Tuberculosis xxx (2015) 1e10

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Genetic and chemical validation identifies Mycobacterium tuberculosis topoisomerase I as an attractive anti-tubercular target Sudha Ravishankar a, *, Anisha Ambady a, Disha Awasthy a, Naina Vinay Mudugal a, Sreenivasaiah Menasinakai a, Sandesh Jatheendranath a, Supreeth Guptha a, Sreevalli Sharma a, Gayathri Balakrishnan a, Radha Nandishaiah a, Vasanthi Ramachandran a, Charles J. Eyermann b, Folkert Reck b, Suresh Rudrapatna a, Vasan K. Sambandamurthy a, Umender K. Sharma a a b

AstraZeneca India Pvt. Ltd., Bellary Road, Hebbal, Bangalore 560024, India AstraZeneca Infection, Innovative Medicines, 35 Gatehouse Drive, Waltham, MA 02451, United States

a r t i c l e i n f o

s u m m a r y

Article history: Received 11 February 2015 Received in revised form 30 April 2015 Accepted 13 May 2015

DNA topoisomerases perform the essential function of maintaining DNA topology in prokaryotes. DNA gyrase, an essential enzyme that introduces negative supercoils, is a clinically validated target. However, topoisomerase I (Topo I), an enzyme responsible for DNA relaxation has received less attention as an antibacterial target, probably due to the ambiguity over its essentiality in many organisms. The Mycobacterium tuberculosis genome harbors a single topA gene with no obvious redundancy in its function suggesting an essential role. The topA gene could be inactivated only in the presence of a complementing copy of the gene in M. tuberculosis. Furthermore, down-regulation of topA in a genetically engineered strain of M. tuberculosis resulted in loss of bacterial viability which correlated with a concomitant depletion of intracellular Topo I levels. The topA knockdown strain of M. tuberculosis failed to establish infection in a murine model of TB and was cleared from lungs in two months post infection. Phenotypic screening of a Topo I overexpression strain led to the identification of an inhibitor, thereby providing chemical validation of this target. Thus, our work confirms the attractiveness of Topo I as an antimycobacterial target. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Mycobacterium tuberculosis Topoisomerase I Essential Knockdown Vulnerable Anziaic acid Phenotypic screening

1. Introduction Tuberculosis (TB) is a leading cause of mortality worldwide and accounts for nearly two million deaths each year [1]. The treatment options to control tuberculosis are dwindling because of the increase in the incidence of drug resistant TB cases [2]. Poor patient compliance, and co-morbidities like HIV, diabetes and immunosuppressive treatments, have increased the complexity of TB treatment. In recent years, several new anti-mycobacterial agents have been discovered and are undergoing clinical trials for the treatment of TB [3]. Additionally, new combinations of anti-TB drugs are being developed to reduce the duration of therapy [4]. Given the global burden of multi drug resistant tuberculosis (MDRTB), it is important to avoid cross-resistance due to existing

* Corresponding author. E-mail address: [email protected] (S. Ravishankar).

mechanisms. One approach to overcome this challenge is to develop new drugs with a novel mechanism of action, inhibiting growth via a new, unexploited target or pathway. Bacterial DNA topoisomerases regulate the level of DNA topological states in the cell. Both DNA supercoiling and relaxation activities, catalyzed by DNA gyrase and Topo I respectively, are essential for the cells to perform transcription, replication, DNA damage repair and recombination [5]. While the type I topoisomerases catalyze their reaction by cleaving and rejoining a single strand of DNA, the type II topoisomerases act on both strands simultaneously [5]. Both enzymes make transient covalent complexes with the cleaved DNA, termed ‘cleavable complex’, during the process of strand passage. Inhibitors like fluoroquinolones and camptothecins, which bind to such complexes irreversibly, result in accumulation of damaged DNA in the cell, thereby, culminating in the induction of the SOS response and cell death [6,7]. The clinical success of fluoroquinolones has unequivocally proven DNA gyrase

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Please cite this article in press as: Ravishankar S, et al., Genetic and chemical validation identifies Mycobacterium tuberculosis topoisomerase I as an attractive anti-tubercular target, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.05.004

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S. Ravishankar et al. / Tuberculosis xxx (2015) 1e10

as a valid antibacterial target [8]. Although, several inhibitors of human Topo I are available for the treatment of cancer, none of them inhibit bacterial Topo I, probably owing to structural and functional differences between the two enzymes [9]. There has been limited exploration of bacterial Topo I as an antibacterial drug target [10,11]. This could be due to the ambiguity over its essentiality in some Gram negative bacterial species [12e14]. The in vitro essentiality of topA in Escherichia coli has been contentious with reports suggesting that topA could only be inactivated in the presence of suppressor mutations [15,16] or when Topo III (the other type I topoisomerase) is over-expressed [17,18]. However, in the Gram-positive pathogen Streptococcus pneumoniae, inhibitors of Topo I have been shown to suppress bacterial growth suggesting Topo I to be an essential enzyme in S. pneumoniae [11]. Genomic and biochemical evidence suggests that mycobacterial Topo I is similar in structure and in overall function to other bacterial type 1A topoisomerases, and is different from its human homolog, type 1B topoisomerase [19,20]. The Mycobacterium tuberculosis genome encodes a single topA gene and the bioinformatics analysis suggests no obvious redundancy in DNA relaxation function, making it a potential target for the development of antimycobacterial agents. Annamalai et al. and Godbole et al. have described the purification of M. tuberculosis Topo I and characterization of its enzymatic activity [21,22]. Transposon mutagenesis studies in M. tuberculosis H37Rv [23,24] and knockdown (down regulation of expression) studies in M. tuberculosis H37Ra [25] have indicated the essentiality of this gene in M. tuberculosis. However, essentiality information for this gene using a gene knockout and/or a gene knockdown in M. tuberculosis H37Rv, the causative agent of tuberculosis is lacking. While the data from gene knockout experiments provide a reliable proof regarding the essentiality of a gene, target depletion experiments using conditional expression or knockdown (KD) strain enables study of the effect of lowering target protein levels on cellular viability both in vitro and in vivo [26e33]. Knockdown strains also offer the flexibility of performing such studies under a variety of physiological conditions in vitro which mimic the physiological state of M. tuberculosis during its course of infection in humans. Here, we report that topA is essential for the survival of M. tuberculosis H37Rv both in vitro and in vivo. Depletion of intracellular protein levels upon down regulation of topA expression led to loss of viability of M. tuberculosis H37Rv in vitro and in vivo. Phenotypic screening of a compound library using a Topo I overexpression strain of M. tuberculosis resulted in the identification of a Topo I inhibitor. The essential and vulnerable nature of Topo I demonstrated in this study makes this an attractive target for discovering novel drugs against M. tuberculosis. 2. Materials and methods

