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Development of an ESI-LC-MS-Based Assay for Kinetic Evaluation of Mycobacterium tuberculosis Shikimate Kinase Activity and Inhibition Johayra Simithy,† Gobind Gill,‡ Yu Wang,‡ Douglas C. Goodwin,‡ and Angela I. Calderón*,† †

Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, 4306 Walker Building, Auburn, Alabama 36849, United States ‡ Department of Chemistry and Biochemistry, Auburn University, 179 Chemistry Building, Auburn, Alabama 36849, United States

ABSTRACT: A simple and reliable liquid chromatography−mass spectrometry (LC-MS) assay has been developed and validated for the kinetic characterization and evaluation of inhibitors of shikimate kinase from Mycobacterium tuberculosis (MtSK), a potential target for the development of novel antitubercular drugs. This assay is based on the direct determination of the reaction product shikimate-3-phosphate (S3P) using electrospray ionization (ESI) and a quadrupole time-of-flight (Q-TOF) detector. A comparative analysis of the kinetic parameters of MtSK obtained by the LC-MS assay with those obtained by a conventional UV-assay was performed. Kinetic parameters determined by LC-MS were in excellent agreement with those obtained from the UV assay, demonstrating the accuracy, and reliability of this method. The validated assay was successfully applied to the kinetic characterization of a known inhibitor of shikimate kinase; inhibition constants and mode of inhibition were accurately delineated with LC-MS.

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RoundUp.4 Aside from its activity against the plant form of EPSPS, this compound has also proven to be active in vitro against the malaria parasite.5 This validates the importance of the shikimate pathway enzymes as targets for the design of potentially selective antimicrobial drugs.6 Shikimate kinase (SK) (EC 2.7.1.71) is the fifth enzyme of the shikimate pathway. It belongs to the family of nucleoside monophosphate (NMP) kinases, and it catalyzes the ATPdependent phosphorylation of the 3-hydroxyl group of shikimate to form shikimate-3-phosphate (S3P) and adenosine diphosphate (ADP) (Scheme 1).7 Gene disruption studies have shown that shikimate kinase is essential for the survival of Mtb. As such, M. tuberculosis shikimate kinase (MtSK) is an attractive target for the development of a new generation of antitubercular drugs.8 Because of the indistinct UV absorption of the substrates (ATP and shikimate) and products (ADP and S3P) of the reaction catalyzed by MtSK (Scheme 1), the activity of this enzyme is usually measured by a spectrophotometric coupled assay involving the enzymes pyruvate kinase (PK) and lactate

uberculosis (TB) is the second most common cause of death due to a single infectious agent. According to the World Health Organization (WHO), approximately nine million people are infected and two million people die from this disease every year.1 The emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) has highlighted the need for the development of faster acting and more effective anti-TB drugs. Preferably, these will belong to new structural classes and possess new mechanisms of action that can target multidrugresistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB).2 For the development of new drugs to treat TB, it is advantageous to find drug-targets that are not present in the human host. One pathway that offers such drug-targets is the shikimate pathway. This pathway is a seven step metabolic route utilized by fungi, higher plants, and bacteria for the biosynthesis of essential aromatic amino acids. The pathway is absent from mammals.3 A great deal of research focusing on the development of inhibitors of the enzymes of this pathway has been carried out in the last several years. For example, glyphosate, a selective inhibitor of the sixth enzyme of the pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19), is the active ingredient in the wide-spectrum herbicide known as © XXXX American Chemical Society

Received: August 26, 2014 Accepted: January 15, 2015

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DOI: 10.1021/ac503210n Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

as the launch point for our development of the straightforward, sensitive and reproducible LC-MS MtSK assay for the kinetic characterization of MtSK activity and inhibition described here. The current method is also based on the detection of the reaction product shikimate-3-phosphate using ESI and a quadrupole time-of-flight (Q-TOF) detector. The method was validated for linearity, intra- and interday precision, limit of detection (LOD) and limit of quantitation (LOQ) using an S3P external standard, and it was successfully applied to the kinetic characterization of a previously reported inhibitor of shikimate kinase. A comparative analysis of this new LC-MS assay with a conventional coupled assay was also performed. Kinetic parameters of MtSK (KM and kcat), as well as the inhibition constants (Ki and αKi) of the positive reference compound determined by the LC-MS assay, were in good agreement with those determined using the coupled assay and with those reported in the literature.11,27 Furthermore, a noncompetitive mechanism of inhibition was delineated, verifying the accuracy and reliability of the LC-MS assay. To the best of our knowledge, this is the first report describing a direct enzymatic assay for the kinetic characterization and evaluation of inhibitors of shikimate kinase. The selectivity and enhanced sensitivity of this method over conventional UV−vis spectroscopy methods, demonstrates that LC-MS represents a viable alternative to accurately monitor steady-state kinetics of any enzymatic reaction where chromogenic substrates or products are not available.

