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Tuberculosis 90 (2010) 354e360

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Tuberculosis

journal homepage: http: / / int l .e lsevierhealth.com/journals / tube

DRUG DISCOVERY AND RESISTANCE

Leads for antitubercular compounds from kinase inhibitor library screens

Sophie Magnet a,b,h, j, Ruben C. Hartkoorn a,b,h, j, Rita Székely b,c,h, János Pató b,c, James A. Triccas a,i,Patricia Schneider a,b, Csaba Szántai-Kis b,c, László }Orfi b,c, f, Marc Chambon d, Damiano Banfi d,Manuel Bueno d, Gerardo Turcatti d, György Kéri b,c,e,f,g, Stewart T. Cole a,b,*

aGlobal Health Institute, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, SwitzerlandbNew Medicines for Tuberculosis (NM4TB) Consortium, SwitzerlandcVichem Chemie Research Ltd., Herman Ottó. 15, H-1022 Budapest, HungarydBiomolecular Screening Facility, Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerlande Semmelweis University, Department of Medical Chemistry, Pathobiochemistry Research Group of Hung.Acad.Sci. H-1085 T}uzoltó u. 37, Budapest, Hungaryf Semmelweis University, Department of Pharmaceutical Chemistry, H-1092 H}ogyes Endre u. 9, Budapest, Hungaryg Semmelweis University, Rational Drug-Design Laboratory CRC, H-1085 Üll}oi út. 26, Budapest, Hungary

a r t i c l e i n f o

Article history:Received 17 June 2010Received in revised form20 August 2010Accepted 7 September 2010

Keywords:TuberculosisScreeningKinase inhibitorQuinoxalineSerine/threonine protein Kinase (STPK)DprE1

* Corresponding author. EPFL, SV-GHI-UPCOL, StaSwitzerland. Tel.: þ41 21 693 1851; fax: þ41 21 693 1

E-mail addresses: sophie.magnet@epfl.ch (S. Mag(R.C. Hartkoorn), rita.szekely@vichem.hu (R. Székely), jj.triccas@usyd.edu.au (J.A. Triccas), patricia.schneider@szantai-kis@vichem.hu (C. Szántai-Kis), laszlo.orfi@chambon@epfl.ch (M. Chambon), damiano.banfi@epfl.epfl.ch (M. Bueno), gerardo.turcatti@epfl.ch (G. Turcattistewart.cole@epfl.ch (S.T. Cole).

h These authors contributed equally.i Permanent address: Discipline of Medicine, U

Australia.j www.nm4tb.org.

1472-9792/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.tube.2010.09.001

s u m m a r y

Discovering new drugs to treat tuberculosis more efficiently and to overcome multidrug resistance isa world health priority. To find antimycobacterial scaffolds, we screened a kinase inhibitor library of morethan 12,000 compounds using an integrated strategy involving whole cell-based assays with Coryne-bacterium glutamicum and Mycobacterium tuberculosis, and a target-based assay with the protein kinasePknA. Seventeen “hits” came from the whole cell-based screening approach, from which three displayedminimal inhibitory concentrations (MIC) against M. tuberculosis below 10 mM and were non-mutagenicand non-cytotoxic. Two of these hits were specific for M. tuberculosis versus C. glutamicum and none ofthem was found to inhibit the essential serine/threonine protein kinases, PknA and PknB present in bothbacteria. One of the most active hits, VI-18469, had a benzoquinoxaline pharmacophore while another,VI-9376, is structurally related to a new class of antimycobacterial agents, the benzothiazinones (BTZ).Like the BTZ, VI-9376 was shown to act on the essential enzyme decaprenylphosphoryl-b-D-ribose 20-epimerase, DprE1, required for arabinan synthesis.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

While short course chemotherapy for drug-susceptible tuber-culosis (TB) remains effective, it is relatively inefficient by modernpharmaceutical standards and universally menaced by the disse-mination of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains ofMycobacterium tuberculosis.1 Thus, as first

tion 19, CH-1015 Lausanne,790.net), ruben.hartkoorn@epfl.chanos.pato@vichem.hu (J. Pató),epfl.ch (P. Schneider), csaba.vichem.hu (L. }Orfi), marc.ch (D. Banfi), manuel.bueno@), gykeri@vichem.hu (G. Kéri),

niversity of Sydney, NSW,

All rights reserved.

declared seventeen years ago by the World Health Organization,finding new drugs to treat TB is an urgent priority.