supernatant was used for separation of pristinamycin components, a depsipeptide (P1) and a macrolide (P2). The separation was performed using Gilson Auto Purification System 845Z with Agilent PDA on a Sunfire 150 mm
300 mm, C18, 10 mM column (Waters Corporation) with a gradient of 10 mM Ammonium Acetate in water pH 4.0 (solvent A) and Acetonitrile (solvent B). The pristinamycin components were identified by analyzing the peaks for required mass using UPLCMS (Waters Acquity UPLCMS system with PDA). Pooling and concentrating of P1 and P2 fractions resulted in a yield of about 52 mg of polyketide and about 48 mg of polypeptide. The purity of the compounds was determined using HPLC (Agilent 1100 Series with PDA). The structure of pristinamycin components were confirmed by recording 1H NMR using 500 MHz Bruker NMR instrument. 2.3. Culture conditions LB medium was used for growing E. coli. Middlebrook 7H9 broth supplemented with 10% Albumin, Dextrose, NaCl (ADN) supplement (Delta Biologicals, India), 0.2% glycerol (SIGMA) and 0.05% Tween80 (SIGMA) was used for culturing mycobacteria. LB agar supplemented with appropriate antibiotics was used when necessary. 7H11 plates were used to plate mycobacteria with appropriate antibiotics and inducer as needed. The media were supplemented with appropriate antibiotics as necessary. Unless mentioned otherwise, the topA knockdown strain was grown in 7H9 broth supplemented with 50 mg/ml hygromycin and 50 ng/ml P1. Hygromycin was used at a concentration of 150 mg/ml and kanamycin at 50 mg/ml for E. coli cultures where necessary. 2.4. Plasmid constructs Plasmids used in this study are listed in Table 1. Briefly, the recombination plasmid containing a deletion in the topA gene of M. tuberculosis H37Rv was constructed by sub-cloning the mutant gene with flanking sequences into a suicide vector, pAZI0290 [34] at the NcoI and BglII sites. The resulting plasmid pAZI0298, had a 299 bp deletion in the topA gene with 804 bp upstream flanking and 800 bp downstream flanking sequences. The complementation construct pBAN1240 was generated by cloning the full length topA gene amplified from M. tuberculosis H37Rv genomic DNA into pAZI0333, a non-replicating vector with attP-int for integration of the plasmid into the chromosome and Ptrc promoter [35] to drive the expression of cloned gene. pBAN0303, the M. tuberculosis topA conditional recombinant plasmid (KD construct) was generated by cloning 690 bp of topA gene from its 50 end, amplified from M. tuberculosis H37Rv genomic DNA into NcoI and SphI sites of pAZI9479 [30]. Topo I over-expression strain was prepared by transforming a recombinant plasmid harboring full length M. tuberculosis topA gene cloned in pMV261 plasmid [36].

2.1. Bacterial strains, media, chemicals and reagents Bacterial strains used in this study are described in Table 1. Restriction enzymes, 1 kb DNA ladder were from New England Biolabs, USA; Hygromycin B was obtained from Roche, USA; pristinamycin (pyostacine) from Sanofi-Aventis, France. Hybond membrane and chemiluminiscence Western and Southern blot kits were from GE Healthcare, USA; 0.1 mm Zirconia beads and Minibead-beater were from Biospec products, USA. 2.2. Purification of pristinamycin components Commercially available 250 mg tablet of pyostacine was crushed using a pestle and agate mortar. The resulting powder was resuspended in methanol and centrifuged at 14000 rpm. The

2.5. Generation of M. tuberculosis topA gene knockout (KO), knockdown (KD) and overexpression (OE) strains The recombinant plasmids were electroporated into M. tuberculosis H37Rv by the procedure described by Wards and Collins [37]. The gene knockout was performed by using the twostep method as described by Parish and Stoker [38]. At each step, the strains {single cross over (SCO), merodiploid or double cross over (DCO)} were selected in the presence of appropriate antibiotic markers and confirmed by a set of PCRs using the primers listed in Table S1. The allelic exchange substrate for topA gene KO on plasmid pAZI0298 consisted of a mutant topA gene with a 299 bp markerless deletion (302e600 bp) with 804 bp upstream and 800 bp downstream fragments flanking the deleted region. The SCO

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Table 1 Strains and plasmids used in this study. Strains/plasmids

Description of strain/plasmid

Source

pAZI0290 pBAN0333 pAZI0298 pBAN1240

Gene KO vector (suicide) with E.coli ori, sacB, hygromycinR Integrating mycobacterial expression vector with Ptrc promoter M. tuberculosis H37Rv topA knockout substrate in pAZI0290 M. tuberculosis H37Rv topA full length gene cloned downstream of Ptrc promoter in pAZI0333 (complementation construct) A conditional expression vector with pristinamycin inducible promoter system M. tuberculosis H37Rv topA full length gene cloned in pMV261 M. tuberculosis topA conditional expression plasmid Replicating E. coli e mycobacterial shuttle vector with hsp60 promoter Plasmid DNA used as substrate in relaxation assay M. tuberculosis H37Rv topA conditional expression strain M. tuberculosis topA SCO recombinant strain M. tuberculosis H37Rv topA merodiploid strain M. tuberculosis H37Rv topA gene knockout strain in the merodiploid background M. tuberculosis H37Rv topA over-expression strain endA1, hsdR17, supE44, recA1, relA1, (lacZYA-argF) Virulent strain of M. tuberculosis H37Rv Non-pathogenic mycobacterial strain.

