Phosphoglycerate dehydrogenase/PHGDH
3-phosphoglycerate dehydrogenase (PHGDH) [1]
IC50 = 33 ± 12 μM (in vitro enzymatic assay) [1]
Ki = 50 ± 20 μM (with respect to substrate 3-PG) [1]
Ki = 50 ± 3 μM (with respect to cofactor NAD+) [1]

CBR-5884 (15 or 30 μM; 3-5 days) specifically prevents breast cancer and melanoma cell lines that have a strong propensity for serine synthesis from proliferating.

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CBR-5884 inhibits de novo serine synthesis in cancer cells. In an acute (3-hour) treatment assay using uniformly labeled 13C6-glucose tracing in Carney cells, CBR-5884 (30 μM) decreased the fraction of newly synthesized (M+3) serine by approximately 30%. This reduction was specific to serine and its precursor 3-PG, with no significant effects on glycolytic end products pyruvate and lactate. [1]
CBR-5884 selectively inhibits the proliferation of breast cancer and melanoma cell lines with high intrinsic serine biosynthetic activity (high PHGDH expression). For example, in serine-replete media, 30 μM CBR-5884 inhibited the growth of high-PHGDH lines (MDA-MB-468, MDA-MB-436, HCC70, Hs578T) by 35% to 60%, while having no effect on low-PHGDH lines (MDA-MB-231, MCF10A). The growth inhibitory effect was significantly enhanced when cells were cultured in serine-deplete media. [1]
Mechanistically, CBR-5884 is a noncompetitive inhibitor of PHGDH with respect to both its substrates (3-PG and NAD+). It exhibits time-dependent inhibition, with potency increasing upon pre-incubation with the enzyme (IC50 decreased to ~7 μM after 4h pre-incubation). The compound disrupts the native tetrameric state of PHGDH, shifting the equilibrium towards dimers. [1]

In vivo studies revealed that CBR-5884 significantly delayed tumor growth, with histological analysis indicating the safety profile of CBR-5884. Finally, the patient-derived organoid (PDO) models were utilized to explore the preclinical efficacy of CBR-5884 against EOC cells, and the results unveiled that CBR-5884 impeded proliferation and downregulated the expression of ITGB4 in EOC PDO models. [2]

Screening for Small Molecule Inhibitors of PHGDH. [1]
An in vitro enzymatic assay for PHGDH activity amenable to high-throughput screening (HTS) was developed by coupling the production of NADH, upon 3-PG oxidation, to the reduction of resazurin to resorufin using diaphorase as the coupling enzyme. Thus, resorufin fluorescence served as a proxy for PHGDH activity. The assay was miniaturized to a 1,536-well format with a Z-factor of >0.75, indicating a high quality assay. A library of 800,000 small molecules was screened in single point format at 13 μM. Setting a threshold Z score of −3, corresponding to at least 50% PHGDH inhibition, gave a 0.5% hit rate, yielding 3,906 hits. Putative hits were reassayed in triplicate and counter-screened against diaphorase to rule out false positives targeting diaphorase. The counter screen eliminated 3,498 compounds, giving 408 PHGDH inhibitors.
A triaging strategy based on hit potency and selectivity was designed. We reasoned that inhibitors specific to PHGDH would minimize general cellular toxicity compared with compounds that hit a variety of dehydrogenases. Thus, half maximal inhibitory concentrations (IC50) were determined for a panel of NAD(P)+-dependent dehydrogenases that included PHGDH, isocitrate dehydrogenase (IDH1), malate dehydrogenase (MDH1), and 3α-hydroxysteroid dehydrogenase (3α-HSD). Compounds at least fourfold more selective for PHGDH were progressed for further analysis. Based on this triaging, seven of the most potent PHGDH inhibitors were selected as lead compounds for evaluation in cell-based assays; selected structures are shown in Fig. 1E. A number of these compounds are likely to target sulfhydryl groups and may therefore react with a PHGDH cysteine residue. For example, both CBR-5807 and CBR-6936 contain sulfhydryl-reactive disulfide centers. Interestingly, CBR-5807 (Disulfiram) is an approved drug dosed in humans to treat alcoholism and known to inhibit aldehyde dehydrogenase by reacting with sulfhydryl groups.[1]