During the last ten years, pharmaceutical companies have basedtheir strategy on target-based approaches that have met with verypoor outcomes2,3 as all TB drug candidates that are currently inclinical trials were first selected on the basis of their antibacterialactivity.4e8 In consequence, whole cell-based screening approachesare being reconsidered as a means of identifying new active scaf-folds2,9 as well as finding new targets.10 In other therapeutic areas,such as oncology, medicinal chemistry efforts have resulted in vastkinase inhibitor libraries that could be used as a source of newantimycobacterial agents. Among the resources available, theNested Chemical Library� (NCL) of Vichem Chemie Ltd. containsinhibitors of more than 100 eukaryotic kinases including EGFR,PDGFR, VEGFR, SRC, PLK, or JNK.11

As in eukaryotes, signaling pathways in prokaryotes are alsocontrolled by protein kinases. Strikingly, actinobacteria contain anunusually high proportion of Serine/threonine Protein Kinases(STPK) compared to two component systems.12 In M. tuberculosisthree conserved STPK were considered as attractive drug targets:PknG, required for intracellular bacterial survival in the host,13,14

S. Magnet et al. / Tuberculosis 90 (2010) 354e360 355

and two essential transmembrane receptor-kinases involved in theregulation of cell division, PknA and PknB.15,16

Attempts to identify inhibitors of mycobacterial STPK havefocused on target-based approaches using PknB and PknG.14,17,18

Recently, a sub-library of the NCL was screened against PknB andgave hits with nanomolar median inhibitory concentration(IC50).19,20 However, none of the kinase inhibitors identified so farinhibits M. tuberculosis growth (MIC >50 mM).19,20 The lack ofcorrelation between the IC50 and the MIC values is not understood.The compounds may not cross the complex mycobacterial cell wallor the accessibility of the inhibitor binding site may be different invivo. On the other hand, a few examples of compounds derivedfrom protein kinase pharmacophores have been shown to inhibitnon-kinase antibacterial targets that use ATP as a cofactor, such asD-alanine-D-alanine ligase 21 or biotin carboxylase kinase.22 Thus,kinase inhibitor libraries can potentially be a source of inhibitors fora wide range of bacterial enzymes.

In this study we screened two kinase inhibitor libraries with theaim of identifying new scaffolds active against M. tuberculosis. TheNCL was screened by an extensive in vitro antibacterial assay withCorynebacterium glutamicum, which has orthologs of PknA, PknBand PknG, and by a more limited screen with M. tuberculosis, inconjunction with an inhibition assay of PknA activity, an STPKwhich has been unexplored thus far. Comparison of the datasets ledto the identification of new scaffolds effective against M. tubercu-losis, without correlation with STPK inhibition.

2. Materials and methods

2.1. Compounds

The compounds tested during this work belong to the NestedChemical Library� (NCL) of Vichem Chemie Research Ltd (Buda-pest).11 The NCL contains 12,100 compounds (organized on 108 corestructures and more than 500 scaffolds), of which most are smallmolecule ATP-binding site inhibitors. The Extended ValidationLibrary (EVL) is a sub-libraryof theNCL,which contains around1000proven kinase inhibitor leads. These molecules represent a broadspectrum of potentially active chemical structures. The log P values(logarithm of the ratio of the concentrations of un-ionized solute insolvents) were calculated with JChem for Excel v1.1.2 (Chemaxon).

2.2. Bacterial strains and growth conditions

Escherichia coli BL21(DE3)pLysS and E. coli PQ3723 were grown at37 �C in Luria broth (LB) supplemented with chloramphenicol30 mg/ml and ampicillin 50 mg/ml, respectively. C. glutamicum(ATCC 13032) was grown in LB at 30 �C. Mycobacterium smegmatismc2155 was grown in LB containing 0.05% tween 80 at 37 �C.Mycobacterium bovis BCG Pasteur and M. tuberculosis H37Rv weregrown in Middlebrook 7H9 supplemented with 10% ADC enrich-ment (albumin, dextrose, catalase; Becton Dickinson), 0.05% tween80, and 0.2% glycerol. The benzothiazinone (BTZ)-resistant mutantsM. smegmatisMN47,M. smegmatisMN84,M. bovis BCG Pasteur BN2were obtained from Prof. G. Riccardi (Pavia, Italy).10 PS1, a BTZ-resistant mutant was obtained by plating C. glutamicum ATCC13032 wild type on LB-agar plates containing BTZ043 (10 mg/ml)and incubating at 30 �C for two weeks.