(34*) (35*) This study This study

pAZI9479 pBAN0446 pBAN0303 pMV261 pJT519 topA/KD topA/SCO topA/mero topA/KO topA/OE E.coli DH5a M. tuberculosis H37Rv ATCC27294 M. smegmatis mc2155 *

(30*) This study This study (36*) (41*) This study This study This study This study This study Lab. Stock Lab. Stock Lab. Stock

References.

recombinants obtained by transformation of M. tuberculosis H37Rv with the allelic exchange substrate pAZI0298 were confirmed by PCR using primers listed in Table S1. Primers were designed in such a way that they would amplify only a single product closest to the recombination site and the second locus being too far because of the integration of the entire plasmid would not get amplified under the conditions used for PCR. One of the SCO recombinants showing integration in the proper locus, topA/SCO, was allowed to undergo DCO recombination event. For generating a merodiploid strain, topA/SCO was transformed with pBAN1240 carrying M. tuberculosis topA gene under the control of Ptrc promoter which had a lac operator sequence adjacent to the Ptrc promoter and the lacI expressed from the same plasmid [35]. The integration of pBAN1240 into the chromosome of the topA/SCO was confirmed by amplification of the kanamycin resistance gene and the resulting strain was designated topA/mero. This strain was grown in 7H9 broth without hygromycin supplementation before plating for DCO selection on 7H9 plates containing 2% sucrose. As the Ptrc promoter was found to have significant leaky expression, it was not necessary to include IPTG to induce topA gene expression. topA/mero strain was allowed to undergo DCO recombination event to generate topA/KO which was confirmed both by PCR and Southern blot analysis. Conditional expression or the knockdown strain, topA/KD, was generated by electroporating M. tuberculosis H37Rv with the recombinant plasmid pBAN0303. Transformants were selected on 7H9-agar plates {with 10% Albumin, Dextrose, NaCl (ADN) supplement (Delta Biologicals, India), 0.2% glycerol (SIGMA) and 0.05% Tween 80 (SIGMA)} containing 50 mg/ml hygromycin and 100 ng/ ml of P1. A set of PCRs were performed to confirm the genotype of the recombinant strain topA/KD i.e., presence of truncated topA gene downstream of the native promoter and full length topA gene downstream of Pptr promoter. The sequences of the primers used in this study are listed in Table S1. M. tuberculosis topA overexpression strain, topA/OE was generated by selecting transformants of M. tuberculosis H37Rv electroporated with plasmid pBAN0446 on 7H9 plates containing 25 mg/ml of kanamycin followed by PCR screening to confirm the presence of Kanamycin resistance gene. 2.6. PCR screening of knockout strains Screening of SCO and DCO recombinants in M. tuberculosis were done by PCR using Taq DNA polymerase. Single colonies were picked from plates, resuspended in 50 ml TE (10 mM Tris, 0.1 mM

EDTA pH 8.0) and boiled for 20 minutes. 5 ml of these cell lysates were used as template in PCR in a 25 ml reaction volume. The denaturation and extension reactions were performed at 94  C and 72  C respectively. The annealing temperature was based on the melting temperatures of the primer pair used (generally 5  C below Tm). The extension time of PCR was based on the length of the PCR product amplified (~1 min/kb).

2.7. Southern blot analysis Genomic DNA isolated from wild-type M. tuberculosis H37Rv and the topA/KO strains were digested with ApaI and PvuII restriction enzymes. The digested DNA was blotted onto Hybond nitrocellulose membrane and probed with a 260 bp PCR product corresponding to 614th to 354th bp upstream of topA gene (Fig. S1A). The DNA bands hybridizing with the probe were detected using ECL chemiluminiscence kit following the method recommended by the manufacturer (GE Healthcare).

2.8. Growth kinetics of M. tuberculosis topA/KD strain Inducer dependency of the conditional expression strain for growth was determined by plating dilutions of a culture of topA/KD strain on 7H11 plates with and without P1. In brief, topA/KD culture was grown in 7H9 broth supplemented with 50 mg/ml of hygromycin and 100 ng/ml P1 till mid-log phase. The culture was washed three times with plain 7H9 broth, resuspended and dilutions plated on 7H11 plates supplemented with 0e100 ng/ml of P1. Wild-type M. tuberculosis H37Rv culture was processed in a similar manner to see the effect of the inducer on its growth. For studying growth kinetics in broth, cells harvested from a mid-log phase culture of topA/KD were washed three times and resuspended in fresh 7H9 broth. The washed culture concentrate was used as inoculum for both in vitro growth kinetic and in vivo phenotyping experiments. For in vitro growth kinetic experiments, a master culture having 106 CFU/ml was prepared in 7H9 broth supplemented with 50 mg/ ml hygromycin. This culture was split into four parts and P1 was added to final concentrations of 0, 12, 25, 50 ng/ml. Growth of these cultures were measured by checking OD600 at regular intervals and viability was assessed by plating culture dilutions on 7H11 plates supplemented with 50 ng/ml P1. The same cultures were also plated on 7H11 plates without P1 supplement to ensure that they had not reverted during the course of the experiment. Culture samples on

