PHGDH/3-phosphoglycerate dehydrogenase (IC50 = 2.5 µM)[1]

NCT-503, a PHGDH (3-phosphoglycerate dehydrogenase) inhibitor, prevents cells from synthesizing 3-phosphoglycerate in a serine manner (IC50=2.5 μM). NCT-503 exhibited negligible cross-reactivity among a panel of 168 GPCRs and was inactive against a panel of other dehydrogenases. A non-competitive mechanism of 3-phosphoglycerate (3-PG) and NAD+ co-substrate inhibition was found in NCT-503 competition assays. NCT-503 has an EC50 of 8–16 μM against PHGDH-supporting cell lines, an EC50 of 6–10 times higher against MDA-MB-231 cells, and no toxicity toward other PHGDH non-supporting cell lines [1].

NCT-503 possesses favorable qualities for metabolism, excretion, folding, and absorption (ADME). Following intraperitoneal spice administration, NCT-503 exhibits considerable distribution, good absorption, a half-life of 2.5 hours, and a Cmax of 20 μM in the belly. The growth and weight of PHGDH-dependent MDA-MB-468 xenografts are reduced by NCT-503 therapy, whereas the growth and weight of PHGDH-independent MDA-MB-231 xenografts are unaffected [1].

Enzyme assays[1]
PHGDH assay buffer contained 50 mM TEA pH 8.0, 10 mM MgCl2, 0.05% BSA, and 0.01% Tween-20. PHGDH enzyme buffer consisted of assay buffer with 20 nM PHGDH and 0.2 mg/mL diaphorase. PHGDH substrate buffer contained 0.3 mM NAD+, 1.25 mM glutamate, 0.1 mM 3-phosphoglycerate, 0.2 mM resazurin, 1 µM PSAT1, and 1 µM PSPH. qHTS was performed in 1536-well plates dispensed with a BioRAPTR FRD. Each well contained equal volumes of substrate buffer and assay buffer. Plates were read at 0 minutes and 20 minutes at room temperature with a ViewLux uHTS Microplate Imager. Follow-up assays were performed in black 384-well plates in 20 µL of enzyme buffer to which compounds were added in dose-response with an HP D300 digital dispenser, followed by addition of 20 µL of substrate buffer. Plates were incubated at room temperature (25 °C) and read at 0 and 20 minutes with a Spectramax M5 plate reader in fluorescence intensity mode with a λex=550 nm and λem=600 nm (emission cutoff=590 nm). Inhibition models were identified using DynaFit 33. The enzyme concentration was fixed at 10 nM and the Km, Ki, and Vmax were allowed to vary. Data were fit to competitive, uncompetitive, noncompetitive, and mixed inhibition models of complex equilibria. The Km values were not greatly affected, and by F-test, the probability of fit to a noncompetitive model was higher than all other models (91.5% for 3-PG and 84.6% for NAD+).
GAPDH substrate buffer contained 210 mM Tris pH 7.4, 2.5 mM NaH2PO4 pH 7.4, 2 mM DL-Glyceraldehyde 3-phosphate, 1.75 mM MgCl2, 0.01 mM NAD+, 0.11 mM resazurin, 0.2 mg/mL BSA, and 0.01% Tween-20. GAPDH enzyme buffer contained 50 mM Tris pH 7.4, 100 mM NaCl, 0.02 mM TCEP, 0.01 mM EDTA, 0.1 mg/mL BSA, 0.42 mg/mL diaphorase, and 2.5 nM GAPDH. The GAPDH, GPD1, and GPD1L assays were run in 384-well plates using the same protocol and readout as the PHGDH assay. GPD1 substrate buffer contained 114 mM Tris pH 7.4, 0.25 mM DHAP, 1.14 mM MgCl2, 0.011 mM NADH, 0.06 mg/mL BSA, and 0.011% Tween-20. GPD1 enzyme buffer contained 50 mM Tris pH 7.4, 100 mM NaCl, 0.08 mM TCEP, 0.4 mg/mL BSA, 0.03 µM EDTA, and 0.8 nM GPD1. 70 µL of substrate buffer were mixed with 10 µL enzyme buffer.
GPD1L substrate buffer contained 200 mM Tris pH 7.4, 0.05 mM sn-G3-P, 2 mM MgCl2, 0.04 mM NAD+, 0.11 mM resazurin, 0.1 mg/mL BSA, and 0.02% Tween-20. GPD1L enzyme buffer contained 50 mM Tris pH 7.4, 100 mM NaCl, 0.02 mM TCEP, 0.42 mg/mL diaphorase, 0.1 mg/mL BSA, 0.008 µM EDTA, and 16 nM GPD1L. 40 µL of substrate buffer were mixed with 40 µL of enzyme buffer.
The GAPDH and GPD1L assays were read using the same protocol as the PHGDH assay. The GPD1 assay was read using loss of NADH fluorescence (λex=340 nm and λem=460 nm).
PHGDH inhibition data was analyzed by calculating delta RFUs (increase in resorufin fluorescence between T=0 and T=30 mins) and normalizing to the delta RFU values of 0× and 1× PHGDH enzyme controls as 100% and 0% PHGDH inhibition, respectively. 32 wells of each 1536-well assay plate were dedicated to each of these 0× and 1× PHGDH controls. These normalized dose-response values were plotted in GraphPad Prism and fit using a Sigmoidal dose response (variable slope) equation. IC50s for each replicate were then averaged to determine average IC50s with standard deviations.