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A high-throughput screening (HTS) assay was developed to identify PHGDH inhibitors. The assay couples the NADH produced by PHGDH-catalyzed oxidation of 3-phosphoglycerate (3-PG) to the reduction of resazurin to fluorescent resorufin using diaphorase. Resorufin fluorescence serves as a readout for PHGDH activity. Phosphoserine aminotransferase (PSAT1) is included in the reaction to prevent product feedback inhibition. The assay was miniaturized to a 1,536-well format. [1]
Mechanistic studies to determine the inhibition modality and constants (Ki) were performed. Initial reaction rates (V) were measured at varying concentrations of one substrate (3-PG or NAD+) while holding the other constant, in the presence of several fixed concentrations of CBR-5884. Data were fitted to a noncompetitive inhibition model to derive Vmax, Km, and Ki values. [1]
Time-dependent inhibition was assessed by pre-incubating CBR-5884 with PHGDH for different durations (0.5, 1, 4 hours) before initiating the enzymatic reaction with substrates. IC50 values were determined for each pre-incubation time. [1]
The effect on PHGDH oligomerization was evaluated by cross-linking. PHGDH protein was incubated with CBR-5884 or vehicle, then treated with a cross-linking reagent. The reaction mixtures were analyzed by SDS-PAGE to visualize the oligomeric states (monomer, dimer, tetramer) of PHGDH. [1]

Cell Proliferation Assay[1]
Cell Types: Breast cancer and melanoma cell lines
Tested Concentrations: 15 or 30 μM
Incubation Duration: 3-5 days
Experimental Results: Inhibited proliferation of melanoma and breast cancer cell lines.

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An acute inhibitor treatment assay combined with stable isotope tracing was used to measure the effect on de novo serine synthesis. Cells were pre-treated with CBR-5884 or vehicle for 1 hour, then the medium was replaced with one containing 13C6-glucose (tracer) and the drug for an additional 2 hours. Cells were quickly washed and flash-frozen. Polar metabolites were extracted and analyzed by gas chromatography-mass spectrometry (GCMS) to determine the isotopic enrichment (M+3 fraction) of serine and other metabolites. [1]
Proliferation assays were conducted to evaluate the growth inhibitory effects. Cells were plated in serine-containing media. The next day, media were replaced with fresh serine-replete or serine-deplete media containing CBR-5884 (typically 15 and 30 μM) or vehicle. Cells were grown for 3-5 days with daily media/drug changes, and relative cell numbers were quantified at the end of the assay. [1]
An acute toxicity assay was performed to rule out general cytotoxicity during short-term treatment for metabolic tracing. Cells were treated with CBR-5884 (1 to 40 μM) for 3 hours. The drug-containing media were then replaced with drug-free media, and cell viability was assessed using luminescence- or fluorescence-based viability assays. [1]

The study utilized 12 female BALB/c nude mice aged 4–6 weeks, procured from the Experimental Animal Center of Southern Medical University. These mice were housed in autoclaved, ventilated cages and provided with autoclaved water. All mice used were bred in the specific pathogen free laboratory. The mice received a subcutaneous injection of 1 × 106 ID8 cells in the right armpit. When the tumors reached an average volume of 65 mm3, the mice were randomly divided into two groups using the random number method: (1) an experimental group, wherein the mice received intragastric administration of CBR-5884 (70 mg/kg, qd; n = 6); and (2) a control group, wherein the mice received intragastric administration of an equal volume of vehicle (corn oil) (n = 6). CBR-5884 treatment lasted for 12 consecutive days, with continuous drug administration. Tumors were resected when the average tumor volume in the control group reached 400 mm3. Tumor growth was tracked using caliper measurements, and tumor volume was calculated using the formula: length × width2/2. Subsequently, images of mice and tumors were captured, and tumors were weighed. Liver, spleen, kidney, and tumors were subjected to histological analysis after hematoxylin–eosin (H&E) staining. The maximal tumor size/burden permitted by our institutional review board is 10% of body weight and mean tumor diameter = or > 15 mm in adult mice (~ 25 g). The maximal tumor size/burden permitted by our institutional review board was not exceeded.[2]

CBR-5884 is an ethyl ester prodrug. Its corresponding carboxylic acid derivatives retain similar in vitro enzyme inhibitory activity and selectivity for PHGDH, suggesting that intracellular hydrolysis of the esterase may not be essential for its activity. However, the reduced cellular activity of the carboxylic acid derivatives may be due to their poor membrane permeability. [1] CBR-5884 is unstable in mouse plasma. [1]

