Leads for antitubercular compounds from kinase inhibitor library screens

Tuberculosis 90 (2010) 354e360

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Tuberculosis journal homepage: http://intl.elsevierhealth.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, } b, c, f, Marc Chambon d, Damiano Banfi d, Patricia Schneider a, b, Csaba Szántai-Kis b, c, László Orfi Manuel Bueno d, Gerardo Turcatti d, György Kéri b, c, e, f, g, Stewart T. Cole a, b, * a

Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland New Medicines for Tuberculosis (NM4TB) Consortium, Switzerland c Vichem Chemie Research Ltd., Herman Ottó. 15, H-1022 Budapest, Hungary d Biomolecular Screening Facility, Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland e } zoltó u. 37, Budapest, Hungary Semmelweis University, Department of Medical Chemistry, Pathobiochemistry Research Group of Hung.Acad.Sci. H-1085 Tu f }gyes Endre u. 9, Budapest, Hungary Semmelweis University, Department of Pharmaceutical Chemistry, H-1092 Ho g }i út. 26, Budapest, Hungary Semmelweis University, Rational Drug-Design Laboratory CRC, H-1085 Üllo b

a r t i c l e i n f o

s u m m a r y

Article history: Received 17 June 2010 Received in revised form 20 August 2010 Accepted 7 September 2010

Discovering new drugs to treat tuberculosis more efficiently and to overcome multidrug resistance is a world health priority. To find antimycobacterial scaffolds, we screened a kinase inhibitor library of more than 12,000 compounds using an integrated strategy involving whole cell-based assays with Corynebacterium glutamicum and Mycobacterium tuberculosis, and a target-based assay with the protein kinase PknA. Seventeen “hits” came from the whole cell-based screening approach, from which three displayed minimal inhibitory concentrations (MIC) against M. tuberculosis below 10 mM and were non-mutagenic and non-cytotoxic. Two of these hits were specific for M. tuberculosis versus C. glutamicum and none of them was found to inhibit the essential serine/threonine protein kinases, PknA and PknB present in both bacteria. 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. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Tuberculosis Screening Kinase inhibitor Quinoxaline Serine/threonine protein Kinase (STPK) DprE1

1. Introduction While short course chemotherapy for drug-susceptible tuberculosis (TB) remains effective, it is relatively inefficient by modern pharmaceutical standards and universally menaced by the dissemination of multidrug-resistant (MDR) and extensively drugresistant (XDR) strains of Mycobacterium tuberculosis.1 Thus, as first

* Corresponding author. EPFL, SV-GHI-UPCOL, Station 19, CH-1015 Lausanne, Switzerland. Tel.: þ41 21 693 1851; fax: þ41 21 693 1790. E-mail addresses: sophie.magnet@epfl.ch (S. Magnet), ruben.hartkoorn@epfl.ch (R.C. Hartkoorn), [email protected] (R. Székely), [email protected] (J. Pató), [email protected] (J.A. Triccas), patricia.schneider@epfl.ch (P. Schneider), csaba. } [email protected] (C. Szántai-Kis), laszlo.orfi@vichem.hu (L. Orfi), marc. chambon@epfl.ch (M. Chambon), damiano.banfi@epfl.ch (D. Banfi), manuel.bueno@ epfl.ch (M. Bueno), gerardo.turcatti@epfl.ch (G. Turcatti), [email protected] (G. Kéri), stewart.cole@epfl.ch (S.T. Cole). h These authors contributed equally. i Permanent address: Discipline of Medicine, University of Sydney, NSW, Australia. j www.nm4tb.org. 1472-9792/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tube.2010.09.001

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 based their strategy on target-based approaches that have met with very poor outcomes2,3 as all TB drug candidates that are currently in clinical trials were first selected on the basis of their antibacterial activity.4e8 In consequence, whole cell-based screening approaches are being reconsidered as a means of identifying new active scaffolds2,9 as well as finding new targets.10 In other therapeutic areas, such as oncology, medicinal chemistry efforts have resulted in vast kinase inhibitor libraries that could be used as a source of new antimycobacterial agents. Among the resources available, the Nested Chemical LibraryÔ (NCL) of Vichem Chemie Ltd. contains inhibitors of more than 100 eukaryotic kinases including EGFR, PDGFR, VEGFR, SRC, PLK, or JNK.11 As in eukaryotes, signaling pathways in prokaryotes are also controlled by protein kinases. Strikingly, actinobacteria contain an unusually high proportion of Serine/threonine Protein Kinases (STPK) compared to two component systems.12 In M. tuberculosis three conserved STPK were considered as attractive drug targets: PknG, required for intracellular bacterial survival in the host,13,14

