 µM
3
3
3
3
5c
6c
5d
6d
cis
100 %
100 µM
100 %
100 %
n.t.
n.t.
n.t.
n.t.
cis
trans
trans
3
3
5e
6e
trans 2
trans 2
100 %
100 %
n.t.
n.t.
3
5f
trans 2
trans 2
100 %
100 %
n.t.
n.t.
3
6f
a
Residual activity of the enzyme at 100 µM of the tested compound. Novobiocin was used as a positive control
b
(
IC = 0.17 µM on GyrB from E. coli and IC = 0.04 µM on GyrB from S. aureus); neg – control 1% DMSO;
5
0
5
0
c
NB – novobiocin as a positive control. n.a. – non active; n.t. – not tested; * non-specific binding.
Some of the compounds display a moderately potent inhibition of DNA Gyrase B. Therefore, reported
inhibitors support the postulated requirements exerted by the binding site and present a good starting point for
optimisation campaign towards antibacterial activity. While the structures of reported inhibitors incorporating a
terminal carboxylate are not ideal for antibacterial activity, multiple optimisation approaches can be undertaken.
Together with further on-target potency optimisation, bioisosteric replacement and incorporation or
modification of ionisation centres as well as optimisation of compounds against efflux pump affinity could be
undertaken [57][58]. By antimicrobial testing using permeabilized (impA) and efflux pump knockout (ꢁtolC) E.
coli strains, we have previously shown that similar rigidized-scaffold pyrrole-2-carboxamides are efflux pump
substrates and optimisation of structures in this context could indeed be beneficial for further development of
reported compounds [45]. Furthermore, similar compounds to pyrrole-2-carboxamides are reported with activity
against Gramm-positive, Gramm-negative and Mycobacteria pathogens. All accounts thus support the potential
of reported scaffolds in further antibacterial research [59][60].
3
.
Conclusions
In this extensive coverage of simple and synthetically accessible compounds with piperazine,
aminopiperidine and cyclohexyl central linkers as inhibitors of DNA gyrase B, we successfully identified the
3
key structural features and the most suitable linker for in vitro inhibition of E. coli DNA gyrase B. The sp -rich
linkers were introduced to allow better fit to a binding site of DNA gyrase B that allows accommodation of
more bulky linkers, and permits more spatial arrangement of additional substituents, as witnessed by in silico
studies. We corroborated previous research on involvement of conserved water molecule and presented a novel
approach towards conserved water identification, which we believe is a valuable tool in similar research
problems. Among identified novel inhibitors of DNA gyrase B, several compounds with 4-aminopiperidine
linker proved to be modest to potent inhibitors of E. coli DNA gyrase B with inhibitory constants in low
micromolar or high nanomolar range, with some of these being active on S. aureus DNA gyrase B as well. The
work grants a solid foundation for future development, especially on the line of compounds with increased
potency on DNA gyrase B from multiple bacterial strains, which could ultimately lead to novel class of broad
spectrum antibacterials.
4
.
Experimental

ACCEPTED MANUSCRIPT
4
.1 In vitro enzyme inhibition measurement
Potency evaluation and determination of half maximal inhibitory concentrations (IC ) on E. coli and S.
