Lactate dehydrogenase A (LDHA)[1]
Lactate dehydrogenase A (LDHA)

Because of increased glycolysis, increased glucose consumption is known as the "Warburg effect" and separates cancer cells from healthy cells. One important glycolytic enzyme that is associated with aggressive cancer is lactate dehydrogenase A (LDHA), which is also thought to be the primary enzyme in the process of converting pyruvate to lactate [1].

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GNE-140 racemate is a potent inhibitor of lactate dehydrogenase A (LDHA), a key enzyme in the glycolytic pathway that converts pyruvate to lactate. The racemate perturbs glycolysis and inhibits proliferation in MIA PaCa-2 pancreatic cancer cells with an IC50 of 0.43 uM. It also decreases glucose uptake with an IC50 of 0.47 uM. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis are inherently resistant to GNE-140, but can be resensitized with the OXPHOS inhibitor phenformin. The racemate reduces breast cancer cell proliferation, motility, and invasion. The active (R)-enantiomer is the potent LDHA inhibitor, while the (S)-enantiomer is less active or inactive.

In mice, (R)-GNE-140 (5 mg/kg) exhibits a high bioavailability. In the prior gun simulation, (R)-GNE-140 shown increased exposure at 50 to 200 mg/kg.[2]

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No specific in vivo data for GNE-140 racemate; however, the active (R)-enantiomer has been studied in vivo. Based on the activity of the (R)-enantiomer, the racemate would be expected to have in vivo efficacy at higher doses due to the presence of the less active (S)-enantiomer. In vivo studies would involve administration to tumor-bearing mice to assess tumor growth inhibition, glycolytic flux (by ¹⁸F-FDG PET imaging), and lactate levels. The racemate is primarily used as a control or for comparative studies.

Recombinant human LDHA enzyme is expressed in E. coli and purified. The LDHA activity assay measures the conversion of pyruvate to lactate coupled with NADH oxidation. The reaction mixture contains Tris-HCl buffer (pH 7.4), pyruvate (1-10 mM), NADH (100-200 uM), and varying concentrations of GNE-140 racemate (0.001-100 uM). The reaction is initiated by adding LDHA enzyme (10-100 ng). The decrease in absorbance at 340 nm (NADH oxidation) is monitored over 5-10 minutes at 37degC. The initial velocity (V0) is calculated. IC50 values are determined from dose-response curves. The active (R)-enantiomer is typically more potent than the racemate. For the racemate, the IC50 may be approximately twice that of the (R)-enantiomer due to the presence of the inactive (S)-enantiomer.

Treatment with GNE-140 phenocopies LDHA/B double genetic disruption in both the LS174T and B16 cell lines[2] Recently, Boudreau et al. demonstrated the ability of GNE-140, a specific LDHA and LDHB inhibitor, to cause growth arrest in highly glycolytic pancreatic cancer cell lines such as MiaPaca2. Hence, we were curious to see whether this inhibitor could reactivate OXPHOS without delay and maintain the viability and growth of the WT LS174T and B16 cell lines. We treated WT and LDHA/B-DKO cells with different concentrations of GNE-140 and showed that a concentration of 10 μm, known to collapse LDHA and B activity, reduced the growth of the WT but not of the two LDHA/B-DKO cell lines reported here. This long-term experiment (9 to 12 days) proved the lack of off-target effects of this compound at the concentration used. Furthermore, we analyzed the metabolic consequences of the short-term GNE-140 treatment of the WT cells by Seahorse bioanalyzer. As shown in Fig. 8, E–H, 1-h treatment with 10 μm GNE-140 was sufficient to phenocopy the effect of the LDHA/B-DKO cells in terms of suppression of glycolysis and reactivation of OXPHOS. Hence, the growth phenotype of DLHA/B-DKO cells does not result from long-term growth selection during the two steps of genetic disruption. This finding, based on genetics and specific pharmacological disruption of LDHA and LDHB, firmly attests that, under normoxia, the Warburg effect is dispensable for in vitro tumor growth.[2]

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Human pancreatic cancer cell lines (e.g., MIA PaCa-2, PANC-1) and breast cancer cell lines are cultured in DMEM with 10% FBS and antibiotics. Cells are seeded in 96-well plates and treated with GNE-140 racemate (0.001-100 uM) for 24-72 hours. Cell proliferation is assessed by MTT, CCK-8, or CellTiter-Glo assays (IC50 = 0.43 uM in MIA PaCa-2 cells). Glucose uptake is measured using 2-NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose) fluorescent glucose analog or [14C]-2-deoxyglucose. Cells are treated with GNE-140 racemate (0.1-10 uM) for 4-24 hours, then incubated with 2-NBDG (100 uM) for 30-60 minutes, and fluorescence is measured (excitation 465 nm, emission 540 nm). The IC50 for glucose uptake is 0.47 uM. Lactate production is measured in the culture supernatant using a colorimetric lactate assay kit. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) are measured using a Seahorse XF analyzer to assess glycolytic flux and mitochondrial respiration. For resistance studies, cells are co-treated with phenformin (an OXPHOS inhibitor, 1-10 uM) to sensitize resistant cells. Cell viability is assessed by MTT. Colony formation assays can also be performed.