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and two essential transmembrane receptor-kinases involved in the regulation of cell division, PknA and PknB.15,16 Attempts to identify inhibitors of mycobacterial STPK have focused on target-based approaches using PknB and PknG.14,17,18 Recently, a sub-library of the NCL was screened against PknB and gave hits with nanomolar median inhibitory concentration (IC50).19,20 However, none of the kinase inhibitors identified so far inhibits M. tuberculosis growth (MIC >50 mM).19,20 The lack of correlation between the IC50 and the MIC values is not understood. The compounds may not cross the complex mycobacterial cell wall or the accessibility of the inhibitor binding site may be different in vivo. On the other hand, a few examples of compounds derived from protein kinase pharmacophores have been shown to inhibit non-kinase antibacterial targets that use ATP as a cofactor, such as 21 D-alanine-D-alanine ligase or biotin carboxylase kinase.22 Thus, kinase inhibitor libraries can potentially be a source of inhibitors for a wide range of bacterial enzymes. In this study we screened two kinase inhibitor libraries with the aim of identifying new scaffolds active against M. tuberculosis. The NCL was screened by an extensive in vitro antibacterial assay with Corynebacterium glutamicum, which has orthologs of PknA, PknB and PknG, and by a more limited screen with M. tuberculosis, in conjunction with an inhibition assay of PknA activity, an STPK which has been unexplored thus far. Comparison of the datasets led to the identification of new scaffolds effective against M. tuberculosis, without correlation with STPK inhibition. 2. Materials and methods 2.1. Compounds The compounds tested during this work belong to the Nested Chemical LibraryÔ (NCL) of Vichem Chemie Research Ltd (Budapest).11 The NCL contains 12,100 compounds (organized on 108 core structures and more than 500 scaffolds), of which most are small molecule ATP-binding site inhibitors. The Extended Validation Library (EVL) is a sub-library of the NCL, which contains around 1000 proven kinase inhibitor leads. These molecules represent a broad spectrum of potentially active chemical structures. The log P values (logarithm of the ratio of the concentrations of un-ionized solute in solvents) 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 at 37  C in Luria broth (LB) supplemented with chloramphenicol 30 mg/ml and ampicillin 50 mg/ml, respectively. C. glutamicum (ATCC 13032) was grown in LB at 30  C. Mycobacterium smegmatis mc2155 was grown in LB containing 0.05% tween 80 at 37  C. Mycobacterium bovis BCG Pasteur and M. tuberculosis H37Rv were grown in Middlebrook 7H9 supplemented with 10% ADC enrichment (albumin, dextrose, catalase; Becton Dickinson), 0.05% tween 80, and 0.2% glycerol. The benzothiazinone (BTZ)-resistant mutants M. smegmatis MN47, M. smegmatis MN84, M. bovis BCG Pasteur BN2 were obtained from Prof. G. Riccardi (Pavia, Italy).10 PS1, a BTZresistant mutant was obtained by plating C. glutamicum ATCC 13032 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 the resazurin reduction microtiter assay (REMA) as previously described.24 Single dose drug susceptibility of C. glutamicum was

