ML285 affects reactive oxygen species’ inhibition of pyruvate kinase M2

Brimacombe KR, Anastasiou D, Hong BS, et al.

Publication Details

The ability of all cells to regulate levels of reactive oxygen species (ROS) is vital for controlling many aspects of proliferation and survival and we have discovered that pyruvate kinase M2 (PKM2) is important for cancer cell biology. PKM2 is directly oxidized on Cys358 to inhibit its catalytic activity, which allows for diversion of glucose-6-phosphate into the pentose phosphate pathway. This, in turn, allows the synthesis of NADPH, which is critical for generating reduced glutathione, necessary for ROS detoxification. In a cellular context, our PKM2 activator, ML285 protects the enzyme from oxidation by ROS and results in sensitization to oxidative stress and increased apoptosis.

Screening Center Name & PI: NIH Chemical Genomics Center, Christopher P. Austin

Chemistry Center Name & PI: NIH Chemical Genomics Center, Christopher P. Austin

Assay Submitter & Institution: Matthew G. Vander Heiden, Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology and Dana Farber Cancer Institute, Harvard Medical School

PubChem Summary Bioassay Identifier (AID): 602359

Probe Structure & Characteristics

Image ml285fu1

Recommendations for Scientific Use of the Probe

ML285 is a potent activator of PKM2 in both biochemical (AC50 = 82 nM) and cell-based assays with high selectivity over PKM1, PKR and PKL. The compound was found to protect PKM2 from oxidation on Cys358 by ROS and result in increased apoptosis under high levels of ROS. Treatment of cells with ML285 followed by ROS induction reduces the shunt of glucose through the pentose phosphate pathway and reduces the levels of reduced glutathione available for detoxification.

1. Introduction

Conditions necessary for increased cellular proliferation and tumorigenesis are often associated with increased levels of reactive oxygen species (ROS). Growth factor stimulation, matrix detachment, and hypoxia all lead to increased levels of ROS and cancer cells often have mutations or altered gene expression that enable survival under these conditions18. Though many cancer cells are able to tolerate these highly oxidative conditions, they still require significant reducing power to avoid apoptosis. The major cellular reductant is glutathione and the reduced form (GSH) is supplied by nicotinamide adenine dinucleotide phosphate (NADPH).9 A significant source of NADPH is provided by the pentose phosphate pathway through the action of phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase (G6PD). The ability of G6PD to generate NADPH and subsequent production of GSH is vital for survival under oxidative stress.1013 The increased uptake of glucose, its fate in aerobic glycolysis, and its role in providing reducing equivalents of NADPH are key components of an altered metabolism exhibited in cancer. Another distinguishing feature is the expression of the M2 isoform of pyruvate kinase (PKM2) in all cancer cells and tumors studied to date.14 With our discovery of small molecule PKM2 activators, we were poised to investigate PKM2’s function in some of the above settings. We have described the high throughput screening and medicinal chemistry optimization on three unique M2 isoform selective activators1519, one of these activators role in inducing tetramerization,20,21 its activity in a mouse xenograft model,20,21 and the discovery of PKM2’s function in supporting the shunt of glucose towards the pentose phosphate pathway.22 With details of our activators effect on PKM2 in biochemical, cell-based, and in vivo systems already reported, this report will focus on their ability to elucidate PKM2’s role in enabling cancer cells to cope with ROS.

Prior Art

We have previously described 3 unique chemotypes capable of potent and isoform selective in vitro activation of PKM2 including a bis-sulfonamide series,16,18,19,21,22 a thieno-pyrrole-pyridazinone series15,18,1921 and a tetrahydroquinoline-6-sulfonamide series (Figure 1).17,19 Extensive structure activity relationships (SAR) and numerous analogs with potencies <100 nM were described for all series and three probes (ML203, ML202 and ML170) and one extended probe, ML265 (based on the ML202 scaffold) have been declared. At the time the studies described herein were initiated, only the bis-sulfonamide series was available and a comparative study with the other 2 series was not done.

Figure 1. Previously reported PKM2 activator ML probes.

Figure 1

Previously reported PKM2 activator ML probes.

2. Materials and Methods

2.1. General Chemistry Methods

All air or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents such as dichloromethane (DCM), N,N-dimethylforamide (DMF), acetonitrile, methanol, triethylamine, and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich. Preparative purification was performed on a Waters semi-preparative HPLC system. The column used was a Phenomenex Luna C18 (5 micron, 30 × 75 mm) at a flow rate of 45 mL/min. The mobile phase consisted of acetonitrile and water (each containing 0.1% trifluoroacetic acid). A gradient of 10% to 50% acetonitrile over 8 minutes was used during the purification. Fraction collection was triggered by UV detection (220 nM). Analytical analysis was performed on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA). Method 1: A 7 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8 minute run time at a flow rate of 1 mL/min. A Phenomenex Luna C18 column (3 micron, 3 × 75 mm) was used at a temperature of 50 °C. Method 2: A 3 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with a 4.5 minute run time at a flow rate of 1 mL/min. A Phenomenex Gemini Phenyl column (3 micron, 3 × 100 mm) was used at a temperature of 50 °C. Purity determination was performed using an Agilent Diode Array Detector for both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. 1H NMR spectra were recorded on Varian 400 MHz spectrometers. Chemical Shifts are reported in ppm with undeuterated solvent (DMSO-h6 at 2.49 ppm) as internal standard for DMSO-d6 solutions. All of the analogs tested in the biological assays have a purity greater than 95% based on both analytical methods. High resolution mass spectrometry was recorded on Agilent 6210 Time-of-Flight LC/MS system. Confirmation of molecular formulae was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02).

