Through a mechanism like that of the endogenous activator FBP, TEPP-46 and DASA-58 activate PKM2. Pre-treating cells with DASA-58 or TEPP-46 inhibits the suppression of PKM2 activity caused by pervanadate. Moreover, acetyl-coA, lactate, ribose phosphate, and serine intracellular levels are decreased by TEPP-46[1]. TEPP-46 suppresses the expression of several other Hif-1α-dependent genes as well as IL-1β and Hif-1α that are stimulated by LPS. Treatment with TEPP-46 dramatically reduces the expression of Cxcl-10 and Il12p40, two M1 indicators. PKM2 activation with TEPP-46 dramatically reduces the expression of Il1b mRNA produced by CpG and FSL-1. TEPP-46 has no effect on Tnf levels but increases Mtb-induced levels of Il10 mRNA and suppresses Mtb-induced levels of Il1b mRNA[2].
PKM2 (pyruvate kinase M2) – promotes tetramer formation and activates pyruvate kinase enzymatic activity [2]

Through a mechanism like that of the endogenous activator FBP, TEPP-46 and DASA-58 activate PKM2. Pre-treating cells with DASA-58 or TEPP-46 inhibits the suppression of PKM2 activity caused by pervanadate. Moreover, acetyl-coA, lactate, ribose phosphate, and serine intracellular levels are decreased by TEPP-46[1]. TEPP-46 suppresses the expression of several other Hif-1α-dependent genes as well as IL-1β and Hif-1α that are stimulated by LPS. Treatment with TEPP-46 dramatically reduces the expression of Cxcl-10 and Il12p40, two M1 indicators. PKM2 activation with TEPP-46 dramatically reduces the expression of Il1b mRNA produced by CpG and FSL-1. TEPP-46 has no effect on Tnf levels but increases Mtb-induced levels of Il10 mRNA and suppresses Mtb-induced levels of Il1b mRNA[2].

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TEPP-46 is a potent and selective activator of recombinant PKM2, with an AC90 of 470 nM and an AC50 of 92 nM, and does not activate recombinant PKM1 in vitro[1]
TEPP-46 promotes the association of PKM2 subunits into stable tetramers, as demonstrated by sucrose gradient ultracentrifugation and size exclusion chromatography[1]
TEPP-46 (1 µM) prevents pervanadate-induced inhibition of PKM2 activity in A549 cells[1]
TEPP-46 (25 µM, 36 hours) decreases intracellular concentrations of lactate, ribose phosphate, and serine in H1299 cells[1]
TEPP-46 (30 µM) has no significant effect on cell proliferation under normoxic (21% O2) conditions but decreases the proliferation rate of H1299 cells under hypoxic (1% O2) conditions[1]
TEPP-46 treatment (concentration not specified in the context) restores the ability of a PKM2 mutant (K305Q) to co-precipitate endogenous PKM2, indicating it stabilizes the tetramer at the A-A' interface[1]

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TEPP-46 (50-100 μM) promoted tetramerization of PKM2 in BMDMs and RAW macrophages, as evidenced by DSS crosslinking showing a high molecular weight PKM2-containing complex of approximately 250 kDa, and by FPLC size exclusion chromatography showing a shift to higher molecular weight (elution volume 10-11 ml). [2]
TEPP-46 (50-100 μM) inhibited LPS-induced nuclear translocation of PKM2 in BMDMs. [2]
TEPP-46 (50 μM, 30 min pretreatment) inhibited LPS-induced pro-IL-1β protein expression in BMDMs and peritoneal cells. [2]
TEPP-46 (100 μM, 1 hr pretreatment) inhibited LPS-induced binding of both PKM2 and Hif-1α to the IL-1β promoter as measured by oligo pulldown assay and sequential ChIP. [2]
TEPP-46 (50 μM) inhibited LPS-induced Hif-1α protein expression in BMDMs and peritoneal cells. [2]
TEPP-46 (50 μM) inhibited LPS-induced glycolysis (ECAR) in BMDMs. [2]
TEPP-46 (50 μM, 30 min pretreatment) inhibited FSL-1 (TLR2/6 ligand) and CpG (TLR9 ligand)-induced Il1b mRNA expression in BMDMs. [2]
TEPP-46 (25-50 μM, 30 min pretreatment) inhibited heat-inactivated Mycobacterium tuberculosis (Mtb)-induced pro-IL-1β and Hif-1α protein, as well as TNFα and IL-6 protein, while boosting Mtb-induced IL-10 production in BMDMs. [2]
TEPP-46 (25 μM, 30 min pretreatment) inhibited live Mtb (H37Ra)-induced Il1b mRNA, had no effect on Tnf mRNA, boosted Il10 mRNA, inhibited IL-1β and TNFα protein, and increased IL-10 protein in infected BMDMs. [2]
TEPP-46 (25 μM, 30 min pretreatment) increased intracellular bacterial load at 72 hours post-Mtb infection in BMDMs. [2]
TEPP-46 (25 μM, 30 min pretreatment) prior to S. typhimurium infection (MOI 10, 4h) increased intracellular bacterial load in BMDMs. [2]
In PKM2 conditional knockout BMDMs (PKM2-/-), TEPP-46 (50 μM) had no further inhibitory effect on LPS-induced Il1b expression beyond that seen in PKM2-/- cells alone, confirming on-target activity. [2]