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determined in a 384 well plate format (20 mL assay) using clear flat bottom polystyrene microplates (Corning). Compounds were mixed with an exponential phase culture diluted to OD600 nm of 0.001 and incubated overnight at 30  C. Resazurin was added (2 mL of 0.025% w/v) and plates were incubated for 4 h. Bacterial growth was determined following resazurin reduction monitored by fluorescence (excitation 570 nm, emission 590 nm). Single dose drug susceptibility of M. tuberculosis was determined similarly, but in a 96 well plate format (100 mL assay) in clear flat bottom polystyrene microplates (Corning). To prevent evaporation plates were sealed with an adhesive polyester film. After 7 days incubation at 37  C, resazurin was added and incubated for 20 h at 37  C before fluorescence reading. Compounds were dispensed with a ZephyrÒ Liquid Handling Workstation (Caliper LifeSciences). Bacteria and resazurin were added either manually, in the case of M. tuberculosis or using an automated liquid dispenser (EL406, BioTek), in the case of C. glutamicum. Fluorescence was measured with a Tecan Infinite 500 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. tuberculosis REMA plates contained 8 wells of each control. 2.4. MIC determination MIC, defined as the minimum concentration of drug that inhibits more than 99% of growth of a liquid culture with an initial OD600 nm ¼ 0.001, was determined in transparent 96 well plates (Corning) using REMA. Compounds were serially diluted two-fold from 200 to 0.62 mM for the molecules selected from the PknA kinase assay screen, or from 100 to 0.16 mM for the molecules selected for their antibacterial activity. A rifampin control (1 mM to 1 nM) was included in every plate. 2.5. Genotoxicity assay The genotoxicity of the compounds was evaluated by the SOS chromotest on LB-agar plate.23 This colorimetric assay is based on the induction of the SOS function SfiA by DNA damaging agents, whose level of expression is monitored by means of a sfiA::lacZ operon fusion in E. coli PQ37. Briefly, compounds (10 mL of a 10 mM solution) were spotted on a LB-agar plate containing ampicillin 50 mg/ml, 0.006% bromo-chloro-indolyl-galactopyranoside (X-Gal) and E. coli PQ37 at OD600 nm ¼ 0.04. Isoniazid and 4-nitroquinoline N-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 two distinct human cell lines: the human lung epithelial cell line; A549, and the human leukemic monocyte cell line; THP-1. Cells were seeded in 48 well plates (2  104 cells per well, in 500 mL of DMEM containing 10% Fetal Bovine Serum) in the presence of increasing concentrations of compound (from 1.5 to 100 mM), for 3 days at 37  C with 5% CO2. To determine THP-1 viability, resazurin was added directly to the cell culture (final concentration ¼ 0.0025%), whilst for A549 viability determination media was aspirated and phosphate buffered saline containing resazurin (0.0025%) was added. Plates were incubated for 2 more hours and cell viability was determined by fluorescence (excitation 570 nm, emission 590 nm). Assays were performed in duplicate and the minimal cytotoxic concentration (MCC) defined as the minimal drug concentration associated with decrease in fluorescence or major morphologic alteration was determined.

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2.7. Protein purification Truncated versions of the kinases PknA and PknB from M. tuberculosis H37Rv were produced with an N-terminal His6-tag in E. coli BL21(DE3)pLysS. The catalytic domain of PknB (residues 1e279) was produced and purified as previously described.25 To produce PknA (residues 1e336, including the catalytic domain plus the juxtamembrane domain), BL21(DE3)pLysS harboring pET28UpknA was grown in LB containing kanamycin 50 mg/ml and chloramphenicol 30 mg/ml at 30  C. Expression of pknA was induced at OD600 nm ¼ 0.6 with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) for 3.5 h at 30  C. Cells were lysed by sonication and the recombinant protein purified from the soluble extract in two steps using fast protein liquid chromatography (FPLC). The protein was first captured by affinity chromatography on a Ni-NTA column, eluted with an imidazole gradient (0e500 mM) and further purified by size exclusion on a Superdex75 column. The protein was stored at 20  C in 12.5 mM HEPES pH ¼ 8, 250 mM NaCl, 0.5 mM dithiothreitol (DTT) and 50% glycerol. Recombinant GarA protein was purified by affinity chromatography and size exclusion as previously described,26 for use as a kinase substrate. A third additional step of purification was performed by affinity chromatography using TALONÒ metal affinity resin (Clontech) to remove traces of a protein contaminant that interferes with fluorescent kinase assays.

negative control and the signal difference between the positive control 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 duplicates was greater than 3-fold the standard deviation determined for the negative control were not taken into account. 2.10. Radiometric kinase assay The radiometric assay was carried out in 10 mL final volume of reaction buffer (20 mM Hepes pH ¼ 7.5, 1 mM DTT, 1 mM MnCl2, 5% Glycerol, 0.01% Brij35) containing 1% DMSO, 45 mM ATP including 3 mCi of [g-33P]ATP, 40 mM GarA and 10 mM or 100 mM compound. The reaction was initiated by the addition of 1.5 mM PknA and stopped after 2 hours incubation at room temperature by the addition of 50 mM EDTA. The samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) on 4e12% gradient gels and, after drying, the incorporation of radiolabelled phosphate into GarA was visualized and quantified with a PhosphorImager system (Storm, Molecular Dynamics) and the software Image Quant. The negative control contained no compound and the positive control contained 50 mM EDTA. 3. Results 3.1. Whole cell-based screening

2.8. Fluorescent kinase assay The resazurin reduction assay, REMA, was used to screen the kinase library for bacterial growth inhibition. For both C. glutamicum and M. tuberculosis the statistical parameter, Z0 -factor27 was greater than 0.9 indicating an excellent assay quality. The whole NCL was first tested against C. glutamicum and the resulting active compounds or “hits” were subsequently tested on M. tuberculosis strain H37Rv. Of the 12,100 compounds, 184 showed activity against C. glutamicum (score > 0.3) of which 14 also displayed activity against M. tuberculosis (score > 0.3) (Figure 1, Table 1). Examination of the REMA results from the EVL sub-library indicated that 32 compounds showed antibacterial activity against C. glutamicum (score > 0.3) two of which also had anti-tuberculosis activity. The EVL was also screened directly on M. tuberculosis. Five compounds exhibited a score >0.5 including the two compounds active on M. tuberculosis that were preselected from the whole library using C. glutamicum (Figure 1). This strategy led to a total of 17 hits with activity against M. tuberculosis.