2.3. Antibacterial assay

The in vitro activity of compounds (10 mM) against C. glutamicum(ATCC 13032) and M. tuberculosis H37Rv was determined using theresazurin reduction microtiter assay (REMA) as previouslydescribed.24 Single dose drug susceptibility of C. glutamicum was

determined in a 384 well plate format (20 mL assay) using clear flatbottom polystyrene microplates (Corning). Compounds weremixed with an exponential phase culture diluted to OD600 nm of0.001 and incubated overnight at 30 �C. Resazurin was added (2 mLof 0.025% w/v) and plates were incubated for 4 h. Bacterial growthwas determined following resazurin reduction monitored by fluo-rescence (excitation 570 nm, emission 590 nm). Single dose drugsusceptibility of M. tuberculosis was determined similarly, but ina 96 well plate format (100 mL assay) in clear flat bottom poly-styrene microplates (Corning). To prevent evaporation plates weresealed with an adhesive polyester film. After 7 days incubation at37 �C, resazurin was added and incubated for 20 h at 37 �C beforefluorescence reading. Compounds were dispensed with a Zephyr�

Liquid Handling Workstation (Caliper LifeSciences). Bacteria andresazurin were added either manually, in the case ofM. tuberculosisor using an automated liquid dispenser (EL406, BioTek), in the caseof C. glutamicum. Fluorescence was measured with a Tecan Infinite500 microplate reader using the bottom measurement mode.C. glutamicum REMA plates contained 32 positive (rifampin, 1 mM)and 32 negative (no drug) control wells, whilst M. tuberculosisREMA plates contained 8 wells of each control.

2.4. MIC determination

MIC, defined as the minimum concentration of drug thatinhibits more than 99% of growth of a liquid culture with an initialOD600 nm¼ 0.001, was determined in transparent 96 well plates(Corning) using REMA. Compounds were serially diluted two-foldfrom 200 to 0.62 mM for the molecules selected from the PknAkinase assay screen, or from 100 to 0.16 mM for the moleculesselected for their antibacterial activity. A rifampin control (1 mM to1 nM) was included in every plate.

2.5. Genotoxicity assay

The genotoxicity of the compounds was evaluated by the SOSchromotest on LB-agar plate.23 This colorimetric assay is based onthe induction of the SOS function SfiA by DNA damaging agents,whose level of expression is monitored by means of a sfiA::lacZoperon fusion in E. coli PQ37. Briefly, compounds (10 mL of a 10 mMsolution) were spotted on a LB-agar plate containing ampicillin50 mg/ml, 0.006% bromo-chloro-indolyl-galactopyranoside (X-Gal)and E. coli PQ37 at OD600 nm¼ 0.04. Isoniazid and 4-nitroquinolineN-oxide were used as negative and positive controls, respectively.Genotoxicity of compounds was evaluated colorimetrically.

2.6. Cytotoxicity assay

The compound cytotoxicity was investigated by REMA on twodistinct human cell lines: the human lung epithelial cell line; A549,and the human leukemic monocyte cell line; THP-1. Cells wereseeded in 48 well plates (2�104 cells per well, in 500 mL of DMEMcontaining 10% Fetal Bovine Serum) in the presence of increasingconcentrations of compound (from 1.5 to 100 mM), for 3 days at37 �C with 5% CO2. To determine THP-1 viability, resazurin wasadded directly to the cell culture (final concentration¼ 0.0025%),whilst for A549 viability determination media was aspirated andphosphate buffered saline containing resazurin (0.0025%) wasadded. Plateswere incubated for 2more hours and cell viability wasdetermined by fluorescence (excitation 570 nm, emission 590 nm).Assays were performed in duplicate and the minimal cytotoxicconcentration (MCC) defined as the minimal drug concentrationassociated with decrease in fluorescence or major morphologicalteration was determined.

