nucl. acids res. 2013 biswas e56

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A novel non-radioactive primasepyrophosphataseactivity assay and its application to the discoveryof inhibitors of Mycobacterium tuberculosis

primase DnaGTapan Biswas1, Esteban Resto-Roldan1,2, Sean K. Sawyer1, Irina Artsimovitch3 and

Oleg V. Tsodikov1,*

1Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, 428 Church Street,

Ann Arbor, MI 48109, USA, 2Research Experiences for Undergraduates Program, College of Pharmacy,

University of Michigan, 428 Church Street, Ann Arbor MI 48109, USA and 3Department of Microbiology,

Ohio State University, 105 Biological Sciences Building, 484 West 12th Avenue, Columbus, OH 43210, USA

Received August 31, 2012; Revised November 10, 2012; Accepted November 12, 2012

ABSTRACT

Bacterial DNA primase DnaG synthesizes RNA

primers required for chromosomal DNA replication.

Biochemical assays measuring primase activity

have been limited to monitoring formation of radio-

actively labelled primers because of the intrinsically

low catalytic efficiency of DnaG. Furthermore, DnaG

is prone to aggregation and proteolytic degradation.

These factors have impeded discovery of DnaG

inhibitors by high-throughput screening (HTS).

In this study, we expressed and purified the previ-

ously uncharacterized primase DnaG from

Mycobacterium tuberculosis (Mtb DnaG). Bycoupling the activity of Mtb DnaG to that of

another essential enzyme, inorganic pyropho-

sphatase from M. tuberculosis (Mtb PPiase), we

developed the first non-radioactive primase

pyrophosphatase assay. An extensive optimization

of the assay enabled its efficient use in HTS (Z0 = 0.7in the 384-well format). HTS of 2560 small molecules

to search for inhibitory compounds yielded several

hits, including suramin, doxorubicin and ellagic

acid. We demonstrate that these three compounds

inhibitMtb DnaG. Both suramin and doxorubicin are

potent (low-mM) DNA- and nucleotide triphosphate-

competitive priming inhibitors that interact withmore than one site on Mtb DnaG. This novel assay

should be applicable to other primases and ineffi-

cient DNA/RNA polymerases, facilitating their char-

acterization and inhibitor discovery.

INTRODUCTION

Primases, essential enzymes in all domains of life, synthe-size primers for DNA replication (1). Bacterial primases(DnaG) are highly conserved, and they are distinct fromtheir eukaryotic and archaeal counterparts (215).Therefore, DnaG is a novel and attractive antibacterialdrug target. By using single-stranded DNA (ssDNA) asa template, DnaG synthesizes short (

6 nt), so that they form a stable duplexwith DNA, needed for robust PicoGreen fluorescence en-hancement. A shortcoming of fluorometric assays in theiruse in HTS is a possible interference of aromatic or non-polar compounds with the signal because of their inter-actions with the fluorescent label.

*To whom correspondence should be addressed. Tel: +1 734 936 2676; Fax: +1 734 647 8430; Email: [email protected]

Published online 24 December 2012 Nucleic Acids Research, 2013, Vol. 41, No. 4 e56doi:10.1093/nar/gks1292

The Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which

permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact

[email protected].


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HTS using radioactivity demands rigorous safetymeasures, generates large amounts of liquid waste and iscostly. For these reasons, use of radioactivity in mostacademic HTS facilities is not feasible. A non-radioactiveand quantitative primase assay, as the one we have de-veloped in this study, is highly desirable for HTS as wellas for a more facile characterization of primases and other

inefficient nucleic acid polymerases.To develop the primase assay, we chose the previously

uncharacterized DnaG from Mycobacterium tuberculosis(Mtb DnaG), the deadliest bacterial pathogen. A highlyoptimized purification procedure forMtbDnaG and iden-tification of conditions that maximize its steady-statenucleotidyl transferase activity reported here enabled usto develop a novel robust primase activity assay. In thisassay, we use another essential bacterial protein, inorganicpyrophosphatase (PPiase) (27) as a coupled enzyme.PPiase selectively cleaves pyrophosphate (PPi) into twophosphates (Pi) and does not hydrolyse nucleotide triphos-phates, thus allowing us to monitor PPi release throughdetection of Pi (28,29). In vivo, the cleavage of PPi by

PPiase is used to drive otherwise reversible nucleotidetransfer reactions, in which a nucleotide monophosphateis transferred from a nucleotide triphosphate (NTP) ontoa substrate with the release of PPi. These types of reactionsinclude chromosomal DNA synthesis as well as the fattyacid adenylation, a critical biochemical step in fatty acidmetabolism. In fact, inorganic pyrophosphatases fromyeast and Escherichia coli were previously used incoupled assays with other enzymes, such as proteinprenyltransferases (30), adenylate cyclase (31), acetyl-CoA synthetase (32) and aminoacyl-tRNA synthetase(33). In our assay, we use PPiase from Mtb (162 aminoacid residues), which shares a modest (30%) amino acidresidue sequence identity to its human counterpart, PPA1

(289 residues). The considerable divergence between PPA1andMtb PPiase, including residue differences in the activesites of these two enzymes (34), implies a possibility ofdiscovering an inhibitor selective for Mtb PPiase.

