LDHA/B
GNE-140 racemate targets lactate dehydrogenase A (LDHA), the enzyme that catalyzes the conversion of pyruvate to lactate in the final step of glycolysis. LDHA is a key glycolytic enzyme that is often overexpressed in aggressive cancers and is considered a marker of the Warburg effect. The racemic mixture contains both (R)- and (S)-enantiomers of GNE-140, which inhibit LDHA activity. LDHA plays a critical role in maintaining NAD+/NADH balance and supporting the high glycolytic flux characteristic of many cancer cells. Inhibition of LDHA by GNE-140 disrupts this metabolic pathway and leads to alterations in cellular energy metabolism.

The "Warburg effect" refers to the increased glycolysis that causes greater glucose consumption, which separates cancerous cells from healthy cells. The primary enzyme thought to be in charge of converting pyruvate into lactate is lactate dehydrogenase A (LDHA), a crucial glycolytic enzyme and a marker of aggressive cancer [1].

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GNE-140 racemate potently inhibits LDHA enzymatic activity. In MIA PaCa-2 human pancreatic cancer cells, GNE-140 rapidly impacts overall metabolism following LDHA inhibition, although cell death occurs only after two days of continuous LDHA inhibition. Pancreatic cancer cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis are intrinsically resistant to GNE-140, but this resistance can be reversed by the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 is driven by activation of the AMPK-mTOR-S6K signaling pathway, which leads to increased OXPHOS and can be prevented by inhibitors targeting this pathway. The compound effectively disrupts glycolytic metabolism and induces metabolic reprogramming in sensitive cancer cells.

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.

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In vivo studies with GNE-140 have demonstrated that LDHA inhibition can suppress tumor growth. The combination of LDHA inhibitors such as GNE-140 with compounds targeting mitochondrial function or the AMPK-S6K signaling axis can expand the clinical utility of LDHA inhibitors beyond glycolysis-dependent tumors and reduce the emergence of resistance. In xenograft models, the efficacy of GNE-140 is influenced by the metabolic phenotype of the tumor, with OXPHOS-dependent tumors showing intrinsic resistance. The compound has been used to study the role of LDHA in tumor growth and to explore combination strategies for overcoming resistance to glycolysis-targeted therapies. Detailed in vivo efficacy data including specific tumor growth inhibition rates are described in the primary literature.

In vitro drug treatment experiments.[1]
All cell lines were obtained from our in-house tissue culture cell bank (original source was ATCC). Lines were authenticated by short tandem repeat (STR) and genotyped upon re-expansion. Cells were maintained in RPMI 1640 media supplemented with 10% FBS. Cells were plated using optimal seeding densities in 384-well plates using RPMI, 5% FBS, 100 ug/ml penicillin, 100 units/ml streptomycin. Optimal seeding densities were established for each cell line in order to reach 75-80% confluence at the end of the assay. The following day, cells were treated with compound 29 using a 6 pt dose titration scheme. After 72 hours, cell viability was assessed using the CellTiter-Glo® Luminescence Cell Viability assay. Absolute inhibitory concentration (IC) values were calculated using four-parameter logistic curve fitting.

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The in vitro enzyme/receptor binding (non-cell-based) assay for GNE-140 racemate typically measures LDHA enzymatic activity using a biochemical assay. Recombinant human LDHA enzyme is incubated with varying concentrations of GNE-140 racemate (typically ranging from nanomolar to micromolar) in the presence of the substrate pyruvate and the cofactor NADH. The enzymatic reaction is monitored by measuring the decrease in absorbance at 340 nm as NADH is consumed during the conversion of pyruvate to lactate. The IC50 value is determined by fitting dose-response curves to the inhibition data. The compound is dissolved in DMSO and diluted in assay buffer to achieve the desired final concentrations, with the DMSO concentration kept constant across all wells. Appropriate positive controls (known LDHA inhibitors) and negative controls (DMSO vehicle) are included in each assay run.

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.

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The in vitro cellular assay for GNE-140 racemate is performed using human pancreatic cancer cell lines such as MIA PaCa-2, which are highly glycolytic and sensitive to LDHA inhibition. Cells are cultured in appropriate medium supplemented with fetal bovine serum and antibiotics. For treatment, cells are plated in multi-well plates and allowed to adhere overnight. The following day, cells are treated with varying concentrations of GNE-140 racemate (typically 0.1-100 μM) or vehicle control (DMSO) for specified time points (e.g., 24, 48, or 72 hours). Cellular metabolic activity is assessed using assays such as MTT, CellTiter-Glo, or Seahorse extracellular flux analysis to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Cell viability and proliferation are quantified, and the effects of LDHA inhibition on glycolysis and OXPHOS are evaluated.