50
aureus DNA Gyrase was performed using a commercial kit purchased from Inspiralis. For the assay black
streptavidin coated 96-well microtiter plates from Thermo Scientific Pierce were used. The plates were
rehydrated with the supplied wash buffer (20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.01% (w/v) BSA, 0.05%
(
v/v) Tween 20). Biotinylated oligonucelotide was then immobilized onto the well-walls and surplus
oligonucleotide washed off. The final assay reaction volume of 30 µL in buffer (35 mM Tris × HCl (pH 7.5), 24
mM KCl, 4 mM MgCl2, 2 mM DTT, 1.8 mM spermidine, 1 mM ATP, 6.5 % (w/v) glycerol, 0.1 mg/mL
albumin) contained 1.5 U of gyrase enzyme from E. coli or S. aureus, 0.75 µg of relaxed pNO1 plasmid and 3
µL of inhibitor solution in 10% DMSO and 0.008% Tween 20. Assay reactions were incubated for 30 min at 37
°C when the addition of the TF buffer (50 mM NaOAc (pH 5.0), 50 mM NaCl and 50 mM MgCl2) terminated
the enzymatic reaction. Incubation was continued for further 30 min at room temperature to allow triplex
formation (biotin-oligonucleotide-plasmid). After the incubation period, the unbound plasmid was washed off
using TF buffer and the solution of SybrGOLD stain in T10 buffer (10 mM Tris × HCl (pH 8.0) and 1 mM
EDTA) was added. After mixing the fluorescence (excitation: 485 nm, emission: 535 nm) was read using a
BioTek's Synergy H4 microplate reader. Preliminary screening was performed at inhibitor concentrations of 100
and 10 µM. For potent compounds, IC50 was determined with the cascading panel of 7 inhibitor concentrations.
IC50 value was calculated from multiple independent measurements (2, 3) using Prism software from GraphPad
and represent the concentration of inhibitor where the residual activity of the enzyme is 50%. Novobiocin (IC50
=
0,17 µM (lit. 0,08 µM)32 for E. coli gyrase and IC = 0,041 µM (lit. 0,01 µM) for S. aureus gyrase) was used
50
as internal control [61].
4
.2 Surface Plasmon Resonance (SPR) Measurements
Surface plasmon resonance measurements were performed on a Biacore (GE Healthcare) T100 instrument
using a versatile sensor chip for immobilization via -NH , -SH, -CHO, -OH or -COOH functional groups
2
(
0
CM5). The system was primed twice with a running buffer (10 mM Hepes, 150 mM NaCl, 3 mM EDTA,
.005% surfactant P20 (pH 7.4)). The first flow cell was activated with a carbodiimide (EDC) and N-
hydroxysuccinimide reagents and subsequently deactivated with a flow of ethanolamine. This cell served as a
reference cell for further measurements. Second flow cell was used for G24 protein immobilisation. In the
immobilisation protocol carboxymethylated dextran layer was activated with 7 min pulse of EDC and N-
hydroxysuccinimide mixed at 1:1 ratio. Protein was diluted to the final concentration of 50 µg/mL in 10 mM
sodium acetate (pH 4.5) and injected in two short pulses to reach the final immobilization level around 18.000
response units. Finally, the rest of the surface was deactivated with 7 min flow of ethanolamine. Analytes were
prepared as DMSO 100× stock solutions and were diluted with a running buffer prior to the injection. They
were injected at a flow rate of 30 µL/min for 90 s, and dissociation was monitored for additional 120 s. The
dissociation of analytes from the ligand was rapid and no regeneration protocol was needed. For the titration of
analytes, the 1% of the DMSO was added to the running buffer in order to diminish the difference in a refractive
index between the samples and running buffer. All analytes were tested in at least eight different concentrations
in three parallel titrations. The sensorgrams were analyzed using Biacore BiaEval software. The equilibrium
binding responses were determined from the binding levels 5 s before the stop of the injection. K values were
d
determined by the fitting of the data to 1:1 steady state binding model. The activity of the chip was tested and
confirmed using novobiocin as a standard [62].
Acknowledgments
This work was supported by the Slovenian Research Agency and by the EU FP7 Integrated Project MAREX
(
Project No. FP7-KBBE-2009-3-245137). We thank Dr. Dušan Žigon (Mass Spectrometry Center, Jožef Stefan
Institute, Ljubljana, Slovenia) for mass spectra. We also thank OpenEye Scientific Software, Santa Fe, New
Mexico, USA) for the academic license for their software solutions.
Conflict of Interest
The authors declare they have no conflict of interest.
Supplementary data

ACCEPTED MANUSCRIPT
Supplementary data related to this article can be found at http://
References
[
1] I.K. Murni, T. Duke, S. Kinney, A.J. Daley, Y. Soenarto, Reducing hospital-acquired infections and
improving the rational use of antibiotics in a developing country: an effectiveness study, Arch. Dis.