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day 8 were subjected to Western blot analysis to measure the intracellular Topo I levels. 2.9. Western blot analysis 10 ml cultures of wild-type M. tuberculosis H37Rv and topA/KD strains were harvested on day 8. The cell pellets were washed twice in 1X PBS and re-suspended in 250e500 ml of 1X PBS containing a cocktail of protease inhibitors (Roche). Cell suspensions were transferred to 2 ml screw cap tubes containing about 0.1 g of 0.1 mm Zirconia beads (Biospec Products). The cells were lysed by bead beating the cell suspension twice at 4500 rpm for 20 s each in a Mini-bead-beater (Biospec Products). The clarified lysate obtained after centrifuging at 10,000 rpm for 5 min was used for protein estimation using Bradford reagent (PIERCE Biotechnology Inc.). Unless otherwise mentioned, 2 mg total protein of each sample was subjected to SDS-PAGE and the proteins were blotted onto a Hybond nitrocellulose membrane. 1:100,000 diluted Rabbit polyclonal anti-Topo I sera (Abexome Biosciences, India) and 1:100,000 diluted anti-SigA sera were used for probing the blots. Blots were further developed using ECL advance Western Blotting kit. 2.10. Infection in mice The AstraZeneca Animal Ethics Committee, registered with the Government of India (registration no. CPCSEA 99/5) approved all animal experimental protocols and usage. The culture stocks for in vivo work were prepared as described above for growth kinetic studies. Balb/c mice (8e10 weeks old) were infected via aerosol route with about 103 CFU of either M. tuberculosis H37Rv or topA/KD strain. Three mice per group were sacrificed on designated days to monitor the course of infection. The lung homogenates from M. tuberculosis H37Rv infected mice were plated on 7H11 plates while those from topA conditional expression strain infected mice were plated on 7H11 plates supplemented with 50 mg/ml hygromycin and 50 ng/ml of P1. The plates were incubated at 37  C for 30 days and the colony forming units (CFU) were enumerated. Undiluted lung homogenates of topA/KD infected mice were also plated on 7H11 plates without P1 supplementation to ensure that the strain had not reverted during the course of infection. 2.11. MIC determination Wild-type M. tuberculosis H37Rv, Mycobacterium smegmatis mc2155 and the topA/OE strains were used to test the MIC of compounds using the standard RBMA detection method [39]. The starter cultures were grown till mid log phase as described earlier and MIC was set up with diluted culture having a starting inoculum of 105 CFU/ml. MICs were also set up for test compounds in presence of fixed concentrations of two efflux inhibitors (32 mg/ml of PABN, 256 mg/ml of verapamil) and two permeability enhancers (64 mg/ml each of colistin and polymyxin B). Minimum bactericidal concentration (MBC) was determined using M. smegmatis mc2155 as described in Shirude et al. [40]. 2.12. Topoisomerase I enzyme assay Recombinant M. tuberculosis H37Rv Topo I was purified (manuscript in preparation) using a protocol similar to the one described by Annamalai et al. [21]. A 30 ml assay was set up in 96 well half area black plate using supercoiled pJT519 [41] plasmid DNA as substrate and M. tuberculosis Topo I. Test wells received 1 ml of 30-fold concentrated compound solutions. While the positive control (complete reaction) well received 1 ml of DMSO, the negative control well received 1 ml of quench buffer (0.5 M Sodium

chloride and 0.5 M Sodium acetate). 30 ml reaction mix consisting of 2 mg of supercoiled pJT519 were incubated with 0.45 nM of M. tuberculosis Topo I in a reaction buffer containing 50 mM Tris-Cl pH 8.0, 100 mM potassium glutamate, 10 mM MnCl2, 1 mM DTT and 0.005% Brij-35. The assay plate was incubated at 37  C for 100 min. At the end of incubation period, reaction was stopped by the addition of SDS-Proteinase K solution and further incubated for 1 h at 37  C to arrest enzyme activity. 4 ml of the reaction mix (~50 ng DNA) was analyzed using 0.8% agarose gel in TBE buffer overnight at 30 V. Gels were stained with 0.5 mg/ml of ethidium bromide and the bands were visualized using Fuji phosphorimager. 2.13. DNA gyrase assay DNA supercoiling assay was performed as previously described [42] to test the inhibitory activity of anziaic acid on M. tuberculosis DNA gyrase. 3. Results 3.1. topA is an essential gene in M. tuberculosis H37Rv For establishing in vitro essentiality of M. tuberculosis topA, we attempted gene knockout (KO) in wild-type and merodiploid strains of M. tuberculosis H37Rv. The ability to inactivate topA only in the presence of an additional copy of the gene in the merodiploid strain and not in the wild-type M. tuberculosis strain was taken as evidence of gene essentiality. The methodology of gene inactivation by homologous recombination using a deleted copy of the gene has been described in detail under materials and methods. The DCO recombinants obtained from topA/SCO (Figure S1B panel a) were analysed by PCR for the presence of sacB and hygromycin genes. Despite showing a high frequency of recombination (70%), none of the colonies showed the presence of a mutant copy of topA in the genome (data not shown). This indicated that the topA gene is likely to be essential for the survival of M. tuberculosis H37Rv in vitro. In order to confirm this hypothesis experimentally, the topA knockout was attempted in a merodiploid background (Figure S1B panel b). The frequency of DCO recombination in the merodiploid strain was in a similar range as seen with the SCO strain earlier. However, in contrast with topA/SCO, the DCO recombination in topA/mero resulted in replacement of the wild-type topA gene by a deleted copy in 20% of the colonies, (18 of 88; Figure S1B panel c). This data proved that topA is an essential gene in M. tuberculosis H37Rv and at least one copy is required for the survival of the cells in vitro. Southern blot analysis was performed to further confirm the genotype in the DCO. As seen in Figure S1C, the genomic DNA from topA/KO strain clearly showed presence of deleted copy of topA gene. 3.2. Generation of M. tuberculosis topA conditional expression strain topA/KO strain was generated with a complementing copy of topA gene driven by IPTG inducible Ptrc promoter. However, due to the leaky expression from this promoter, the strain couldn't be used like a conditional expression strain to study the phenotype under target depleted conditions. Therefore, we utilized the tightly repressible pristinamycin inducible promoter system [30] for generating a topA/KD strain in M. tuberculosis H37Rv. Pristinamycin used for the treatment of Staphylococcal and Streptococcal infections, is a streptogramin class of antibiotic produced by S. pristinaespiralis. The two components P1, a depsipeptide and P2, a macrolide are known to be less potent than their mixture which work synergistically [43]. Due to its high potency against