Cell lines, cell culture, virus preparations, PKM2 activator and oxidant treatments

293T and A549 cells were obtained from ATCC and cultured in DMEM (Mediatech) supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. H1299 (ATCC) cells were cultured in RPMI (Mediatech) supplemented as above. All cells were cultured in a humidified incubator at 37° C/5% CO2 unless otherwise stated. Glucose concentration in the media was 25 mM (4.5 g/L), unless otherwise stated. Diamide [1,1′ Azobis(N,Ndimethylformamide), D3648] and H2O2 (H1009) were from Sigma and used as described elsewhere.22 Hypoxia treatments were performed using an InVivo2 400 humidified workstation (Ruskinn, Pencoed, UK). For all hypoxia treatments (and corresponding normoxic control cultures), the media were supplemented with 20 mM HEPES buffer. For the experiment in Figure 2, A549 cells were washed once with PBS (37 °C), the culture medium was replaced with medium containing 5.6 mM glucose, and the cells were placed for 3 hr under 21% O2 or 1% O2. For experiments where 5.6 mM glucose was used in any part of the experiment, DMEM without glucose and without sodium pyruvate (Invitrogen-11966025) or RPMI medium without glucose (Invitrogen-1187920) were used, and supplemented with antibiotics as above, 10% dialyzed FCS (Invitrogen-26400044), and D-(+)-glucose (Sigma-G7021) at the indicated final concentrations. Cells expressing specific Flag-tagged isoforms of mouse pyruvate kinase M, or mutants thereof, in the absence of endogenous PKM2 were derived by first infecting cells with retroviruses to express the relevant cDNA, followed by shRNA mediated knock-down of endogenous PKM2 with a lentivirus-expressed shRNA. Retroviruses were produced in 293T cells by co-transfection of a plasmid expressing the amphotropic receptor gene and pLHCX-based vectors expressing the cDNA of interest fused to the C-terminus of a sequence encoding the Flag-epitope. Point mutations were introduced by two-step PCR. Viral 2 supernatants were harvested at 48 hr post-transfection, supplemented with 4 μg/mL polybrene and applied to target cells for 6–8 hr before replacing with normal growth media. Infection was repeated with fresh viral supernatants the following day after which cells were allowed to recover in normal medium for 12–16 hr prior to selection with hygromycin (300 μg/mL) for at least 10 days. Lentiviruses were produced in 293T cells by co-transfection of plasmids expressing gag/pol, rev and vsvg with a pLKO vector encoding a short hairpin targeting human PKM2. Selection was achieved with puromycin (2 μg/mL) for at least 4 days. PKM2 activators were described in Figure 1. ML285 at 10 μM was used in all experiments unless otherwise stated.

Figure 2. A) Hypoxia and exogenous oxidants increase ROS levels.

Figure 2

A) Hypoxia and exogenous oxidants increase ROS levels. B) Oxidative stress in A549 cells was induced by either B) Diamide and H2O2 or C) hypoxia and these conditions resulted in inhibition of PKM2 activity. This inhibitory action can be partially reversed (more...)

Cell harvesting, lysis, SDS-PAGE, and Western blotting

Cells attached to culture dishes were quickly washed once with a large volume (20–30 mL) of ice-cold PBS, snap-frozen in a liquid nitrogen bath, and stored at − 80 °C until further processing. Cells were lysed in PK lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% Igepal-630) supplemented freshly prior to usage with protease inhibitors [10 μg/mL phenymethylsulfonyl fluoride, 4 μg/mL aprotinin, 4 μg/mL leupeptin, and 4 μg/mL pepstatin (pH 7.4)] and 1 mM dithiothreitol (DTT) where applicable. For the detection of oxidized PKM2 by SDS-PAGE, cells were lysed in de-gassed lysis buffer without reducing agents until electrophoresis within 1 hr post-lysis. PK activity assays from total lysates of normally growing cells indicate that within this time frame (<1 hr), PKM2 activity is not significantly affected by exposure to ambient oxygen concentrations, based on the fact that DTT does not enhance PKM2 activity from untreated (no oxidants) cells. For reducing SDS-PAGE, lysates were mixed with SDS-PAGE loading buffer (50 mM Tris-HCl pH 8.8, 1% w/v SDS, 2.5% glycerol, 0.001% w/v bromophenol blue and 143 mM β-mercaptoethanol, final concentrations) and boiled for 10 min. For non-reducing SDS-PAGE, β-mercaptoethanol was omitted from the gel loading buffer and samples were not boiled. Antibodies for western blotting were: PKM1/2 (goat, 1:2000, Abcam-cat. # ab6191-5), PKM2 (rabbit, 1:1000, Cell Signaling Technology-cat. # 4053), and Flag (mouse, 1:5000, Sigma-cat. # F1804).