With a lengthy half-life, low clearance, and a good volume of distribution, TEPP-46 demonstrates strong oral bioavailability and exhibits metrics that indicate drug exposure in tumor tissues. In A549 xenograft tumors, TEPP-46 at 150 mg/kg easily reaches maximal PKM2 activation[1].

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TEPP-46 (50 mg/kg, orally, twice daily) inhibits the growth of H1299 human non-small cell lung cancer xenograft tumors in immunocompromised (nu/nu) mice, delaying tumor emergence and reducing final tumor weight[1]
TEPP-46 treatment (50 mg/kg, 16 and 4 hours before sacrifice) promotes PKM2 tetramer formation and lowers concentrations of lactate, ribose phosphate, and serine in H1299 xenograft tumors[1]

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Mice were injected i.p. with TEPP-46 (50 mg/kg) or vehicle (20% 2-Hydroxypropyl-β-cyclodextrin) for 1 hour, followed by LPS (15 mg/kg, i.p.) for 2 hours. TEPP-46 dramatically reduced pro-IL-1β levels in peritoneal cells and serum IL-1β levels, while serum IL-6 levels were unchanged and serum IL-10 levels were greatly increased compared to LPS alone. [2]
Mice were injected i.p. with TEPP-46 (50 mg/kg) 1 hour prior to infection with S. typhimurium (1×10⁶ CFU, i.p.) for 2 hours. TEPP-46 significantly reduced pro-IL-1β in peritoneal cells, had no effect on serum IL-6 or IL-18 levels, and greatly enhanced serum IL-10 levels compared to infection alone. [2]
Mice infected with S. typhimurium (1×10⁶ CFU, i.p.) and sacrificed 24h post-infection showed significantly increased bacterial load (log CFU) in both spleens and livers when pretreated with TEPP-46 (50 mg/kg, i.p., 1 hr prior to infection). [2]

Pyruvate kinase activity was measured in cell or tissue lysates by monitoring the conversion of phosphoenolpyruvate (PEP) to pyruvate, with activity linked to NADH oxidation via a coupled reaction with lactate dehydrogenase[1]
For inhibitor studies, cells were treated with 100 µM pervanadate for 10 minutes prior to lysis to acutely increase tyrosine-phosphorylated protein levels[1]
For phosphoryrosine peptide experiments, peptides corresponding to the optimal PKM2 interaction motif (e.g., M2tide and P-M2tide) were used[1]

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Crosslinking assay: BMDMs or RAW 264.7 macrophages were treated with 50 μM TEPP-46 or DMSO. Crosslinking was performed using 500 μM disuccinimidyl suberate for 30 minutes. Lysates were analyzed by Western blot to detect high molecular weight PKM2 complexes (~250 kDa). [2]
Size exclusion chromatography: 2-3 mg protein from RAW 264.7 cells treated with ±10 μM TEPP-46 or DMSO (1 hr) followed by LPS (100 ng/ml, 24 hrs) was loaded on a Superdex 200 column and eluted with buffer. 250 μL fractions were collected and analyzed by SDS-PAGE and Western blot for PKM2. TEPP-46 caused a shift to higher molecular weight (elution volume 10-11 ml) corresponding to tetrameric PKM2. [2]
Oligo pulldown assay: BMDMs were treated with ± TEPP-46 (50 μM, 60 min) followed by LPS (24 hrs). An oligonucleotide specific to the Hif-1α binding site of the IL-1β promoter was incubated with cell lysates. Samples were probed for Hif-1α and PKM2 by Western blot. [2]
Sequential Chromatin Immunoprecipitation (ChIP): BMDMs were treated with ± TEPP-46 (50 μM, 30 min) prior to LPS (100 ng/ml, 24 hrs). Lysates were first immunoprecipitated with anti-HIF-1α antibody, then the precipitated sample was re-probed for PKM2 binding. qRT-PCR was performed using primers for the IL-1β promoter consensus HIF1α binding site (-408). Data calculated as percent of input. [2]