The fluorescent kinase assays monitor the formation of ADP by means of fluorescence changes. The ADP detection step was carried out either by homogenous time resolved fluorescence (HTRF) using the HTRFÒ Transcreener ADP kit (Cisbio) or by fluorescence polarization (FP) using the TranscreenerÒ ADP2 FP kit (BellBrooks Labs). These detection methods are based on an antibody specific for ADP which competes for both native ADP produced by the kinase reaction and a fluorophore-coupled ADP present in the reaction mixture. The assays were carried out in duplicate, in black, low volume, non-binding surface, 384 well plates (Corning). In the case of PknA, the kinase reaction was performed in 10 mL reaction buffer A (20 mM HEPES pH ¼ 7.5, 1 mM DTT, 1 mM MnCl2, 5% glycerol, 0.01% Brij35) containing 1% dimethyl sulfoxide (DMSO), 1.5 mM PknA, 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 reaction was performed in 10 mL buffer B (50 mM HEPES pH ¼ 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 was incubated at 22  C for 30 min. Two negative control columns (no compound) and two positive control columns (containing 50 mM EDTA) were included in every assay plate. For the screen, compounds (2.5 mL at 40 mM) were dispensed with a ZephyrÒ Liquid Handling Workstation (Caliper LifeSciences), enzyme and substrates were added successively in two manual steps, while buffer and detection reagents were added using a EL406 dispenser (BioTek). The HTRF signal was measured with a Tecan Infinite 500 microplate reader and the FP signal with an Analyst GT microplate reader (Molecular Devices).

The 17 compounds that displayed anti-tuberculosis activity were tested for inhibition of PknA and PknB using the TranscreenerÒ ADP2 FP kit or radiometric assay (Figure 1 and data not shown). Compounds VI-13292, VI-11955 and VI-9502, which contain a charged guanidino group, inhibited both kinases, most likely due to promiscuous28 activity as they were previously found to inhibit various unrelated targets (our own unpublished data). Beside this non-specific effect, none of the 14 remaining compounds significantly affected PknA or PknB activity.

2.9. Screening data analysis

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

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

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

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

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radiometric kinase assay (Figure 2A) however, significant inhibitory effects (>50%) could only be seen at 100 mM. Interestingly, three compounds VI-16319, VI-16317 and VI-15739, shared a 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 on M. tuberculosis H37Rv by REMA, none was found to affect bacterial growth at concentrations up to 200 mM. 3.4. Mutagenicity, cytotoxicity and MIC of compounds active on M. tuberculosis The 17 compounds selected by whole cell-based assay screening were further examined for their potential genotoxic and cytotoxic activities and their MIC against M. tuberculosis H37Rv was determined (Table 1). The structures of the three compounds that were found to be non-mutagenic, non-cytotoxic and displayed an MIC <10 mM against M. tuberculosis H37Rv are shown in Figure 3. We then conducted hit expansion and structureeactivity relationship (SAR) studies with the two quinoxaline containing scaffolds namely, VI-9376 (7-bromo-2-methyl-5-nitro-3-phenylquinoxaline) and VI-18469 (N-(3-bromophenyl)-3-(thiophen-2-yl)benzo[g]quinoxalin-2-amin). 3.5. Activity of VI-18469 derivatives

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

Compound VI-18469 was identified by screening the EVL against M. tuberculosis. More than 450 derivatives of VI-18469 present in the NCL were subsequently tested against M. tuberculosis H37Rv at a single dose (10 mM), and the MIC of the active compounds was determined (data not shown). Most of the derivatives were inactive at 10 mM. The MIC of the active derivatives indicated that the position and the identity of the halogen group substituting the N-phenyl do not affect the activity of the compounds. The replacement of the bromophenyl by 1-ethylpiperidine or 4-ethylmorpholine resulted in increased activity associated with mutagenic and cytotoxic effects. Interestingly, there is some structural similarity between VI-18469 and the known leprosy drug, clofazimine, a riminophenazine compound,29 which also shows some activity against M. tuberculosis.30 3.6. Activity of VI-9376 derivatives