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

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In vivo animal experiments with GNE-140 racemate are typically conducted using immunocompromised mice bearing human cancer xenografts. MIA PaCa-2 human pancreatic cancer cells or other cancer cell lines are implanted subcutaneously into the flank of nude or SCID mice. When tumors reach a predetermined size (e.g., 100-200 mm³), animals are randomized into treatment and control groups. GNE-140 is administered via oral gavage or intraperitoneal injection at various doses and schedules. Tumor volume is measured twice weekly using calipers, and body weight is monitored to assess tolerability. At the end of the study, tumors are harvested for analysis of LDHA activity, metabolic markers, and signaling pathway proteins (e.g., AMPK, mTOR, S6K phosphorylation). Combination studies with phenformin or AMPK-S6K pathway inhibitors are also performed to evaluate synergistic effects.

Detailed pharmacokinetic (PK) parameters for GNE-140 racemate are not extensively documented in publicly available sources. As a small-molecule inhibitor with a molecular weight of 499.04, the compound is expected to have moderate oral bioavailability and reasonable tissue distribution. GNE-140 racemate is soluble in DMSO and other organic solvents for formulation purposes. For in vivo administration, the compound is typically formulated in vehicles such as PEG300, Tween 80, or carboxymethyl cellulose (CMC) to ensure adequate solubility and stability. The compound should be stored at -20°C in a dry, dark environment for long-term stability. Detailed PK parameters including half-life, clearance, volume of distribution, and maximum concentration (Cmax) require further investigation from primary literature sources.

Comprehensive toxicological data for GNE-140 racemate are not extensively documented in publicly available sources. As a research-grade compound, GNE-140 racemate is intended for laboratory research purposes only and is not approved for human therapeutic use. Standard laboratory safety practices should be followed when handling this compound, including the use of appropriate personal protective equipment and working in a well-ventilated area. In animal studies, GNE-140 has been reported to be generally well-tolerated at the doses tested, with no overt toxicity observed in preliminary studies. However, comprehensive toxicological profiling (e.g., LD50, maximum tolerated dose, organ-specific toxicity) is not available in the current search results and would require consultation of the primary literature or safety data sheets.

[1]. ACS Med Chem Lett.2016 Aug 26;7(10):896-901.
[2]. 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.
[3]. Nat Chem Biol.2016 Oct;12(10):779-86;

series of trisubstituted hydroxylactams were identified as potent enzymatic and cellular inhibitors of human lactate dehydrogenase A. Several inhibitors with IC50 values less than 10 μM for lactate in the MiaPaca2 cell line were discovered using structure-based design and physical property optimization. After optimization of this series of compounds, compound 29 was obtained, which is a potent cellularly active molecule (MiaPaca2 IC50 = 0.67 μM) and also showed good exposure when administered orally to mice. [1]

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GNE-140 racemate is a research compound developed for studying LDHA inhibition and tumor metabolism. The compound has been used to demonstrate that LDHA inhibition disrupts glycolytic metabolism and induces metabolic reprogramming in cancer cells. The discovery that OXPHOS-dependent cancer cells are intrinsically resistant to GNE-140, and that this resistance can be overcome by combining LDHA inhibitors with OXPHOS inhibitors or AMPK-S6K pathway inhibitors, has important implications for the clinical development of glycolysis-targeted therapies. GNE-140 racemate is not currently in clinical trials nor approved for therapeutic use; it remains an investigational tool compound for preclinical cancer metabolism research. The compound is available from various chemical suppliers for research purposes.

C25H23CLN2O3S2

499.044722795486

498.083

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

1809794-70-4

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

118384502

White to off-white solid powder

4.8

2

6

5

33

739

0

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

SUFXXEIVBZJOAP-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/h1-9,14,16,29H,10-13,15H2,(H,27,30)

3-((2-Chlorophenyl)thio)-4-hydroxy-6-(4-morpholinophenyl)-6-(thiophen-3-yl)-5,6-dihydropyridin-2(1H)-one

GNE-140 racemic; GNE-140; GNE-140 racemic; 1809794-70-4; CHEMBL4436150; 3-((2-Chlorophenyl)thio)-4-hydroxy-6-(4-morpholinophenyl)-6-(thiophen-3-yl)-5,6-dihydropyridin-2(1H)-one; 5-(2-chlorophenyl)sulfanyl-4-hydroxy-2-(4-morpholin-4-ylphenyl)-2-thiophen-3-yl-1,3-dihydropyridin-6-one; 3-[(2-chlorophenyl)sulfanyl]-4-hydroxy-6-[4-(morpholin-4-yl)phenyl]-6-(thiophen-3-yl)-1,2,5,6-tetrahydropyridin-2-one; R-GNE-140; (2~{r})-5-(2-Chlorophenyl)sulfanyl-2-(4-Morpholin-4-Ylphenyl)-4-Oxidanyl-2-Thiophen-3-Yl-1,3-Dihydropyridin-6-One; GNE 140; GNE140.

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 : ≥ 50 mg/mL (~100.19 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.)