Luminescent pyruvate kinase activation assay

The primary pyruvate kinase activation assay was performed in 1536-well white microtiter plates using 4 μL/well assay volume with final concentrations of 0.1 nM human PKM2, 0.4 nM PKM1, 3 nM PKL, or 0.6 nM PKR, 0.5 mM PEP, and 0.1 mM ADP in assay buffer containing 50 mM Imidazole pH 7.2, 50 mM KCl, 7 mM MgCl₂, 0.01% Tween, and 0.05% BSA. Compounds were dissolved in DMSO and 23 nL were transferred with a 1536-well pintool. The enzymatic reaction was allowed to proceed for 60 minutes, and then ATP was detected using a bioluminescent detection reagent containing D-luciferin and firefly luciferase (KinaseGlo Plus, Promega). Luminescence was then measured on the Perkin Elmer ViewLux using an exposure of 1 sec (with 1× binning). Kinase activity levels were graphed using GraphPad Prism to determine initial velocities (V₀), which were then plotted for each set of conditions.

Confirmatory LDH kinetic assay

All compounds were also tested in a kinetic mode by coupling the generation of pyruvate by pyruvate kinase to the depletion of NADH through lactate dehydrogenase. For PKM2, 3 μL of substrate mix (final concentration, 50 mM Tris-Cl pH 8.0, 200 mM KCl, 15 mM MgCl₂, 0.1 mM PEP, 4.0 mM ADP, and 0.2 mM NADH) were dispensed into Kalypsys black-solid 1,536 well plates using the Aurora Discovery BioRAPTR Flying Reagent Dispenser (FRD; Beckton-Dickenson, Franklin Lakes, NJ) and 23 nL of compounds were delivered via a Kalypsys pin tool; then, 1 μL of enzyme mix (final concentrations, 10 nM PKM2 and 1 μM of LDH) was added. Plates were immediately placed in ViewLux (Perkin Elmer) and NADH fluorescence was determined at 30 second exposure intervals for between 3 and 6 minutes. Data were normalized to the uninhibited and EC₁₀₀ activation using known activators, such as fructose-1,6-bis-phosphate. The data has been deposited in PubChem (AID 602377).

PKM2 K_m determination

M2 substrate K_m values were determined using a modified time course version of the primary luminescence assay. The assay was performed in 1536-well white microtiter plates using 4 μL/well assay volume with final concentrations of 5 nM human PKM2, and 4 mM of either PEP or ADP in assay buffer containing 50 mM Tris pH 7.5, 100 mM KCl and 5 mM MgCl₂. The non-titrated substrate in each reaction was included at non-limiting concentrations (4 mM), and the opposing substrate was diluted 1:2 in assay buffer to give an 8-pt titration (final concentration range 6.0 mM - 0.02 mM). Activator (either 1,6-FBP, ML265, or DMSO control) was added to the PKM2 solution at a final concentration of 10 μM, and the resulting solution was then added to the substrate solution to start the reaction. The PKM2 reactions were stopped (and ATP levels quantified), at 0, 5 and 10 min timepoints by addition of KinaseGlo Plus bioluminescent detection reagent. Luminescence was then measured on the Perkin Elmer ViewLux using an exposure of 1 sec (with 1× binning). M2 activity levels at each timepoint were graphed using GraphPad Prism to determine initial velocities (V₀), which were then plotted for each set of conditions. Curves were fit and K_m values calculated using an allosteric sigmoidal nonlinear regression algorithm in Prism.

Cell lines

293T (human embryonic kidney) and A549 (human non-small cell lung carcinoma) 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 (human non-small cell lung carcinoma), T.T (human medullary thyroid carcinoma), SN12C (human renal cell carcinoma), and SKMel28 (human melanoma) were obtained from ATCC and 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. 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. Cells expressing specific Flag-tagged isoforms of mouse pyruvate kinase M, or mutants thereof, in the absence of endogenous PKM2 were derived as described⁸.

