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Purity: ≥98%
(R)-GNE-140, the S-enantiomer of GNE-140, is a novel and potent lactate dehydrogenase (LDHA) inhibitor with potential anticancer activity. It inhibits LDHA/B with IC50s of 3 nM and 5 nM, respectively. Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.
| Targets |
LDHA (IC50 = 3 nM); LDHB (IC50 = 5 nM)
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| ln Vitro |
At a dose of 5 μM, (R)-GNE-140 demonstrated cell proliferation in 37 out of 347 pancreatic lineages that were evaluated. With an IC50 of 0.8 μM, (R)-GNE-140 inhibits two chondroma (bone) hexane lines expressing IDH1 dimming [1].
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| ln Vivo |
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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| Enzyme Assay |
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. |
| Cell Assay |
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. |
| References |
[1]. Purkey HE, et al. Cell Active Hydroxylactam Inhibitors of Human Lactate Dehydrogenase with Oral Bioavailability in Mice. 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. |
| Molecular Formula |
C25H23CLN2O3S2
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|---|---|
| Molecular Weight |
499.0447
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| Exact Mass |
498.08386
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| Elemental Analysis |
C, 60.17; H, 4.65; Cl, 7.10; N, 5.61; O, 9.62; S, 12.85
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| CAS # |
2003234-63-5
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| Related CAS # |
GNE-140 racemate;1802977-61-2;(S)-GNE-140;2003234-64-6; 1809794-70-4 (racemate)
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| Appearance |
Light yellow to yellow solid
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| LogP |
4.8
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| tPSA |
115Ų
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| SMILES |
O=C1C(SC2=CC=CC=C2Cl)=C(O)C[C@](C3=CSC=C3)(C4=CC=C(N5CCOCC5)C=C4)N1
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| InChi Key |
SUFXXEIVBZJOAP-RUZDIDTESA-N
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| InChi Code |
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)/t25-/m1/s1
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| Chemical Name |
(R)-3-((2-Chlorophenyl)thio)-4-hydroxy-6-(4-morpholinophenyl)-6-(thiophen-3-yl)-5,6-dihydropyridin-2(1H)-one
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| Synonyms |
R-GNE-140; (R)-GNE-140; GNE 140; GNE140.
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ≥ 50 mg/mL (~100.19 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.01 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. Solubility in Formulation 2: 2.5 mg/mL (5.01 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.01 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 2.5 mg/mL (5.01 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 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. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0038 mL | 10.0192 mL | 20.0385 mL | |
| 5 mM | 0.4008 mL | 2.0038 mL | 4.0077 mL | |
| 10 mM | 0.2004 mL | 1.0019 mL | 2.0038 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
 Overlay of previously disclosed X-ray structures of LDHA/diketone-containing inhibitor complexes 4QO7 (cyan) and 4QO8 (white).Hydrogen bonds from 4QO7 are shown as yellow dashed lines.ACS Med Chem Lett.2016 Aug 26;7(10):896-901. |
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 Compound9(cyan) cocrystallized with LDHA (white) [PDB: 5IXS]. The NADH cofactor is shown in green sticks, the crystallographic water as a red sphere, and hydrogen bonds are yellow dashed lines.ACS Med Chem Lett.2016 Aug 26;7(10):896-901. |
 Overlay of the crystal structures29(white) [PDB: 4ZVV] and30(cyan) [PDB: 5IXY] bound to LDHA. Hydrogen bonds are shown as yellow dashed lines.ACS Med Chem Lett.2016 Aug 26;7(10):896-901. |
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