Conceptually novel anti-tuberculosis drugs are acutelyneeded in clinics because of the alarming spread ofmultidrug-resistant strains ofMtb. Herein, we report thedevelopment of a new coupled primasepyrophosphataseassay that exploits two novel attractive bacterial targets,Mtb DnaG and Mtb PPiase, for inhibitor discovery.

MATERIALS AND METHODS

Cloning and purification ofMtb

DnaGThe primase genednaG(locus tag: Rv2343c) was amplifiedby polymerase chain reaction from Mtb H37Rv genomicDNA (BEI Resources, NIAID, NR-14865) by usingprimers (50-AGTTAGCACATATGTCCGGCCGGATCTCCG-30) and (50-CCGCTCGAGTCACGCGGTGAGATCG-30) and cloned between NdeI and XhoI sites of amodified pET19b vector (35), encoding an N-terminaldecahistidine tag separated from the recombinantprotein by a PreScission protease (GE Healthcare,Piscataway, NJ, USA) cleavage site. The construct ex-pressingMtb DnaG E268Q was generated by mutagenesis

of the aforementioned construct with a QuikChange Kit(Qiagen, Valencia, CA, USA) by using primers 50-CATCAGGCCGTCGTCGTCCAGGGCTACACCGATGTCATG-30 and 50-CA TGACATCGGTGTAGCCCTGGACGACGACGGCCTGATG-3 0. The wild-type and the mutantproteins were expressed and purified analogously. Proteinexpression was carried out in BL21 (DE3) cells cultured in

LB broth. The culture was induced with 0.5 mM of IPTGat an attenuance of 0.2 and then incubated for 16 h at18C. All purification steps were carried out at 4C. Thecell pellet from a 4 l culture was suspended in 100 mlof buffer A [40 mM TrisHCl pH 8.0, 600 mM of NaCl,10% of glycerol, 1 mM of PMSF, 0.5 mM of adenosinetriphosphate (ATP), 2 mM of MgCl2 and 2mM of b-mercaptoethanol] containing two tablets of completeethylenediaminetetraacetic acid (EDTA)-free protease in-hibitor cocktail (Roche Applied Science, Indianapolis, IN,USA). The cells were disrupted by sonication on ice andclarified by centrifugation at 38 000g for 60min. Thesupernatant was filtered through a 0.45 mm Millex-HVPVDF filter (Millipore, Billerica, MA, USA) and applied

to a 5 ml Ni-IMAC HisTrap FF column (GE Healthcare)equilibrated with buffer A. The column was washed with120 ml of buffer A containing 50 mM imidazole, and thenprotein was eluted with 11 ml of buffer A containing500mM imidazole. The eluate was adjusted to NaCl of200 mM, with buffer A prepared without NaCl anddigested overnight at 4C with PreScission protease. Thedigested protein was purified on a size-exclusion S-200column (GE Healthcare) equilibrated in buffer B (40mM TrisHCl pH 8.0, 600 mM of NaCl, 10% ofglycerol, 0.25mM of ATP, 2 mM of MgCl2 and 2 mM ofb-mercaptoethanol), and the protein-containing fractionswere pooled and concentrated using an Amicon Ultra-15centrifugal filter device (Millipore) to 5 mg/ml. Theenzyme was stored on ice for all assays. The recombinantuntagged DnaG was highly homogeneous (Coomassieblue staining indicated >95% purity) and remainedstable and active, while on ice, for 2 weeks. One hourbefore the assays, the storage buffer was exchanged byMicroSpin G-25 columns (GE Healthcare) into buffer C(40 mM of HEPESNaOH pH 7.5, 600 mM of NaCl, 10%of glycerol, 2 mM of MgCl2, 2mM ofb-mercaptoethanoland 0.1 of mM ATP). The presence of ATP in the proteinbuffer results in 10mM ATP in the reaction mixture.This low concentration of ATP is negligible, as it isbelow Km for ATPases and other ATP-interactingenzymes, including Mtb DnaG. Partial degradation ofsuch a small amount of ATP, even if it occurred, wouldbe below or at the detection limit of our assay.

Purification ofMtbPPiase

The pJ411 vector (DNA2.0, Menlo Park, CA, USA)encoding Mtb PPiase bearing an N-terminalhexahistidine-tag was a generous gift from Dr LuizPedro Carvalho. Mtb PPiase was expressed similarly toMtb DnaG. All purification steps were carried out at4C. The cell pellet was resuspended in 20 mM of TEAbuffer pH 7.8. The cells were disrupted sonicationand clarified by centrifugation at 25 000g for 30 min.

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The supernatant was applied to a 5 ml Ni-IMAC HisTrapFF column (GE Healthcare) equilibrated with buffer A(20 mM TEA pH 7.8, 50 mM of imidazole and 300 mMof NaCl). The column was washed with 220 ml of bufferA, and protein was eluted with a linear gradient of 50500 mM of imidazole in buffer A (570 ml). The fractionscontaining Mtb PPiase were pooled, concentrated and

passed through a size-exclusion S-200 column (GEHealthcare) equilibrated in 40 mM of TrisHCl pH 8.0,100 mM of NaCl. The fractions containing protein werepooled and concentrated using an Amicon Ultra-15 cen-trifugal filter device (Millipore) to 750 mM. The recombin-ant His-tagged Mtb PPiase was highly homogeneous(Coomassie blue staining indicated >98% purity) andstable at 4C for 6 months.

Coupled colorimetric primasepyrophosphatase assay(optimization of conditions)

In all coupled primasepyrophosphatase assays that werecarried out during the optimization phase to determine

DNA template (sequence and length), divalent metaltype and concentration, buffer type and pH and in timecourse experiments, we mixed DNA (Integrated DNATechnologies, Coralville, IA, USA), buffer, NTP(Promega, Madison, WI, USA) and pyrophosphatase onice and then addedMtbDnaG, so that the reagent mixtureis at twice the intended concentration of all reagents. Themixture was then diluted two-fold with water to give thefinal concentration ofMtb DnaG (0.7 mM or as specified)DNA (1.25mM or as specified), NTP (110mM or asspecified), NaCl (50 mM), KGlu (150mM), buffer(20 mM of CAPS pH 8.8 or as specified) and divalentmetals (2mM of Mn2+ and 1mM of Mg2+ or asspecified).Mtb DnaG alone is prone to severe aggregation

and rapidly becomes inactive at salt concentrations


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where Av(pos) and SD(pos) are the signal (absorbance)and its standard deviation for the positive controls(EDTA-containing reactions), and Av(neg) and SD(neg)are the respective values for negative controls (DMSO-containing reactions).

Doseresponse assays

For the mode of inhibition studies, the reactions werecarried out under conditions similar to those used in theHTS, except the volumes of the colour reagents wereslightly modified to adapt it to the 96-well plate and tofurther increase the stability and consistency of the colourformation. In these assays, 90 ml of MGR was added to a30 ml reaction mixture followed by 30 ml of 10% sodiumcitrate.

Reactions were carried out with [MtbDnaG] = 0.5 mM,[DNA] = 1.25 mM, [NTP] = 110 mM at different concen-trations of inhibitors, as specified in the text. Relativeactivities were calculated by dividing the concentrationsof PPireleased in the priming reactions in the presence ofinhibitors by that released in its absence. The followingdoseresponse equation was used to fit these data withSigmaPlot 9.0 (SysStat Software, San Jose, CA, USA),with IC50 and the Hill coefficient n as the fitting parameters:

f 11+

I IC50

n

Analysis of mode of inhibition assays

The assays were performed with Mtb DnaG (0.5 mM) andMtb PPiase (50 nM) in the same reaction buffer as thatused in the HTS assays. The steady-state rate of PPi

release was calculated based on measurements of the con-centration of released PPiin 20-min reactions, as describedearlier in the text. Two sets of reactions were run: (i) atconstant [DNA] = 1.25mM at different concentrations ofNTP (31.25, 62.5, 125, 250 and 500 mM) and (ii) atconstant [NTP] = 110mM at different concentrations ofDNA (0.19, 0.38, 0.75 and 1.5 mM). We observed inhib-ition of Pi generation by NTP and DNA at higher NTPand DNA concentrations beyond the aforementionedranges; therefore, they were not used in this analysis.For each pair of DNA and NTP concentrations, differentconcentrations of the inhibitors (suramin and doxorubi-cin) were used (0, 1.25, 2.5, 5, 10, 20 and 40 mM). As asimplifying approximation, to analyse the data, we used

an ordered sequential rapid equilibrium mechanism ofRNA synthesis by DnaG, in which DNA binds freeDnaG first, followed by binding of the first NTP and sub-sequent binding and incorporation of an average of mnucleotides (releasing m PPi molecules) and dissociationof DnaG from DNA:

KDNA KNTP kcat

DnaG+DNA!DnaGDNA+NTP!DnaGDNANTP!DnaG+DNAprimer+mPPi+mNTP

Here, KDNA and KNTP are equilibrium constantsfor binding primed DNA and NTP, respectively, bothdefined for the dissociation direction. This mechanism

assumes that the concentration of NTP affects only initi-ation of primer synthesis. If the first NTP incorporation isslow (primer nucleation) relative to the m-1 subsequentincorporation steps (elongation), then kcat is the catalyticrate constant of the slow nucleation step.

Because the data exhibit competitive inhibition withrespect to both DNA and NTP and the inhibitor:target

stoichiometry higher than 1:1 (model-independent obser-vations), we considered the simplest competitive inhib-ition mechanism, in which an inhibitor (I) can bindnon-cooperatively to n binding sites on Mtb DnaG:

DnaG+I! DnaGIDnaGI+I! DnaGI2. . .

DnaGIn1+I! DnaGInWe designate as Kian intrinsic equilibrium constant for

binding of an inhibitor molecule to a site on DnaG,defined for the dissociation direction. This mechanism

yields the following equation for the steady-state rate ofPPi release (V) in large excess of NTP and inhibitor, butnot DNA, over Mtb DnaG:

Vkcat,appDnaG DNA NTP KDNAKNTP

1

Because one nucleation event leads to release ofm PPimolecules, kcat,app= mkcat. The concentrations of freeMtb DnaG, [DnaG] and all DNA species not bound toDnaG, [DNA], are obtained from the conservation ofmaterial equations for DnaG and DNA and from theaforementioned equilibria as:

DnaG b+ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffib2

4acp

2a , 2

where

a KNTP+ NTP KNTPKDNA

1+

I Ki

n, 3

b 1+ I Ki

n+

KNTP+ NTP KNTPKDNA

DNA TDnaG T

,

4c DnaG T, 5

DNA DnaG 1+ I Ki

n+ DNA TDnaG T 6

Here, [DnaG]Tand [DNA]Tare the total concentrationsofMtb DnaG and DNA in the reaction, respectively. Thisrate law was globally fit to all the data collected for eachinhibitor by using non-linear regression with SigmaPlot9.0, withkcat,app,KDNA,KNTPandKias fitting parametersfor different fixed integer values of n. A satisfactory fitwas not obtained for n =1 or n = 2 for either suraminor doxorubicin, consistent with the model-independentdoseresponse analysis. For suramin, the data were wellfit for n = 4, whereas for doxorubicin, an equally good fitwas produced for n = 3, yielding the best fit parameter



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values and the theoretical curves generated by using theseas presented in the Results section. The data could be fitequally well by a more complex model, in which the in-hibitors bind Mtb DnaG with high cooperativity, withn =3 and n = 2 for suramin and doxorubicin, respect-ively. However, because the cooperative binding modelcontains too many fitting parameters to be resolved by

the data, and because it lacks support by other experimen-tal evidence, it is not presented here in further detail.

DNA-binding assay

DNA affinity ofMtb DnaG for ssDNA was measured atthe conditions of the priming activity assays, either in theabsence of NTPs or in the presence of all four NTPs, at[NTP] = 110 mM. We used a single-stranded 28-merssDNA: 50-CCGAATCAGTCCGACGACGCATCAGCAC-30, labelled at the 50-end with 6-carboxyfluorescein(6FAM; Integrated DNA Technologies, Coralville, IA,USA). Titrations were set-up at the constant concentra-tion of ssDNA of 20 nM at different concentrations ofMtb DnaG in separate 50 ml reaction mixtures and wereequilibrated for 20 min at 22C. Fluorescence anisotropywas monitored by a SpectraMax M5 plate reader(Molecular Devices, Sunnyvale, CA, USA), set at the ex-citation and emission wavelengths of 495 and 520 nm, re-spectively. To obtain equilibrium protein-DNA bindingconstant Kd, non-linear least-squares regression datafitting to the equilibrium 1:1 binding isotherm was per-formed with SigmaPlot 9.0 as previously described (38).No significant difference was observed between titrationsin the presence or absence of NTPs; therefore, these datawere averaged together.

RESULTS

Purification ofMtbDnaG

Expression and purification ofMtb DnaG have not beenpreviously reported. Initially, we tested a variety of expres-sion and purification protocols previously reported forother DnaG proteins; these efforts yielded inactiveaggregated protein. During the extensive optimization ofthe purification procedure, we found that ATP (0.25 mM),NaCl (600 mM) and glycerol (10%) all need to be

present during all purification steps to prevent MtbDnaG from aggregation. As a result, we obtained pureand active Mtb DnaG. The purified protein (Figure 1A)is homogeneous and monomeric as judged by S-200 size-exclusion chromatography (Figure 1B).

Development of a novel non-radioactive coupledprimasephosphatase assay

Primase activity has been historically monitored by visu-alization and quantification of radiolabelled RNA primerson a denaturing gel. We sought to develop a novel activityassay that would be robust (even at room temperature),would not require the use of radioactivity and would notrequire accessory factors, such as ssDNA-binding proteinor DnaB helicase, which have been used previously toenhance primase activity. Such an assay would be highlysuitable for high-throughput applications.

We developed a colorimetric coupled primasepyrophosphatase assay satisfying these requirements(Figure 2). Inorganic pyrophosphatase (PPiase, type I) is

used to cleave the pyrophosphate (PPi) liberated in thepriming reaction into phosphate (Pi). To measure the Piconcentration, we use the MGR (36), whose absorbance at620 nm increases with increasing concentration of Pi,changing its colour from yellow to green. PPiase cleavesexclusively PPi and does not exhibit any cleavage activityon NTP (29), a property that we also confirmed for yeastandMtb PPiases. The concentration of PPiase was chosento be three-fold higher than the minimum concentrationrequired for cleavage of all released PPi.

