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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].
(R)-GNE-140 proved to possess the optimal combination of strong enzymatic and MiaPaca2 cellular potency, moderate permeability, and reduced plasma protein binding compared to other potent compounds in this series. |
| 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. |
| Animal Protocol |
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 |
| ADME/Pharmacokinetics |
(R)-GNE-140 was stable in liver microsomes with predicted hepatic clearances (Clp) of 3.3, 8.1, and 14 mL/min/kg, based on human, rat, and mouse microsomes, respectively. We carried out in vivo pharmacokinetic experiments in mice with (R)-GNE-140 and were gratified to observe that this compound presented a low Clp, consistent with the predicted hepatic Cl, and also had high bioavailability when dosed orally at 5 mg/kg (Table 5; concentration vs time plots in Figure S2). At higher oral doses, ranging from 50 to 200 mg/kg, (R)-GNE-140 displayed greater exposure (Table 5 and Figure S3), confirming the possibility of (R)-GNE-140 serving as a tool compound for in vivo efficacy studies in mice.[1]
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| References | |
| Additional Infomation |
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.[1]
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| Molecular Formula |
C25H23CLN2O3S2
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|---|---|
| Molecular Weight |
499.0447
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| Exact Mass |
498.083
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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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| PubChem CID |
121225870
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| Appearance |
Light yellow to yellow solid
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| LogP |
4.8
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
33
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| Complexity |
739
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1COCCN1C2=CC=C(C=C2)[C@]3(CC(=C(C(=O)N3)SC4=CC=CC=C4Cl)O)C5=CSC=C5
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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; (R)-GNE-140; 2003234-63-5; (R)-3-((2-chlorophenyl)thio)-4-hydroxy-6-(4-morpholinophenyl)-6-(thiophen-3-yl)-5,6-dihydropyridin-2(1H)-one; (2R)-5-(2-chlorophenyl)sulfanyl-4-hydroxy-2-(4-morpholin-4-ylphenyl)-2-thiophen-3-yl-1,3-dihydropyridin-6-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; inhibitor 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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