Scheme 1. Reaction Catalyzed by Shikimate Kinase

dehydrogenase (LDH) (Scheme 2). This coupled assay is based on the conversion of ADP (produced by MtSK) to ATP by Scheme 2. Pyruvate Kinase (PK)/Lactate Dehydrogenase (LDH) Coupled Assay for MtSK Activity

pyruvate kinase (PK) in the presence of phosphoenol pyruvate (PEP). The pyruvate generated in the PK reaction is then reduced to lactate by lactate dehydrogenase (LDH) in the presence of NADH, which is oxidized to NAD+.9 NADH has an absorption maximum at 340 nm, a wavelength that NAD+ does not absorb. Consequently, the activity of MtSK can be monitored by measuring the decrease in absorbance at 340 nm. While this assay has been widely used for the determination of kinetic parameters of ATP-dependent kinases including SK from different species,10−13 it presents major drawbacks and limitations. First, by monitoring the disappearance of NADH, this assay measures a decrease in signal rather than an increase, resulting in restrictions on the dynamic range of the assay if large amounts of NADH are used. Sensitivity is also compromised because of the relatively low extinction coefficient of NADH. Additionally, the presence of coupling enzymes and additional substrates adds more reaction steps, making the assay more difficult to optimize.14 Finally, and most importantly, this assay may have limitations for the screening and validation of compounds with inhibitory activity. Assay interferences mediated by intrinsic absorbance/fluorescence of compounds can often lead to aberrant readouts of methods employing optical detection.15−17 Also, compounds that can inhibit or act as substrates of the coupling enzymes are considered a primary source for false positives. This emphasizes the need for secondary or orthogonal assays to truly assess the inhibitory activity of a compound against a given target.18,19 Soft ionization methods, such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) coupled to LC have been utilized as an alternative to spectrophotometric methods for the study of enzyme kinetics and inhibitor discovery.20−25 MS methods can directly detect and quantify the formation of products and/or the consumption of substrates, eliminating the need for secondary enzymatic reactions unrelated to the target enzyme. Our research group has previously reported an ultrafiltrationliquid chromatography−mass spectrometry (UF-LC-MS)based ligand binding assay and an LC-MS-based functional assay for the screening of inhibitors of MtSK.26 This has served

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MATERIALS AND METHODS Reagents. Imidazole, ampicillin (AMP), chloramphenicol (CAM), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma (St. Louis, MO). Isopropyl-β-Dthiogalactopyranoside (IPTG) was obtained from Fisher (Pittsburgh, PA). Bugbuster and benzonase nuclease were purchased from Novagen (Madison, WI). All restriction enzymes were purchased from New England Biolabs (Beverly, MA). All oligonucleotide primers were purchased from Invitrogen (Carlsbad, CA). The E. coli strains (BL-21-Gold [DE3] pLysS and XL-1 Blue), Pfu polymerase, and T4 DNA ligase were purchased from Agilent (La Jolla, CA). Nickelnitrilotriacetic acid (Ni-NTA) resin was purchased from Qiagen (Valencia, CA). Desalting 10DG chromatography columns were purchased from Bio-Rad (Hercules, CA). Dimethyl sulfoxide (DMSO), shikimate-3-phosphate (S3P) used for the calibration curve, the substrates adenosine-5′-triphosphate (ATP), shikimate, reduced nicotinamide adenine dinucleotide (NADH), phosphoenol-pyruvate (PEP) and the enzymes pyruvate kinase (PK) and lactate dehydrogenase (LDH) used in the coupled assay were purchased from Sigma-Aldrich (St. Louis, MO). The reference standard for inhibition, compound 1, (3-methoxy-4-{[2-({2-methoxy-4-[(4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene) methyl] phenoxy} methyl) benzyl]oxy} benzaldehyde), was purchased from ChemBridge (San Diego, CA). All organic solvents were HPLC or LC-MS grade and were purchased from Thermo Fisher (Hanover Park, IL). All buffers and media were prepared using water purified by a MilliQ purification system (Millipore, Billerica, MA) or by a Barnstead EasyPure II system (18.2 MΩ/cm resistivity). Construction of MtSK Plasmid. The MtSK gene (aroK) was PCR amplified from the pProEX-MtSK plasmid provided by Southern Research Institute (Birmingham, AL) and subcloned into pET-20b(+) expression vector (Novagen, B


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Analytical Chemistry Madison, WI) using its Nde I-Xho I restriction sites. Two synthetic oligonucleotide primers were designed to amplify the MtSK gene and to introduce an Xho I restriction site at its 3′ end. The sequences for these primers were: 5′-Phos-ATGGCACCCAAAGCGGTTCTCGTCGGCC-3′ (sense) and 5′TATCTCGAGTGTGGCCGCCTCGCTGGGGCTG-3′ (antisense). For amplification of the expression vector sequence, the primers designed were: 5′ACCTGCTCGAGCACCACCACC-3′ (sense) and 5′-Phos-ATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGAAACC-3′ (antisense). Both PCR products were digested with Xho I followed by heat inactivation and ligation using T4 DNA Ligase (Quick Ligation kit) according to manufacturer’s instructions. PCR products of the gene and the vector carrying Xho I cohesive ends were ligated together and the 5′ end of MtSK gene was blunt-end ligated with the Nde I site of the expression vector. The resulting construct was used to transform E. coli (XL-1 Blue) by heat shock according to manufacturer’s instructions. Transformants were selected on the basis of ampicillin resistance, and the plasmids from candidate colonies were isolated and verified by diagnostic restriction digest with endonucleases Nde I and Xho I. A DNA sequence analysis was performed (Davis Sequencing, Davis, CA) to confirm the identity of MtSK and to verify that no unintended mutations were introduced during PCR procedures. The plasmid carrying the verified MtSK sequence was used to transform E. coli (BL21-Gold [DE3] pLysS). Transformants were selected on the basis of ampicillin and chloramphenicol resistance. MtSK Expression and Purification. MtSK expression was carried out in E. coli (BL-21-Gold [DE3] pLysS) using Luria− Bertani broth supplemented with ampicillin (100 μg/mL) and chloramphenicol (0.034 mg/mL). Cells were grown at 37 °C with constant agitation (250 rpm) to mid log phase (OD600 = 0.4) and induced by IPTG (1 mM). The cells were then grown for an additional 4 h postinduction, harvested by centrifugation at 5,000 rpm for 15 min at 4 °C and stored at −80 °C until purification. Expression analysis was carried out using a trichloroacetic acid (TCA) precipitation protocol as described by Varnado et al. except that 11% acrylamide SDS-PAGE gels were used.28 A solubility test protocol was followed to determine the proper method of purification. Expression analyses revealed the presence of a protein with the anticipated molecular weight in the soluble and insoluble fractions. Consequently, protocols were developed to purify the protein from its soluble and insoluble (i.e., inclusion body) states. For soluble protein purification, cell pellets were resuspended in buffer A (50 mM Tris-HCl, pH 7.4; 0.5 M NaCl) in the presence of PMSF (0.1 mM). The mixture was homogenized with a dounce homogenizer and lysed by sonication using a Branson Sonifier 250 (Branson Ultrasonics Corp., Danbury, CT, USA). Sonication was carried out with an output control of 3.5 and a 30% duty cycle for 8 intervals of 42 s each with cooling on ice between each sonication. Following sonication, benzonase nuclease (250 U) was added, and the mixture was incubated for 2 h at 4 °C with gentle stirring. The cell lysate was centrifugated at 10 000 rpm for 15 min at 4 °C, the supernatant was collected and the pellets were stored at −80 °C for further purification under denaturing conditions. The supernatant was loaded onto a column with Ni-NTA resin by recirculating the solution through the column at 1 mL/min overnight. After loading, the column was washed in succession with 50 mM Tris, pH 8.0, followed by buffer A supplemented