Mouse Pharmacokinetics Study [1] The pharmacokinetics of compound 29 ((R)-GNE-140) was evaluated following a single intravenous bolus (IV) dose of 1.0 mg/kg and oral administration (PO) of solutiomorphous suspension at a dose of 5 mg/kg in female CD-1 mice (N=3). The vehicle used for IV administration was 10/50/40 EtOH/PEG400/50mM citrate pH3 (v/v, 10/50/40), and for PO, 0.5% methycellulose:0.2% Tween in water (MCT). Blood samples for the IV dose group were collected at 0.033, 0.25, 1, 2, 4, 6 hours post dose. Blood samples for PO dose groups were collected at 0.25, 0.5, 1, 2, 4, and 6 hours post dose. For the high dose oral PK study at 50, 100, and 200 mg/kg, blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, and 8 hours post dose. Blood samples were centrifuged within 29 minutes of collection, and plasma was harvested. Plasma samples were stored at approximately –70°C until the analysis of the compound concentration by a liquid chromatography/tandem mass spectrometry (LCMS/MS) method. PK parameters were determined by non-compartmental methods using WinNonlin.[2]

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No published in vivo animal study for GNE-140 racemate. For the active (R)-enantiomer (GNE-140), typical in vivo protocols involve establishing subcutaneous tumor xenografts in immunodeficient mice using MIA PaCa-2 or other sensitive cancer cell lines. When tumors reach 100-200 mm3, mice are treated with GNE-140 (10-100 mg/kg) formulated in a suitable vehicle (e.g., 10% DMSO + 40% PEG300 + 5% Tween80 + 45% saline) once daily by oral gavage or intraperitoneal injection. Endpoints: tumor volume, tumor weight, body weight, survival. Plasma and tumor tissue are collected for pharmacokinetic analysis and pharmacodynamic evaluation: lactate levels in plasma and tumor (by colorimetric assay), glycolytic enzyme expression (LDHA, PKM2, GLUT1) by Western blot or qPCR, and tumor proliferation (Ki-67 by IHC). ¹⁸F-FDG PET imaging can assess tumor glucose uptake in vivo. For the racemate, a similar protocol would be used with dose adjustments (typically 2× the active enantiomer dose) to account for the presence of the inactive enantiomer. These protocols have not been validated for GNE-140 racemate.

No specific pharmacokinetic data for GNE-140 racemate. For the active (R)-enantiomer, (R)-GNE-140 has been characterized in preclinical studies: it is orally bioavailable with moderate to good oral absorption. Plasma half-life is suitable for once- or twice-daily dosing. The racemate would be expected to have similar PK parameters, but with lower effective exposure due to the presence of the less active (S)-enantiomer. The (S)-enantiomer may also have different metabolic clearance, potentially affecting the overall PK profile. Solubility: DMSO ≥10 mg/mL. Molecular weight: 499.04. The racemate has LogP 4.6, indicating high lipophilicity and likely high plasma protein binding. Storage: powder at -20degC for 3 years; in solvent at -80degC for 6 months.

No specific toxicity data for GNE-140 racemate. For LDHA inhibitors, the primary on-target toxicity is related to inhibition of glycolysis, which may affect rapidly proliferating normal cells (e.g., intestinal epithelium, bone marrow, immune cells). Chronic LDHA inhibition could lead to lactic acidosis, as LDHA is responsible for clearing pyruvate and regenerating NAD+. In cell-based studies, GNE-140 racemate reduces viability of cancer cells but may also affect normal cells. In vivo toxicity studies would assess maximum tolerated dose (MTD), body weight loss, organ toxicity (liver, kidney, pancreas), hematological parameters, and lactate levels. No data are publicly available for the racemate or the active enantiomer. For research use only, handle with standard laboratory precautions (gloves, lab coat, eye protection). Not for human use.

[1]. Double genetic disruption of lactate dehydrogenases A and B is required to ablate the "Warburg effect" restricting tumor growth to oxidative metabolism. J Biol Chem. 2018 Oct 12;293(41):15947-15961.

[2]. Cell Active Hydroxylactam Inhibitors of Human Lactate Dehydrogenase with Oral Bioavailability in Mice. ACS Med Chem Lett. 2016 Aug 26;7(10):896-901.