By monitoring synthesis of radiolabelled primers, wedirectly established that at the minimal conditions ofour novel coupled assay, Mtb DnaG displays robustMn2+-dependent primer synthesis activity and has no de-

tectable activity in Mg2+ (Figure 3). This activity is nothighly processive, as evidenced by several short (


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DNA template (Figure 4, top inset). This template doesnot contain thymidine nucleotides in its 50-half, so thatATP is not needed for the synthesis of complementaryRNA; as a result, we could omit ATP in the reaction.Indeed, we obtained a robust DNA-dependent signalfrom the primer synthesis activity by Mtb DnaG in thisreaction set-up with only CTP, GTP and UTP ( Figure 4).An Mtb DnaG point mutant E268Q of a conserved

divalent metal-coordinating residue (39) that is absolutelyrequired for catalysis (21,40) was used as a control toensure that the observed signal resulted specifically fromthe primer synthesis activity ofMtb DnaG. No signal wasobserved without the inorganic pyrophosphatase. We thenperformed the assay with a variety of divalent metal salts(Figure 5). The results confirmed the lack of detectableactivity with Mg2+ and demonstrated that Mn2+ wasthe only divalent metal that supported robust activityamong the seven metals tested. Mn2+ was previouslyreported to increase DnaG activity without significantlyaffecting other properties of DnaG (21). In a recentlydetermined crystal structure of the catalytic core ofStaphylococcus aureusDnaG in complex with nucleotides,

a catalytic Mn

2+

ion was shown to be interchangeablewith Mg2+ (39).

Optimization of assay conditions

The colorimetric primasepyrophosphatase assay pro-vides a quantitative measure of kinetics of NTP incorp-oration into growing RNA by measuring PPi release(Figure 6A). At these conditions ([Mtb DnaG]= 0.7 mM,[DNA] = 1.25 mM, [NTP (CTP, GTP, UTP)] = 110 mMeach), the linear steady-state accumulation of PPipersists until 30 min from the start of the reaction,after which the signal starts to reach the plateau, likelybecause of accumulation of stable RNADNA duplexesor because of protein aggregation. Therefore, to obtain a

robust signal in the steady-state measurements, a reactiontime of 2030 min (as specified) was chosen for furthermeasurements. To find conditions that maximize theprimer synthesis activity in our assay, we tested a varietyof solution parameters. When measured as a function ofthe concentration of the divalent metal of choice, Mn2+,Mtb DnaG exhibited a maximum activity near 2 mM ofMn2+ (Figure 6B). Mg2+ (1 mM) is used in the reaction toensure robust activity of PPiase, which is not highly activein Mn2+ alone (34). Potassium glutamate (KGlu) waschosen for the assay, as it is the major electrolyte in vivo(41), and it significantly enhances proteinDNA

Figure 3. Synthesis of radiolabelled RNA primers by Mtb DnaGand its inhibition by 10mM doxorubicin (D) and suramin (S).Phosphorimager analysis of a 12% of ureapolyacrylamide gel electro-phoresis showing RNA products of the priming reaction. The primingreaction was performed with 1 mM of Mg2+, 2mM of Mn2+ and bothon a 38-mer ssDNA template 50-CTGGTGGGCCCAAACTTGATGCTCTAATACCGACGCGT-3 0 in the presence of a mixture of the fournucleotides containing a-32P-GTP.

Figure 4. Representative coupled primasepyrophosphatase reactions(a 30-min time point) with wild-type (wt) and E268Q mutant of MtbDnaG.

Figure 2. A schematic of the coupled colorimetric primase (DnaG)pyrophosphatase (PPiase) assay.



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interactions in vitro (42). The activity was optimal in thephysiological KGlu concentration range, 150200 mM(Figure 6C). The primase was active in a variety of buffersand with pH values in the range 6.09.0 (Figure 6D);20 mM of CAPS pH 8.8 was chosen, as it supported thehighest activity of Mtb DnaG among the tested buffersand pHs.