with 2 mM, 20 mM, 50 mM, 0.1 M, 0.5 M, and 1 M imidazole. The eluted fractions were collected and analyzed by SDSPAGE. The fraction containing the protein (0.1 M imidazole) was concentrated by ultrafiltration with a 3 kDa molecular cutoff filter (Amicon, Billerica, MA) and subjected to buffer exchange using a 10DG size exclusion column (BioRad, Hercules, CA) equilibrated with buffer A. For denaturing purification, pellets reserved from the soluble purification procedure were resuspended in buffer B (50 mM Tris-HCl, 0.5 M NaCl, and 8 M Urea) and homogenized at room temperature. The mixture was centrifuged at 10 000 rpm for 15 min and the supernatant was collected and mixed with Ni-NTA resin pre-equilibrated with buffer B. The protein/resin mixture was allowed to bind overnight with gentle agitation at room temperature. On the following day, this mixture was loaded onto an empty column, washed with 25 mL of buffer B supplemented with 20 mM imidazole and the protein was then eluted off the column with 25 mL of buffer B supplemented with 400 mM imidazole. Urea and imidazole were removed by dialysis against buffer A for 20 h, replacing the dialysis buffer every 4−6 h. The concentration of MtSK was measured using the Pierce BCA Protein Assay (Pierce, Rockford, IL) according to manufacturer’s instructions using bovine serum albumin as a standard. Enzyme purified by denaturing conditions was employed for the results presented in this report. ESI-LC-MS Analysis of Intact MtSK. Analyses of intact MtSK were performed on an Agilent (Little Falls, DE) 6520 Accurate-Mass Q-TOF mass spectrometer coupled to an Agilent 1200 RRLC system. ESI was conducted using a capillary voltage of 4000 V. Nitrogen was used as the nebulizing gas (25 psig) and drying gas (10 L/h; 350 °C). The TOF fragmentor and skimmer were set to 180 and 65 V, respectively. Samples were injected onto a 2.1 × 100 mm; 3.5-μm Zorbax 300SB-C18 column (Agilent Technologies, Inc.) using a flow rate of 0.4 mL/min. The mobile phase consisted of water with 0.1% (v/v) formic acid (A) and acetonitrile with 0.1% (v/v) formic acid (B) with a gradient elution as follows: 30% B at 0 min, 80% B at 3 min, and 30% B at 5 min. Each run was followed by a 1 min postrun with 30% B. The total run-time analysis was 6 min and the column temperature was 27 °C. Spectra were acquired in the positive (ES+) ion mode, and full scan mass spectra (m/z 300−3200) were collected at a rate of 1 spectrum/sec. Mass data obtained from LC-MS was analyzed using Agilent MassHunter Qualitative Analysis software and Agilent MassHunter BioConfirm software version B.02.00. LC-MS Activity Assay. An end-point LC-MS assay based on the quantitative determination of the product, S3P (m/z = 253.0119 [M-H]−), was employed. All assays were performed at 25 °C in 0.1 M Tris, pH 7.6, 50 mM KCl and 5 mM MgCl2. Reactions were initiated by the addition of purified MtSK (0.2 μM) to a microfuge tube containing assay buffer and the substrates at the following saturating conditions: ATP (1.2 mM) and shikimate (5 mM) to a final volume of 500 μL. After incubation for 30 s, reactions were quenched by the addition of 2 μL of 98% formic acid followed by vortexing. For mass spectrometric analyses, 5 μL of quenched reaction mixtures were injected onto a reversed-phase column (Zorbax Eclipse Plus Phenyl-hexyl, 4.6 × 100 mm 3.5 μm, Agilent Technologies, Inc.). The mobile phase consisted of water with 0.1% (v/v) formic acid (A) and acetonitrile 100% (B) with a gradient elution as follows: 0 min, 2% B held for 4 min, to 30% B in the next 2 min. Each run was followed by a 1 min postrun C


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mL final volume) consisted of 0.1 M Tris pH 7.6, 50 mM KCl, 5 mM MgCl2, 5 mM shikimate, 1.2 mM ATP, 1.5 mM PEP, 0.2 mM NADH, 3 U/mL pyruvate kinase and 2.5 U/mL lactate dehydrogenase. Reactions were initiated by the addition of MtSK (20 nM), and the change of absorption was monitored for 3 min at 340 nm. In this reaction, the oxidation of NADH is stoichiometrically equivalent to the production of ADP and S3P by MtSK because MtSK is the rate limiting factor of the coupled reaction. Steady-state kinetic parameters were determined from initial velocity measurements varying the concentrations of one substrate while keeping the other substrate concentration fixed as described above. Data were collected in triplicate and analyzed using GraphPad Prism 5.02 software (Mountain View, CA). All spectra were obtained at room temperature on a Shimadzu UV-1601 spectrophotometer (Columbia, MD) with a cell path length of 1.0 cm. Kinetic Characterization of Known Inhibitor of MtSK. The inhibitory potency of compound 1, a previously reported inhibitor of SK,13 was evaluated using both the end-point LCMS assay and the coupled assay.

with 2% B. The total run-time analysis was 7 min at a flow rate of 0.4 mL/min and a column temperature of 45 °C. All acquisitions were performed under negative ionization mode using a narrow m/z range (m/z 100−300). For mass spectrometric detection of S3P, the Q-TOF conditions were the same as described above, except that a capillary voltage of 3200 V was used for the ESI source, and the fragmentor voltage was set to 175 V. For quantification of S3P, extracted ion chromatograms (EIC) of m/z 253.0119 [M − H]− were integrated using Agilent MassHunter Workstation Qualitative Analysis software (version B.02.00), and the peak area was used to evaluate enzyme activity. Determination of steady-state kinetic parameters for each substrate was performed using the same conditions described above. To evaluate ATP concentration dependence, ATP was varied (0.05−1.2 mM) against shikimate fixed at 5 mM. To evaluate shikimate concentration dependence, shikimate was varied (0.2−5 mM) against ATP fixed at 1.2 mM. Samples were prepared in triplicate and analyzed two times. The data were averaged and fit to the Michaelis−Menten equation (eq 1) using GraphPad Prism 5.02 software (Mountain View, CA). v0 =

Vmax[S] KM + [S]

Scheme 3. Structure of Reference Compound 1

(1)

Calibration Curve and Method Validation. The optimized LC-MS method was validated using parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ) and precision. The linearity of the method was evaluated by analyzing a series of concentrations (0.0004−1 mM) of a shikimate-3-phosphate (S3P) standard. Standard solutions were prepared by weighing the proper amount of S3P reference standard (corrected for purity) and dissolving it in a solution containing 0.1 M Tris (pH 7.6), 50 mM KCl, 5 mM MgCl2, 1.2 mM ATP, 5 mM shikimate and 0.2 μM MtSK prequenched with 98% formic acid. From this stock solution, serial dilutions were prepared to obtain the desired range of concentrations. Calibration curves were constructed by injecting each standard solution at each concentration level in triplicate. The peak areas were calculated from extracted ion chromatograms (EIC) of the most abundant ion (m/z 253.0119 [M − H]−) and the data were plotted against the corresponding concentration using least-squares linear regression. The parameters of the linear equations were used to obtain concentration values of the S3P formed in each reaction mixture. The LOD and LOQ were defined as the lowest concentration with a signal-to-noise (S/N) ratio of 3 and 10, respectively. The precision of the method was evaluated by determining the intraday and interday relative standard deviations (RSD) of the measured concentrations of S3P in a 100% control experiment (in the absence of inhibitor) and a 50% control experiment (in the presence of reference compound 1 at its Ki concentration). Each sample was injected 10 times the same day and on four consecutive days under the same experimental conditions. Spectrophotometric Coupled Assay. The enzymatic activity of MtSK was also assayed by a double coupled assay involving the enzymes pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) following procedures described by Rosado et al.11 In this assay, the generation of ADP by the MtSK-catalyzed reaction leads to the oxidation of NADH to NAD+; the decrease in NADH concentration is monitored at 340 nm (ε = 6220 M−1 cm−1). Assay mixtures (1

For the LC-MS assay, compound 1 dissolved in DMSO was tested at four different concentrations ranging from 0−10 μM. All reactions were carried out as described above. For each concentration of reference compound, five ATP concentrations ranging from 0.05 to 1.2 mM were used while keeping the shikimate constant at a saturating concentration (5 mM). Conversely, five different concentrations of shikimate ranging from 0.2 to 5 mM were used keeping ATP constant at a saturating concentration (1.2 mM). A total of 50 reaction mixtures (500 μL each) were prepared. Each reaction was initiated by the addition of 0.2 μM MtSK, incubated for 30 s, and quenched by the addition of 2 μL of 98% formic acid and vortexing. All quenched samples were analyzed by LC-MS for quantification of S3P following the procedures described above. Samples in the absence of reference compound were used as negative controls. The concentration of DMSO in each sample was kept lower than 2% (v/v), a concentration that was found not to affect the enzymatic activity of MtSK. Compound 1 was evaluated by the coupled assay in the same manner as described above. The A340 values at each compound 1 concentration (0−10 μM) were background corrected and initial velocities were measured in the presence of various concentrations of compound as a function of substrate concentration. Each reaction was started by the addition of MtSK (20 nM) and monitored for 3 min as described above. All data were analyzed using GraphPad Prism 5.02 software (Mountain View, CA). Steady-state inhibition constants for compound 1 were calculated by nonlinear fitting of the data to the noncompetitive inhibition equation (eq 2) where [I] is the D


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Analytical Chemistry inhibitor concentration in μM, Ki is the dissociation constant for the inhibitor−enzyme complex, and αKi is the dissociation constant for the inhibitor−enzyme−substrate complex. Ki values were determined by a minimum of two independent experiments. v=

Vmax[S]

(

[S] 1 +

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Among all the columns evaluated, the ZORBAX Eclipse Plus Phenyl-Hexyl column (Agilent) provided a better desalting, separation between the eluting peaks and a relatively shorter retention time. Different mobile-phases were also evaluated using various proportions of organic modifier/aqueous phases. The best mobile phase consisted of an acidic water/acetonitrile combination (pH 3.5) which favored the neutral state of the ionizable groups present in S3P and improved the retention time. A representative extracted ion chromatogram (EIC) of a typical MtSK reaction mixture acquired in the negative ionization mode is shown in Figure 2. Reproducible separation

[I] αK i

) + K (1 + ) M

[I] Ki

(2)

RESULTS AND DISCUSSION Recombinant MtSK LC-MS Analysis. The deconvoluted ESI-MS spectrum of MtSK showed two species with an average molecular mass of 19 648.71 Da and 19 517.54 Da (Figure 1).