To assess pyruvate kinase complex formation in cells, Flag-tagged PKM1 or PKM2 was expressed, immunoprecipitated with Flag antibodies, and co-precipitating endogenous PKM2 was detected by western blot[1]
Cell proliferation was assessed by periodic measurement of cell mass accumulation over six days using crystal violet staining or by using commercial cell viability assays (e.g., MTS assay)[1]
For metabolic measurements, lactate production was assayed in cell incubation medium using a biochemistry analyzer[1]
Glucose incorporation into lipids was measured by incubating cells with [6-14C]-glucose, extracting cellular lipids, and quantifying incorporated 14C by scintillation counting[1]
Intracellular metabolite concentrations (e.g., lactate, ribose phosphate, serine) were determined using targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)[1]
To assess PKM2 multimeric state in cells, lysates were subjected to size exclusion chromatography, and fractions were analyzed by western blot for PKM2[1]
For sucrose gradient ultracentrifugation, recombinant PKM2 was incubated with compounds, layered on a gradient, centrifuged, and fractions were analyzed by SDS-PAGE and staining[1]

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BMDMs (1×10⁶ cells/ml) from C57BL/6 mice were treated with ± TEPP-46 (50-100 μM, 30-60 min pretreatment) followed by LPS (100 ng/ml) for various times (6-48 hrs). Pro-IL-1β, Hif-1α, PKM2, and β-actin protein expression were analyzed by Western blotting. [2]
BMDMs were treated with ± TEPP-46 (100 μM, 1 hr) followed by LPS (24 hrs). RNA was extracted and Il1b, Il6, Ldha, Il12p40, Cxcl-10, Arg-1, Mrc-1, and Pkm2 mRNA expression were measured by qRT-PCR. [2]
BMDMs were treated with ± TEPP-46 (50 μM, 30 min) followed by LPS (24 hrs). IL-6, TNFα, and IL-10 protein in supernatants were measured by ELISA. [2]
BMDMs were treated with ± TEPP-46 (50 μM, 30 min) followed by FSL-1 (100 ng/ml) or CpG (1 μg/ml) for 24 hrs. Il1b mRNA was analyzed by qRT-PCR. [2]
BMDMs were treated with ± TEPP-46 (25-50 μM, 30 min) followed by heat-inactivated or live Mtb (H37Ra, MOI 5) for 3-24 hrs. PKM2, IL-1β, Hif-1α, and β-actin protein were analyzed by Western blot; Pkm2, Il1b, Tnf, Il10 mRNA by qRT-PCR; and IL-10, TNFα, IL-6 protein by ELISA. [2]
BMDMs from PKM2fl/fl mice were treated with ± 600 nM tamoxifen for 72 hrs to delete PKM2, then treated with ± TEPP-46 (50 μM) prior to LPS (100 ng/ml, 24 hrs). Il1b mRNA was measured by qRT-PCR. No further inhibition was observed in PKM2-/- cells. [2]
Glycolysis measurement: BMDMs were plated at 200,000 cells/well, treated with ± 50 μM TEPP-46 (1 hr) followed by LPS (24 hrs), and extracellular acidification rate (ECAR) was measured using a flux analyzer. Results normalized to cell number. [2]

150 mg/kg
Mice

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For xenograft efficacy studies, H1299 cells (5×10^6) were injected subcutaneously into immunocompromised (nu/nu) mice[1]
Mice were randomly divided into cohorts and treated with either vehicle or TEPP-46 (50 mg/kg) administered orally twice daily for the duration of the experiment (over 7 weeks)[1]
For acute pharmacodynamic studies, mice bearing H1299 xenografts received bolus oral doses of TEPP-46 (e.g., 150 mg/kg or 50 mg/kg at specified times before sacrifice)[1]
Tumor growth was monitored, and mice were sacrificed at the indicated times for tumor collection and analysis[1]