In acute (3-hour) treatment experiments, CBR-5884 at concentrations up to 40 μM did not cause significant cytotoxicity in Carney cells, as confirmed by two independent cell viability assays. [1] In long-term proliferation experiments (3–5 days), 30 μM CBR-5884 significantly inhibited the growth of PHGDH-dependent cancer cell lines, indicating that CBR-5884 has cytotoxic or inhibitory effects under serine synthesis-dependent conditions. [1]

[1]. Identification of a small molecule inhibitor of 3-phosphoglycerate dehydrogenase to target serine biosynthesis in cancers Proc Natl Acad Sci U S A, 2016 Feb 16, 113(7):1778-83.

Reprogramming of the serine biosynthesis pathway (SSP) is closely associated with the progression of epithelial ovarian cancer (EOC). CBR-5884 is a selective small molecule inhibitor targeting phosphoglycerate dehydrogenase (PHGDH), effectively inhibiting de novo serine synthesis in cancer cells. This study aimed to evaluate the inhibitory effect of CBR-5884 on EOC cells and elucidate its specific mechanism, thereby proposing a novel strategy for treating EOC. This study used RNA sequencing and a serine detection kit to assess the inhibition of serine biosynthesis after CBR-5884 treatment. The results showed that CBR-5884 effectively downregulated serine biosynthesis in EOC cells, especially in cells with high PHGDH expression. In vitro studies demonstrated that CBR-5884 has significant antitumor effects, inhibiting the migration and invasion of ovarian cancer cells by downregulating the ITGB4/ERK/EMT signaling pathway. In addition, CBR-5884 can reduce the stemness of ovarian cancer cells and improve their sensitivity to chemotherapy. In vivo studies have shown that CBR-5884 can significantly delay tumor growth, and histological analysis has also confirmed the safety of CBR-5884. Finally, the preclinical efficacy of CBR-5884 against ovarian cancer cells was explored using a patient-derived organoid (PDO) model. The results showed that CBR-5884 can inhibit the proliferation of ovarian cancer cells and downregulate the expression of ITGB4. Our results support the anti-cancer properties of CBR-5884 in ovarian cancer cells with high PHGDH expression, which are manifested by inhibiting cell proliferation, migration and invasion, while enhancing chemosensitivity, indicating that CBR-5884 is expected to become an effective strategy for the treatment of ovarian cancer [2].

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CBR-5884 is a tool compound discovered from a high-throughput screening of 800,000 compounds. It represents a proof of concept for the pharmacological inhibition of PHGDH. [1]
Due to its plasma instability and the need to improve cellular efficacy, this compound is more suitable as a starting point for medicinal chemistry optimization rather than a direct drug candidate. [1]
Its mechanism of action involves non-competitive, time-dependent inhibition of PHGDH and disruption of its active tetrameric form. [1] PHGDH is a metabolic enzyme that is overexpressed in certain cancers (e.g., breast cancer, melanoma, non-small cell lung cancer) and is essential for the de novo synthesis of serine. Inhibition of PHGDH is a potential therapeutic strategy for cancers that depend on this pathway. [1] The selectivity of CBR-5884 has been evaluated against a range of other NAD(P)+ dependent dehydrogenases (e.g., LDH, MDH1, IDH1, 3α-HSD), and the results show that its selectivity for PHGDH is at least 4 times that of the other enzymes. [1]

C14H12N2O4S2

336.386

336.023

C, 49.99; H, 3.60; N, 8.33; O, 19.02; S, 19.06

681159-27-3

4674993

Typically exists as light yellow to yellow solids at room temperature

1.4±0.1 g/cm3

378.1±42.0 °C at 760 mmHg

182.5±27.9 °C

0.0±0.9 mmHg at 25°C

1.628

4.15

1

7

6

22

479

0

O=C(C1=C(C)C(SC#N)=C(NC(C2=CC=CO2)=O)S1)OCC

QBVIRPJBDIZKBC-UHFFFAOYSA-N

InChI=1S/C14H12N2O4S2/c1-3-19-14(18)11-8(2)10(21-7-15)13(22-11)16-12(17)9-5-4-6-20-9/h4-6H,3H2,1-2H3,(H,16,17)

ethyl 5-(furan-2-carboxamido)-3-methyl-4-thiocyanatothiophene-2-carboxylate

CBR-5884; CBR 5884; CBR5884.

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 (~148.64 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.18 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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