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

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

DOI:

10.1016/j.ejmech.2016.09.040

Reference:

EJMECH 8906

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.

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Graphical abstract

ACCEPTED MANUSCRIPT Linker-switch approach towards new ATP binding site inhibitors of DNA Gyrase B Marko Jukič1, Janez Ilaš1, Matjaž Brvar2, Danijel Kikelj1, Jožko Cesar1* and Marko Anderluh1* 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 *

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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: [email protected]; [email protected].

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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 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-2carboxamides while predicting unfavourable interactions with the active site for piperazine pyrrole-2carboxamides. 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-dibromo1H-pyrrole-2-carboxamide)cyclohexyl)glycine and 4-(4-(3,4-dichloro-5-methyl-1H-pyrrole-2carboxamido)piperidin-1-yl)-4-oxobutanoic acid with an IC50 values of 16 and 0.5 µM respectively. Keywords: antibiotics, antibacterials, inhibitor, DNA Gyrase B, ATP binding site, pyrrole-2-carboxamides, structure-based drug design, ligand-based drug design

1.

Introduction

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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

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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]].

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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]].

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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 highresolution 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

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2.1 Computational studies and design

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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-2carboxamide 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 that employ a sterically rigid linker between pyrrole-2-carboxamide and acidic group, we focused on simple sp3rich 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]]. b)

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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.

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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 (Figure 4, a, b) for further studies [[26]]. Docking protocol validation was successful with an RMSD value of 0.16 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]].

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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 12GyrB 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]].

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

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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.

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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 4DUH) [[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.

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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: 4DUH; 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

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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). b)

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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).

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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).

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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).

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Visual inspection of MD trajectories corroborates binding mode predicted by docking studies where pyrrole-2carboxamide 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 9, a) adopts a conformation that is kinked and interacts with diphosphate binding site. Interaction diagrams 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 13 (Figure 9, a) buries halogenated pyrrole in adenine pocket too, but is kinked towards diphosphate binding 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.

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2.3 Chemistry

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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-(3dimethylaminopropyl)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.

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Scheme 1. Reagents and conditions: (a) EDC, HOBt, NMM, DMF, 0 °C → r.t., 16 h; (b) H2, Pd/C, AcOHgl, MeOH, THF, r.t., 4h; (c) methyl malonyl chloride, DIPEA, CH2Cl2, 0 °C → r.t., 3 h; (d) LiOH, THF, H2O, r.t., 4h.

Compounds bearing piperidin-1-yl pyrrole-2-carboxamide and indole-2-carboxamide scaffolds 28, 29, 30 and 31 were synthesized according to the Scheme 2. N1-BOC protected 4-aminopiperidine 23 was alkylated via 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.

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Scheme 2. Reagents and conditions: (a) benzaldehyde, sodium triacetoxyborohydride (Na(Ac)3BH), CH2Cl2, 0 °C → r.t., 1 h (b) allyl bromide, K2CO3, CH2Cl2, 0 °C → r.t., 15 h; (c) EDC, HOBt, NMM, DMF, 0 °C → r.t., 16 h; (d) Trifluoroacetic acid (TFA), CH2Cl2, 50 °C, 3 h; (e) acid fragment, TBTU, NMM, CH2Cl2, DMF, r.t. → 40 °C , 16 h; (f) acyl chloride, DIPEA or Et3N, CH2Cl2, 0 °C → r.t., 1 h; (g) alkyl halogenide, Cs2CO3, Acetonitrile (CH3CN), r.t., 5 h; (h) LiOH, THF, H2O, r.t., 2-8 h.

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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 (SOCl2), MeOH, 40 °C, 2h; (b) EDC, HOBt, NMM, DMF, 0 °C → r.t., 16 h; (c) LiOH, THF, H2O, r.t., 2-8 h; (d) amino acid methyl ester, TBTU, NMM, CH2Cl2, DMF, r.t. → 40 °C , 16 h.