Child. (2014) 1 6.
2] D.M. Livermore, Bacterial Resistance: Origins, Epidemiology, and Impact, Clin. Infect. Dis. 36 (2003)
23.
–
[
11–
[
[
3] S.B. Levy, Antibiotic resistance—the problem intensifies, Drug Deliv. Rev. 57 (2005) 1446–1450.
4] J.F. Fisher, S.O. Meroueh, S. Mobashery, Bacterial Resistance to β-Lactam Antibiotics:ꢂ Compelling
Opportunism, Compelling Opportunity, Chem. Rev. (2005) 395–424.
[
[
5] W.D. Wright, Bacterial resistance to antibiotics: Enzymatic degradation and modification, Adv. Drug
Deliv. Rev. 57 (2005) 1451–1470.
6] A. Kumar, H.P. Schweizer, Bacterial resistance to antibiotics: Active efflux and reduced uptake, Drug
Deliv. Rev. 57 (2005) 1486–1513.
[
[
7] K. Lewis, Platforms for antibiotic discovery, Nat. Rev. Drug. Discov. 12 (2013) 371
8] Z. Gao, H. Li, H. Zhang, X. Liu, L. Kang, X. Luo, W. Zhu, K. Chen, X. Wang, H. Jiang, PDTD: a
web-accessible protein database for drug target identification, BMC Bioinformatics 9 (2008) 1 7.
9] E.D. Brown, G. D. Wright, New targets and screening approaches in antimicrobial drug discovery,
Chem. Rev. 105 (2005) 759 774.
10]T.F. Molinski, D.S. Dalisay, S.L. Lievens, J.P. Saludes, Drug development from marine natural
products, Nat. Rev. Drug Dis. 8 (2009) 69 85.
11]F. Collin, S. Karkare, A. Maxwell, Exploiting bacterial DNA gyrase as a drug target: current state and
perspectives, Appl. Microbiol. Biotechnol. 92 (2011) 479 497.
12]J. J. Champoux, DNA topoisomerases: Structure, function, and mechanism, Annu. Rev. Biochem. 70
2001) 369−413.
13]C. Sissi, M. Palumbo, In front of and behind the replication fork: bacterial type IIA topoisomerases,
Cell Mol. Life Sci. 67 (2010) 2001 2024.
14]J. S. Wolfson, D. C. Hooper, The fluoroquinolones: structures, mechanisms of action and resistance,
and spectra of activity in vitro, Antimicrob. Agents Chemother. 28 (1985) 581 586.
15]A. Maxwell, The interaction between coumarin drugs and DNA gyrase, Mol. Microbiol. 9 (1993) 681–
86.
16]M. Oblak, M. Kotnik, T. Solmajer, Discovery and Development of ATPase Inhibitors of DNA Gyrase
as Antibacterial Agents, Curr. Med. Chem. 14 (2007) 2033 2047.
17]T. Tomašić, L. Peterlin Mašič, Prospects for Developing New Antibacterials Targeting Bacterial Type
IIA Topoisomerases, Curr. Top. Med. Chem. 14 (2014) 130 151.
–387.
–
[
[
[
–
–
–
[
[
(
–
[
–
[
[
6
–
[
[
[
–
18]C. Mayer, Y.L. Janin, Non-quinolone Inhibitors of Bacterial Type IIA Topoisomerases: A Feat of
Bioisosterism, Chem. Rev. 114 (2015) 2313−2342.
19]G.S. Bisacchi, J.I. Manchester, A New-Class AntibacterialAlmost. Lessons in Drug Discovery and
Development: A Critical Analysis of More than 50 Years of Effort toward ATPase Inhibitors of DNA
Gyrase and Topoisomerase IV, ACS Infect. Dis. 1 (2015) 4−41.
[
20]F.T.F. Tsai, O.M.P. Singh, T. Skarzynski, A.J. Wonacott, S. Weston, A. Tucker, R.A. Pauptit, A.L.