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M. tuberculosis H37Rv with a MIC of 0.12 mg/ml (Table S2), the use of pristinamycin containing both components as inducer to regulate bacterial gene expression in M. tuberculosis could be challenging. Therefore, we purified component P1 to homogeneity as described in materials and methods. A 250 mg tablet of pyostacine was crushed and subjected to HPLC purification (Figure S2A). The pure fractions characterized by 500 MHz 1H NMR (Figure S2B) were used for bacteriological testing. MIC of these individual components against M. tuberculosis H37Rv was determined to be in the range of 2e16 mg/ml by the standard RBMA method (Table S2). pAZI9479 (Figure S3) is a non-replicating vector containing Pptr promoter-operator and the PIP repressor. The gene for PIP repressor from Streptomyces coelicolor present on this plasmid is constitutively expressed from M. tuberculosis PfurA102 promoter. The transcription block created by the binding of repressor to the operator region gets relieved when the PIP repressor binds to pristinamycin or component P1 allowing expression of genes cloned downstream of Pptr promoter. This system has been evaluated in M. tuberculosis for its ability to regulate expression in the absence of inducer and to induce expression upon addition of inducer by Forti et al. [30]. For generating a topA/KD strain, M. tuberculosis H37Rv was transformed with the suicide plasmid pBAN0303 carrying a truncated copy of topA under pristinamycin inducible promoter. The SCO recombinants generated by integration of the plasmid into the topA locus were selected on 7H9 agar plates containing 50 mg/ml of hygromycin and varying concentrations (10e1000 ng/ml) of P1. A bonafide recombinant strain was expected to have the full length topA gene downstream of the inducible Pptr promoter and the cloned truncated topA gene downstream of the topA native promoter. The gene arrangement of the resultant SCO recombinants were confirmed by a set of PCRs as depicted in the schematic (Figure S4A) using primers listed in table S1. Two of the transformants #36 and #41 appeared to have appropriate genotype (Figure S4B) and were dependent on P1 supplementation for growth (data not shown). The recombinant colony #41 which got designated as topA/KD was used for all the phenotyping experiments. 3.3. Growth of topA conditional expression strain is inducer dependent Growth of a conditional expression strain would be inducer dependent if the gene under study is an essential one for the survival of M. tuberculosis provided the regulatory system is tightly controlled and can achieve ‘zero’ level of expression in the absence of inducer. To confirm that the growth of topA/KD conditional expression strain was inducer dependent, different dilutions of a culture of topA/KD strain were plated on 7H11 agar plates containing 50 mg/ml hygromycin and 0, 5, 10, 25, 50, 100 ng/ml of P1. Growth could not be detected below 25 ng/ml of P1 on agar plates indicating that the KD strain is dependent on P1 for its growth. A concentration of 50 ng/ml of P1 was found to be optimum for growth of topA/KD strain (Figure 1A). P1 did not have any effect on the growth of wild-type M. tuberculosis strain at this concentration (data not shown). Based on this observation, the recombinant strain was always grown in the presence of 50 ng/ml P1 (minimum inducer required) unless otherwise mentioned. 3.4. Depletion of intracellular Topo I is bactericidal for M. tuberculosis Although depletion or inhibition of essential proteins in bacteria results in inhibition of growth, it may not necessarily lead to loss of cellular viability [26e28]. Hence, in order to develop cidal drugs it is important to identify targets whose depletion or inhibition will

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lead to cidality. In order to study the effect of depletion of Topo I on growth and viability of M. tuberculosis, we analyzed the growth and survival of topA/KD strain in the presence of increasing concentrations of P1 ranging from 0 to 50 ng/ml for a period of 10 days. During the study, the cells were monitored for growth by measuring cell viability at regular intervals by plating on 7H11 plates containing 50 ng/ml of P1 (Figure 1B). Non viability of these same culture samples on 7H11 plates without P1 confirmed the strain had not reverted during the course of the experiment. As seen in Figure 1B, complete withdrawal of inducer resulted in a phenotypic effect which was biphasic in nature. Until day 4, the growth rates of the topA/KD strain without and with different inducer concentrations were not significantly different. This probably is due to the pre-existing Topo I in the cell synthesized during the inoculum preparation when the strain was grown with 50 ng/ ml P1. It could be also due to the expression of Topo I in these 4 days from the P1 accumulated inside the cells during the inoculum preparation. However, between day 4 and day 10, more than two log reduction in CFU was observed in the culture without P1. This could be attributed to the depletion in the total intracellular Topo I levels when probably the pre-existing Topo I is completely degraded and there is no fresh synthesis due to the absence of inducer in the growth medium. However, during this 7 day period (day 4 to day 10), the growth rates of topA/KD strain supplemented with 12, 25 and 50 ng/ml of P1 did not seem to vary significantly. Day 8 samples were also analyzed for intracellular levels of Topo I protein by Western blot analysis. Assessment of intracellular Topo I levels in cells sampled on day 8 from the kinetic study showed significant reduction in Topo I levels in cultures without P1 as compared to the levels seen in the wild-type cells (Figure 1C). Topo I levels were the same as in wild-type strain or higher in the rest of the topA/KD cultures. In contrast, the Sigma 70 (SigA) levels used as internal control were found to be same as in all the samples irrespective of the concentration of P1 used in the culture. This data clearly demonstrated a direct correlation between the inducer concentration in the culture medium and the intracellular Topo I levels which is in turn correlated with cell viability of the topA/KD strain. 3.5. Topo I is required for the survival of M. tuberculosis H37Rv in mice Conditional expression strains have been used to evaluate the impact of target depletion on the ability of a pathogenic strain to establish infection and survive in an animal model [32,33]. MIC of P1 is about 4 mg/ml against M. tuberculosis H37Rv while the topA/KD strain needed a minimum inducer concentration of about 50 ng/ml for sufficient gene induction (Figure 1A, B, C). Therefore, it was essential to maintain plasma levels of P1 within these limits over a period of time for optimal gene expression in vivo to study the survival kinetics of topA/KD in mice. However, the pharmacokinetic data (Figure S5) generated after oral administration of P1 at 10 and 30 mg/kg dose in mice indicated poor plasma concentration due its short half-life (~1 h). Such low levels of P1 would not allow sufficient exposure to maintain topA gene expression during the course of infection. Therefore, the in vivo phenotypic analysis of the topA/ KD strain was performed in the absence of inducer. The inoculum for mouse infection was prepared by growing the topA/KD strain in the presence of 50 ng/ml of P1. The cells were washed to remove the inducer and used as inoculum for aerosol infection. In this study, we compared the course of infection of the conditional expression strain with that of the wild-type M. tuberculosis H37Rv. As seen in Figure 2, aerosol infection of both wild-type and the KD strains resulted in seeding of 103 CFU per mouse lung at 24 h post infection. With the wild-type strain,

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Figure 1. Growth and survival of M. tuberculosis topA/KD strain in vitro. A. Determination of minimum inducer concentration for growth of topA/KD strain. A mid log phase culture of topA/KD strain grown with 50 mg/ml hygromycin and 50 ng/ml P1 was harvested, washed three times and several dilutions plated on 7H11-hygromycin plates supplemented with 0, 10, 25, 50 or 100 ng/ml of P1. Colony forming units (CFU) are the average numbers of colonies obtained from 4 different dilution plating. B. Growth kinetics of topA/KD strain. topA/KD strain was grown in 7H9 broth supplemented with 50 mg/ml hygromycin and 50 ng/ml P1. The cells harvested were washed 3 times and resuspended in fresh 7H9 broth to use as inoculum in growth kinetic experiment. A master stock was prepared by diluting the washed culture preparation to achieve a CFU of about 106/ml. This was split into many roller bottles each of which received a different inducer concentration. The growth was monitored by plating culture samples for CFU on days 0, 4, 8 and 10 by plating them on 7H11 plates containing 50 ng/ml P1 for survivors and on plates without P1 for revertants. Data shown here is a representative of data from three independent experiments. WT: wild-type M. tuberculosis H37Rv. C. Western blot analysis. 2 mg equivalent of total protein loaded per lane was resolved by SDS-PAGE followed by Western blotting. The blots were probed with either Topo I or SigA (internal control) antisera. 0, 12, 25 and 50 ng/ml: P1 concentration to which the topA/KD strain culture was exposed to. P: purified Topo I and Rv: M. tuberculosis H37Rv cell lysate.

there was an exponential increase in the bacterial cell numbers up to 20 days followed by steady numbers in the bacterial CFU up to 60 days. In contrast, the topA/KD strain did not show any increase in CFU until day 10. However, beyond day 10, the KD strain showed

continuous decline in CFU with 2 log drop by day 20. By day 60, the KD strain was completely cleared from the lungs as no CFU could be detected even after plating the entire lung homogenate (Figure 2). This data clearly indicated that topA/KD cells were unable to survive in the lungs of mice in the absence P1 supplementation suggesting the requirement for Topo I expression during the course of infection. 3.6. Chemical validation of M. tuberculosis Topo I

Figure 2. Survival kinetics of topA/KD strain in mice. Bacterial load in lungs of mice infected with M. tuberculosis H37Rv (open squares) and topA/KD strain (open circles) was monitored by plating for survivors on 7H11 plates for M. tuberculosis H37Rv and 7H11 plates supplemented with 50 ng/ml of P1 for topA/KD strain.

Two approaches were taken to chemically validate M. tuberculosis Topo I. The first approach was to test a reported bacterial Topo I inhibitor for its inhibitory action against M. tuberculosis H37Rv. The second approach was to identify a Topo I inhibitor with a phenotypic screen using Topo I overexpression strain of M. tuberculosis H37Rv (topA/OE) with the rationale that we would be able to identify inhibitors of Topo I with different mechanisms of action which would ultimately lead to growth inhibition. The process of relaxation of supercoiled DNA catalyzed by Topoisomerase I goes through a series of key steps like DNA binding, cleavage, strand passage and religation. The cleaved DNA is covalently held by the enzyme until the strand passage occurs and religation step is initiated [6]. Although compounds inhibiting any of these steps would be effective, an inhibitor of religation step is considered detrimental for cells as it results in accumulation of

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S. Ravishankar et al. / Tuberculosis xxx (2015) 1e10