Immunoprecipitation

Cells were harvested as above and lysed in 700 μL PK lysis buffer supplemented with protease inhibitors and 1 mM DTT, where applicable. Lysates were centrifuged (20,000 xg, 10 min, at 4 °C), supernatants were transferred to fresh eppendorf tubes containing 20 μL of 50% Flag-agarose (Sigma- A2220) bead slurry in PK lysis buffer, and incubated rotating at 4 °C for 1 hr. Under these conditions, lysates were immunodepleted of detectable Flag-tagged proteins. Immunoprecipitates were washed 4 times with PK lysis buffer (1 mL = 100 bead-volumes per wash) then eluted from beads with 3x Flag peptide (150 μg/mL final concentration, Sigma, F4799, dissolved in 50 mM Tris-HCl pH 7.4, 150 mM NaCl) for 30 min rotating at 4 °C. Following a brief centrifugation of the beads, eluates were transferred to fresh Eppendorf tubes, supplemented with SDS-PAGE loading buffer and analyzed by SDS-PAGE.

Biotin labeling of oxidized PKM2

Cells were lysed for 15 min. on ice in biotin labeling lysis buffer (BLLB: 50 mM Tris-HCl pH 7.0, 5 mM EDTA, 120 mM NaCl, 0.5% Igepal-630) containing protease inhibitors (as above) and 100 mM maleimide (Sigma-129585). Insoluble material was then removed by centrifugation at 20,000 xg for 10 min. at 4 °C. The cleared supernatant was transferred to a fresh Eppendorf tube and protein concentration was determined by the Bradford assay. Protein concentration was adjusted to 1 μg/μL with BLLB, SDS was added from a 10% stock to a final concentration of 1%, and the cell lysates were incubated at room temperature for 2 hr while rotating. To remove unreacted maleimide, proteins were subsequently precipitated by adding 5 volumes of acetone pre-equilibrated at −20 °C and incubated for 20 min at −20 °C. The preparations were centrifuged at 20,000 xg for 10 min. at 4 °C, supernatants removed, and discarded and precipitated protein pellet was air-dried. The pellet was then resuspended in 200 μL BLLB containing 1% SDS, 10 mM DTT, and 0.1 mM biotin-maleimide (Sigma-B1267, stock dissolved in dimethylformamide) to reduce the remaining, previously oxidized, sulfhydryl groups and allow their reaction with biotin maleimide. Proteins were again precipitated with 5 volumes of methanol (−20 °C) as above, the dried pellet was resuspended in 500 μL of BLLB, incubated with 10 μL of a 50% slurry of streptavidin-sepharose beads (GE Healthcare-17511301) rotating at 4 °C for 2 hr. The beads were then washed 4 times with BLLB and resuspended in SDS-PAGE loading buffer for SDS-PAGE analysis and western blotting with the indicated antibodies.

Pyruvate kinase activity assays

PK activity was measured by monitoring pyruvate-dependent conversion of NADH to NAD+ by lactate dehydrogenase (LDH). Cells were lysed as above and protein concentration was determined by the Bradford assay. Immediately prior to start of the assay, 1 μg of total protein was mixed with 1x pyruvate kinase reaction buffer [50 mM Tris-HCl pH 7.5, 100 mM KCl, and 5 mM MgCl2 containing 0.5 mM PEP (Sigma-P0564), 0.6 mM ADP (Sigma-A5285), 180 μM NADH (Sigma-N8129), 0.015% Brij, 8 units LDH (Sigma-L1254), 1 mM DTT (where applicable), and 200 μM FBP (Fluka- 47810, where applicable). The final reaction volume was 100 μL/well in 96 well plates. For the experiment in Figure 4, recombinant PKM2 was produced in E. coli and purified and after treatments, catalase (Sigma-C1345, 1 mg/mL stock in 50 mM KPO4 at pH 7.0) was used at 10 μg/mL.

Figure 4. Purified recombinant PKM2 was treated with either A) DMSO or B) 1 μM ML285 followed by H2O2 for 30 minutes, diluted 100-fold and PK activity was assayed.

Figure 4

Purified recombinant PKM2 was treated with either A) DMSO or B) 1 μM ML285 followed by H2O2 for 30 minutes, diluted 100-fold and PK activity was assayed.

Metabolite analysis by targeted liquid-chromatography tandem mass spectrometry (LC-MS/MS)

Prior to each experiment, 2.5 × 105 cells were seeded in 6 cm dishes in media without sodium pyruvate, containing 10% dialyzed FCS, 2 mM glutamine, 100 U/mL penicillin and 100 μg/ml streptomycin. Media were changed at 24 hr and 46 hr. After 48 hr incubation, diamide was added directly to the media, where applicable, at a final concentration of 250 μM and cells were harvested at the indicated time points. Media were aspirated and metabolites were extracted with 1.5 mL of 4:1 v/v MeOH/H2O equilibrated at −80 °C. The extract and cells were scraped and collected into 15 mL conical tubes and centrifuged for 5 min at 690xg and solvent in the resulting supernatant was evaporated using a speed-vac. Samples were re-suspended in 20 μL HPLC-grade water for mass spectrometry. 85 μL were injected and analyzed using a 5500 QTRAP triple quadrupole mass spectrometer (AB/Sciex) coupled to a Prominence UFLC system (Shimadzu) via selected reaction monitoring (SRM) of 249endogenous water-soluble metabolites. Some metabolites were targeted in both positive and negative ion mode for a total of 298 SRM transitions. ESI voltage was +4900 V in positive ion mode and –4500 V in negative ion mode. The dwell time was 5 ms per SRM transition and the total cycle time was 2.09 sec. Approximately 8–10 data points were acquired per detected metabolite. Samples were delivered to the MS via normal phase chromatography using a 2.0 mm i.d. × 15 cm Luna NH2 HILIC column (Phenomenex) at 285 μL/min. Gradients were run starting from 85% buffer B (HPLC grade acetonitrile) to 42% B from 0–5 min; 42% B to 0% B from 5–16 min; 0% B was held from 16–24 min; 0% B to 85% B from 24–25 minutes; 85% B was held for 7 min to re-equilibrate the column. Buffer A was comprised of 20 mM ammonium hydroxide/20 mM ammonium acetate (pH=9.0) in 95:5 water:acetonitrile. Peak areas from the total ion current for each metabolite SRM transition were integrated using MultiQuant v1.1 software (AB/Sciex).

Pentose phosphate pathway (PPP)-dependent glucose oxidation to CO2

PPP activity was measured using an adaptation of previously published procedures (Figure 7).2,3 More specifically, 4,500 cells were seeded in 96-well plates 24 hr prior to the experiment. Media were supplemented with 5 μCi/mL of [1-14C]-glucose (specific activity 45–60 mCi/mmol) or [6-14C]-glucose (specific activity 50–62 mCi/mmol) and treatment compounds (diamide and PKM2 activator), in a final volume of 100 μL. The wells were overlaid with 3mm Whatman paper which had been impregnated just prior to use in a saturated Ba(OH)2 solution (prepared with boiled water) and blotted dry. Released 14CO2 was captured immediately above each well by forming insoluble Ba14CO3 on the filter. The plate lid was placed on top of the filter; the plate was sealed with parafilm and was incubated at 37%/5% CO2 for 3 hr. The Whatman paper was then removed, placed in an acetone bath, air-dried and incubated at 110 °C for 5 min. The filter was then cut into pieces each corresponding to a well of the plate, placed in a scintillation vial containing scintillation fluid and radioactivity was measured in a Beckman LS6000SC scintillation counter. The release of 14CO2 from [1-14C]-glucose, provided a quantitative measure of flux through the PPP enzyme, 6-phosphogluconate dehydrogenase, a parallel experiment which measured 14CO2 release from [6-14C]-glucose provided a quantification of TCA cycle-dependent CO2 production from glucose. PPP-dependent CO2 production was calculated as the difference between 14CO2 derived from [1-14C]-glucose and 14CO2 derived from [6-14C]-glucose.

Figure 7. A) [1-14C]glucose flux through PPP to generate 14CO2 in response to diamide was measured in DMSO and ML285 treated cells.

Figure 7

A) [1-14C]glucose flux through PPP to generate 14CO2 in response to diamide was measured in DMSO and ML285 treated cells. B) Changes in G-6-P levels were measured in response to diamide treatment and were measured in DMSO and ML285 treated cells. C) GSH (more...)

ROS and GSH measurements

For ROS measurements, the medium was aspirated, cells were washed 1x with PBS and incubated with PBS containing 1 μM chloromethyl-H2DCFDA (CM-H2DCFDA, Invitrogen-C6827) in DMSO for 30 min at 37 °C in 5% CO2. The dye was then removed and media containing H2O2 were added at the indicated concentrations and times. For ROS measurements under hypoxia (Figure 2A), cells were washed 1x with warm PBS and incubated for 2 hr under 1% O2 in media containing 5.6 mM glucose. The media were then removed and retained, and cells were loaded with 1 μM CM-H2DCFDA in PBS for 30 min, at which point the PBS was removed and the same media were replaced on the cells. All buffers and media used for the experiments had been pre-equilibrated under hypoxic conditions, at least overnight. Following these procedures, cells were trypsinized, centrifuged, resuspended in 500 μL PBS, and maintained on ice in the dark, until analysis by flow cytometry (FACScan, BD Biosciences). For GSH measurements, the medium was aspirated, cells were washed 1x with PBS, and incubated with PBS containing 12.5 μM ThiolTrackerTM Violet (Molecular Probes-T10095) for 30 min at 37 °C in 5% CO2. ThiolTrackerTM Violet conjugates to reduced (GSH) but not to oxidized glutathione, therefore ThiolTrackerTM Violet fluorescence corresponds to intracellular GSH concentration. Cells were subsequently harvested by trypsinization and processed for flow cytometry as above.

MTS cell viability assay

2,000 cells were seeded in 96-well plates 24 hr prior to treatment start. CellTiter96® AQueous (Promega-G5421) was used according to the manufacturer’s protocol to assess cell viability following oxidant and PKM2 activator combination treatments. MTS: (3-(4,5-dimethylthiazol-2 yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).

Statistical analysis

For metabolomics analyses, prior to any statistical computation, the metabolite measurement data were log2-transformed and normalised using the quantile approach implemented in the limma package in R 2.12, which ensures that the intensities of all metabolite measurements have the same empirical distribution across different sample runs. The empirical Bayes (eBayes) shrinkage of the standard errors towards a common value approach4 was used to identify the metabolites whose levels were significantly different between ML285 and control (DMSO) treatments at each of the respective time points (Figure 4). For all other statistical analyses, two-way ANOVA (GraphPad Prism® 5.03) or unpaired Student’s t-test (Excel) were used as indicated in the respective figure legends.

Cloning, Expression, and Purification of Full Length PKM2 for Crystallization Experiments

The cDNA sequence of full-length human PKM2 was amplified by PCR and cloned into the expression plasmid pET28a-LIC, and the construct was verified by DNA sequencing. The resultant plasmid was transformed into Escherichia coli strain BL21(DE3) and the cells were grown in Terrific Broth at 37 °C with 50 μg/mL kanamycin. When the OD 600 reached 1.0, the cells were induced by the addition of 0.5 mM isopropyl β-D-thiogalactoside for overnight at 18 °C and the recombinant PKM2 was expressed as a histag fusion protein containing 19 N-terminal amino acid residues (MGSSHHHHHHSSGLVPRGS). Cells were harvested by centrifugation, frozen in liquid nitrogen, and stored at −80 °C. The thawed cell pellet was resuspended in binding buffer (10 mM HEPES, pH 7.5; 300 mM KCl; 5 mM imidazole, 5 mM MgCl2; 5% glycerol; 2.5 mM TCEP), supplemented with protease inhibitor cocktail (Sigma) and 0.5% CHAPS, and then lysed by sonication. The lysate was clarified by centrifugation, and the supernatant was loaded onto DE52 column (Whatman) pre-equilibrated with binding buffer. The collected flow-through was loaded again onto Ni-NTA gravity flow column (Qiagen) equilibrated with binding buffer, and the bound histagged PKM2 protein was then eluted with elution buffer (10 mM HEPES, pH 7.5; 300 mM KCl; 5 mM imidazole, 5 mM MgCl2; 5% glycerol; 2.5 mM TCEP). The fractions eluted from Ni-NTA column were pooled, concentrated, and loaded onto a HiLoad 26/60 Superdex 200 column (Amersham Biosciences) pre-equilibrated with 10 mM HEPES, pH 7.5; 100 mM KCl; 5 mM MgCl2; 5% glycerol; 2.5 mM TCEP. The fractions containing PKM2 protein were collected, concentrated to 20 mg/mL using Amicon Ultra-15 centrifugal filter device (Millipore), and stored at −80°C until use. The final protein purity was confirmed by SDS-PAGE.

Crystallography methods

For co-crystallization, PKM2 was incubated overnight at room temperature in the presence of 5 mM activators (TEPP-46 or DASA-58) and crystallization trays were set up using the sitting-drop vapor diffusion method with droplets of protein solution (0.5 μl) and reservoir solution (0.5 μL). The best diffracting crystals were obtained from a reservoir solution containing 25% P3350, 0.2 M NH4OAc and 0.1 M Bis-Tris pH 6.5. Data collection was carried out at the Advanced Photon Source beamline 23ID-B. Data were reduced with HKL-200033 (DASA-58) or HKL-300034 (TEPP-46). Structures were solved by direct replacement with the isomorphous Protein Data Bank (PDB)35 entry 3GQY. Activator geometry restraints were obtained at the PRODRG36 server. Iterations of model rebuilding, refinement and geometry validation were performed with COOT,37 REFMAC,38 and MOLPROBITY,39 respectively.

For each complex crystal 360 0.5° oscillation images were collected in a continuous sweep. Crystals belonged to space group P21. Structures were solved by direct replacement with the isomorphous Protein Data Bank (PDB)23 entry 3GR4. Activator geometry restraints were obtained at the PRODRG24 server. Iterations of model rebuilding, refinement and geometry validation were performed with COOT,25 REFMAC,26 and MOLPROBITY,27 respectively. The refined models were deposited28 in the PDB.

Cell doubling time

20,000 cells were seeded in 12-well plates at day -1 in media containing 5.6 mM D-glucose and incubated at the indicated O2 concentrations. In all cases, media contained 10% dialyzed FCS and 20 mM HEPES pH 8.0. At day 0 the media were replaced with fresh media that had been equilibrated since the time of cell seeding at 37 °C under the corresponding oxygen concentrations. At days 0, 2, 4 and 6 cells were fixed with 10% formalin and at the end of the experiment stained with 0.1% w/v crystal violet in 20% methanol, shaking for 15 min at room temperature, and washed with water twice for 10 min each; the plates were then air dried. Cell-bound crystal violet was solubilized in 1 mL 10% v/v acetic acid and, because the amount of dye bound to cells is proportional to the number of cells, accumulation of cell mass was assessed by measurement of crystal violet absorbance at 595 nm in a spectrophotometer. Doubling times were calculated using exponential regression (http://www.doubling-time.com/compute.php).

LC/MS/MS Tandem mass spectrometry

For all mass spectrometry (MS) experiments, pyruvate kinase immunoprecipitates were separated using SDS-PAGE, the gel was stained with Coomassie blue, destained, and the pyruvate kinase band was excised. Samples were washed with 50% acetonitrile, subjected to reduction with 10 mM DTT for 30 min, alkylation with 55 mM iodoacetamide with 45 minutes, and in-gel digestion with TPCK modified trypsin (Promega) or chymotrypsin (Princeton Scientific) overnight at pH 8.3, followed by reversed-phase microcapillary/tandem mass spectrometry (LC/MS/MS). LC/MS/MS was performed using an EASY-nLC splitless nanoflow HPLC (Thermo Fisher Scientific) with a self-packed 75 μm id × 15 cm C18 Picofrit column (New Objective) coupled to a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific) in data-dependent positive ion mode at 300 nL/min with one full MS-FT scan followed by six MS/MS-IT scans via collision induced dissociation. MS/MS spectra were searched against the concatenated target and decoy (reversed) Swiss-Prot protein database using Sequest (Proteomics Browser Software (PBS), Thermo Fisher Scientific) with differential modifications for Met oxidation (+15.99), deamidation of Asn and Gln (+0.984) and fixed Cys alkylation (+57.02). Peptide sequences were identified if they initially passed the following Sequest scoring thresholds against the target database: 1+ ions, Xcorr ≥ 2.0 Sf ≥ 0.4, P ≥ 5; 2+ ions, Xcorr ≥ 2.0, Sf ≥ 0.4, P ≥ 5; 3+ ions, Xcorr ≥ 2.60, Sf ≥ 0.4, P ≥ 5 against the target protein database. Passing MS/MS spectra were manually inspected to be sure that all b- and y- fragment ions aligned with the assigned sequence. False discovery rates (FDR) of peptide hits were estimated below 1.5%, based on reversed database hits.

2.2. Probe Chemical Characterization

*Purity >95% as determined by LC/MS and 1H NMR analyses.

*Purity >95% as determined by LC/MS and 1H NMR analyses

1-(2,6-difluorophenylsulfonyl)-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazine (10).1H NMR (400 MHz, CDCl3) δ: 7.55 (m, 1H), 7.24 (m, 2H), 7.00 (m, 3H), 4.33 (m, 4H), 3.38 (m, 4H), 3.13 (m, 4H). LC/MS: Method 1, retention time: 5.781 min; Method 2, retention time: 3.889 min. HRMS: m/z (M+) = 460.0570 (Calculated for C18H18F2N2O6S2 = 460.0574).

2.3. Probe Preparation

Preparation of ML285

Scheme 1. Preparation of: 1-(2,6-difluorophenylsulfonyl)-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazine (ML285).

Scheme 1Preparation of: 1-(2,6-difluorophenylsulfonyl)-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazine (ML285)

Step 1

1-Boc-piperazine (250 mg, 1.34 mmol, 1 equiv.) was dissolved in dichloromethane (2.5 mL) and cooled in an ice bath under nitrogen atmosphere. Triethylamine (375 μL, 2.68 mmol, 2.0 equiv.) was added followed by portion-wise addition of 2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl chloride (346 mg, 1.48 mmol, 1.1 equiv.). The reaction was stirred in the ice bath for one hr, quenched with saturated aqueous ammonium chloride (~3 mL). The organic layer was washed twice with saturated ammonium chloride, once with brine, dried over sodium sulfate, concentrated in vacuo and then purified on silica gel chromatography using a 95/5 – 5/95, hexane/EtOAc (v/v) gradient to give 1-boc-4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazine as a white powder (516 mg, 89% yield).

Step 2

1-Boc-4-(2,3-dihydrobenzo[b][1,4]dioxine-6-sulfonyl)piperazine (400 mg, 1.04 mmol) was dissolved in dichloromethane (1 mL) and cooled in an ice bath. Trifluoroacetic acid (1 mL) was then added and the solution was stirred in the ice bath. The reaction was monitored by TLC and showed completion after one hr. The solution was removed from the ice bath and the solvents removed on in vacuo to yield the TFA salt of 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazine, which was carried onto the next step without purification. The oily residue was dissolved in dichloromethane (2 mL) and cooled in an ice bath. Triethylamine (580 μL, 4.16 mmol, 4 equiv.) was added followed by portionwise addition of 2,6-difluorobenzene-1-sulfonyl chloride (242 mg, 1.14 mmol, 1.1 equiv.). The progress was monitored by TLC and showed completion after 1 hr. The reaction was quenched with saturated aqueous ammonium chloride (~3 mL). The organic layer was washed twice with saturated ammonium chloride, once with brine, dried over sodium sulfate and concentrated in vacuo and then dissolved in DMSO and purified by reverse phase HPLC to yield pure ML285 as a white powder.

3. Results

ROS inhibits PKM2 activity via Oxidation of C358

To determine the effect of ROS on pyruvate kinase activity, we first tested whether exogenous oxidants and hypoxia had an effect on ROS levels and then determined the resultant PKM2 activity (Figure 2). As expected, diamide, hydrogen peroxide and hypoxia all induced elevated levels of ROS in A549 cells (Figure 2A). Confirming previous reports on the effect of oxidants on pyruvate kinase,2932 the above conditions decreased PKM2 activity and this inhibition could be partially rescued by addition of the thiol reducing agent DTT (Figures 2B and 2C). Interestingly, in cells engineered to exclusively express PKM1, diamide had no effect on pyruvate kinase activity (Figure 2D). Realizing that cysteine, a thiol containing amino acid, is particularly prone to oxidation, and that DTT, a strong thiol reducing agent, can reverse the PKM2 inhibition of the oxidants, we investigated whether a covalent modification was responsible for the aforementioned activity. Thus, diamide treated and untreated A549 cells were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under both reducing and non-reducing conditions (Figure 3A). Under reducing SDS-PAGE conditions, single PKM2 bands were obtained for both treated and untreated cells. Under non-reducing SDS-PAGE conditions, the diamide treated cells exhibited a faster migrating PKM2 band not observed in any other conditions. Thus, it was hypothesized that cysteine residues that could interfere with tetramer formation or PKM2’s enzymatic function might be the target of this oxidation. To this end, A549 cells expressing Flag-PKM2 mutants C31S, C424S (both located at the subunit interaction interface, C358S (located in a β barrel containing residues responsible for catalytic activity) or K433E (amino acid key for phosphotyrosine peptide binding33) were made and subjected to the above conditions (Figure 3B). C31S, C424S, and K433E mutant cells showed no difference compared to wild type (WT) and all exhibited a single PKM2 band under reducing conditions, but contained the faster migrating band after diamide treatment with non-reducing SDS-PAGE. Interestingly, the C358S mutant showed only a single PKM2 band in all conditions, indicating it may be the target of oxidation resulting in inhibition of catalytic activity. Next, cells were lysed under denaturing conditions, reduced cysteines were blocked with maleimide followed by reduction of oxidized cysteines and then reduced thiols were labeled with biotinylated maleimide. This allowed for streptavidin pull-down and identification of oxidized proteins (Figure 3C). Flag-PKM2 was detected in lysates of diamide treated WT cells, but not control treated or C358S mutant cells. The lack of a Flag-PKM2 band in the diamide treated C358S cells indicated that the oxidation of C358 is primarily responsible ROS induced inhibition of PKM2 activity.

Figure 3. A) SDS-PAGE run under non-reducing conditions gives a single band for PKM2 in control treated cells, but diamide treated cells two bands for PKM2.

Figure 3

A) SDS-PAGE run under non-reducing conditions gives a single band for PKM2 in control treated cells, but diamide treated cells two bands for PKM2. When both conditions are run under reducing SDS-PAGE conditions, a single band for PKM2 is observed. B) (more...)

ML285 Protects PKM2 from ROS Induced Oxidation of C358

To determine the affect our activator had on this inhibitory mechanism we treated purified recombinant PKM2 with either DMSO or ML285 followed by H2O2 and measured PKM2 activity (Figure 4).

This peroxide treatment showed a dose-response inhibition of PKM2 with DMSO control (Figure 4A), but this inhibition was prevented by ML285 treatment (Figure 4B). Extension to a cellular context showed that ML285 activated PKM2 activity in A549 cells in a dose-response manner (Figure 5A). Significant PKM2 activation was seen with ML285 treatment and this activity was slightly enhanced by addition of DTT (Figure 5B). Cells that were diamide treated and followed by addition of activator after cell lysis, did not see as significant of an activation of PKM2 activity (Figure 5C). This led us to believe that ML285 induced a conformation that prevented ROS induced oxidation of C358.

Figure 5. A) ML285 shows a dose-response activation of PKM2 in A549 cells.

Figure 5

A) ML285 shows a dose-response activation of PKM2 in A549 cells. B) Diamide treatment inhibits PKM2 activity in A549 cells and this inhibition can be prevented by DTT treatment. ML285 activates PKM2 activity in these A549 cells in both diamide treated (more...)

To help elucidate a potential mechanism for ML285’s ability to protect PKM2 from oxidation, we were able to generate a co-crystal structure of ML285 bound to human PKM2. This structure showed that the PKM2 tetramer contained two equivalents of ML285 each bound at the interface between the A domains of each dimer (Figure 6). The binding site of ML285 was over 30Å away from both the 1,6-fructose bisphosphate and ADP binding pockets. The ML285 binding pocket was lined with equivalent sets of residues provided by each of the PKM2 monomers forming the interface where the activator was accommodated through polar and van der Waals interactions. Two water-mediated hydrogen bonds were observed between the sulfonamide oxygen and the backbone nitrogen of Tyr390. Importantly, Cys358 was located in a β barrel that is not solvent exposed. This suggests that ML285 induction of this tetramer may prevent oxidants from accessing Cys358 and maintain PKM2 in the active conformation.