Breeze, J.P. Poyser, R. Obrien, J.E. Ladbury, D.B. Wigley, The high-resolution crystal structure of a
2
4-kDa gyrase B fragment from E. coli complexed with one of the most potent coumarin inhibitors,
clorobiocin, Proteins 28 (1997) 41 52.
–
[
[
21]D. Lafitte, V. Lamour, P.O. Tsvetkov, A.A. Makarov, M. Klich, P. Deprez, D. Moras, C. Briand, R.
Gilli, DNA gyrase interaction with coumarin-based inhibitors: the role of the hydroxybenzoate
isopentenyl moiety and the 5'-methyl group of the noviose, Biochemistry 41 (2002) 7217
22]M. Schechner, F. Sirockin, R.H. Stote, A.P. Dejaegere, Functionality Maps of the ATP Binding Site of
DNA Gyrase B: Generation of a Consensus Model of Ligand Binding, J. Med. Chem. 47 (2004) 4373
390.
–7223.
–
4

ACCEPTED MANUSCRIPT
[
23]G.A. Holdgate, A. Tunnicliffe, W.H.J. Ward, S.A. Weston, G. Rosenbrock, P.T. Barth, I.W.F. Taylor,
R.A. Pauptit, D. Timms, The entropic penalty of ordered water accounts for weaker binding of the
antibiotic novobiocin to a resistant mutant of DNA gyrase: a thermodynamic and crystallographic
study. Biochemistry 36 (1997) 9663
24]V. Škedelj, T. Tomašić, L. Peterlin Mašič, A. Zega, ATP-binding site of bacterial enzymes as a target
for antibacterial drug design, J. Med. Chem. 54 (2011) 915 929.
–9673.
[
[
–
25]M. Oblak, S.G. Grdadolnik, M. Kotnik, R. Jerala, M. Filipič, T. Solmajer, In silico fragment-based
discovery of indolin-2-one analogues as potent DNA gyrase inhibitors, Bioorg. Med. Chem. Lett. 15
(
2005) 5207
26]M. Brvar, A. Perdih, M. Renko, G. Anderluh, D. Turk, T. Solmajer, Structure based discovery of
substituted 4,5'-bithiazoles as novel DNA gyrase inhibitors, J. Med. Chem. 55 (2012) 6413 6426.
–5210.
[
[
–
27]P.S. Charifson, A.L. Grillot, T.H. Grossman, J.D. Parsons, M. Badia, S. Bellon, D.D. Deininger, J.E.
Drumm, C.H. Gross, A. LeTiran, Y.S. Liao, N. Mani, D.P. Nicolau, E. Perola, S. Ronkin, D. Shannon,
L.L. Swenson, Q. Tang, P.R. Tessier, S.K. Tian, M. Trudeau, T.S. Wang, Y.Y. Wei, H. Zhang, D.
Stamos, Novel dual-targeting benzimidazole urea inhibitors of DNA gyrase and topoisomerase IV
possessing potent antibacterial activity: intelligent design and evolution through the judicious use of
structure guided design and stucture-activity relationships, J. Med. Chem. 51 (2008) 5243–5263.
[
[
[
[
28]J. Zhang, Q. Yang, J.B. Cross, J.A.C. Romero, K.M. Poutsiaka, F. Epie, D. Bevan, B. Wang, Y. Zhang,
A. Chavan, X. Zhang, T. Moy, A. Daniel, K. Nguyen, B. Chamberlain, N. Carter, J. Shotwell, J.
Silverman, C.A. Metcalf, D. Ryan, B. Lippa, R.E. Dolle, Discovery of Azaindole Ureas as a Novel
Class of Bacterial Gyrase B Inhibitors, J. Med. Chem. 58 (2015) 8503–8512.
29]J. Zhang, Q. Yang, J.A.C. Romero, J. Cross, B. Wang, K.M. Poutsiaka, F. Epie, D. Bevan, Y. Wu, T.