Table 1 Selection of compounds active against M. tuberculosis H37Rv. Compound Screening score M. tuberculosis C. glutamicum H37Rv ATCC 13032 VI-18678 VI-18679 VI-3992 VI-13292 VI-3521 VI-9376y VI-7777y VI-18469y VI-9502 VI-12955 VI-17918 VI-11955 VI-6378 VI-14072 VI-17416 VI-12029 VI-12121 * y

1 0.9 1 1 1 1 0.9 0.5 0.9 0.8 0.5 0.8 0.8 0.5 0.7 1 1

1.1 1.1 1 0.9 0.7 0.5 0.1 0.2 1.1 1.1 0.6 1.1 1 0.5 0.9 1.1 0

Genotoxicity Cytotoxicity MIC at 10 mM* (mM) MCC (mM)

M M M e M e e e M M e e e e e e e

M: Mutagenic. Compound selected for further investigations.

>100 >100 >0.8 12.5 >0.8 >100 >100 100 50 3.1 3.1 100 50 >100 >100 25 12.5

<0.2 3.1 3.1 3.1 3.1 3.1 3.1 6.25 6.25 6.25 6.25 12.5 12.5 12.5 12.5 25 25

Amongst the compounds active on M. tuberculosis, we noticed that the structure of VI-9376 (2-methyl-3-phenyl-5-nitro-7-bromoquinoxaline) 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 -epimerase involved in the synthesis of arabinan.10 For this reason, VI-9376 was tested against several BTZ-resistant mutants of mycobacteria and C. glutamicum. The MIC results (Table 2) revealed cross-resistance between the BTZ lead compound, BTZ043, and VI-9376. Forty derivatives of VI-9376 were subsequently synthesized and tested against M. tuberculosis H37Rv. The SAR and MIC data obtained for the derivatives showed that the nitro group at the fifth position of the quinoxaline scaffold is absolutely required for activity. Replacement of the bromine at the fourth position by a trifluoromethyl group increased the potency of the scaffold, whilst modifications at positions 2 or 3 did not lead to a significant improvement of activity against M. tuberculosis (Figure 4B). 4. Discussion In this work, we took advantage of an existing resource, a library of compounds generated by medicinal chemistry as eukaryotic

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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 from the 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 the negative control [(SCPD-SDMSO)/(SEDTA-SDMSO)]. Inhibition (%) of PknA activity by 100 mM compound was determined by radioactive assay. (B) Chemical structure of the 4 compounds that inhibit PknA.

kinase inhibitors, to identify new antimycobacterial scaffolds. These compounds are predominantly small molecule ATP-binding site inhibitors, but the library includes a series of allosteric inhibitors as well. Influenced by the lack of success of target-based approaches in the discovery of antimycobacterial agents, we favored whole cell-based assays with C. glutamicum and M. tuberculosis over further target-based screens with purified STPK. The rationale for using the fast-grower C. glutamicum in this study stems from the fact that it has a similar cell wall structure,31 a much smaller genome than M. tuberculosis, and as opposed to M. smegmatis, contains only four STPK, including PknA and PknB.15,16,32,33 By adopting this strategy we expected to identify antimycobacterial agents that could inhibit enzymes that use a substrate or cofactor related to ATP, possibly essential STPK. We first screened the compound library for anticorynebacterial activity, and subsequently checked whether any of the hits acted by targeting STPK essential for mycobacterial growth. From the whole library, 184 compounds (1.5%) inhibited growth of C. glutamicum, of which 14 were subsequently found to inhibit M. tuberculosis (Figure 1). To further evaluate the approach and to check that no interesting scaffold was missed, which would act on M. tuberculosis but not on C. glutamicum, a sub-library (EVL) representative of the chemical diversity was also screened directly on M. tuberculosis.

Interestingly, direct screening of the EVL identified five molecules with activity against M. tuberculosis, of which only two were also active against C. glutamicum (Figure 1). The low agreement in inhibitory activity against the two species revealed by this study would argue in favor of a direct screen on M. tuberculosis when feasible, to avoid missing hits in future screening work. In total, whole cell-based screening revealed 17 compounds with inhibitory activity against M. tuberculosis. Testing these compounds for their in vitro activity against PknA and PknB showed that none of them specifically inhibited the STPK activity. To see whether there was no inhibitor of PknA in the library or whether they did not display antibacterial activity, we screened the EVL sub-library against PknA in a kinase assay. Amongst the four confirmed hits that inhibit PknA, compound VI-16444 was previously known to inhibit PknB from M. tuberculosis (IC50 ¼ 5 mM) as well as the eukaryotic kinase, Cdk2 (unpublished data). Interestingly, the three remaining hits (VI-16317, VI-16319, VI-15739, Figure 2B) share a similar core structure N-{3-[2-amino-3-cyano-6-(2-hydroxyphenyl)pyridin-4yl]phenyl}-3-(piperidin-1-yl)propanamide, which represents a novel, inhibitor scaffold (Figure 2B). Comparison of the structures of these hits with structurally similar but inactive compounds present in the library indicated that the piperidine ring is important for activity as well as the length of the carbon chain between the

Figure 3. Structure and physico-chemical properties of non-mutagenic compounds active against M. tuberculosis.