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Thermal shift assays[1]
Differential scanning fluorimetry assays were carried out in 20 mM TEA pH 8, 100 mM NaCl, 1× SYPRO Orange, and 2 µM PHGDH in a volume of 10 µL. Unfolding was monitored using a LightCycler 480 II real-time PCR instrument (λex=465 nm and λem=580 nm) over a linear 20 to 85°C gradient. Compounds were dispensed with an HP D300 digital dispenser and DMSO concentration was normalized to 1% in all samples. Plots of the first derivative of fluorescence vs. temperature were generated in LightCycler software.

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High-Throughput PAMPA Protocol[1]
The stirring double-sink PAMPA method was employed to determine the permeability of compounds via PAMPA passive diffusion. The artificial membrane contained a proprietary lipid mixture and dodecane, optimized to predict gastrointestinal tract (GIT) passive diffusion permeability. Membranes were immobilized on a plastic matrix of a 96 well “donor” filter plate placed above a 96 well “acceptor” plate. A pH 7.0 solution was used in both “donor” and “acceptor” wells. A 10 mM stock of compound in DMSO was diluted to 0.05 mM in aqueous buffer (pH 7.4), with a final concentration of DMSO was 0.5. During the 30-minute permeation period at room temperature, the test samples in the “donor” compartment were stirred. Compound concentrations in the “donor” and “acceptor” compartments were measured using an UV plate reader (Nano Quant, Infinite® 200 PRO, Tecan Inc., Männedorf, Switzerland). Permeability calculations were performed using Pion Inc. software and were expressed in the unit of 10−6cm/s. All samples were tested in duplicate, and control compounds (ranitidine, dexamethasone and verapamil) were included in each run.

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High-Throughput Kinetic Solubility Test Protocol[1]
Compounds were evaluated using a µSOL assay for kinetic solubility determination that adapts classical saturation shake-flask solubility method to a 96-well microtiter plate format with a co-solvent method using n-propanol. Test compounds were prepared in 10 mM DMSO solutions, and diluted with the co-solvent before being spiked to the aqueous solution (pH 7.4). The final drug concentration in the aqueous solution was 150 µM. Samples were incubated at room temperature for 6 hours and filtered to remove precipitate. The drug concentration in the filtrate was determined by direct UV measurement (λ: 250–498 nm). The reference drug concentration of 17 µM in a co-solvent was used for quantitation of unknown drug concentration in filtrate. Spectroscopically pure n-propanol used as the co-solvent suppressed precipitation in the reference solutions. Kinetic solubility (µg/mL) was calculated using µSOL Evolution software. All samples were tested in duplicate and control compounds (albendazole, phenazolpyridine and furosemide) were included in each run.