The observed steady-state rate of PPi release measuredas a function of DNA concentration at [NTP] = 110mMdisplayed a saturating behaviour indicative of tightbinding of Mtb DnaG to DNA (a nearly linear increaseof activity until half-saturation), with Km,obs3s) of the negative controls onthe same plate (Figure 7B). Three compounds(disopyramide phosphate, oleandomycin phosphate andfosfosal) displayed increase in absorbance. This was notbecause of enzyme activation, but because the first two

compounds were phosphate salts and because phosphatewas released on rapid hydrolysis of fosfosal at the acidicpH of the MGR (43). We performed doseresponse meas-urements with three strong hits, suramin (7.2s), doxorubi-cin (3.7s) and ellagic acid (5.1s) (Figure 8). These datayielded IC50values of 6.3 0.2mM and 7.7 0.5 mM forsuramin and doxorubicin, respectively, indicating theirpotent inhibitory effects. Ellagic acid displayed approxi-mately three-fold weaker inhibition (IC50 = 2 2 3mM)and was not characterized further. None of the three com-pounds inhibited Mtb PPiase as assayed separately withinorganic pyrophosphate as a substrate, indicating thatthese compounds are bona fide Mtb DnaG inhibitors(the other six hits remain to be tested for DnaG andPPiase inhibition). The doseresponse curves for suraminand doxorubicin could be well modelled only by using theHill coefficients of 2.9 0.1 and 2.0 0.2, respectively(Figure 8), which indicates that the target containsmultiple inhibitor-binding sites (44).

Mode of inhibition ofMtbDnaG by suramin anddoxorubicin

We first confirmed directly that suramin and doxorubicinare potent Mtb DnaG inhibitors by observing their effecton the synthesis of radioactively labelled RNA productson the same DNA template at conditions similar to thoseof the colorimetric assay (Figure 9) and in assays with a

different template DNA and with all four NTPs presentin the reaction (Figure 3). To investigate the inhibitorymechanisms of suramin and doxorubicin in more detail,we measured the effect of these compounds on thesteady-state accumulation of PPi in the primer synthesisreaction. To determine whether suramin and doxorubicininterfere with interactions ofMtb DnaG with DNA, NTPor both, we performed measurements of the steady-staterate of PPirelease as a function of inhibitor concentrationfor different concentrations of DNA and NTP. One set ofmeasurements was performed at a fixed concentration ofDNA ([DNA] = 1.25 mM) with different concentrations of

Figure 5. Activity of Mtb DnaG in the presence of different divalentmetals, at 2 mM. The grey bars indicate the absorbance at time 0 in thereaction.

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Figure 6. Optimization of conditions of the primasepyrophosphatase assay. (A) A representative time course of the priming reaction, monitored byquantifying released PPi. (B) Dependence of activity ofMtb DnaG on the concentration of MnCl2. (C) Activity ofMtb DnaG as a function of theconcentration of potassium glutamate. (D) Activity ofMtb DnaG in a variety of buffers (20 mM) at different pH in the range 6.09.0. The grey barsindicate the absorbance at time 0 in the reaction. (E) The steady-state rate of PPi release during primer synthesis by Mtb DnaG as a function ofDNA concentration. The reactions were carried out for 30 min at [MtbDnaG]= 0.7mM, [NTP]= 110mM. (F) The steady-state rate of PPi release byMtb DnaG as a function of NTP concentration. The reactions were carried out for 30 min at [Mtb DnaG]= 0.7mM, [DNA] = 1.25 mM.

Figure 7. High-throughput coupled Mtb DnaG-MtbPPiase assays. (A) A control assay used to calculate the Z0 score. The last two columns of wellscontain 30 mM of EDTA, used as a positive inhibition control in the HTS. (B) The scatterplot of the pilot assay with 2560 diverse compounds. Redsquares indicate full inhibition by 30 mM of EDTA (as in panel A). Compounds that do not exhibit inhibitory properties (serving as negativecontrols) are shown by the blue squares, and the rest of the compounds are shown by the green squares. The red line indicates a 3s level ofinhibition, where s is the standard deviation of the negative controls.



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NTP (Figure 10A and C) and the other set at a fixedconcentration of NTP ([NTP] = 110mM) with differentconcentrations of DNA (Figure 10B and D). Thecommon y-axis intercepts in the LineweaverBurk plotsof these data (insets in Figure 10) show that bothsuramin (Figure 10A and B) and doxorubicin (Figure10B and C) are competitive inhibitors with respect toboth NTP and DNA. These data effectively containmultiple doseresponse curves, which, similarly to thedoseresponse curves presented in the previous section,

indicate the presence of multiple binding sites for bothsuramin and doxorubicin. To obtain the equilibriumbinding constant Ki and the number of sites on MtbDnaG for each inhibitor, we performed a global non-linear regression analysis of these data. We used asimple assumption of multiple non-cooperative inhibitorbinding sites. In addition, we used a simple approximationof an ordered sequential mechanism of priming, where anssDNA molecule binds free Mtb DnaG first followed bybinding of an NTP, slow primer nucleation and its rapidelongation, incorporating m NTP molecules per DNA-binding event. This minimal kinetic mechanism is consist-ent with the observed first-order kinetics of thesteady-state activity of Mtb DnaG with respect to NTP

concentration (Figure 6F) and with other data (Figure 10);the ordered binding of DNA and NTP was previ-ously proposed for a eukaryotic primase (45). Thisanalysis yields the values of the mechanistic parametersfor Mtb DnaG: the catalytic rate constant kcat,app=5.70.2min1, the effective equilibrium constants forbinding of ssDNA to Mtb DnaG KDNA=2410n Mand for binding of an NTP to the primed DNA-MtbDnaG complex KNTP=17010mM. To obtain an inde-pendent measurement ofMtb DnaG affinity for ssDNA,we performed a fluorimetric assay, in which increase influorescence polarization of a carboxyfluorescein label

on ssDNA was monitored as a function of concentrationof Mtb DnaG (Supplementary Figure S1). This assayyields equilibrium binding constant Kd =3616nM, ingood agreement with the KDNA value obtained from thekinetic assays.