2.4 DNA gyrase B inhibition assays

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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

compound

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R2

20 21

22d 22e

DNA Gyrase S. aureus 100 % 0.041

78 %

n.t.

1080 µM

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100 %

n.t.

100 %

n.t.

100 %

n.t.

100 %

n.t.

100%

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Kd determined by surface plasmon resonance; b Residual activity of the enzyme at 100 µM of the tested compound. Novobiocin was used as a positive control (IC50 = 0.17 µM on GyrB from E. coli and IC50 = 0.04 µM on GyrB from S. aureus); c neg – control 1% DMSO; d NB – novobiocin as a positive control. n.a. – non active; n.t. – not tested.

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Biological evaluation of compounds with piperazin-1-yl and piperazin-1-yl pyrrole-2-carboxamide scaffold

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 IC50 in the low micromolar range. 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 Kd value of 12.6 µM. Exchange of aliphatic terminal fragments with sterically rigid methyl 4-methylbenzoate 29b, 31b or methyl 4carbonylbenzoate moieties 29a, 31a afforded free acids that displayed potency in the low micromolar range.

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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 IC50s in low micromolar range. Next, we examined dibromopyrrole exchange and noticed a 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-1Hpyrrol-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

compound

R1

R2

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Biological evaluation of compounds with piperidin-4-yl pyrrole-2-carboxamide scaffold

IC50 or (Kd) a [µM] or RAb [%]

DNA Gyrase E. coli 100 % 0.17

DNA Gyrase S. aureus 100 % 0.041

>50 µM

n.t.

4.35 µM

74 %

49.2 µM

n.t.

13.7 µMe

n.t.

10.8 µM

80 %

20.9 µM

n.t.

1.55 µM (SPR 12.6 µM)

93.8 µM

11.2 µM

n.t.

3.18 µM

71 %

100 %

n.t.

29a

480 µMf

n.t.

31a

14.9 µM (SPR 256 µM)

84 %

29b

100 %

n.t.

M AN U

negc NBd 25 28a

28c 30c 28d 30d 27a

EP

30b

AC C

28b

TE D

30a

ACCEPTED MANUSCRIPT 63.1 µM (SPR *)

100 %

29c

100 %

n.t.

31c

49.5 µM (SPR 11.2 µM)

100 %

27b

2.84 µM

61.4 µM

27c

8.08 µM

28e

20.0 µM

30e

0.48 µM

28f

100 %

RI PT

31b

n.t.

SC

n.t.

n.t.

M AN U

n.t.

100 %

30f

Kd determined by surface plasmon resonance; b Residual activity of the enzyme at 100 µM of the tested compound. Novobiocin was used as a positive control (IC50 = 0.17 µM on GyrB from E. coli and IC50 = 0.04 µM on GyrB from S. aureus); c neg – control 1% DMSO; d NB – novobiocin as a positive control; e Primary test result against E. coli (ATCC 25922) is 10,2 % inhibition after 24h (50 µM); f Primary test result against E. coli (ATCC 25922) is 11,5 % inhibition after 24h (50 µM). n.a. – non active; n.t. – not tested; * non-specific binding.

TE D

a

n.t.

Table 3

compound

EP

Biological evaluation of compounds with piperidin-4-yl indoloamide scaffold and bulky pyrrole-2-carboxamides

R1

R2

IC50 or (Kd) a [µM] or RAb [%]

DNA Gyrase S. aureus 100 % 0.041

100 %

n.t.

88 %

n.t.

31d

100 %

n.t.

29e

100 %

n.t.

31e

100 µM

n.t.

26

29d

AC C

DNA Gyrase E. coli 100 % 0.17

negc NBd

ACCEPTED MANUSCRIPT 100 %

n.t.

31f

100 %

n.t.

27d

150 µM

n.t.

29g

100 %

n.t.

31g

100 %

29h

100 %

31h

100 %

29i

36 %

RI PT

29f

n.t.

SC

n.t.

M AN U

n.t.

31i

29j

29 %

n.t.