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High-Throughput Rat Liver Microsomal Stability Determination Protocol[1]
Microsomal stability of test articles was determined in a 96-well plates at a single time point. Compound concentrations were determined by LC/MS/MS and used to calculate in vitro half-life. All samples were tested in duplicate, and six standard controls were tested in each run: buspirone and propranolol (short half-life), loperamide and diclofenac (short to medium half-life), and carbamazepine and antipyrine (long half-life). The assay incubation system consisted of 0.5 mg/mLmicrosomal protein, 1.0 µM drug concentration, and NADPH regeneration system (containing 0.650 mM NADP+, 1.65 mM glucose 6-phosphate, 1.65 mM MgCl2, and 0.2 unit/mL G6PDH) in 100 mM phosphate buffer at pH 7.4. Incubation was carried out at 37 °C for 15 min and quenched by adding 555 µL of acetonitrile (~1:2 ratio) containing 0.28 µM albendazole (internal standard). After a 20 min centrifugation at 3000 rpm, 30 µL of the supernatant was transferred to an analysis plate and was diluted 5-fold using 1:2 v/v acetonitrile/water before the samples were analyzed by LC/MS−MS.

Cytotoxicity experiments[1]
Cells were seeded in white 96-well plates at a density of 2000 cells/well (MDA-MB-468, BT-20, MT-3) or 1000 cells/well (all other cell lines) and allowed to attach for 24 hours. Compounds were prepared in DMSO and dispensed using an HP D300 compound dispenser. Cell viability was assessed with Cell Titer-Glo at four days following treatment and luminescence measured with a SpectraMax M5 Plate Reader. Luminescence was normalized to an untreated control in identical medium. For rescue experiments, RPMI was supplemented with 40 µM adenosine, uridine, guanosine, cytidine, deoxyadenosine, thymidine, deoxyguanosine, and deoxycytidine and the medium was replaced daily.

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Oxygen consumption measurements[1]
Oxygen consumption of intact MDA-MB-468 cells was measured using an XF24 Extracellular Flux Analyzer. 85,000 cells were plated in RPMI media and exposed to compounds at 10 and 50 µM. Each measurement represents the average of six independent wells.

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Metabolite profiling: Steady-state and labeling experiments[1]
Cells were evenly seeded at 400,000 cells per well of a 6-well plate and allowed to attach for 24 hours. Prior to all labeling experiments, cells were pretreated with 10 µM compound or an equivalent volume of DMSO in RPMI for 1 hour. For steady-state metabolite concentrations, cells were washed with PBS prior to pretreatment and treatment in RPMI lacking serine and glycine. For labeling experiments, U-13C-glucose, U-13C-serine, or U-13C-glycine replaced the corresponding unlabeled RPMI component. Cells were washed in 4 °C 0.9% (w/v) NaCl in LCMS-grade water and extracted in 1 mL/well of 80:20 (v/v) methanol:water with 0.01 ng/mL Val-d8 and Phe-d8 as internal extraction standards. The extraction solvent was dried under nitrogen gas and metabolite samples were stored at −80 °C until analysis. Triplicate identically seeded and treated wells were trypsinzed and analyzed with a Multisizer Coulter Counter to obtain cell counts and total cell volumes for normalization.

Mouse orthotopic xenografts[1]
Female NOD.CB17-Prkdcscid/J mice, 6–8 weeks old, were obtained from Jackson Laboratories. All animals were provided with food ad libitum for the duration for the duration of the experiment. The animals were allocated randomly for induction with MDA-MB-231 or MDA-MB-468 tumors and tumor group was assigned blindly. 500,000 MDA-MB-231 or MDA-MB-468 cells were injected into the 4th mammary fat pad of each mouse. After 30 days, the tumors were palpable, and the mice were pooled by tumor type and divided randomly to two groups, which were assigned blindly to vehicle or NCT-503 treatment. Each arm contained 10 mice for a total of forty mice in all arms. NCT-503 was prepared in a vehicle of 5% ethanol, 35% PEG 300, and 60% of an aqueous 30% hydroxypropyl-β-cyclodextrin solution, and injected intraperitoneally once daily. Dose was adjusted to mouse weight, and the volume of injection did not exceed 150 µL. Caliper measurements were obtained twice weekly and tumor volumes were calculated with the modified ellipsoid formula: volume = 0.5 × width2 × length.
For quantitation of necrotic regions, fixed tumors were embedded and sections stained with hematoxylin and eosin. Slides were scanned with a Leica Aperio AT2 brightfield scanner. Tumor and necrotic cross-sectional regions were manually delineated and measured using Leica ImageScope software to calculate the percentage of necrosis.

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Glucose infusions in mice[1]
Chronic catheters were surgically implanted into the jugular veins of normal or tumor bearing animals 3–4 days prior to infusions. Animals were fasted for 6 hours (morning fast) and infusions were performed in free-moving, conscious animals at 1:00pm for all studies to minimize metabolic changes associated with circadian rhythm. Following administration of either vehicle or NCT-503 at 30 mg/kg, a constant infusion of U-13C-glucose (30 mg/kg/min) was administered for a 3-hour duration. Animals were terminally anesthetized with sodium pentobarbital and all tissues were fully harvested in less than 5 minutes to preserve the metabolic state. Tumors and adjacent lung tissue were carefully dissected and rapidly frozen using a BioSqueezer to ensure rapid quenching of metabolism throughout the tissue section. Tissues were stored at −80C and extracted with 80:20 (v/v) methanol:water in the same manner as cells prior to LCMS analysis.

[1]. Pacold ME, et al. A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate. Nat Chem Biol. 2016 Jun;12(6):452-8.

Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.[1]

C20H23F3N4S

408.4872

408.1596

C, 58.81; H, 5.68; F, 13.95; N, 13.72; S, 7.85

1916571-90-8

1916571-90-8

118796328

Typically exists as off-white to light yellow solids at room temperature

1.3±0.1 g/cm3

474.3±55.0 °C at 760 mmHg

240.6±31.5 °C

0.0±1.2 mmHg at 25°C

1.611

2.61

63.5Ų

S=C(N1CCN(CC2=CC=C(C(F)(F)F)C=C2)CC1)NC3=NC(C)=CC(C)=C3

PJNSZIQUFLWRLH-UHFFFAOYSA-N

InChI=1S/C20H23F3N4S/c1-14-11-15(2)24-18(12-14)25-19(28)27-9-7-26(8-10-27)13-16-3-5-17(6-4-16)20(21,22)23/h3-6,11-12H,7-10,13H2,1-2H3,(H,24,25,28)

N-(4,6-dimethylpyridin-2-yl)-4-(4-(trifluoromethyl)benzyl)piperazine-1-carbothioamide

NCT-503; NCT 503; NCT-503; 1916571-90-8; N-(4,6-dimethylpyridin-2-yl)-4-(4-(trifluoromethyl)benzyl)piperazine-1-carbothioamide; N-(4,6-dimethylpyridin-2-yl)-4-[[4-(trifluoromethyl)phenyl]methyl]piperazine-1-carbothioamide; NCGC00351958-05; N-(4,6-dimethylpyridin-2-yl)-4-{[4-(trifluoromethyl)phenyl]methyl}piperazine-1-carbothioamide; CHEMBL4099051; SCHEMBL17927258; NCT503.

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 : ~50 mg/mL (~122.41 mM)
Ethanol : ~13.33 mg/mL (~32.63 mM)

Solubility in Formulation 1: 2.5 mg/mL (6.12 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.12 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 10 mg/mL (24.48 mM) in 50% PEG300 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.

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Solubility in Formulation 4: 2 mg/mL (4.90 mM) in 0.5% methylcellulose 0.2% Tween 80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)