Moy, A. Daniel, B. Chamberlain, N. Carter, J. Shotwell, A. Arya, V. Kumar, J. Silverman, K. Nguyen,
C.A. Metcalf, D. Ryan, B. Lippa, R.E. Dolle, Discovery of Indazole Derivatives as a Novel Class of
Bacterial Gyrase B Inhibitors, Med. Chem. Lett. 6 (2015) 1080–1085.
30]V.U. Jeankumar, J. Renukaa, V.K. Pulla, V. Soni, J.P. Sridevi, P. Suryadevara, M. Shravana, R.
Medishetti, P. Kulkarni, P. Yogeeswari, D. Sriram, Development of novel N-linked aminopiperidine-
based mycobacterial DNA gyrase B inhibitors: Scaffold hopping from known antibacterial leads, Int. J.
Antimicrob. Ag. 43 (2014) 269–278.
31]B.A. Sherer, K. Hull, O. Green, G. Basarab, S. Hauck, P. Hill, J.T. Loch, G. Mullen, S. Bist, J. Bryant,
A. Boriack-Sjodin, J. Read, N. DeGrace, M. Uria-Nickelsen, R.N. Illingworth, A.E. Eakin,
Pyrrolamide DNA gyrase inhibitors: optimization of antibacterial activity and efficacy, Bioorg. Med.
Chem. Lett. 21 (2011) 7416–7420.
[
32]R.J. Lewis, O.M. Singh, C.V. Smith, T. Skarzynski, A. Maxwell, A.J. Wonacott, D.B. Wigley, The
nature of inhibition of DNA gyrase by the coumarins and the cyclothialidines revealed by X-ray
crystallography, EMBO J. 15 (1996) 1412–1420.
[
[
33]J.J. Champoux, DNA topoisomerases: Structure, function, and mechanism, Annu. Rev. Biochem. 70
(
2001) 369−413.
34]L.W. Tari, X. Li, M. Trzoss, D.C. Bensen, Z. Chen, T. Lam, J. Zhang, S.J. Lee, G. Hough, D.
Phillipson, S. Akers-Rodriguez, M.L. Cunningham, B.P. Kwan, K.J. Nelson, A. Castellano, J.B. Locke,
V. Brown-Driver, T.M. Murphy, V.S. Ong, C.M. Pillar, D.L. Shinabarger, J. Nix, F.C. Lightstone, S.E.
Wong, T.B. Nguyen, K.J. Shaw, J. Finn, Tricyclic GyrB/ParE (TriBE) inhibitors: a new class of broad-
spectrum dual-targeting antibacterial agents, PLoS One 8 (2013) 1–13.
[
[
35]L.W. Tari, M. Trzoss, D.C. Bensen, X. Li, Z. Chen, T. Lam, J. Zhang, C.J. Creighton, M.L.
Cunningham, B. Kwan, M. Stidham, K.J. Shaw, F.C. Lightstone, S.E. Wong, T.B. Nguyen, J. Nix, J.
Finn, Pyrrolopyrimidine inhibitors of DNA gyrase B (GyrB) and topoisomerase IV (ParE). Part I:
Structure guided discovery and optimization of dual targeting agents with potent, broad-spectrum
enzymatic activity, Bioorg. Med. Chem. Lett. 23 (2013) 1529–1536.
36]M. Trzoss, D.C. Bensen, X.M. Li, Z.Y. Chen, T. Lam, J.H. Zhang, C.J. Creighton, M.L. Cunningham,
B. Kwan, , M. Stidham, K. Nelson, V. Brown-Driver, A. Castellano, K.J. Shaw, F.C. Lightstone, S.E.
Wong, T.B. Nguyen, J. Finn, L.W. Tari, Pyrrolopyrimidine inhibitors of DNA gyrase B (GyrB) and
topoisomerase IV (ParE), Part II: development of inhibitors with broad spectrum Gram-negative
antibacterial activity, Bioorg Med. Chem. Lett. 23 (2013) 1537–1543.

ACCEPTED MANUSCRIPT
[
[
37]K. Mdluli, Z. Ma, Mycobacterium tuberculosis DNA Gyrase as a Target for Drug Dicovery, Infect.
Disord. Drug. Targets. 7 (2007) 1–10.