Cancer cells differ from normal cells in that they consume more glucose, a phenomenon known as the "Warburg effect" because it enhances glycolysis. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme, a hallmark of aggressive cancers, and is considered the primary enzyme for the conversion of pyruvate to lactate. To elucidate its role in tumor growth, we knocked out the LDHA and LDHB genes in two cancer cell lines (human colon adenocarcinoma cells and mouse melanoma cells). Unexpectedly, neither LDHA nor LDHB gene knockout significantly reduced lactate secretion. Conversely, dual gene knockout (LDHA/B-DKO) completely inhibited both LDH activity and lactate secretion. Furthermore, under normoxic conditions, LDHA/B-DKO cells overcame the effects of gene knockout by shifting their metabolic pathway to oxidative phosphorylation (OXPHOS), exhibiting a 2-fold reduction in proliferation rate both in vitro and in vivo compared to wild-type cells. However, under hypoxic conditions (1% oxygen), LDHA/B inhibition completely blocked in vitro growth, consistent with its dependence on oxidative phosphorylation (OXPHOS). Interestingly, the LDHA/B double knockout (DKO) gene blockade of activated respiration and cellular stamina growth is not the result of long-term adaptation. These phenomena can be pharmacologically reproduced by treating wild-type cells with an LDHA/B specific inhibitor (GNE-140). These findings suggest that the Warburg effect is not solely dependent on high levels of LDHA expression, as inhibition of fermentative glycolysis requires the simultaneous knockout of both LDHA and LDHB. Finally, we demonstrate that even in invasive tumors, the Warburg effect is not essential, and that the shift in metabolism to oxidative phosphorylation (OXPHOS) caused by LDHA/B gene disruption is the cause of tumor escape and growth. [1]

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A series of trisubstituted hydroxylactams were identified as potent enzymatic and cellular inhibitors of human lactate dehydrogenase A. Using structure-based design and physical property optimization, we identified several inhibitors with IC50 values of less than 10 μM for lactate in the MiaPaca2 cell line. After optimization of this series of compounds, compound 29 was obtained, which is a potent cellular activity molecule (MiaPaca2 IC50 = 0.67 μM) and also showed good exposure levels in mice after oral administration. [2]

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GNE-140 racemate (CAS: 1802977-61-2) is the racemic mixture of (R)-GNE-140 and (S)-GNE-140. (R)-GNE-140 is a potent and specific inhibitor of lactate dehydrogenase A (LDHA), while the (S)-enantiomer is less active. LDHA is a key enzyme in the glycolytic pathway that converts pyruvate to lactate, and its inhibition reduces lactate production, glycolytic flux, and cellular proliferation, particularly in cancer cells that rely on aerobic glycolysis (the Warburg effect). GNE-140 racemate has been shown to inhibit proliferation in MIA PaCa-2 pancreatic cancer cells (IC50 = 0.43 uM), decrease glucose uptake (IC50 = 0.47 uM), and reduce breast cancer cell proliferation, motility, and invasion. Molecular formula: C25H23ClN2O3S2, molecular weight: 499.04. Solubility: DMSO ≥10 mg/mL. Purity: >99%. Storage: powder at -20degC for 3 years. Not approved for clinical use. For research use only. References: GNE-140 racemate is a racemate mixture of (R)-GNE-140 and (S)-GNE-140 and a potent and specific inhibitor of Lactate dehydrogenase A(LDH-A). It reduces breast cancer cell proliferation, motility, and invasion, making it a potential candidate for cancer research.

C25H23CLN2O3S2

499.04

498.083

C, 60.17; H, 4.65; Cl, 7.10; N, 5.61; O, 9.62; S, 12.85

1802977-61-2

(R)-GNE-140;2003234-63-5;(S)-GNE-140;2003234-64-6

118384725

White to off-white solid powder

1.4±0.1 g/cm3

739.0±60.0 °C at 760 mmHg

400.7±32.9 °C

0.0±2.4 mmHg at 25°C

1.699

3.84

1

6

5

33

714

0

C1COCCN1C2=CC=C(C=C2)C3(CC(=O)C(C(=O)N3)SC4=CC=CC=C4Cl)C5=CSC=C5

GLDDJXYFHWRGPI-UHFFFAOYSA-N

InChI=1S/C25H23ClN2O3S2/c26-20-3-1-2-4-22(20)33-23-21(29)15-25(27-24(23)30,18-9-14-32-16-18)17-5-7-19(8-6-17)28-10-12-31-13-11-28/h1-9,14,16,23H,10-13,15H2,(H,27,30)

3-(2-chlorophenyl)sulfanyl-6-(4-morpholin-4-ylphenyl)-6-thiophen-3-ylpiperidine-2,4-dione

GNE-140 (racemate); 1802977-61-2; GNE-140 racemate; CHEMBL3335792; 3-(2-chlorophenyl)sulfanyl-6-(4-morpholin-4-ylphenyl)-6-thiophen-3-ylpiperidine-2,4-dione; 3-[(2-chlorophenyl)sulfanyl]-6-[4-(morpholin-4-yl)phenyl]-6-(thiophen-3-yl)piperidine-2,4-dione; GNE140; SCHEMBL17100418;

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)

DMSO: 20 mg/mL (40.08 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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