cleaved DNA leading to induction of SOS response culminating in bactericidal effect. This mechanism of inhibition (poisoning) would be very similar to that of fluoroquinolones on DNA gyrase and the anticancer agent camptothecin on human Topo I [7]. We hypothesized that Topo I overexpressing M. tuberculosis will display an increased sensitivity to Topo I poisons akin to the increased sensitivity demonstrated by human Topo I overexpressing cells for camptothecin [44]. Topo I over-expressing cells have also been shown to have increased sensitivity to DNA damaging agents probably due to the accumulation of replication mediated double strand DNA breaks and stalling of transcription complexes [45,46]. Addition of inhibitors like camptothecin which traps the Topo IDNA complex would increase such DNA damage several fold making it impossible for the cellular DNA damage repair systems to counter the situation leading to cell killing. On the other hand, nonpoison inhibitors of M. tuberculosis Topo I would be less potent on the overexpression strain as compared to the potency on wild-type strain. An inhibitor identified by either approach would not only indicate the mode of action of the inhibitor but also chemically validate the target. An overexpression strain of M. tuberculosis Topo I was generated as described in materials and methods in which topA overexpression was confirmed by Western blot analysis (Figure 3A). In the first approach towards M. tuberculosis Topo I chemical validation, anziaic acid (Figure 3B), an inhibitor reported for E. coli and B. subtilis Topo I [47,48] was synthesized (Figure S6) and tested for anti-tubercular activity and mycobacterial Topo I inhibitory activity. MIC assay was set up both in the absence and the presence of efflux pump inhibitors like verapamil, PABN and permeability enhancers like colistin, polymixin B. Some of these agents are known to potentiate the antibiotic activity of mycobacterial species [49,50]. Anziaic acid had a potent MIC of about 6 mM against M. tuberculosis, but its MIC remained the same irrespective of the absence or the presence of efflux pump inhibitors and permeability enhancers (Table 2) indicating absence of any major efflux and permeability issues for this molecule. Since anziaic acid was reported as a Topo I poison, it was tested for MIC against both wildtype M. tuberculosis and topA/OE strains. We hoped that the topA/ OE cells would display increased sensitivity to anziaic acid as is the case with camptothecin in human Topo I overexpressing cells [44] and imipramine, norclomipramine in case of mycobacteria [51]. However, MIC of anziaic acid was the same (Table 2) both in wildtype and topA/OE strains suggesting a different mechanism of action. All Topo I inhibitors, irrespective of their mechanism of action should inhibit a DNA relaxation assay in a cell-free system. In order to confirm this, we tested anziaic acid in a DNA relaxation assay catalyzed by M. tuberculosis Topo I. Even in this assay, we didn't observe any inhibitory activity even at 250 mM (Figure 3C). The above results were intriguing as anziaic acid was inhibiting the growth of M. tuberculosis but not through inhibition of topoisomerase I. To check if anziaic acid would inhibit the only other topoisomerase in M. tuberculosis, we tested it in mycobacterial gyrase assay and found that it didn't inhibit DNA gyrase also (Figure 3D). We therefore concluded that anziaic acid is not an inhibitor of any of the M. tuberculosis topoisomerases and that the growth inhibition observed is due to a different mechanism which needs to be unraveled. Thus this approach was unable to provide a positive proof towards chemical validation of M. tuberculosis Topo I. In the second approach, we screened for small molecule inhibitors of M. tuberculosis Topo I using a Topo I overexpression strain (topA/OE). A focused library of about 1500 small molecules put together from AstraZeneca's corporate compound collection was screened in parallel against both wild-type M. tuberculosis H37Rv and M. tuberculosis topA/OE strains. Unlike the conventional 10 point MIC assay with two fold changes in compound

7

concentration, the primary screen was performed in a 5 point MIC assay with four fold changes in compound concentrations. Screening of 1500 small molecule compounds collection resulted in a shortlist of ten compounds displaying altered MIC in the topA/OE strain as compared to the wild-type strain. Conventional 10 point MIC assays were subsequently performed with these ten compounds to confirm the observation made in the primary screen. While a number of initial hits failed to reproduce the results in the secondary screen, one compound - dihydrobenzofuranyl urea (compound S, Figure 3E) consistently showed 4e8 fold increase in MIC in the Topo I overexpressing strain (Table 2). No modulation in MIC was observed with M. tuberculosis strain harboring the parent plasmid pMV261. Also, none of the other standard inhibitors tested showed any MIC modulation in topA/OE as compared to either the wild-type strain or pMV261 harboring M. tuberculosis H37Rv (Table S3). These results indicated that compound S is a Topo I inhibitor, but probably not a Topo I poison. However, as mentioned before, all Topo I inhibitors with differing mechanisms of action, are expected to inhibit the DNA relaxation activity. We tested all the 10 compounds in a Topo I enzyme assay in which only compound S inhibited the enzyme assay with an IC50 of about 60 mM (Figure 3F). The compound also had MIC of 4 mM and MBC (minimum bactericidal concentration) of 60 mM against the related non-pathogenic strain M. smegmatis. Thus, the phenotypic screen was able to provide a compound, although not very potent, enabling chemical validation of M. tuberculosis Topo I suggesting that the target is vulnerable to chemical inhibition as well. Further medicinal chemistry efforts are needed to fine tune the SAR to improve the potency of this molecule both on the enzyme and on M. tuberculosis culture. 4. Discussion The increased incidence of multi drug resistance has prompted the search for new drugs with a novel mechanism of action for the treatment of TB. Exploitation of new targets or pathways offers better probability of finding novel drugs with the ability to work against multi drug resistant strains of M. tuberculosis. However, a new target requires thorough validation before launching a fullfledged drug discovery program [52]. The targets of most of the antibiotics are essential proteins and inactivation of corresponding genes is known to result in loss of bacterial viability. Hence, target essentiality has generally been considered a critical attribute for a target to design and develop specific antibacterial inhibitors. Even though chemical inhibition of an essential target is expected to result in growth inhibition, in many instances, potent enzyme inhibitors have failed to have any effect on bacterial growth or viability [53]. Cell permeability issues, efflux or compound modification in bacterial cells could be some of the reasons for this disconnect. However, with certain targets like dihydro folate reductase (DHFR), growth inhibition or loss of viability may require a near complete inhibition or depletion of the target [28]. Hence, in recent years efforts have been focused to identify targets in M. tuberculosis whose depletion leads to loss of viability. Target depletion studies using KD strains have proven to be useful tools for identifying such vulnerable targets [25e33]. Unlike DNA gyrase, Topo I has not been considered as a target for developing inhibitors against bacteria. The reason could be its non-essential nature in a number of Gram negative bacterial species [12,13,54,55]. Although, initial studies highlighting the essentiality of topA in E. coli had been debated, later studies showed that, topA could be inactivated in E. coli with mutants showing a cold sensitive phenotype [17]. It appeared that RNAse H could complement the function of Topo I in these mutants, as E. coli mutants lacking both Topo I and RNAse H were found to be non-viable. The loss of Topo I function in E. coli

Please cite this article in press as: Ravishankar S, et al., Genetic and chemical validation identifies Mycobacterium tuberculosis topoisomerase I as an attractive anti-tubercular target, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.05.004