S. Magnet et al. / Tuberculosis 90 (2010) 354e360356

2.7. Protein purification

Truncated versions of the kinases PknA and PknB fromM. tuberculosis H37Rv were produced with an N-terminal His6-tagin E. coli BL21(DE3)pLysS. The catalytic domain of PknB (residues1e279) was produced and purified as previously described.25 Toproduce PknA (residues 1e336, including the catalytic domain plusthe juxtamembrane domain), BL21(DE3)pLysS harboringpET28UpknA was grown in LB containing kanamycin 50 mg/ml andchloramphenicol 30 mg/ml at 30 �C. Expression of pknA wasinduced at OD600 nm¼ 0.6 with 1 mM isopropyl b-D-1-thio-galactopyranoside (IPTG) for 3.5 h at 30 �C. Cells were lysed bysonication and the recombinant protein purified from the solubleextract in two steps using fast protein liquid chromatography(FPLC). The protein was first captured by affinity chromatographyon a Ni-NTA column, eluted with an imidazole gradient(0e500 mM) and further purified by size exclusion on a Super-dex75 column. The protein was stored at �20 �C in 12.5 mM HEPESpH¼ 8, 250 mM NaCl, 0.5 mM dithiothreitol (DTT) and 50% glyc-erol. Recombinant GarA protein was purified by affinity chroma-tography and size exclusion as previously described,26 for use asa kinase substrate. A third additional step of purification was per-formed by affinity chromatography using TALON� metal affinityresin (Clontech) to remove traces of a protein contaminant thatinterferes with fluorescent kinase assays.

2.8. Fluorescent kinase assay

The fluorescent kinase assays monitor the formation of ADP bymeans of fluorescence changes. The ADP detection step was carriedout either by homogenous time resolved fluorescence (HTRF) usingthe HTRF� Transcreener ADP kit (Cisbio) or by fluorescence polar-ization (FP) using the Transcreener� ADP2 FP kit (BellBrooks Labs).These detection methods are based on an antibody specific for ADPwhich competes for both native ADP produced by the kinasereaction and a fluorophore-coupled ADP present in the reactionmixture. The assays were carried out in duplicate, in black, lowvolume, non-binding surface, 384 well plates (Corning). In the caseof PknA, the kinase reactionwas performed in 10 mL reaction bufferA (20 mM HEPES pH¼ 7.5, 1 mM DTT, 1 mM MnCl2, 5% glycerol,0.01% Brij35) containing 1% dimethyl sulfoxide (DMSO), 1.5 mMPknA, 40 mM GarA, 45 mM ATP (Km value) and compounds (10 mM),and was incubated at 22 �C for 2 h. For PknB, the kinase reactionwas performed in 10 mL buffer B (50 mM HEPES pH¼ 7, 1 mM DTT,0.5 mM MnCl2, 5% Glycerol, 0.01% Brij35) containing 1% DMSO,1 nM PknB, 2.25 mM ATP (Km value) and 10 mM GarA, and wasincubated at 22 �C for 30 min. Two negative control columns (nocompound) and two positive control columns (containing 50 mMEDTA) were included in every assay plate. For the screen,compounds (2.5 mL at 40 mM) were dispensed with a Zephyr�

Liquid Handling Workstation (Caliper LifeSciences), enzyme andsubstrates were added successively in two manual steps, whilebuffer and detection reagents were added using a EL406 dispenser(BioTek). The HTRF signal was measured with a Tecan Infinite 500microplate reader and the FP signal with an Analyst GT microplatereader (Molecular Devices).

2.9. Screening data analysis

The quality of the assay was assessed by determining the Z0-factor (Z0 ¼ 1� (3� SD of positive controlþ 3� SD of negativecontrol)/jmean of positive control-mean of negative controlj) for anassay plate containing half negative control wells and half positivecontrol wells (38). The single dose results were expressed as theratio of the signal difference between a given compound and the

negative control and the signal difference between the positivecontrol and the negative control [(SCPD� SDMSO)/(Spositive� SDMSO)].The final score corresponds to the mean value of the duplicates.Compounds for which the variability between the two duplicateswas greater than 3-fold the standard deviation determined for thenegative control were not taken into account.