S. Magnet et al. / Tuberculosis 90 (2010) 354e360 Table 2 Effect of BTZ-resistance mutations in DprE1 on susceptibility to VI-9376 and BTZ043. Strain

Amino acid 387*

Amino acid 347*

MIC of VI-9376 (mM)

MIC of BTZ043 (mM)

M. smegmatis mc2155 M. smegmatis MN47 M. smegmatis MN84

Cys Gly Ser

Phe Phe Phe

3.1 >100 >100

0.009 9.2 37.1

M. bovis BCG M. bovis BCG BN2

Cys Ser

Phe Phe

3.1 50

0.005 37.1

C. glutamicum ATCC 13032 C. glutamicum PS1

Cys Cys

Phe Ser

3.1 12.5

0.144 0.58

*

Position according to M. tuberculosis H37Rv DprE1 sequence.

amide and the piperidine. However, none of the hits was particularly potent, being unable to fully inhibit PknA activity at 100 mM and, not surprisingly, none showed activity against M. tuberculosis. Previous studies performed with the same library, as well as our unpublished work, to find antimycobacterials that target STPK never resulted in an MIC below our cut-off (i.e. <10 mM) despite generating inhibitors with IC50 in the nanomolarepicomolar range. Finding several compounds with antimycobacterial activity in the kinase inhibitor library suggests that some compounds initially designed for eukaryotic targets can also penetrate the unique cell wall of mycobacteria. While this work was in progress, Pfizer published the findings obtained from a similar study22 in which their corporate library was screened for inhibitors with whole cell antibacterial activity. This resulted in the identification of a family of pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. The pyridopyrimidines were shown to target the ATP-binding site of biotin carboxylase, the enzyme which catalyzes the first step in fatty acid biosynthesis. Taken together with our findings, this argues strongly in favor of cell-based screening of collections of drug-like molecules as being a privileged route in anti-infective discovery. However, identification of the target of the hits is then crucial to understand the mechanism of action of the compounds and rationalize further medicinal chemistry work on the leads. Strikingly, the structure of one of our best hits VI-9376 (2-methyl-3-phenyl-5-nitro-7-bromoquinoxaline) resembled that of the benzothiazinones (BTZ)10 and dinitrobenzamides (DNB),34 two new classes of compounds active against M. tuberculosis. Both BTZ and

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DNB target DprE1, a subunit of the decaprenylphosphoryl-b-Dribose 20 -epimerase involved in the synthesis of arabinan.10,34 To determine whether VI-9376 also targeted DprE1, we investigated the effect of BTZ-resistance mutations in dprE1, on the susceptibility to VI-9376. The key mutations in DprE1 that confer resistance to BTZ occur in Cys387 and to a lesser extent Phe347. It was found that mutations in either residue in M. smegmatis, M. bovis or C. glutamicum (Table 2) also caused resistance to VI-9376, and the impact of each type of mutation on MIC correlated perfectly with the results obtained for BTZ.10 Furthermore, it is also known that the nitro group of BTZ043 is pivotal for its anti-tuberculosis activity and removal of the nitro group from VI-9376 showed exactly the same SAR, abolishing its activity. Thus, our results strongly suggest that VI-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 whole cell-based screens have now identified compounds targeting DprE1 recently both in vitro or ex vivo.10,34 This highlights the vulnerability of DprE1 and its importance as an antimycobacterial target and shows that whole cell screens can overcome the difficulties associated with the development of high throughput assays using membrane-bound proteins 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 VI18469 and VI-7777 are specific for M. tuberculosis (Table 1). Derivatives of VI-18469 that were screened only against C. glutamicum were selected from the NCL and tested against M. tuberculosis, but none of the derivatives showed a significant improvement in anti-TB activity without associated cytotoxic effects. Target identification and chemistry programs focusing on anti-TB activity are now required to rationalize the synthesis of new derivatives and study the SAR of these compounds. However, the structural similarity between VI-18469 and clofazimine, a leprosy drug that is occasionally used for the treatment of MDR-TB, opens avenues for target assessment. It will be interesting to see if clofazimineresistant mutants show cross-resistance to VI-18469. In conclusion, this work, and that of Miller et al.,22 demonstrates that kinase inhibitor libraries, originally designed to target eukaryotic kinases, represent a valuable source of new antibacterial leads. Hopefully, this insight will encourage pharmaceutical companies to make their compound libraries available for screening against M. tuberculosis and thus contribute to combating the growing menace of XDR-TB.