Figure 2. (A) Extracted ion chromatogram (EIC) of MtSK reaction mixture separated on a ZORBAX Eclipse Plus phenyl-hexyl column. The peaks are (1) buffer salts, (2) shikimate-3-phosphate, and (3) shikimate.

was achieved, with buffer salts, shikimate-3-phosphate, and shikimate eluting at 2.2, 2.6, and 3.0 min, respectively, in a 6 min run using a gradient mode. The linearity of the assay in terms of S3P concentration versus detector signal was evaluated using a series of concentrations of S3P reference standard in a prequenched MtSK reaction mixture. The MS signal was linear (y = [3 × 107]x − 25714; R2 = 0.999) over a range of concentrations (0.0004−1 mM) sufficient to cover S3P formed in the wide variety of reactions performed. During the assay optimization process, it was observed that the presence of salts (especially Tris-HCl) in the samples decreased the sensitivity of S3P by ion suppression. Different desalting methods including desalting tips and spinning columns were evaluated, but these resulted in substantial loss of the product S3P. Ammonium acetate, an MS-friendly buffer was also evaluated as an alternative matrix for the reactions and increased sensitivity was observed for S3P (LOD 0.007 μg/mL and LOQ 0.05 μg/ mL) compared to Tris-HCl buffer (LOD 0.03 μg/mL and LOQ 0.1 μg/mL). Unfortunately, ammonium acetate produced diminished rates of MtSK catalysis. With the limit of detection obtained when using Tris-HCl, the lowest reaction rate that could be measured with the LC-MS assay was 0.2 min−1 at 0.2 μM enzyme. In comparison, the limit of detection for the coupled assay using UV detection has been reported as 0.2 min−1 at 1 μM enzyme.32 Given that sensitivity was not being sacrificed even when ion suppression was observed, we decided to use Tris-HCl to run all the reactions reported in this manuscript. The precision of the LC-MS assay was evaluated by determination of RSD of 100% (in the presence of DMSO) and 50% (in the presence of compound 1 at its K i concentration) control samples. These samples were analyzed ten times in the same analytical run (intraday) and on separate

Figure 1. Deconvoluted ESI-MS spectrum showing both modified (19517.54) and unmodified (19648.71) recombinant MtSK.

The mass difference (131.2 Da) is consistent with the posttranslational removal of N-terminal methionine, a modification performed by the enzyme methionine aminopeptidase (MAP). This modification is often observed during expression of recombinant proteins by E. coli in which the second amino acid is alanine, glycine, proline, serine, threonine or valine.29,30 The average molecular mass for intact MtSK (19 648.71) was in good agreement with the theoretical mass calculated from the amino acid sequence (19 648.59 Da). Furthemore, the absence of N-terminal methionine has been demostrated in the crystallographic structure of MtSK (PDB accession number 1WE2).31 This result thereby demonstrates the identity and purity of the recombinant MtSK used for this study. LC-MS Assay Development and Validation. We have developed and validated an LC-MS enzymatic assay based on the quantitation of the reaction product shikimate-3-phosphate (m/z = 253.0119 [M − H]−) formed during the MtSK catalyzed reaction. For the chromatographic separation of shikimate-3-phosphate from the rest of the reaction components, different experiments were carried out to optimize the mobile and stationay phases. Several reversed-phase columns (Zorbax Eclipse XDB-C18, Zorbax Eclipse Plus C18, Poroshell 300SB-C18, Zorbax 300SB-C8 and Zorbax Eclipse Plus PhenylHexyl) with different stationary phases, lengths, diameters, and particle sizes were tested. S3P is highly hydrophilic, and it is retained poorly by conventional reversed-phase columns. Indeed, it coelutes with the substrate shikimate and with other salts present in the reaction mixture. A normal phase column (Polaris 3 NH2), consisting of an aminopropylsilanecovered ultrapure silica that can offer polar selectivity under reversed phase and normal phase conditions was also evaluated. E


