In H1299 and MDA-MB231 cells, inhibition of GDH1 activity by R162 treatment led to decreased intracellular fumarate levels, decreased GPx activity, increased ROS levels, and decreased cell proliferation. These effects were verified by methyl-α-KG treatment and anti-oxidize NAC. R162 inhibits the potential for tumor growth and cell proliferation in human dyes [1].

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Enzyme Inhibition: R162 is a potent inhibitor of GDH1 enzyme activity. It was identified as a purpurin analog with improved cell permeability due to its allyl group. In vitro GDH activity assays using purified GDH1 protein showed that R162 effectively inhibits GDH1 with Ki of 1.9 ± 0.26 μM [1]
.
- Binding Mechanism: Thermal melt shift assay demonstrated that incubating GDH1 with increasing concentrations of R162 raised the melting temperature (Tm) in a dose-dependent manner, indicating direct binding. Lineweaver-Burk plot analysis showed that R162 acts as a mixed model inhibitor of GDH1 [1]
.
- Cellular Effects - GDH Activity: Treatment of cancer cells (H1299 and MDA-MB231) with R162 resulted in significantly decreased mitochondrial GDH activity and elevated ROS levels, similar to effects observed with GDH1 knockdown [1]
.
- Cellular Effects - Metabolites: R162 treatment decreased intracellular fumarate levels and attenuated GPx (glutathione peroxidase) activity in cancer cells [1]
.
- Cellular Effects - ROS: R162 treatment increased reactive oxygen species (ROS) levels in cancer cells, which could be significantly rescued by treatment with methyl-α-KG (cell-permeable GDH1 product) or the antioxidant N-acetylcysteine (NAC) [1]
.
- Anti-proliferative Effects: R162 dramatically attenuated cell viability in a panel of human cancer cell lines including lung cancer (H1299, A549), breast cancer (MDA-MB231, SKBR3), and leukemia cells (HEL, KG1a, Molm14, K562). In contrast, R162 did not affect cell viability of non-malignant proliferating human cells including HaCaT (keratinocytes), MRC-5 (fetal lung fibroblasts), and HFF (foreskin fibroblasts) [1]
.
- Primary Patient Cells: R162 treatment decreased cell viability of primary leukemia cells from patients with myeloid leukemia (AML and CML), but did not affect cell viability of peripheral blood mononucleocytes from healthy human donors [1]
.
- Rescue Experiments: The effects of R162 on fumarate levels, GPx activity, ROS levels, and cell proliferation were significantly rescued by treatment with methyl-α-KG (1 mM) or NAC (3 mM), confirming that these effects are mediated through GDH1 inhibition [1]
.

R162 (30 mg/kg/d, i.p.) did not modify the constructed hematological parameters in xenograft models or result in significant histopathological changes between the vehicle- and R162-treated groups. R162 (20 mg/kg/d) successfully inhibited GDH1 activity in xenografted naked tumors and dramatically decreased tumor development and bulk in mice as compared to control mice [1].

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Toxicity Study: For initial in vivo toxicity studies, R162 was administered to mice at 30 mg/kg/day for 30 days by intraperitoneal injection. Chronic R162 treatment did not result in significant histopathological changes in tissues compared to vehicle-treated groups, nor did it alter complete blood counts or hematopoietic properties, suggesting minimal toxicity in vivo [1]
.
- Xenograft Tumor Model: H1299 lung cancer cells were xenografted into nude mice. One day after injection, mice were divided into two groups (n=8/group) and treated with either R162 (20 mg/kg/day) or control DMSO for 35 days by intraperitoneal injection. R162 treatment resulted in significantly decreased tumor growth and tumor masses compared to control mice [1]
.
- Pharmacodynamic Effect in Tumors: R162 effectively inhibited GDH1 activity in resected tumors from xenograft nude mice, confirming target engagement in vivo [1]
.

GDH Activity Assay: GDH enzyme activity was measured by monitoring the oxidation of NADPH as a decrease in absorbance at 340 nm. The reaction mixture contained 50 mM triethanolamine pH 8.0, 100 mM ammonium acetate, 100 μM NADPH, 2.6 mM EDTA, and 20 μg of total cell lysates or 100 ng of purified GDH1. The reaction was initiated by adding α-KG [1]
.
- Thermal Shift Assay: Thermal shift assay was performed using the Protein Thermal Shift Dye Kit. 10 μM of purified GDH was incubated with different concentrations of R162. Fluorescence was recorded using Real-Time PCR Systems and data were analyzed using Protein Thermal Shift Software v1.0 [1]
.
- Enzyme Kinetics: 2 μM purified GDH1 was incubated with different concentrations of R162 in 50 mM Tris-Cl buffer (pH 7.5). Intrinsic tryptophan fluorescence (Ex: 280 nm/Em: 350 nm) was measured for binding assay. Nonlinear regression analysis was performed using Prism 6 to calculate dissociation constant (Kd). Inhibition constant (Ki) was determined by GDH activity assay with different concentrations of substrate α-KG [1]
.
- GPx Activity Assay: Glutathione peroxidase enzyme activity was determined using commercially available kits from Biovision according to the manufacturer's instructions [1]
.

Cell Culture: H1299, A549, HEL, KG1a, Molm14, K562 cells were cultured in RPMI 1640 with 10% FBS. 293T, MDA-MB231, SKBR3, HaCaT, HFF, and MRC-5 cells were cultured in DMEM with 10% FBS [1]
.
- Cell Proliferation/Viability Assay: 5×10⁴ adherent cells or 1×10⁵ leukemia cells were seeded in 6-well plates. Cells were treated with different concentrations of R162, and cell numbers were determined by trypan blue exclusion using TC10 automated cell counter [1]
.
- Intracellular ROS Measurement: Total intracellular ROS was determined by staining cells with carboxy-H₂DCFDA (Invitrogen). 2×10⁵ cells were incubated with 10 μM carboxy-H₂DCFDA for 30 minutes at 37°C, washed, and analyzed by flow cytometry [1]
.
- Mitochondrial ROS Measurement: Mitochondrial ROS level was determined using MitoPY1, a specific mitochondrial H₂O₂ probe. 2×10⁵ cells were incubated with 10 μM MitoPY1 for 30 minutes at 37°C and analyzed by flow cytometry [1]
.
- Intracellular Metabolite Measurements: Intracellular levels of fumarate were determined using commercial kits (Biovision). 2×10⁶ cells were homogenized in PBS, proteins removed using 10kD Amicon Ultra Centrifugal Filters, and the flow-through containing metabolites was used for measurement [1]
.
- Primary Patient Cell Assay: Primary leukemia cells from patients with myeloid leukemia and peripheral blood mononucleocytes from healthy donors were treated with R162, and cell viability was assessed [1]
.

Toxicity Study:** R162 was administered to mice at 30 mg/kg/day for 30 days by intraperitoneal injection. Vehicle control groups received 50% DMSO in PBS. After 30 days, histopathological analysis of tissues and complete blood counts were performed to assess toxicity [1]
.
- **Xenograft Study:** Nude mice (athymic nu/nu, female, 4-6 weeks old) were subcutaneously injected with 1×10⁷ H1299 cells. One day after injection, mice were divided into two groups (n=8/group) and treated with either R162 (20 mg/kg/day) or control DMSO for 35 days by daily intraperitoneal injection. Tumor growth was recorded by measurement of two perpendicular diameters and tumor size was calculated using the formula 4π/3 × (width/2)² × (length/2). Tumors were harvested and weighed at the experimental endpoint. