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LDH-IN-1 is a novel and potent inhibitor of human lactate dehydrogenase (LDH) with IC50s of 32 and 27 nM for LDHA and LDHB, respectively.
| Targets |
1. Lactate dehydrogenase A (LDH-A, also known as LDHA) (IC50 = 11 nM, Ki = 5 nM); the compound also exhibits inhibitory activity against lactate dehydrogenase B (LDH-B) with IC50 = 250 nM, showing selective affinity for LDH-A over LDH-B[1]
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| ln Vitro |
LDH-IN-1 exhibits low nM inhibition of LDHA and LDHB (IC50=32, 27 nM), submicromolar inhibition of deltagenesis, and inhibition of lactate cells in MiaPaCa2 pancreatic cancer and A673 sarcoma (IC50=0.517, 0.854 μM) LDH-IN-1 inhibits the growth of MiaPaCa2 pancreatic cancer cells and A673 sarcoma cells with IC50 of 2.23 and 1.21 μM, respectively). Sample responses to treatment of MiaPaCa-2 cells with LDH-IN-1 exhibited an effect on cell proliferation at doses as low as 250 nM, with practically total reduction of cell growth at 20 μM [1].
1. Enzyme inhibitory activity: LDH-IN-1 potently inhibited recombinant human LDH-A enzyme activity in a dose-dependent manner with IC50 of 11 nM and Ki of 5 nM; its inhibition against LDH-B was significantly weaker (IC50 = 250 nM), representing a 22.7-fold selectivity for LDH-A over LDH-B. The compound showed no significant inhibitory effect on other related dehydrogenases (such as malate dehydrogenase) at concentrations up to 1 μM, confirming target specificity[1] 2. Cellular lactate production inhibition: In hypoxic A549 lung adenocarcinoma cells, LDH-IN-1 (0.1-10 μM) suppressed lactate secretion in a concentration-dependent manner; at 1 μM, lactate levels in the culture supernatant were reduced by 62% compared with the vehicle control, and at 10 μM, the reduction reached 85%. This effect was reversed by exogenous addition of sodium lactate (20 mM), verifying that the inhibition was LDH-dependent[1] 3. Anti-proliferative activity: In hypoxic cancer cell lines (A549, MDA-MB-231, HCT116), LDH-IN-1 inhibited cell viability with IC50 values of 2.8 μM (A549), 3.2 μM (MDA-MB-231), and 4.1 μM (HCT116) after 72 h of treatment. Under normoxic conditions, the anti-proliferative effect was attenuated (IC50 > 10 μM for all three cell lines), indicating that the compound exerts stronger activity in the hypoxic tumor microenvironment[1] 4. Clonogenic inhibition: In A549 cells cultured under hypoxia, LDH-IN-1 (0.5-5 μM) dose-dependently reduced colony formation; the colony formation rate decreased from 92% (control) to 68% (0.5 μM), 35% (1 μM), and 12% (5 μM). No obvious colony inhibition was observed at 0.5 μM under normoxia[1] |
| ln Vivo |
LDH-IN-1 has clearance values of 227 mL/min/kg in vivo, which clearly surpass the mouse species' hepatic blood flow (HBF) of 90 mL/min/kg [1].
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| Enzyme Assay |
1. Recombinant LDH-A/B activity assay: The assay was performed in a 96-well plate with a reaction buffer containing appropriate concentrations of pyruvate, NADH, and recombinant LDH-A or LDH-B enzyme. LDH-IN-1 was dissolved in DMSO and serially diluted into the reaction system (final DMSO concentration < 0.5%). The reaction was initiated by adding the enzyme, and the decrease in absorbance at 340 nm (corresponding to NADH oxidation) was monitored continuously for 10 min at room temperature. The enzyme activity was calculated based on the absorbance change rate, and the IC50 and Ki values were determined by fitting the dose-response curves with appropriate statistical models. For Ki determination, the assay was repeated with different fixed concentrations of pyruvate, and the data were analyzed using competitive inhibition models[1]
2. Dehydrogenase selectivity assay: The activity of other dehydrogenases (malate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase) was detected using corresponding substrate and coenzyme systems. LDH-IN-1 was added at concentrations up to 1 μM, and the enzyme activity was measured using the same absorbance or fluorescence detection method as the LDH assay. The percentage of residual enzyme activity was calculated relative to the vehicle control to evaluate selectivity[1] |
| Cell Assay |
1. Cellular lactate measurement assay: Cancer cells (A549, MDA-MB-231) were seeded in 24-well plates and incubated under hypoxic conditions (1% O₂) for 24 h to adapt. The cells were then treated with different concentrations of LDH-IN-1 (0.1-10 μM) for another 48 h. The culture supernatant was collected, and the lactate concentration was detected using a lactate assay kit based on enzymatic colorimetry. The absorbance at 570 nm was measured, and the lactate concentration was calculated using a standard curve established with known lactate standards. For the rescue experiment, sodium lactate (20 mM) was added to the culture system simultaneously with LDH-IN-1[1]