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(Figure 3-inset) and corresponded to reaction times where less than 10% of the substrate had been consumed. Thus, a quench time of 30 s was considered suitable to avoid complications due to substrate depletion and inhibition by accumulated product.33 Initial velocities were then calculated by dividing the concentration of S3P by the reaction quenching time (V0 = [S3P]/tq). To report values in terms of active site catalytic cycles per unit time, V0 was divided by enzyme concentration (i.e, V0/[E]T). Consequently, the asymptotic maximum V0/ [E]T, approached at saturating concentrations of substrate was indicative of the enzyme’s kcat. KM and kcat for Shikimate and ATP by LC-MS. To determine the steady-state kinetic parameters for each substrate by LC-MS, one substrate was kept at a fixed saturated concentration while the other substrate concentration was varied in each reaction. On the basis of the KM values (112 μM for ATP and 650 μM for shikimate) previously reported in the literature for MtSK,11 the concentration of the substrate to be varied in the experiment ranged from 0.5−10 times the reported KM. For the substrate to be fixed at saturating concentration, at least 10−15 times the reported KM was included. Initial velocities were measured for each reaction and plotted against each substrate concentration using nonlinear regression to obtain the respective KM and kcat values. A hyperbolic response of MtSK reaction rate was observed with increasing concentrations of ATP (Figure 4A, blue line) and shikimate (Figure 4B, blue line). A nonlinear least-squares fit of both data sets to a Michaelis−Menten rate equation produced an apparent KM with respect to ATP of 0.20 mM (Figure 4A) and an apparent KM with respect to shikimate of 0.53 mM (Figure 4B). Both of these values were in good agreement with those reported in the literature, indicating the accuracy and precision of the LC-MS method. The kcat values obtained from each plot were highly similar to one another (68 s−1 for ATP and 65 s−1 for shikimate) indicating that the maximum catalytic output of the enzyme was closely approached in both experiments. Likewise, the kcat was nearly identical to the values obtained by Rosado et al.11 and Gu et al.27 using spectrophotometric methods. Kinetic Characterization of MtSK by Spectrophotometric Coupled Assay. Determination of the optimum

runs for 4 days (interday). Excellent intraday and interday precision for the 100% control sample (RSD % 2.7 and 3.2, respectively) and for the 50% control sample (RSD % 3.8. and 4.4, respectively) was observed when the samples were kept at 4 °C. This allows for the analysis of numerous samples without significant loss of signal. Kinetic Characterization of MtSK by LC-MS Assay. Prior to measuring the kinetic parameters of MtSK by the endpoint LC-MS assay, determination of the optimum reaction conditions in terms of enzyme concentration and quenching time was performed. Optimal enzyme concentration was determined by measuring the amount of S3P generated over a period of time for five different concentrations of MtSK (0.1− 1 μM), at saturating concentrations of shikimate and ATP. The concentration of enzyme at which the production of S3P was linear over time (0.2 μM) was selected for subsequent experiments. The appropriate reaction quenching time was determined by plotting the amount of S3P formed versus time at a constant concentration of MtSK (0.2 μM) (Figure 3). The

Figure 3. MtSK reaction progress curve to determine optimum quenching time. [ATP] = 1.2 mM, [Shikimate] = 5 mM, [MtSK] = 0.2 μM, pH = 7.6. The inset shows an expansion of the first 120 s of the curve. The dashed red line is from linear regression analysis of data collected over the first 30 s of the reaction.

reaction was quenched at different time intervals from 0−4000 s. Production of S3P over the first 30 s was linear with time

Figure 4. MtSK initial velocity plots obtained by the LC-MS assay (blue lines) and the UV coupled assay (red lines) with respect to (A) ATP and (B) shikimate concentrations. For both methods, experiments were carried out in triplicate at room temperature, using five different concentrations of each substrate. Concentrations of MtSK were fixed at 0.2 μM and 20 nM for the LC-MS assay and for the coupled assay, respectively. Assays carried out for panel A contained 5 mM shikimate and assays carried out for panel B contained 1.2 mM ATP. F


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a distinct detection method for analysis such as mass spectrometry.46 Previously, we have successfully screened a library of compounds against MtSK using a direct and selective mass spectrometry-based method and provided IC50 values for the active compounds without the need for orthogonal testing.47 For this report, a well characterized inhibitor of shikimate kinase from Helicobacter pylori (HpSK)13 was chosen to validate our assay and to determine if the kinetic data generated by LCMS supported noncompetitive inhibition as previously observed by compound 1. For the kinetic characterization of compound 1 using both the spectrophotometric and the LC-MS assay, each concentration of inhibitor (from 0 to 10 μM) was tested by varying the concentration of one substrate, while the concentration of the other substrate was kept constant as described in Materials and Methods. Plots of enzyme velocity generated as a function of substrate concentrations at varying concentrations of compound 1 displayed a hyperbolic decrease of the enzyme’s apparent kcat, while the apparent value of KM was unaffected, consistent with noncompetitive inhibition (data not shown).46 Fitting of the data to eq 2 was done to determine the inhibition constants Ki and αKi with respect to ATP and shikimate. Data were collected from two separate experiments, and the average dissociation constants obtained from the LC-MS assay and the coupled assay are shown in Table 2. The Ki and αKi values, as

reaction conditions was carried out for the UV coupled assay in the same manner as for the LC-MS assay. To yield valid steadystate kinetic parameters for MtSK using this method, it was necessary to ensure that the rate of NADH consumption was strictly because of MtSK functioning as the rate-limiting step. In order to determine the appropriate concentration of enzyme to be used in the coupled assay, different reactions using varying concentrations of MtSK (0−80 nM) were performed while keeping the concentrations of the rest of the reagents constant. Figure 5 shows that at concentrations of MtSK below 40 nM, a linear increase in activity was observed. Rates of NADH

Figure 5. Effect of MtSK concentration on NADH consumption to determine the optimum rate-limiting enzyme concentration.