The aforementioned value of kcat,app is equal to theintrinsic kcat only if the primase is completely non-processive, dissociating from DNA after incorporationof one NTP, that is, when m = 1. Because primases areat least somewhat processive (46,47), this value overesti-

mates kcat. In the other extreme, if Mtb DnaG rapidlyincorporates on average 30 nt per DNA binding event,we obtain kcat= kcat,app/30 = 0.19 0.01min

1, anunderestimate of kcat. The processivity of Mtb DnaGand the kinetics of individual NTP incorporation stepswill be investigated in detail by future studies. Forsuramin inhibition data, an excellent fit was obtained fora minimum ofn = 4 binding sites onMtb DnaG (bindingto one of which is sufficient for inhibition), with an intrin-sic equilibrium binding constant for each site ofKi =3.20.5 mM (Figure 10A and B). The analogousanalysis of doxorubicin inhibition yielded best-fit valuesof n =3 and Ki =2.50.5 mM and also an excellentagreement with the model mechanism (Figure 10C and

D). The data can also be fit well to a more complexmodel, where three molecules of suramin or two moleculesof doxorubicin bind Mtb DnaG with high cooperativity(see Materials and Methods section). Future studiesare aimed at analysing the mechanism of inhibition ofMtb DnaG by these inhibitors in kinetic and structuraldetail.

DISCUSSION

Pioneering studies of bacterial primase DnaG 40 years ago(48,49) established this enzyme as an indispensable RNA

Figure 8. Doseresponse assays with three hit compounds from the pilot HTS, suramin, doxorubicin and ellagic acid (the chemical structures areshown as insets). The assays were performed with 1.25 mM of DNA, 110 mM of NTP and 0.5 mM of Mtb DnaG.

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polymerase that generates primers for chromosomal DNAsynthesis in vivo. Since these initial studies, the primaseactivity has been monitored either directly, by detectingprimers in gel and by high-performance liquid chromatog-raphy (50) or indirectly, by observing DNA extended fromthe primer by DNA polymerase or by monitoring increasein PicoGreen fluorescence on formation of an RNADNAhybrid (26). The use of radioactivity and gel electrophor-

esis or high-performance liquid chromatography forproduct detection impose limitations on the extent ofquantitative characterization of primases and, more im-portantly, on our ability to exploit primases as drugtargets through HTS. This study reports a novel assaythat overcomes these obstacles.

Primases are intrinsically poorly processive, likelyevolved so to limit the size of primers, which serve astransient substrates for initiating DNA replication, even-tually getting excised by RNase H and replaced by DNA.To observe the weak primase activityin vitro, other stimu-latory DNA replication factors, such as replicative

helicase DnaB and the ssDNA-binding protein havebeen commonly used. With the goal of creating anorthogonal technique for detection and quantitativemeasurements of primase activity, we developed a non-radioactive assay, where robust activity of DnaG wasachieved without its interacting partners at conditionssuitable for HTS.

As Mtb DnaG is an important potential therapeutictarget, we chose this primase and obtained it in a highlypurified monomeric form; this is the first study of a myco-bacterial DnaG. For coupling the primase reaction withinorganic PPiase, we chose Mtb PPiase. Because of itsextreme specificity to PPi, and not NTP, as a substrate,Mtb PPiase is exquisitely suitable for this assay. Being anessential enzyme itself, Mtb PPiase serves simultaneouslyas a second target in inhibitor discovery by HTS.

The only previous study of HTS to search for primaseinhibitors was by researchers from Bristol-Myers SquibbCompany, who reported a radioactive scintillation prox-imity DnaG activity assay with DnaB as an accessoryfactor for increasing activity of DnaG and as a coupled

target (25). As assays using radioactivity cannot be per-formed in most academic screening facilities, ournon-radioactive assay reported here provides a new ac-cessible route to using DnaG as an inhibitory target inHTS. A robust performance of our coupled assay isshowcased by a pilot HTS of 2556 small molecules,which yielded several confirmed hits. Three of these hits,suramin, doxorubicin and ellagic acid efficiently inhibitedDnaG, the first two at a high potency. Both suramin anddoxorubicin (Figure 8) contain aromatic base-like struc-tural groups; therefore, these molecules are likely to bindthe primase at a primase site (or sites) that would blockDNA and/or NTP binding. Suramin was previouslydemonstrated to act as DNA- and NTP-competitive

inhibitor of human DNA polymerase a and primase, re-spectively (51). Owing to its polyanionic character,suramin may interact with some of the same sites onDnaG that contact phosphate groups of the DNAbackbone or incoming NTP. Quantitative analysis of in-hibition by suramin and doxorubicin in this study showsthat both molecules apparently compete with both DNAand NTP. We considered a simple model, where ssDNAmust bind DnaG first, followed by NTP for productiveRNA synthesis, similarly to the ordered bindingproposed for the eukaryotic primase (45). In the frame-work of this model, the observed inhibitor competitionwith both DNA and NTP for binding the primase is in-terpreted as inhibitor binding to Mtb DnaG to block