Figure 4. Preliminary structureeactivity relationship investigation on VI-9376. (A) Core structure of VI-9376 and derivatives. (B) Structural variation versus MIC for several VI-9376 derivatives.

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

Acknowledgments The authors would like to thank A. Lucia Quintana and M. Bellinzoni for their input in PknA purification. We thank P. Alzari, G.S. Besra and G. Riccardi for sharing biological materials. The NM4TB Consortium is funded by the European Commission (LHSP-CT2005-018923). R. Hartkoorn was the recipient of a postdoctoral fellowship from the Heiser Program for Research in Leprosy and Tuberculosis of the New York Community Trust. Funding:

None.

Competing interest: Ethical approval:

None declared. Not required.

References 1. Chan ED, Iseman MD. Multidrug-resistant and extensively drug-resistant tuberculosis: a review. Curr Opin Infect Dis 2008;21:587e95. 2. Balganesh TS, Alzari PM, Cole ST. Rising standards for tuberculosis drug development. Trends Pharmacol Sci 2008;29:576e81. 3. Payne DJ, Gwynn MN, Holmes DJ, Pompliano DL. Drugs for bad bugs: confronting the challenges of antibacterial discovery. Nat Rev Drug Discov 2007;6: 29e40. 4. Matsumoto M, Hashizume H, Tomishige T, Kawasaki M, Tsubouchi H, Sasaki H, et al. OPC-67683, a nitro-dihydro-imidazooxazole derivative with promising action against tuberculosis in vitro and in mice. PLoS Med 2006;3:e466. 5. Andries K, Verhasselt P, Guillemont J, Gohlmann HW, Neefs JM, Winkler H, et al. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science 2005;307:223e7. 6. Stover CK, Warrener P, VanDevanter DR, Sherman DR, Arain TM, Langhorne MH, et al. A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis. Nature 2000;405:962e6. 7. Protopopova M, Hanrahan C, Nikonenko B, Samala R, Chen P, Gearhart J, et al. Identification of a new antitubercular drug candidate, SQ109, from a combinatorial library of 1,2-ethylenediamines. J Antimicrob Chemother 2005;56: 968e74. 8. Rivers EC, Mancera RL. New anti-tuberculosis drugs in clinical trials with novel mechanisms of action. Drug Discov Today 2008;13:1090e8. 9. Ananthan S, Faaleolea ER, Goldman RC, Hobrath JV, Kwong CD, Laughon BE, et al. High-throughput screening for inhibitors of Mycobacterium tuberculosis H37Rv. Tuberculosis (Edinb) 2009;89:334e53. 10. Makarov V, Manina G, Mikusova K, Mollmann U, Ryabova O, Saint-Joanis B, et al. Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis. Science 2009;324:801e4. 11. Keri G, Szekelyhidi Z, Banhegyi P, Varga Z, Hegymegi-Barakonyi B, SzantaiKis C, et al. Drug discovery in the kinase inhibitory field using the Nested Chemical Library technology. Assay Drug Dev Technol 2005;3:543e51. 12. Wehenkel A, Bellinzoni M, Grana M, Duran R, Villarino A, Fernandez P, et al. Mycobacterial Ser/Thr protein kinases and phosphatases: physiological roles and therapeutic potential. Biochim Biophys Acta 2008;1784:193e202. 13. Scherr N, Muller P, Perisa D, Combaluzier B, Jeno P, Pieters J. Survival of pathogenic mycobacteria in macrophages is mediated through autophosphorylation of protein kinase G. J Bacteriol 2009;191:4546e54. 14. Walburger A, Koul A, Ferrari G, Nguyen L, Prescianotto-Baschong C, Huygen K, et al. Protein kinase G from pathogenic mycobacteria promotes survival within macrophages. Science 2004;304:1800e4. 15. Fernandez P, Saint-Joanis B, Barilone N, Jackson M, Gicquel B, Cole ST, et al. The Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth. J Bacteriol 2006;188:7778e84.

