Lactate dehydrogenase A (LDHA)[1]

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].

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]

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]

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 solution/amorphous 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]

[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.

Increased glucose consumption distinguishes cancer cells from normal cells and is known as the "Warburg effect" because of increased glycolysis. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme, a hallmark of aggressive cancers, and believed to be the major enzyme responsible for pyruvate-to-lactate conversion. To elucidate its role in tumor growth, we disrupted both the LDHA and LDHB genes in two cancer cell lines (human colon adenocarcinoma and murine melanoma cells). Surprisingly, neither LDHA nor LDHB knockout strongly reduced lactate secretion. In contrast, double knockout (LDHA/B-DKO) fully suppressed LDH activity and lactate secretion. Furthermore, under normoxia, LDHA/B-DKO cells survived the genetic block by shifting their metabolism to oxidative phosphorylation (OXPHOS), entailing a 2-fold reduction in proliferation rates in vitro and in vivo compared with their WT counterparts. Under hypoxia (1% oxygen), however, LDHA/B suppression completely abolished in vitro growth, consistent with the reliance on OXPHOS. Interestingly, activation of the respiratory capacity operated by the LDHA/B-DKO genetic block as well as the resilient growth were not consequences of long-term adaptation. They could be reproduced pharmacologically by treating WT cells with an LDHA/B-specific inhibitor (GNE-140). These findings demonstrate that the Warburg effect is not only based on high LDHA expression, as both LDHA and LDHB need to be deleted to suppress fermentative glycolysis. Finally, we demonstrate that the Warburg effect is dispensable even in aggressive tumors and that the metabolic shift to OXPHOS caused by LDHA/B genetic disruptions is responsible for the tumors' escape and growth.[1]

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A series of trisubstituted hydroxylactams was identified as potent enzymatic and cellular inhibitors of human lactate dehydrogenase A. Utilizing structure-based design and physical property optimization, multiple inhibitors were discovered with <10 μM lactate IC50 in a MiaPaca2 cell line. Optimization of the series led to 29, a potent cell active molecule (MiaPaca2 IC50 = 0.67 μM) that also possessed good exposure when dosed orally to mice.[2]

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

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.)