DNA binding. A more complex model where eitherDNA or NTP can bind free DnaG on the pathway tonucleotide transfer is also consistent with the data, andsuch a model is expected to yield similar Ki values. Infact, either NTP or DNA can bind to free bacterial,phage and eukaryotic primases (39,40,5254), althoughNTP binding per se need not lead to nucleotide transfer.In any case, because this more complex model containstoo many fitting parameters to be resolved by the data, itwas not used in the analysis. Further studies by this andother assays are needed to clarify the order of NTP andDNA binding. Another interesting observation is that the

Figure 9. Inhibition of Mtb DnaG activity by suramin (S) and doxo-rubicin (D). The assay was performed under similar optimized reactionconditions and with the same DNA template as in the HTS andvisualized and quantified as described in Figure 3.



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dependence of the steady-state activity ofMtb DnaG oninhibitor concentration indicates binding of multiple in-hibitor molecules (four for suramin and three for doxo-rubicin, given the absence of cooperativity). The sameintrinsic affinity is assumed for each subsequent bindingevent for simplicity, to keep the number of fitting param-eters to a minimum. Even though this is an oversimplifi-cation, the low-mM effective Ki values and a complexstoichiometry are model-independent observations. NTPequilibrium binding constants for Mtb DnaG obtained

from this analysis are comparable with those previouslyreported for other primases (55,56). Equilibrium constantsfor primase binding to DNA, similarly to otherDNA-binding proteins, likely strongly depend on the con-centration of salt and the nature of the anion ( 42,57,58);therefore, they are more difficult to compare. As expectedfor the highly optimized conditions of the assay, the Kdvalue forMtbDnaG binding to ssDNA reported here is inthe lower range of values reported for primases. Forexample, E. coli DnaG binds ssDNA with affinity of720 nM (17), whereas S. aureus DnaG binds ssDNAwith a six-fold greater affinity, 112 nM [as measured by

the same group (50)], which is only three-fold weaker thanthat ofMtbDnaG. TheKdvalue that we obtained for MtbDnaG is similar to the recently reported affinity(345nM) ofBacillus anthracis DnaG for an ssDNAoligomer of a similar size (19) and to the DNA-bindingaffinity of eukaryotic primases (45). These measurementsunderscore the applicability of our assay to quantitativekinetic and thermodynamic studies of primases.

In summary, the discovery of potentMtb DnaG inhibi-tors serves as a validation of the robust assay performance

in HTS. We expect that our novel assay will find applica-tions in basic characterization of not only primases butalso other inefficient nucleic acid polymerases and pavethe way to a more facile HTS discovery of potentprimase inhibitors. Discovery of such compounds will cer-tainly be useful in development of clinically useful agents.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online:Supplementary Figure 1.

Figure 10. Measurements of the steady-state rate of PPi release on primer synthesis by Mtb DnaG in the presence of suramin (A and B) anddoxorubicin (C and D). The assays were carried out at a fixed DNA concentration of 1.25 mM as a function of concentration of NTP (A and C) andat a fixed NTP concentration of 110 mM as a function of concentration of DNA (B and D). In all panels, the data collected without inhibitor areshown as open triangles, with 1.25 mM inhibitor as open squares, 2.5 mM inhibitor as open diamonds, 5 mM inhibitor as filled circles, 10 mM inhibitoras filled squares, 20 mM inhibitor as filled diamonds and 40 mM inhibitor as filled triangles. The insets display representative LineweaverBurk plotsof the same data.

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ACKNOWLEDGEMENTS

The authors thank Andrew Pratt for testing inhibition ofMtbPPiase, Dr Luiz Pedro S. de Carvalho for a generousgift of the Mtb PPiase plasmid, Ahmed Mady and ShirinShokooh for assistance with colorimetric activity assays,Tom McQuade and Dr Martha Larsen for assistance withthe HTS and Dr George Garcia and Dr Garry Dotson forsharing microplate readers.

FUNDING

Seed award 2UL1TR000433-06 from the MichiganInstitute for Clinical and Health Research (MICHR)and University of Michigan College of Pharmacystart-up funds (both to O.V.T.). Funding for open accesscharge: University of Michigan College of Pharmacystart-up funds.

Conflict of interest statement. None declared.

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