2.10. Radiometric kinase assay

The radiometric assay was carried out in 10 mL final volume ofreaction buffer (20 mMHepes pH¼ 7.5, 1 mM DTT, 1 mMMnCl2, 5%Glycerol, 0.01% Brij35) containing 1% DMSO, 45 mM ATP including 3mCi of [g-33P]ATP, 40 mMGarA and 10 mMor 100 mM compound. Thereaction was initiated by the addition of 1.5 mM PknA and stoppedafter 2 hours incubation at room temperature by the addition of50 mM EDTA. The samples were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE) on 4e12%gradient gels and, after drying, the incorporation of radiolabelledphosphate into GarA was visualized and quantified with a Phos-phorImager system (Storm, Molecular Dynamics) and the softwareImage Quant. The negative control contained no compound and thepositive control contained 50 mM EDTA.

3. Results

3.1. Whole cell-based screening

The resazurin reduction assay, REMA, was used to screen thekinase library for bacterial growth inhibition. For both C. gluta-micum and M. tuberculosis the statistical parameter, Z0-factor27 wasgreater than 0.9 indicating an excellent assay quality. The wholeNCL was first tested against C. glutamicum and the resulting activecompounds or “hits” were subsequently tested on M. tuberculosisstrain H37Rv. Of the 12,100 compounds, 184 showed activityagainst C. glutamicum (score> 0.3) of which 14 also displayedactivity against M. tuberculosis (score> 0.3) (Figure 1, Table 1).Examination of the REMA results from the EVL sub-library indi-cated that 32 compounds showed antibacterial activity against C.glutamicum (score> 0.3) two of which also had anti-tuberculosisactivity. The EVL was also screened directly on M. tuberculosis. Fivecompounds exhibited a score >0.5 including the two compoundsactive on M. tuberculosis that were preselected from the wholelibrary using C. glutamicum (Figure 1). This strategy led to a total of17 hits with activity against M. tuberculosis.

3.2. Kinase inhibitory activity of hits active on M. tuberculosis

The 17 compounds that displayed anti-tuberculosis activity weretested for inhibition of PknA and PknB using the Transcreener�

ADP2 FP kit or radiometric assay (Figure 1 and data not shown).Compounds VI-13292, VI-11955 and VI-9502, which containa charged guanidino group, inhibited both kinases, most likely dueto promiscuous28 activity as they were previously found to inhibitvarious unrelated targets (our own unpublished data). Beside thisnon-specific effect, none of the 14 remaining compounds signifi-cantly affected PknA or PknB activity.

3.3. Screening of the EVL sub-library on PknA activity

The EVL sub-library was tested at a single concentration(10 mM) against PknA activity in the HTRF fluorescence kinaseassay (Figure 1). The Z0-factor27 for this screen was 0.63, indicatinga good quality assay. Five compounds were found to inhibit PknAwith a score >0.5. The kinase inhibition activity of four of thefive hits was subsequently confirmed by both HTRF and the

Table 1Selection of compounds active against M. tuberculosis H37Rv.

Compound Screening score Genotoxicityat 10 mM*

CytotoxicityMCC (mM)

MIC(mM)

M. tuberculosisH37Rv

C. glutamicumATCC 13032

VI-18678 1 1.1 M >100 <0.2VI-18679 0.9 1.1 M >100 3.1VI-3992 1 1 M >0.8 3.1VI-13292 1 0.9 e 12.5 3.1VI-3521 1 0.7 M >0.8 3.1VI-9376y 1 0.5 e >100 3.1VI-7777y 0.9 �0.1 e >100 3.1VI-18469y 0.5 0.2 e 100 6.25VI-9502 0.9 1.1 M 50 6.25VI-12955 0.8 1.1 M 3.1 6.25VI-17918 0.5 0.6 e 3.1 6.25VI-11955 0.8 1.1 e 100 12.5VI-6378 0.8 1 e 50 12.5VI-14072 0.5 0.5 e >100 12.5VI-17416 0.7 0.9 e >100 12.5VI-12029 1 1.1 e 25 25VI-12121 1 0 e 12.5 25

* M: Mutagenic.y Compound selected for further investigations.

Figure 1. Screening strategy. Schematic representation of the various screens per-formed in this study. The number of compounds tested, the assay used, and thenumber of hits selected after confirmation are indicated for each step. Compoundswere screened at 10 mM.

S. Magnet et al. / Tuberculosis 90 (2010) 354e360 357

radiometric kinase assay (Figure 2A) however, significant inhibi-tory effects (>50%) could only be seen at 100 mM. Interestingly,three compounds VI-16319, VI-16317 and VI-15739, shareda common scaffold: N-{3-[2-amino-3-cyano-6-(2-hydroxyphenyl)pyridin-4-yl]phenyl}-3-(piperidin-1-yl)propanamide (Figure 2B).On testing the antibacterial activity of the four compounds onM. tuberculosis H37Rv by REMA, none was found to affect bacterialgrowth at concentrations up to 200 mM.

3.4. Mutagenicity, cytotoxicity and MIC of compounds active on M.tuberculosis

The 17 compounds selected bywhole cell-based assay screeningwere further examined for their potential genotoxic and cytotoxicactivities and their MIC against M. tuberculosis H37Rv was deter-mined (Table 1). The structures of the three compounds that werefound to be non-mutagenic, non-cytotoxic and displayed an MIC<10 mM against M. tuberculosis H37Rv are shown in Figure 3. Wethen conducted hit expansion and structureeactivity relationship(SAR) studies with the two quinoxaline containing scaffoldsnamely, VI-9376 (7-bromo-2-methyl-5-nitro-3-phenylquinoxaline)and VI-18469 (N-(3-bromophenyl)-3-(thiophen-2-yl)benzo[g]qui-noxalin-2-amin).

3.5. Activity of VI-18469 derivatives

Compound VI-18469 was identified by screening the EVLagainst M. tuberculosis. More than 450 derivatives of VI-18469present in the NCL were subsequently tested againstM. tuberculosisH37Rv at a single dose (10 mM), and the MIC of the activecompounds was determined (data not shown). Most of the deriv-atives were inactive at 10 mM. The MIC of the active derivativesindicated that the position and the identity of the halogen groupsubstituting the N-phenyl do not affect the activity of thecompounds. The replacement of the bromophenyl by 1-ethyl-piperidine or 4-ethylmorpholine resulted in increased activityassociated with mutagenic and cytotoxic effects. Interestingly,there is some structural similarity between VI-18469 and theknown leprosy drug, clofazimine, a riminophenazine compound,29

which also shows some activity against M. tuberculosis.30

3.6. Activity of VI-9376 derivatives

Amongst the compounds active on M. tuberculosis, we noticedthat the structure of VI-9376 (2-methyl-3-phenyl-5-nitro-7-bro-moquinoxaline) partly resembles that of the benzothiazinones(BTZ), a new class of antimycobacterial agent that targets DprE1,a subunit of the decaprenylphosphoryl-b-D-ribose 20-epimeraseinvolved in the synthesis of arabinan.10 For this reason, VI-9376 wastested against several BTZ-resistant mutants of mycobacteria and C.glutamicum. The MIC results (Table 2) revealed cross-resistancebetween the BTZ lead compound, BTZ043, and VI-9376. Fortyderivatives of VI-9376 were subsequently synthesized and testedagainst M. tuberculosis H37Rv. The SAR and MIC data obtained forthe derivatives showed that the nitro group at the fifth position ofthe quinoxaline scaffold is absolutely required for activity.Replacement of the bromine at the fourth position by a trifluoro-methyl group increased the potency of the scaffold, whilst modi-fications at positions 2 or 3 did not lead to a significant improve-ment of activity against M. tuberculosis (Figure 4B).

4. Discussion

In this work, we took advantage of an existing resource, a libraryof compounds generated by medicinal chemistry as eukaryotic

Figure 2. Activity and structure of PknA inhibitors. (A) Results of the screen of EVL for PknA inhibitors and activity of the compounds on PknA, and on M. tuberculosis. Results fromthe screen correspond to the ratio of the signal difference between a given compound and the negative control and the signal difference between the positive control and thenegative control [(SCPD-SDMSO)/(SEDTA-SDMSO)]. Inhibition (%) of PknA activity by 100 mM compound was determined by radioactive assay. (B) Chemical structure of the 4 compoundsthat inhibit PknA.

S. Magnet et al. / Tuberculosis 90 (2010) 354e360358

kinase inhibitors, to identify newantimycobacterial scaffolds. Thesecompounds are predominantly small molecule ATP-binding siteinhibitors, but the library includes a series of allosteric inhibitors aswell. Influenced by the lack of success of target-based approachesin the discovery of antimycobacterial agents, we favored wholecell-based assays with C. glutamicum and M. tuberculosis overfurther target-based screens with purified STPK. The rationale forusing the fast-grower C. glutamicum in this study stems from thefact that it has a similar cell wall structure,31 a much smallergenome than M. tuberculosis, and as opposed to M. smegmatis,contains only four STPK, including PknA and PknB.15,16,32,33 Byadopting this strategy we expected to identify antimycobacterialagents that could inhibit enzymes that use a substrate or cofactorrelated to ATP, possibly essential STPK.

We first screened the compound library for anticorynebacterialactivity, and subsequently checked whether any of the hits acted bytargeting STPK essential for mycobacterial growth. From the wholelibrary, 184 compounds (1.5%) inhibited growth of C. glutamicum, ofwhich 14 were subsequently found to inhibit M. tuberculosis(Figure 1). To further evaluate the approach and to check that nointeresting scaffold was missed, which would act onM. tuberculosisbut not on C. glutamicum, a sub-library (EVL) representative of thechemical diversity was also screened directly on M. tuberculosis.

Figure 3. Structure and physico-chemical properties of non

Interestingly, direct screening of the EVL identified five moleculeswith activity against M. tuberculosis, of which only two were alsoactive against C. glutamicum (Figure 1). The low agreement ininhibitory activity against the two species revealed by this studywould argue in favor of a direct screen on M. tuberculosis whenfeasible, to avoid missing hits in future screening work.

In total, whole cell-based screening revealed 17 compoundswithinhibitory activity againstM. tuberculosis. Testing these compoundsfor their in vitro activity against PknA and PknB showed that none ofthem specifically inhibited the STPK activity. To see whether therewas no inhibitor of PknA in the library or whether they did notdisplay antibacterial activity, we screened the EVL sub-libraryagainst PknA in a kinase assay. Amongst the four confirmed hits thatinhibit PknA, compound VI-16444 was previously known to inhibitPknB from M. tuberculosis (IC50¼ 5 mM) as well as the eukaryotickinase, Cdk2 (unpublished data). Interestingly, the three remaininghits (VI-16317, VI-16319, VI-15739, Figure 2B) share a similar corestructure N-{3-[2-amino-3-cyano-6-(2-hydroxyphenyl)pyridin-4-yl]phenyl}-3-(piperidin-1-yl)propanamide, which representsa novel, inhibitor scaffold (Figure 2B). Comparison of the structuresof these hits with structurally similar but inactive compoundspresent in the library indicated that the piperidine ring is importantfor activity as well as the length of the carbon chain between the

-mutagenic compounds active against M. tuberculosis.

Table 2Effect of BTZ-resistancemutations in DprE1 on susceptibility to VI-9376 and BTZ043.

Strain Aminoacid 387*

Aminoacid 347*

MIC ofVI-9376(mM)

MIC ofBTZ043(mM)

M. smegmatis mc2155 Cys Phe 3.1 0.009M. smegmatis MN47 Gly Phe >100 9.2M. smegmatis MN84 Ser Phe >100 37.1

M. bovis BCG Cys Phe 3.1 0.005M. bovis BCG BN2 Ser Phe 50 37.1

C. glutamicum ATCC 13032 Cys Phe 3.1 0.144C. glutamicum PS1 Cys Ser 12.5 0.58

* Position according to M. tuberculosis H37Rv DprE1 sequence.

S. Magnet et al. / Tuberculosis 90 (2010) 354e360 359

amide and thepiperidine. However, noneof thehitswas particularlypotent, being unable to fully inhibit PknA activity at 100 mMand, notsurprisingly, none showed activity against M. tuberculosis.

Previous studies performed with the same library, as well as ourunpublished work, to find antimycobacterials that target STPKnever resulted in an MIC below our cut-off (i.e. <10 mM) despitegenerating inhibitors with IC50 in the nanomolarepicomolar range.Finding several compounds with antimycobacterial activity in thekinase inhibitor library suggests that some compounds initiallydesigned for eukaryotic targets can also penetrate the unique cellwall of mycobacteria. While this work was in progress, Pfizerpublished the findings obtained from a similar study22 in whichtheir corporate library was screened for inhibitors with whole cellantibacterial activity. This resulted in the identification of a familyof pyridopyrimidines derived from a protein kinase inhibitorpharmacophore. The pyridopyrimidines were shown to target theATP-binding site of biotin carboxylase, the enzyme which catalyzesthe first step in fatty acid biosynthesis. Taken together with ourfindings, this argues strongly in favor of cell-based screening ofcollections of drug-like molecules as being a privileged route inanti-infective discovery. However, identification of the target of thehits is then crucial to understand the mechanism of action of thecompounds and rationalize further medicinal chemistry work onthe leads.

Strikingly, the structure of one of our best hits VI-9376 (2-met-hyl-3-phenyl-5-nitro-7-bromoquinoxaline) resembled that of thebenzothiazinones (BTZ)10 and dinitrobenzamides (DNB),34 two newclasses of compounds active against M. tuberculosis. Both BTZ and

Figure 4. Preliminary structureeactivity relationship investigation on VI-9376. (A) Core struderivatives.

DNB target DprE1, a subunit of the decaprenylphosphoryl-b-D-ribose 20-epimerase involved in the synthesis of arabinan.10,34 Todetermine whether VI-9376 also targeted DprE1, we investigatedthe effect of BTZ-resistancemutations in dprE1, on the susceptibilityto VI-9376. The keymutations in DprE1 that confer resistance to BTZoccur in Cys387 and to a lesser extent Phe347. It was found thatmutations in either residue in M. smegmatis, M. bovis or C. gluta-micum (Table 2) also caused resistance to VI-9376, and the impact ofeach type of mutation on MIC correlated perfectly with the resultsobtained for BTZ.10 Furthermore, it is also known that the nitrogroup of BTZ043 is pivotal for its anti-tuberculosis activity andremoval of the nitro group from VI-9376 showed exactly the sameSAR, abolishing its activity. Thus, our results strongly suggest thatVI-9376 targets DprE1, which uses an adenine containing cofactor(presumably NADH or FAD) related in structure to ATP. Strikingly,with the addition of the present report, three independent wholecell-based screens have now identified compounds targeting DprE1recently both in vitro or ex vivo.10,34 This highlights the vulnerabilityof DprE1 and its importance as an antimycobacterial target andshows that whole cell screens can overcome the difficulties associ-ated with the development of high throughput assays usingmembrane-boundproteins or enzymes that use complex substrates,which are not commercially available, as is the case with DprE1.

The two remaining promising scaffolds that we identified VI-18469 and VI-7777 are specific for M. tuberculosis (Table 1).Derivatives of VI-18469 that were screened only against C. gluta-micum were selected from the NCL and tested against M. tubercu-losis, but none of the derivatives showed a significant improvementin anti-TB activity without associated cytotoxic effects. Targetidentification and chemistry programs focusing on anti-TB activityare now required to rationalize the synthesis of newderivatives andstudy the SAR of these compounds. However, the structural simi-larity between VI-18469 and clofazimine, a leprosy drug that isoccasionally used for the treatment of MDR-TB, opens avenues fortarget assessment. It will be interesting to see if clofazimine-resistant mutants show cross-resistance to VI-18469.

In conclusion, this work, and that of Miller et al.,22 demonstratesthat kinase inhibitor libraries, originally designed to target eukary-otic kinases, represent a valuable source of new antibacterial leads.Hopefully, this insight will encourage pharmaceutical companies tomake their compound libraries available for screening againstM. tuberculosis and thus contribute to combating the growingmenace of XDR-TB.

cture of VI-9376 and derivatives. (B) Structural variation versus MIC for several VI-9376

S. Magnet et al. / Tuberculosis 90 (2010) 354e360360

Acknowledgments

The authors would like to thank A. Lucia Quintana and M. Bel-linzoni for their input in PknA purification. We thank P. Alzari, G.S.Besra and G. Riccardi for sharing biological materials. The NM4TBConsortium is funded by the European Commission (LHSP-CT-2005-018923). R. Hartkoorn was the recipient of a postdoctoralfellowship from the Heiser Program for Research in Leprosy andTuberculosis of the New York Community Trust.

Funding: None.

Competing interest: None declared.

Ethical approval: Not required.

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