Western Blot

Cells or tissues were lysed in RIPA buffer with 2 mM DTT and protease inhibitors and clarified by centrifugation at 21,000 × g. The protein content of supernatants was quantified by Bradford assay, and analyzed by SDS-PAGE followed by Western blot using standard protocols and the following primary antibodies: anti-pyruvate kinase (Abcam, ab6191), anti-PKM1 (Sigma, SAB4200094), anti-PKM2 (Cell Signaling, 4053), anti-FLAG (Sigma, F3165), and anti-actin (Abcam, ab1801). Iso-electric focusing/SDS-PAGE two-dimensional Western blot analysis was performed as described previously²⁰ after incubation of cells with DMSO or drug as indicated for one hour.

Immunoprecipitation

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 700 μL PK lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% Igepal-630) supplemented freshly prior to use 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). Lysates were centrifuged (20,000xg, 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 3xFlag 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.

Xenograft Experiments

H1299 parental and H1299 cells with constitutive expression of a mouse PKM1 cDNA (H1299-PKM1 cells) were propagated in RPMI supplemented with 10% fetal bovine serum, penicillin/streptomycin, 2 mM glutamine, and hygromycin for transgene selection. Cells were harvested, resuspended in sterile PBS, and 5×10⁶ cells were injected subcutaneously into nu/nu mice. Tumor growth was monitored via caliper measurement, the mice were sacrificed and tumors harvested after the time indicated. Tumors were weighed, divided, and either flash-frozen in liquid nitrogen or fixed in formalin for later analysis.

PKM2 activity assay

Pyruvate kinase activity was measured by monitoring pyruvate-dependent conversion of NADH to NAD⁺ by lactate dehydrogenase (LDH). Briefly, for cell line experiments, the medium was replaced with fresh medium 1 hr prior to the start of treatment with DMSO or activator. Also, where indicated, 100 μM pervanadate was added 10 min. prior to cell lysis. Cells were lysed on ice with RIPA buffer containing 2 mM DTT and protease inhibitors and clarified by centrifugation at 21,000 × g. 5 μL of the supernatant was used to assess pyruvate kinase activity²⁶. Pyruvate kinase activity was subsequently normalized for total protein content. For the experiment in Figure 4, the amino acid sequences of the peptides were: GGAVDDDYAQFANGG (M2tide) and GGAVDDDpYAQFANGG (P-M2tide)¹⁷.

Figure 4

ML265 activates PKM2 in the presence of phosphorylated peptides. a) Size exclusion chromatography of recombinant PKM2 after incubation with an M2 binding peptide motif (M2tide) or phosphorylated version of the same peptide motif (pM2tide). b) ML265’s (more...)

Sucrose-gradient ultracentrifugation

Approximately 2.3 mL of a 10–40% sucrose gradient was layered in ultracentrifuge tubes and left to equilibrate overnight. Recombinant PKM2 was incubated with drug or 1 mM FBP for 30 minutes on ice before layering on top of the gradient. The gradients were then spun at 55,000 rpm for 10 hours using a Beckman TLS-55 rotor and fractions collected. The resulting fractions analyzed by SDS-PAGE and Coomassie blue staining. Intensity of staining was quantified using IR fluorescence on a LiCOR instrument.

Size exclusion chromatography

Recombinant protein or ~2 mg of cellular protein from hypotonically lysed cells was separated on a HiPrep 16/60 Sephacryl S-200 HR column (GE) in 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.2. Fractions were analyzed by UV absorbance or SDS-PAGE with Western blot as indicated. For the experiment in Figure 4 the peptides used are the same as described under PKM2 activity assay.

Crystallography methods

For each complex crystal 360 0.5° oscillation images were collected in a continuous sweep. Data were reduced with HKL-2000²⁷ (DASA-58) or HKL-3000²⁸ (TEPP-46). Crystals belonged to space group P2₁. Structures were solved by direct replacement with the isomorphous Protein Data Bank (PDB)²⁹ entry 3GQY. Activator geometry restraints were obtained at the PRODRG³⁰ server. Iterations of model rebuilding, refinement and geometry validation were performed with COOT³¹, REFMAC³², and MOLPROBITY³³, respectively. The refined models were deposited³⁴ in the PDB.

Cloning, Expression, and Purification of Full Length PKM2

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.

Protein Crystallization and structure determination

For co-crystallization, the purified PKM2 was incubated at room temperature for overnight in the presence of 5 mM activators (TEPP-46 or DASA-58) and then set up crystallization trays 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 within 2 days from a reservoir solution containing 25% P3350, 0.2 M NH₄OAc and 0.1 M Bis-Tris pH 6.5. Crystals were cryo-protected with 50% Paratone-N, 50% mineral oil and were frozen directly in liquid nitrogen for x-ray data collection. Data collection was carried out at the Advanced Photon Source beamline 23ID-B.

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 dried on air. 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 dithiothreitol (DTT) for 30 minutes, 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 (CID). 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.

In vivo PK methods

Preparation of (ML265)

Scheme 1Preparation of: 2-methylsulfinyl-4-methyl-6-[(3-aminophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone (ML265)

Step 1: To a solution of 2-bromo-4-methyl-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone (9.5 g, 33.4 mmol) in DMF (120 mL) were added 3-nitrobenzylbromide (14.5 g, 66.9 mmol) and potassium carbonate (10.2 g, 73.6 mmol) and the mixture was heated at 100 °C overnight. After cooling to room temperature, water (100 mL) was added and the mixture was stirred for 10 min to dissolve the inorganic salts. The majority of the desired product was the solid which was filtered, first washed with water (3×50 ml) then EtOAc (3×50 ml). Part of product was still dissolved in the filtrate. The organic layer of the filtrate was separated and washed with brine, dried over Na₂SO₄. After the removal of organic solvent in vacuo, the residue was purified by silica gel chromatography (EtOAc/hexane= 1/4 to 1/1 gradient) to recover the desired product. The total amount of 2-bromo-4-methyl-6-[(3-nitrophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone was 10.5 g (75%).

Step 2: To a solution of 2-bromo-4-methyl-6-[(3-nitrophenyl)methyl]-4H-thieno[3,2-b]-pyrrole[3,2-d]pyridazinone (5.0 g, 11.9 mmol) in EtOH (40 mL) was added SnCl₂.2H₂O (21.5 g, 95.2 mmol). Concentrated HCl (40 mL) was added dropwise with ice/water cooling. The mixture was heated at 35 °C overnight. The pH was adjusted with 1N NaOH to pH = 9 and filtered through a pad of Celite. Excess EtOAc was used to wash the solid and the filtrate was washed with brine, dried over Na₂SO₄. After the removal of EtOAc in vacuo, the crude residue was purified by silica gel chromatography (MeOH/DCM= 1/100 to 1/20 gradient) to give 2-bromo-4-methyl-6-[(3-aminophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone (3.8 g, 82%) as a light yellow solid.

Step 3: To a solution of 2-bromo-4-methyl-6-[(3-aminophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone (200 mg, 0.51 mmol) in DMF (4 mL) in a sealed tube were added copper (I) bromide (74 mg, 0.51 mmol) and sodium thiomethoxide (108 mg, 1.53 mmol) and the sealed tube was purged with N₂ for 1 min then sealed. The mixture was heated at 100 °C overnight. After cooling to room temperature, excess EtOAc was added and the precipitate was filtered through a pad of Celite. The filtrate was washed with brine and the organic layer was separated, dried over Na₂SO₄. After the removal of EtOAc, the crude residue was purified through silica gel chromatography (DCM/MeOH= 1/100 to 1/20) to give 2-methylthio-4-methyl-6-[(3-aminophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone (119 mg, 65%) as a light yellow solid.

Step 4: To a solution of 2-methylthio-4-methyl-6-[(3-aminophenyl)methyl]-4H-thieno[3,2-b]pyrrole[3,2-d]pyridazinone (100 mg, 0.28 mmol) in DCM (2 mL) cooled to −78 °C was added a m-CPBA (77wt%, 69 mg, 0.31 mmol) in DCM (1 ml) solution dropwise within 2 min. The mixture was stirred at this temperature for 1 h. Excess DCM (ca. 10 ml) was added and the reaction mixture was allowed to warm to room temperature. The mixture was washed with 10% aqueous NaHCO₃ solution, brine and the organic layer was dried over Na₂SO₄. After the removal of DCM, the crude residue was purified through silica gel chromatography (MeOH/DCM= 1/100 to 1/10) to give 2-methylsulfinyl-4-methyl-6-[(3-aminophenyl)methyl]-4H-thieno[3,2-b]pyrrole-[3,2-d]pyridazinone (83 mg, 80%) as a light yellow solid.