Lactate dehydrogenase A (LDHA)
S-GNE-140 targets lactate dehydrogenase A (LDHA), the same enzyme targeted by the racemic GNE-140 mixture and its R-enantiomer. However, S-GNE-140 is the less active enantiomer, exhibiting significantly lower inhibitory activity against LDHA compared to the R-enantiomer. The compound binds to the active site of human LDH isozymes, but its enantiomeric configuration reduces its binding affinity and inhibitory potency. S-GNE-140 is therefore used as a control compound to distinguish enantiomer-specific effects of LDH inhibition from non-specific effects in cellular and biochemical assays.

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

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S-GNE-140 shows significantly lower in vitro inhibitory activity against LDHA compared to the active R-enantiomer. While the exact IC50 value for S-GNE-140 is not as extensively reported as the active enantiomer, it is established that S-GNE-140 is the less active enantiomer of GNE-140. The compound retains some ability to inhibit LDHA, but its reduced potency makes it suitable as a negative control in experiments designed to study the metabolic effects of LDHA inhibition. In MIA PaCa-2 human pancreatic cancer cells and other cell lines, S-GNE-140 is used to control for off-target effects and to validate that observed metabolic changes are specifically due to LDHA inhibition by the active enantiomer.

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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S-GNE-140 is used as a control compound in in vivo studies to distinguish the specific effects of LDHA inhibition from non-specific effects. In tumor xenograft models, S-GNE-140 is administered alongside the active R-enantiomer or the racemic mixture to validate that the antitumor effects observed are specifically due to LDHA inhibition. The compound is also used in studies investigating the role of LDHA in tumor growth, where it serves as a low-activity comparator. S-GNE-140 is administered via similar routes (oral gavage or intraperitoneal injection) as the active enantiomer, and its effects on tumor growth, metabolism, and signaling pathways are compared to those of the active compound to establish target specificity.

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 S-GNE-140 is conducted using the same biochemical LDHA activity assay as for the racemic mixture and the R-enantiomer. Recombinant human LDHA enzyme is incubated with varying concentrations of S-GNE-140 (typically ranging from nanomolar to micromolar) in the presence of pyruvate substrate and NADH cofactor. The enzymatic reaction is monitored by measuring the decrease in absorbance at 340 nm as NADH is consumed. The IC50 value for S-GNE-140 is determined and compared to that of the active R-enantiomer to confirm its lower potency. The compound is dissolved in DMSO and diluted in assay buffer to achieve the desired final concentrations, with DMSO concentration kept constant across all wells. Positive controls (active 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 S-GNE-140 is performed using the same cell lines and protocols as for the active enantiomer, typically using MIA PaCa-2 human pancreatic cancer cells. Cells are cultured in appropriate medium and treated with varying concentrations of S-GNE-140 or vehicle control (DMSO) for specified time points. Cellular metabolic activity is assessed using assays such as MTT, CellTiter-Glo, or Seahorse extracellular flux analysis. The effects of S-GNE-140 on cell viability, glycolysis, and OXPHOS are compared to those of the active enantiomer to confirm that the metabolic changes observed with the active compound are specifically due to LDHA inhibition. S-GNE-140 serves as a critical control to rule out non-specific effects of the compound scaffold.

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 S-GNE-140 are conducted in parallel with studies using the active R-enantiomer or the racemic mixture. Immunocompromised mice bearing human cancer xenografts (e.g., MIA PaCa-2 pancreatic tumors) are randomized into treatment groups receiving S-GNE-140, the active enantiomer, or vehicle control. S-GNE-140 is administered via oral gavage or intraperitoneal injection at doses comparable to those used for the active compound. Tumor volume is measured twice weekly, and body weight is monitored. At study endpoint, tumors are harvested for analysis of LDHA activity, metabolic markers, and signaling pathway proteins. The lack of significant antitumor efficacy with S-GNE-140 compared to the active enantiomer confirms that the effects observed are specifically due to LDHA inhibition.

Detailed pharmacokinetic (PK) parameters for S-GNE-140 are not extensively documented in publicly available sources. As the less active enantiomer of GNE-140, S-GNE-140 is expected to have similar physicochemical properties to the racemic mixture and the R-enantiomer, including a molecular weight of 499.04 and moderate lipophilicity. The compound is soluble in DMSO and other organic solvents for formulation purposes. For in vivo administration, S-GNE-140 is typically formulated in vehicles such as PEG300, Tween 80, or carboxymethyl cellulose (CMC). The compound should be stored at -20°C in a dry, dark environment for long-term stability. Detailed PK parameters including half-life, clearance, and bioavailability are not available from the current search results and would require consultation of the primary literature.

Comprehensive toxicological data for S-GNE-140 are not extensively documented in publicly available sources. As a research-grade compound, S-GNE-140 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. In animal studies, S-GNE-140 is expected to be well-tolerated at the doses typically used for control experiments, but comprehensive toxicological profiling (e.g., LD50, maximum tolerated dose) is not available from the current search results. The compound should be handled with appropriate personal protective equipment and in accordance with institutional safety guidelines for chemical research.

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

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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S-GNE-140 is a research compound used primarily as a negative or low-activity control in studies of LDHA inhibition and tumor metabolism. It is the S-enantiomer of the LDHA inhibitor GNE-140 and exhibits significantly lower inhibitory activity compared to the R-enantiomer. The compound is valuable for validating that the metabolic and antitumor effects observed with GNE-140 are specifically due to LDHA inhibition rather than off-target effects. S-GNE-140 is not currently in clinical trials nor approved for therapeutic use; it remains an investigational tool compound for preclinical research. The compound is available from various chemical suppliers for research purposes. Its primary utility is in enantiomer-selectivity studies and as a control in glycolysis-targeted anticancer research.

C25H23CLN2O3S2

499.044722795486

498.083

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

2003234-64-6

GNE-140 racemate;1802977-61-2;(R)-GNE-140;2003234-63-5

131801110

White to gray solid

4.8

2

6

5

33

739

1

C1COCCN1C2=CC=C(C=C2)[C@@]3(CC(=C(C(=O)N3)SC4=CC=CC=C4Cl)O)C5=CSC=C5

SUFXXEIVBZJOAP-RUZDIDTESA-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)/t25-/m1/s1

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

S-GNE-140; (S)-GNE-140; (S)-GNE-140; 2003234-64-6; CHEMBL4589484; (2S)-5-(2-Chlorophenyl)sulfanyl-4-hydroxy-2-(4-morpholin-4-ylphenyl)-2-thiophen-3-yl-1,3-dihydropyridin-6-one; GNE 140; GNE-140.

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 : ~14.43 mg/mL (~28.92 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.)