Table 2. Inhibition Constants of Compound 1 against MtSK Determined by the LC-MS and the UV-Coupled Assay

oxidation deviated from linearity above MtSK concentrations of 40 nM. Therefore, 20 nM of MtSK was used for subsequent coupled assay experiments. All samples were run in triplicate, a molar extinction coefficient (ε) for NADH of 6220 M−1 cm−1 and a path length of 1 cm were used for the assay. Steady-state kinetic parameters for each substrate were determined by the coupled assay using the same range of concentrations for ATP and shikimate described above, 20 nM MtSK and fixed concentrations of the other reagents (1.5 mM PEP, 0.2 mM NADH, 3 U/mL pyruvate kinase and 2.5 U/mL lactate dehydrogenase). The KM and kcat values calculated for ATP were 0.17 mM and 64 s−1, respectively, and for shikimate these values were 0.51 mM and 63 s−1, respectively (Figure 4 red lines). Table 1 shows the kinetic constants calculated from the conventional enzyme-coupled assay and the LC-MS assay, as well as those reported in the literature. Kinetic Evaluation of Inhibitor. MtSK is considered a potential target for the development of novel anti-TB drugs.34 To date, only a few inhibitors of this enzyme have been identified, mostly through computational techniques35−42 or through enzyme-coupled assays.43−45 With coupled assays, there is a need for confirmatory methods to exclude false positives acting on the coupled-enzyme component of the assay, requiring multiple assay formats to help in the verification of hit compounds. Confirmation is usually done by counter-screening against the coupling enzymes, or by using

LC-MS assaya

UV- assaya

literatureb UV-assay

10.90 6.94 10.09

9.87 7.82 8.07

9.48 7.18 10.67

Ki (μM) αKiATP (μM) αKiShikimate (μM) a

Values obtained in this study. bValues reported by Han et al.13

well as the mechanism of inhibition exhibited by compound 1 using both assays were consistent with the inhibition study with HpSK. These results indicate that the LC-MS assay can reveal not only kinetic parameters of bisubstrate enzymatic reactions, but it can also provide information regarding inhibition potency and mode of action of active compounds.

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CONCLUSIONS In this study, we have developed a rapid and straightforward enzymatic assay for the kinetic characterization of MtSK and for the evaluation of inhibitors using LC-MS. Performance of the method was evaluated by determination of the kinetic parameters of MtSK (KM and kcat) and inhibitory potency of a reference compound (Ki and αKi). The results obtained by the LC-MS assay aligned with those obtained from the spectrophotometric coupled assays performed in our laboratory with our preparations of MtSK, as well as those reported in the literature, demonstrating the reliability of this assay. The greater

Table 1. Kinetic Parameters of MtSK Calculated from Both UV-Coupled Assay and the LC-MS Assay UV-coupled assaya KM (mM) kcat (s−1) a

LC-MS assaya

literature (UV-assay)b

literature (UV-assay)c

ATP

shikimate

ATP

shikimate

ATP

shikimate

ATP

shikimate

0.17 ± 0.02 64 ± 2

0.51 ± 0.04 63 ± 1

0.20 ± 0.01 68 ± 2

0.53 ± 0.05 65 ± 2

0.11 ± 0.004 60 ± 8

0.65 ± 0.03 60 ± 8

0.08 ± 0.004 44 ± 2

0.41 ± 0.02 44 ± 2

Values obtained in this study. bValues reported by Rosado et al.11 cValues reported by Gu et al.27 G


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specificity and sensitivity of the LC-MS assay over conventional coupled enzyme assays suggests that it can be applied for the kinetic characterization of enzymatic reactions for which spectrophotometric assays are not viable or as an orthogonal method for the validation of lead compounds identified by high-throughput screening methods.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by an Auburn University Intramural Grants Program (AU-IGP) grant to A.C. and D.G., through the Office of the Vice President for Research (OVPR). J.S. is ́ grateful to the Secretariá Nacional de Ciencia y Tecnologia” (SENACYT) in collaboration with the “Instituto para la Formación de Recursos Humanos” (IFARHU) of the Panamanian government for Ph.D. scholarship. G.G. was supported by an Auburn University Cellular and Molecular Biosciences Undergraduate Summer Research Fellowship.

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ABBREVIATIONS ESI, electrospray ionization; LC-MS, liquid chromatography− mass spectrometry; MtSK, Mycobacterium tuberculosis shikimate kinase; Q-TOF, quadrupole time-of-flight; S3P, shikimate-3phosphate

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REFERENCES

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