16. Kang CM, Abbott DW, Park ST, Dascher CC, Cantley LC, Husson RN. The Mycobacterium tuberculosis serine/threonine kinases PknA and PknB: substrate identification and regulation of cell shape. Genes Dev 2005;19: 1692e704. 17. Drews SJ, Hung F, Av-Gay Y. A protein kinase inhibitor as an antimycobacterial agent. FEMS Microbiol Lett 2001;205:369e74. 18. Wehenkel A, Fernandez P, Bellinzoni M, Catherinot V, Barilone N, Labesse G, et al. The structure of PknB in complex with mitoxantrone, an ATP-competitive inhibitor, suggests a mode of protein kinase regulation in mycobacteria. FEBS Lett 2006;580:3018e22. 19. Hegymegi-Barakonyi B, Szekely R, Varga Z, Kiss R, Borbely G, Nemeth G, et al. Signalling inhibitors against Mycobacterium tuberculosiseearly days of a new therapeutic concept in tuberculosis. Curr Med Chem 2008;15:2760e70. 20. Szekely R, Waczek F, Szabadkai I, Nemeth G, Hegymegi-Barakonyi B, Eros D, et al. A novel drug discovery concept for tuberculosis: inhibition of bacterial and host cell signalling. Immunol Lett 2008;116:225e31. 21. Triola G, Wetzel S, Ellinger B, Koch MA, Hubel K, Rauh D, et al. ATP competitive inhibitors of D-alanine-D-alanine ligase based on protein kinase inhibitor scaffolds. Bioorg Med Chem 2009;17:1079e87. 22. Miller JR, Dunham S, Mochalkin I, Banotai C, Bowman M, Buist S, et al. A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore. Proc Natl Acad Sci U S A 2009;106:1737e42. 23. Quillardet P, Huisman O, D’Ari R, Hofnung M. SOS chromotest, a direct assay of induction of an SOS function in Escherichia coli K-12 to measure genotoxicity. Proc Natl Acad Sci U S A 1982;79:5971e5. 24. Palomino JC, Martin A, Camacho M, Guerra H, Swings J, Portaels F. Resazurin microtiter assay plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2002;46:2720e2. 25. Boitel B, Ortiz-Lombardia M, Duran R, Pompeo F, Cole ST, Cervenansky C, et al. PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP, the cognate phospho-Ser/Thr phosphatase, in Mycobacterium tuberculosis. Mol Microbiol 2003;49:1493e508. 26. Villarino A, Duran R, Wehenkel A, Fernandez P, England P, Brodin P, et al. Proteomic identification of M. tuberculosis protein kinase substrates: PknB recruits GarA, a FHA domain-containing protein, through activation loopmediated interactions. J Mol Biol 2005;350:953e63. 27. Zhang JH, Chung TD, Oldenburg KR. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 1999;4:67e73. 28. Seidler J, McGovern SL, Doman TN, Shoichet BK. Identification and prediction of promiscuous aggregating inhibitors among known drugs. J Med Chem 2003;46: 4477e86. 29. Levy L, Shepard CC, Fasal P. Clofazimine therapy of lepromatous leprosy caused by dapsone-resistant Mycobacterium leprae. Am J Trop Med Hyg 1972;21: 315e21. 30. Cholo MC, Boshoff HI, Steel HC, Cockeran R, Matlola NM, Downing KJ, et al. Effects of clofazimine on potassium uptake by a Trk-deletion mutant of Mycobacterium tuberculosis. J Antimicrob Chemother 2006;57:79e84. 31. Dover LG, Cerdeno-Tarraga AM, Pallen MJ, Parkhill J, Besra GS. Comparative cell wall core biosynthesis in the mycolated pathogens, Mycobacterium tuberculosis and Corynebacterium diphtheriae. FEMS Microbiol Rev 2004;28:225e50. 32. Fiuza M, Canova MJ, Zanella-Cleon I, Becchi M, Cozzone AJ, Mateos LM, et al. From the characterization of the four serine/threonine protein kinases (PknA/ B/G/L) of Corynebacterium glutamicum toward the role of PknA and PknB in cell division. J Biol Chem 2008;283:18099e112. 33. Schultz C, Niebisch A, Schwaiger A, Viets U, Metzger S, Bramkamp M, et al. Genetic and biochemical analysis of the serine/threonine protein kinases PknA, PknB, PknG and PknL of Corynebacterium glutamicum: evidence for nonessentiality and for phosphorylation of OdhI and FtsZ by multiple kinases. Mol Microbiol 2009;74:724e41. 34. Christophe T, Jackson M, Jeon HK, Fenistein D, Contreras-Dominguez M, Kim J, et al. High content screening identifies decaprenyl-phosphoribose 2’ epimerase as a target for intracellular antimycobacterial inhibitors. PLoS Pathog 2009;5: e1000645.