Figure 6. ML285-bound crystal structure of human pyruvate kinase M2 (PDB code: 3GR4).

Figure 6

ML285-bound crystal structure of human pyruvate kinase M2 (PDB code: 3GR4). A. Ribbon representation of the human pyruvate kinase M2 tetramer. Each monomer is highlighted in different color. ML285, FBP, ADP and Cys358 are shown in CPK mode. B. Binding (more...)

ML285 Reduces Cell’s Ability to Shunt Glucose-6-phosphate to the PPP and Sensitizes Cells to Oxidative Stress

Pyruvate kinase functions at the final stage of glycolysis and is important in controlling flux of glycolytic intermediates. The M2 isoform has been shown to be considerably less active34 and has been hypothesized to support anabolic processes.14,3537 In order to understand how ROS induced inhibition of PKM2 may support cancer cell proliferation and/or survival, a study of labeled glucose was undertaken. Cognizant that the pentose phosphate pathway (PPP) is required for reduction of ROS via production of GSH, we analyzed the fate of [1-14C]glucose. Diamide treatment did increase 14CO2 production via the PPP confirming that shunting through this pathway was activated in response to oxidative stress (Figure 7A). Cells that were treated with ML285 prior to diamide treatment did not show a significant change in 14CO2 derived from [1-14C]glucose (Figure 7A). Correlatively, the level of glucose-6-phosphate was significantly increased after diamide treatment, but this increase was prevented by treatment with ML285 (Figure 7B). This further signifies that ROS induced oxidation of PKM2 is important for inhibiting glycolysis and providing substrates for the PPP. As previously mentioned, one of the important functions of the PPP shunt under oxidative stress is to produce the NADPH required to provide GSH for ROS detoxification. To this end, GSH levels were measured in a variety of conditions: WT cells, C358S mutant cells, ML285 treated WT cells and PKM1 expressing cells (Figures 7C and D). Levels of GSH were higher in WT cells compared to C358S mutant and these levels observed in WT cells were reduced to levels similar to the mutant upon treatment with ML285 (Figure 7C). GSH concentrations in PKM1 expressing cells were not affected by ML285 treatment, indicating that these compounds are producing this effect by activation of PKM2 and not an off-target effect (Figure 7D).

To tie in these activators’ effect on reducing GSH, ROS levels and cell survival were studied. As expected, H2O2 produced higher ROS levels in activator treated cells compared to control and again showed a dose-response with increasing levels of H2O2 (Figure 8A). PKM1 expressing cells showed higher levels of ROS after H2O2 treatement, presumably due to their inability to slow down glycolysis and produce GSH for ROS reduction (Figure 8B). ML285 was able to sensitize cells to oxidant treatment and significant cell death was observed after diamide or H2O2 treatment (Figure 8C). The C358S mutant cells were also more sensitive to diamide treatment, presumably due to an inability to respond to ROS via PKM2 inhibition (Figure 8D).

Figure 8. A) Intracellular ROS levels measured with increasing concentrations of H2O2 in either DMSO or ML285 treated A549 cells.

Figure 8

A) Intracellular ROS levels measured with increasing concentrations of H2O2 in either DMSO or ML285 treated A549 cells. B) ROS concentration measured in MEF cell lines expressing either PKM2 or PKM1 with and without H2O2 treatment. C) Cell survival measured (more...)

4. Discussion

With its ubiquitous expression in cancer cells, PKM2 has emerged as an important enzyme to study some of the metabolic rewiring seen in cancer cells. Most fetal tissue expresses PKM2, but upon maturation its splice variant PKM1 is predominantly expressed. Though many of the different regulatory properties and activities between these two enzymes have been known for some time,34,3841 potentially important new roles of these enzymes are currently being discovered.22,36,37,4246 In an entirely new addition to this emerging literature, we have discovered that PKM2 expression is important for cells to respond to ROS by allowing G-6-P shunting through the PPP. This generates the reducing equivalents of NADPH needed to reduce the oxidized glutathione to its reduced form, GSH, which can detoxify the reactive oxygen and allow cell survival. The small molecule PKM2 activator, ML285, was vital for uncovering this activity. We believe that through induction of the tetrameric form of PKM2, ML285 is able to prevent C358 oxidation and the associated inhibition. This sensitizes cells to increased ROS, by not allowing appropriate diversion of G-6-P to generate the requisite reducing powder of GSH. The ability of these small molecules to activate PKM2, induce tetramerization, prevent both phosphorylated peptide and ROS induced inhibition of PKM2, reduce tumor size in a mouse xenograft model and sensitize cells to oxidative stress make ML170, ML202, ML203, ML265, and ML285 useful molecules for studying PKM2 and potentially provide starting points for therapeutic development.

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