38]S. Saxena, J. Renuka, P. Yogeeswari, D. Sriram, Discovery of Novel Mycobacterial DNA Gyrase B
Inhibitors: In Silico and In Vitro Biological Evaluation, Mol. Inf. 33 (2014) 597–609.
[
[
39]L.L. Silver, Challenges of Antibacterial Discovery, Clin. Microbiol. Rev. 24 (2011) 71–109.
40]M. Jukič, R. Frlan, F. Chan, R.W. Kirby, D.J. Madge, J. Tytgat, S. Peigneur, M. Anderluh, D. Kikelj,
Synthesis and biological evaluation of piperazine derivatives as novel isoform selective voltage-gated
sodium (Na ) 1.3 channel modulators, Med. Chem. Res. 24 (2015) 2366–2380.
41]R.P. Walker, D.J. Faulkner, D. Van Engen, J. Clardy, Sceptrin, an antimicrobial agent from the sponge
Agelas sceptrum, J. Am. Chem. Soc. 103 (1981) 6772–6773.
42]P.A. Keifer, R.E. Schwartz, M.E.S. Koker, R.G. Hughes Jr, D. Rittschof, K.L. Rinehart, Bioactive
bromopyrrole metabolites from the Caribbean sponge Agelas conifer, J. Org. Chem. 56 (1991) 2965–
V
[
[
2
975.
43]M. S. Laport, O.C.S. Santos, G. Muricy, Marine Sponges: Potential Sources of New Antimicrobial
Drugs, Curr. Pharm. Biotechnol. 10 (2009) 86 105.
[
[
–
44]N. Zidar, S. Montalvao, Ž. Hodnik, D.A. Nawrot, A. Žula, J. Ilaš, D. Kikelj, P. Tammela, L. Peterlin
Mašič, Antimicrobial activity of the marine alkaloids, clathrodin and oroidin, and their synthetic
analogues, Mar. Drugs 12 (2014) 940–963.
[
[
45]T. Tomasic, S. Katsamakas, Ž. Hodnik, J. Ilaš, M. Brvar, T. Solmajer, S. Montalvao, P. Tammela, M.
Banjanac, G. Ergovic, M. Anderluh, L. Peterlin Mašič, D. Kikelj, Discovery of 4,5,6,7-
Tetrahydrobenzo[1,2-d]thiazoles as Novel DNA Gyrase Inhibitors Targeting the ATP-Binding Site, J.
Med. Chem. 58 (2015) 5501–5521.
46]N. Zidar, H. Macut, T. Tomasic, M. Brvar, S. Montalvão, P. Tammela, T. Solmajer, L. Peretlin Masic,
J. Ilaš, D. Kikelj, N-Phenyl-4,5-dibromopyrrolamides and N-Phenylindolamides as ATP competitive
DNA Gyrase B Inhibitors: Design, Synthesis, and Evaluation, J. Med. Chem. 58 (2015) 6179
47]D.A. Erlanson, R.S. McDowell, T. O’Brien, Fragment-Based Drug Discovery, J. Med. Chem. 47
2004) 3463 3482.
48]R.A. Laskowski, M. B. Swindells, LigPlot+: multiple ligand-protein interaction diagrams for drug
discovery, J. Chem. Inf. Model. 51 (2011) 2778 2786.
49]L. Brino, A. Urzhumtsev, M. Mousli, C. Bronner, A. Mitschler, P. Oudet, D. Moras, Dimerization of
Escherichia coli DNA-gyrase B provides a structural mechanism for activating the ATPase catalytic
center, J. Biol. Chem. 275 (2000) 9468–9475.
–6194.
[
[
[
(
–
–
[
[
50]H. Yu, S.W. Rick, Free energies and entropies of water molecules at the inhibitor−protein interface of
DNA gyrase, J. Am. Chem. Soc. 131 (2009) 6608−6613.
51]J. Konc, D. Janezic, ProBiS–2012: web server and web services for detection of structurally similar
binding sites in proteins, Nucleic. Acids. Res. 40 (2012) W214–W221.