Based on in vitro ADME analysis, TEPP-46 was predicted to have better in vivo drug exposure compared to other analogs [1]. TEPP-46 showed good oral bioavailability, relatively low clearance, long half-life and good volume of distribution in mice [1]. An acute oral dose of 150 mg/kg of TEPP-46 maximally activated PKM2 in A549 xenograft tumors [1]. After a single intravenous, intraperitoneal or oral administration, the changes in plasma drug concentration over time were measured, and pharmacokinetic parameters (Cmax, Tmax, T1/2, AUC) were calculated using a non-compartmental model [1].

A 5-day repeated-dose tolerance study in mice showed that TEPP-46 was well tolerated by oral administration of 50 mg/kg twice daily, with no signs of weight loss [1]. Based on blood cell counts, serum chemistry and histological examination of major organs, no significant toxicity was observed in mice after continuous oral administration (50 mg/kg twice daily) for more than 7 weeks [1].

[1]. Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis. Nat Chem Biol. 2012 Oct;8(10):839-847.

[2]. Pyruvate kinase M2 regulates Hif-1α activity and IL-1β induction and is a critical determinant of the warburg effect in LPS-activated macrophages. Cell Metab. 2015 Jan 6;21(1):65-80.

6-[(3-aminophenyl)methyl]-4-methyl-2-methylsulfinyl-5-thieno[3,4]pyrrolo[1,3-d]pyridazinone is an organosulfur heterocyclic compound, an organonitrogen heterocyclic compound, and an organoheterobicyclic compound. TEPP-46 (also known as ML-265) is a representative compound of thieno[3,2-b]pyrrolo[3,2-d]pyridazinone PKM2 activators [1]. TEPP-46 binds to a pocket at the AA' interface of the PKM2 tetramer, which is different from the binding site of the endogenous activator fructose-1,6-bisphosphate (FBP), and stabilizes the tetramer conformation [1].
The stabilizing effect of TEPP-46 on the PKM2 tetramer makes the enzyme resistant to inhibition of tyrosine-phosphorylated proteins [1]
TEPP-46 alters cancer cell metabolism by reducing glycolytic intermediates and biosynthetic precursors, thereby interfering with the anabolism required for tumor growth [1]

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TEPP-46 is a highly specific small-molecule activator of PKM2 that promotes tetramer formation and boosts PKM2 enzymatic activity, limiting tumor growth in vivo (as previously reported). In this study, TEPP-46 was used as a tool compound to probe PKM2 function in LPS-activated macrophages. It inhibited LPS-induced pro-IL-1β and Hif-1α, attenuated M1 macrophage polarization, inhibited glycolysis and succinate accumulation, and promoted IL-10 production. In vivo, TEPP-46 reduced IL-1β and increased IL-10 in sepsis and S. typhimurium infection models, leading to increased bacterial dissemination. The compound was synthesized in accordance with published methods (Boxer et al., 2010; Jiang et al., 2010). [2]

C17H16N4O2S2

372.464540481567

372.071

C, 54.82; H, 4.33; N, 15.04; O, 8.59; S, 17.22

1221186-53-3

44246499

White to light yellow solid powder

1.6±0.1 g/cm3

711.6±70.0 °C at 760 mmHg

384.2±35.7 °C

0.0±2.3 mmHg at 25°C

1.805

0.61

1

6

3

25

601

0

S1C(=CC2=C1C1C=NN(CC3C=CC=C(C=3)N)C(C=1N2C)=O)S(C)=O

ZWKJWVSEDISQIS-UHFFFAOYSA-N

InChI=1S/C17H16N4O2S2/c1-20-13-7-14(25(2)23)24-16(13)12-8-19-21(17(22)15(12)20)9-10-4-3-5-11(18)6-10/h3-8H,9,18H2,1-2H3

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.87 mg/mL (7.71 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.71 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: 2.5 mg/mL (6.71 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (5.58 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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Solubility in Formulation 5: 10 mg/mL (26.85 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 6: 5 mg/mL (13.42 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)