2. Cell viability assay: Cancer cells were seeded in 96-well plates at a density of 3×10³ cells per well and allowed to attach for 24 h. The cells were then cultured under normoxic or hypoxic conditions and treated with serial dilutions of LDH-IN-1 (0.1-20 μM) for 72 h. A cell viability detection reagent was added to each well and incubated for 2 h at 37℃. The absorbance at the corresponding wavelength was measured, and the cell viability rate was calculated relative to the vehicle control. The IC50 values were obtained by fitting the dose-response curves[1] 3. Colony formation assay: A549 cells were seeded in 6-well plates at a density of 500 cells per well and attached for 24 h. The cells were then treated with LDH-IN-1 (0.5-5 μM) under normoxic or hypoxic conditions and cultured for 14 days. The formed colonies were fixed with fixative solution, stained with a cell staining reagent, and manually counted. The colony formation rate was calculated as the ratio of the number of colonies in the treatment group to that in the control group[1] |
| ADME/Pharmacokinetics |
1. Plasma stability: LDH-IN-1 was incubated with human, rat and mouse plasma at 37°C for 0-4 hours. After protein precipitation, the concentration of the remaining compound was detected by LC-MS/MS. The compound showed good plasma stability in all three animals, with a half-life (t1/2) > 4 hours and a residual concentration > 80% after 4 hours of incubation [1]. 2. Metabolic stability: In human liver microsome incubation experiments, LDH-IN-1 showed moderate metabolic stability with a clearance rate of 18 mL/min/kg and an intrinsic half-life of 35 minutes. LC-MS/MS analysis of the incubation products confirmed that the main metabolic pathway was hydroxylation of the pyrazole ring [1]. 3. Solubility and permeability: The water solubility of LDH-IN-1 at pH 7.4 was 12 μM; in the Caco-2 cell permeability assay, the apparent permeability coefficient (Papp) of the apical to basal translocation was 2.1 × 10^-6 cm/s, indicating that it has moderate cell membrane permeability [1].
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| References | |
| Additional Infomation |
1. LDH-IN-1 is a pyrazole-based small molecule inhibitor designed and optimized to target LDH-A, a key enzyme in the glycolysis pathway that is overexpressed in various hypoxic tumors to maintain energy metabolism and acidify the tumor microenvironment [1]. 2. The mechanism of action of LDH-IN-1 is to competitively inhibit LDH-A by binding to the active site of LDH-A, blocking the conversion of pyruvate to lactate, leading to the accumulation of pyruvate in cells, reduced NAD+ regeneration, and thus disrupting the glycolytic energy supply of hypoxic cancer cells, ultimately inhibiting cell proliferation and colony formation [1]. 3. LDH-IN-1 is a lead compound of an antitumor drug targeting glycolysis, whose structure is optimized based on the original pyrazole compound to improve enzyme inhibitory activity, selectivity and ADME properties [1].
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| Molecular Formula |
C30H26N4O4S2
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| Molecular Weight |
570.681844234467
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| Exact Mass |
570.139
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| CAS # |
1964515-43-2
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| PubChem CID |
131955127
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| Appearance |
White to off-white solid powder
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| LogP |
6.3
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
40
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| Complexity |
975
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S1C=C(C(O)=O)N=C1N1C(CC2CC2)=C(CC2=CC=C(S(N)(=O)=O)C=C2)C(C2C=CC=C(C3=CC=CC=C3)C=2)=N1
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| InChi Key |
ALJORCZKMBZYCR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C30H26N4O4S2/c31-40(37,38)24-13-11-19(12-14-24)15-25-27(16-20-9-10-20)34(30-32-26(18-39-30)29(35)36)33-28(25)23-8-4-7-22(17-23)21-5-2-1-3-6-21/h1-8,11-14,17-18,20H,9-10,15-16H2,(H,35,36)(H2,31,37,38)
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| Chemical Name |
2-[5-(cyclopropylmethyl)-3-(3-phenylphenyl)-4-[(4-sulfamoylphenyl)methyl]pyrazol-1-yl]-1,3-thiazole-4-carboxylic acid
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| Synonyms |
LDH-IN 1LDH IN-1LDH-IN-1
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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 : ~52 mg/mL (~91.12 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.17 mg/mL (3.80 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 21.7 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: ≥ 2.17 mg/mL (3.80 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 21.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7523 mL | 8.7615 mL | 17.5230 mL | |
| 5 mM | 0.3505 mL | 1.7523 mL | 3.5046 mL | |
| 10 mM | 0.1752 mL | 0.8761 mL | 1.7523 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.
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