52]The PyMOL Molecular Graphics System, Version 1.7.6, Schrödinger, LLC.
53]S.L. McGovern, B.T. Helfand, B. Feng, B.K. Shoichet, A Specific Mechanism of Nonspecific
[
[
Inhibition, J. Med. Chem. 46 (2003) 4265
54]M. McGann, FRED and HYBRID docking performance on standardized datasets, J. Comp.-Aid. Mol.
Design 26 (2012) 897 906.
55]M. McGann, FRED Pose Prediction and Virtual Screening Accuracy, J. Chem. Inf. Model. 51 (2011)
78 596.
56]M. McGann, H.R. Almond, A. Nicholls, J.A. Grant, F.K. Brown, Gaussian docking functions.
Biopolymers, 68 (2003) 76 90.
–4272.
[
[
[
[
–
5
–
–
57]N. Thota, M.V. Reddy, A. Kumar, I.A. Khan, P.L. Sangwan, N.P. Kalia, J.L. Koul, S. Koul.
Substituted dihydronaphthalenes as efflux pump inhibitors of Staphylococcus aureus. Eur. J. Med.
Chem. 45 (2010) 3607–3616.
[
[
58]H. Nikaido, J.-M. Pagès. Broad-specificity efflux pumps and their role in multi drug resistance of
Gram-negative bacteria. FEMS Microbiol. Rev. 36 (2012) 340–363.
59]K. Mdluli, Z. Ma. Mycobacterium tuberculosis DNA gyrase as a target for drug discovery. Infect.
Disord. Drug Targets 7 (2007) 159-168.

ACCEPTED MANUSCRIPT
[
[
60]S. Chopra, K. Matsuyama, T. Tran, J.P. Malerich, B. Wan, S.G. Franzblau, S. Lun, H. Guo, M.C.
Maiga, W.R. Bishai, P.B. Madrid. Evaluation of gyrase B as a drug target in Mycobacterium
tuberculosis. J. Antimicrob. Chemother. 67 (2012) 415-421.
61]S. Alt, L.A. Mitchenall, A. Maxwell, L. Heide, Inhibition of DNA gyrase and DNA topoisomerase IV
of Staphylococcus aureus and Escherichia coli by aminocoumarin antibiotics, J. Antimicrob. Chemoth.
6
6 (2011) 2061
62]S.C. Kampranis, N.A. Gormley, R. Tranter, G. Orphanides, A. Maxwell, Probing the binding of
coumarins and cyclothialidines to DNA gyrase, Biochemistry 38 (1999) 1967 1976.
–2069.
[
–

ACCEPTED MANUSCRIPT
Linker-switch approach towards new ATP binding site inhibitors of DNA Gyrase B
1
1
2
1
1*
1*
Marko Jukič , Janez Ilaš , Matjaž Brvar , Danijel Kikelj , Jožko Cesar and Marko Anderluh
1
University of Ljubljana, Faculty of Pharmacy, Department of Medicinal Chemistry, Aškerčeva cesta 7, 1000
Ljubljana, Slovenia
2
National Institute of Chemistry, Laboratory for Biocomputing and Bioinformatics, Hajdrihova ulica 19, 1001
Ljubljana, Slovenia
*
Corresponding authors: Jožko Cesar and Marko Anderluh, University of Ljubljana, Faculty of Pharmacy,
Department of Pharmaceutical Chemistry, Aškerčeva 7, 1000 Ljubljana, Slovenia, Tel: +386-1-47-69-639, Fax:
+
386-1-425-80-31, E-mail: jozko.cesar@ffa.uni-lj.si; marko.anderluh@ffa.uni-lj.si.
Highlights:
•
•
DNA Gyrase B inhibitors with three types of sp3-rich scaffolds were prepared
Key interactions with E. coli DNA Gyrase B binding site are proposed, involving a
conserved water
•
•
•
High-nanomlar inhibitor of DNA Gyrase B is reported
A robust model for in-silico design of DNA Gyrase B inhibitors is reported
A novel approach towards conserved water identification was employed