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Figure 3. Identification and Characterization of Topo I inhibitors. A- Western blot analysis of topA/OE strain: lanes 1. Purified M. tuberculosis Topo I, 2. 0.5 mg M. tuberculosis/ pMV261 lysate (vector control), 3. 0.5 mg of topA/OE lysate; B- Anziaic acid; C- M. tuberculosis Topo I assay: lanes- P. Supercoiled DNA substrate, N. Negative control, X. Positive control, 1e10. 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19 mM of Anziaic acid; D- Mycobacterial DNA gyrase assay: lanes- N. Negative control, X. Positive control, 1e10. 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19 mM of Anziaic acid; E- Compound S; F- M. tuberculosis Topo I assay: lanes- N. Negative control, X. Positive control, 1e10. 250, 125, 62.5, 31.2, 15.6, 7.8, 3.9, 1.9, 0.97, 0.49 mM of Compound S. R: Relaxed Plasmid DNA, SC: Supercoiled plasmid DNA.

could also be compensated by over expressing Topo III [17,18]. Similarly over-expression of Topo IV could compensate for the loss of topA in Shigella flexneri [55]. The ability of Topo III, Topo IV or RNAse H to complement the function of Topo I in mutant strains suggested the existence of functional redundancy to carry out DNA

relaxation in bacteria [14]. Our gene KO data in the present work has established that topA is an essential gene in M. tuberculosis H37Rv. This is also consistent with the reported inability to obtain transposon insertions in topA gene of M. tuberculosis H37Rv [23] and a recent report on KD of topA in an avirulent strain of

Table 2 MIC of compound S and anziaic acid against M. tuberculosis H37Rv. MIC (mM) of M. tuberculosis H37Rv in the presence of

MIC (mM)

Inhibitor

16 mg/ml polymixin B

8 mg/ml PABN

50 mg/ml verapamil

16 mg/ml colistin

M. tuberculosis H37Rv

topA/OE

Anziaic acid Compound S

6.25 ND

3.12 ND

3.12 ND

12.5 ND

6.25 12e25

6.25 100

PABN, Verapamil, Colistin and Polymixin B have MIC of 32, 256, 64 and 64 mg/ml respectively on the wild-type strain. MIC values are an average of the values obtained from 2 independent experiments.

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S. Ravishankar et al. / Tuberculosis xxx (2015) 1e10

M. tuberculosis H37Ra [25]. This can be explained by the absence of Topo III, Topo IV and RNAse H coding genes in the M. tuberculosis genome. Complete lack of growth of topA/KD strain in the absence of added inducer in liquid cultures or agar plates provided further evidence to the fact that Topo I expression is essential for the growth of M. tuberculosis H37Rv in vitro. The growth kinetics data along with Western blot analysis confirmed that the depletion of intracellular Topo I levels resulted in mycobacterial cell death. Given the correlation between protein depletion due to chemical inhibition [28] and in the knockdown strain grown under suboptimal conditions resulting in the decrease of functional enzyme levels, it is likely that a Topo I inhibitor will result in a bactericidal effect. Studies investigating DNA cleavage along with mutation analysis using E. coli and Y. pestis Topo I have shown that similar to DNA gyrase, inhibition of Topo I results in DNA cleavage leading to induction of the SOS response [56,57] and bacterial cell death. Thus, inhibitors that poison Topo I with SOS inducing properties might turn out to be highly bactericidal to M. tuberculosis. Limited attempts were made during the course of this study to identify compounds that inhibit M. tuberculosis Topo I and exhibit antimycobacterial activity to enable us to use them for chemical validation of the target. The phenotypic screening performed on a focussed set of compounds using a Topo I overexpressing strain resulted in the identification of a potential Topo I inhibitor which showed inhibition of DNA relaxation activity in vitro. This evidence is consistent with the cellular killing of M. tuberculosis by this compound being mediated by inhibition of Topo I. Further medicinal chemistry efforts to expand this initial hit and screen a larger library of analogs will enable the discovery of novel M. tuberculosis Topo I inhibitors with potent anti-mycobacterial activity. The severe attenuated phenotype of topA/KD strain observed in the lungs of infected mice suggests that potent antimicrobial inhibitors of Topo I are likely to be efficacious in infected mice. A desirable attribute of a novel inhibitor of M. tuberculosis is its ability to achieve sterilization in infected tissues by virtue of its property to kill non-replicating M. tuberculosis. Hence, proteins that are crucial for maintaining survival under non-replicating conditions are considered good targets for developing inhibitors with the potential for sterilization. The continued basal level of transcription and DNA damage repair are possibly the major mechanisms in the non-replicating state of M. tuberculosis [58,59] which would require the function of Topo I. Hence we propose that presence of Topo I needed for both these activities would be essential for the survival of such population and its depletion by means of a chemical inhibitor would make cells vulnerable. The availability of extensive biochemical data on M. tuberculosis Topo I [21,22] will be valuable in designing inhibitors against this enzyme. Structural studies have shown critical differences between bacterial and human Topo I proteins [60,61] which in turn should allow the identification of specific inhibitors against the M. tuberculosis enzyme. In conclusion, the availability of robust biochemical tools, distinct structural differences between the bacterial and human Topo I enzyme combined with essential nature of this target under in vitro and in vivo growth conditions offers an attractive option to identify novel class of inhibitors against M. tuberculosis. Acknowledgments We thank Abhishek Roy and Haripriya Ramu for the generation of KD vectors, Vishwas and Naveen Kumar for their help in performing animal infection. Drs. Radha Shandil and Sheshagiri Gaonkar for inputs on in vivo study design and Manish Parab and K. N. Mahesh Kumar for pharmacokinetic studies. We thank NM4TB and Dr. Weber for providing the components of pristinamycin

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inducible system. We thank Dr. T.S. Balganesh for continued support during this study. We acknowledge the support provided by Drs. Balasubramanian, Achyut Sinha and Dwarakanath in manuscript preparation. We acknowledge Syngene International Ltd for the supply of pure anziaic acid. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.tube.2015.05.004. Funding:

None.

Competing interests: Ethical approval:

None declared. Not required.

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Please cite this article in press as: Ravishankar S, et al., Genetic and chemical validation identifies Mycobacterium tuberculosis topoisomerase I as an attractive anti-tubercular target, Tuberculosis (2015), http://dx.doi.org/10.1016/j.tube.2015.05.004