Lactate dehydrogenase (LDH)

NCATS-SM1441 is an LDH inhibitor with improved cellular potency, in vitro ADME, and in vivo PK properties.

NCATS-SM1441 demonstrated improved in vivo target engagement, PK properties and LDH activity in vivo.

LDH biochemical assays[1]
Briefly, 3 μL of recombinant human lactate dehydrogenase 5 (LDHA, #A38558H, Meridian Life Science, Inc., Memphis, TN) in LDH assay buffer (200 mM Tris HCl pH 7.4, 100 μM EDTA and 0.01% Tween-20) was added to a black solid-bottom 1536-well assay plate (Greiner Bio-One) using a BioRAPTR FRD dispenser (Beckman Coulter, Brea, CA). A 1536-well pintool dispenser (Wako Automation, San Diego, CA) was used to transfer 23 nL of DMSO-solubilized compound (both library and vehicle controls) to each 1536-well assay plate. Following compound transfer, 1 μL of substrate solution containing NADH and sodium pyruvate (Sigma-Aldrich, St. Louis, MO) in LDH assay buffer was dispensed via BioRAPTR FRD to initiate the reaction. Final concentrations in the 4 μL reaction volume were 2 nM LDHA enzyme, 0.06 mM NADH and 0.2 mM sodium pyruvate. Following a 5 minute incubation period at room temperature, 1 μL of detection reagent (Clostridium kluyveri diaphorase (Sigma-Aldrich) and resazurin sodium salt (Sigma-Aldrich) in LDH assay buffer) was added to a total volume of 5 μL. Final concentrations of detection reagents were 0.133 mg/mL diaphorase and 37 μM resazurin. Plates were immediately transferred to a ViewLux microplate imager (PerkinElmer, Waltham, MA), and any resulting resorufin fluorescence was measured (ex540, em590 nm) at 0 and 20minutes. Fluorescence was normalized using enzyme-free and DMSO-treated control wells on each plate. Human lactate dehydrogenase 1 (LDHB, #A38155H, Meridian Life Science, Inc., Memphis, TN) was assayed as described above for LDHA.

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MDH biochemical assay
Briefly, 3 L of MDH solution (containing 13.33 IU/mL malate dehydrogenase from porcine heart, 0.2 mM NAD, 0.067mg/mL diaphorase and 0.067 mM resazurin in MDH assay buffer (50 mM Tris pH 8.0, 5 mM MgCl2, 0.01% Brij 3)) was added to a black solid bottom 1536-well assay plate (Greiner Bio-One) using a BioRAPTR FRD dispenser (Beckman Coulter, Brea, CA). A 1536-well pintool dispenser outfitted with 20 nL pins (Wako Automation, San Diego, CA) was used to transfer 23 nL of DMSO-solubilized compound (Cherrypick plates) to each 1536-well assay plate. Following compound transfer, plates were incubated in room temperature for 10 min. 1 μL of substrate solution containing malic acid (160uM) was dispensed via BioRAPTR FRD to initiate the reaction. Plates were immediately transferred to a ViewLux microplate imager (PerkinElmer, Waltham, MA), and any resulting resorufin fluorescence was measured (ex540, em590 nm) at 0 and 5 min. Well fluorescence was normalized using enzyme-free and DMSO-treated control wells on each plate, and changes in fluorescence (ΔRFU) were calculated using the difference in fluorescent signal for each well at 5 versus 0 minutes.

Split Nano Luciferase Cellular Thermal Shift Assay. [1]
The SplitLuc CETSA was performed in 1536-well plates as previously described. Briefly, HEK293T cells were transiently transfected with plasmid encoding a CMV-driven LDHA open-reading frame with a carboxy-terminal 86b fusion tag (GS[HiBiT]GS). Cells were transfected in a T175 flask for 24 h using 2.3 × 107 cells, 52.5 μg of plasmid DNA, and 105 μL of Lipofectamine 2000. Cells were lifted, resuspended at 5 × 105 cells/mL (DPBS with CaCl2 and MgCl2 plus 1 g/L glucose), and reseeded into 1536-well cyclic olefin white plates (Aurora, cyclic olefin polymer, cat# EWB041000A) using a Multidrop Combi at 2500 cells per well (5 μL volume). Compounds (23 nL) were added to cells using a pin tool (Wako Automation) and incubated for 1 h at 37 °C. Plates were heated to 63.5 °C for 7.5 min using a custom-machined copper heat block fitted with an internal type-T thermocouple and controlled by a Watlow temperature controller. The plates were removed from the heat block and cooled to room temperature. One microliter of 6% NP40 was added per well and plates were incubated for 30 min to allow cell lysis, followed by addition of 3 μL of substrate containing 11S (final concentration 100 nM) and furimazine (final concentration 0.5×; from Promega 50× stock). The plates were centrifuged and analyzed for luminescence intensity using a ViewLux reader equipped with clear filters. Luminescence values were normalized to an unheated control sample.

Two million A673 cells were injected SQ into each female athymic nude mouse from Taconic (CrTac:NCr-Foxn1nu). The mice were divided into four treatment groups of 16 mice each. Each group received a single IV injection of 50 mg/kg of an LDHA inhibitor. Four mice from each group were sacrificed at 1, 3, 6, and 24 h after dosing. The inhibitors were dissolved in a standard PBS-based vehicle. At the time of sacrifice, frozen samples of tumor and plasma were collected and compound levels were determined through LC–MS/MS measurement against standard curves of each compound. Frozen tumor samples were also collected for LDHA activity measurements as follows. Briefly, 10–50 mg of frozen tumors was pulverized in liquid nitrogen, followed by the addition of 10 volumes of PBS at pH 7.4 containing 0.1% Triton ×−100 and incubated on ice for 1 h. The samples were clarified by centrifugation and cleared tumor lysates (10–20 μg) were measured by UV–vis spectrometry for LDH activity in LDH assay buffer containing 10 mM sodium pyruvate (Sigma-Aldrich, St. Louis, MO). The reactions were initiated by the addition of 15 mM NADH (final concentration 0.3 mM) and monitored for the oxidation of NADH at 340 nM at 37 °C.

[1]. Pyrazole-based lactate dehydrogenase inhibitors with optimized cell activity and pharmacokinetic properties. Journal of medicinal chemistry, 2020, 63(19): 10984-11011.

C31H25FN4O4S3

632.75

1964517-04-1

139465347

Typically exists as solid at room temperature

2

10

10

43

1190

0

CC1=CC=C(S1)C#CC2=CC(=CC=C2)C3=NN(C(=C3CC4=CC(=C(C=C4)S(=O)(=O)N)F)CC5CC5)C6=NC(=CS6)C(=O)O

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)