100 %

n.t.

49.5 µM (SPR 11.2 µM)

n.t.

TE D

31j

n.t.

a

Kd determined by surface plasmon resonance; b Residual activity of the enzyme at 100 µM of the tested compound. Novobiocin was used as a positive control (IC50 = 0.17 µM on GyrB from E. coli and IC50 = 0.04 µM on GyrB from S. aureus); c neg – control 1% DMSO; d NB – novobiocin as a positive control. n.a. – non active; n.t. – not tested; * non-specific binding.

Table 4

AC C

EP

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 IC50 of 15.1 µM.

Biological evaluation of 4-cyclohexyl pyrrole-2-carboxamides

compound

scaffold

Negb NBc 35a

cis

R2

IC50 [µM] or RAa [%]

DNA Gyrase E. coli 100 % 0.17

DNA Gyrase S. aureus 100 % 0.041

100 %

n.t.

ACCEPTED MANUSCRIPT cis

100 %

n.t.

35b

trans

100 %

n.t.

36b

trans

15.1 µM

n.t.

35c

cis

100 %

n.t.

36c

cis

100 µM

n.t.

35d

trans

100 %

36d

trans

100 %

35e

trans 2

100 %

36e

trans 2

100 %

35f

trans 2

100 %

36f

trans 2

RI PT

36a

n.t. n.t.

M AN U

SC

n.t.

100 %

a

n.t. n.t. n.t.

Residual activity of the enzyme at 100 µM of the tested compound. Novobiocin was used as a positive control (IC50 = 0.17 µM on GyrB from E. coli and IC50 = 0.04 µM on GyrB from S. aureus); b neg – control 1% DMSO; c NB – novobiocin as a positive control. n.a. – non active; n.t. – not tested; * non-specific binding.

Conclusions

AC C

3.

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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].

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 key structural features and the most suitable linker for in vitro inhibition of E. coli DNA gyrase B. The sp3-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

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Potency evaluation and determination of half maximal inhibitory concentrations (IC50) on E. coli and S. 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 IC50 = 0,041 µM (lit. 0,01 µM) for S. aureus gyrase) was used as internal control [61]. 4.2 Surface Plasmon Resonance (SPR) Measurements

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Surface plasmon resonance measurements were performed on a Biacore (GE Healthcare) T100 instrument using a versatile sensor chip for immobilization via -NH2, -SH, -CHO, -OH or -COOH functional groups (CM5). The system was primed twice with a running buffer (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20 (pH 7.4)). The first flow cell was activated with a carbodiimide (EDC) and Nhydroxysuccinimide 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 Nhydroxysuccinimide 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. Kd values were 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

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[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) 11–23. [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:  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) 371–387. [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– 686. [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. [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 24-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) 7217–7223. [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– 4390.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

[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) 9663–9673. [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. [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) 5207–5210. [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. [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 aminopiperidinebased 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 broadspectrum 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

AC C

EP

TE D

M AN U

SC

RI PT

[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 (NaV) 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– 2975. [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 4,5,6,7Tetrahydrobenzo[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) 6179–6194. [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. [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) 4265–4272. [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) 578–596. [56] M. McGann, H.R. Almond, A. Nicholls, J.A. Grant, F.K. Brown, Gaussian docking functions. Biopolymers, 68 (2003) 76–90. [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

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[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. 66 (2011) 2061–2069. [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.

ACCEPTED MANUSCRIPT Linker-switch approach towards new ATP binding site inhibitors of DNA Gyrase B

Marko Jukič1, Janez Ilaš1, Matjaž Brvar2, Danijel Kikelj1, Jožko Cesar1*and Marko Anderluh1*

1

University of Ljubljana, Faculty of Pharmacy, Department of Medicinal Chemistry, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia 2

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National Institute of Chemistry, Laboratory for Biocomputing and Bioinformatics, Hajdrihova ulica 19, 1001 Ljubljana, Slovenia

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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: [email protected]; [email protected].

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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

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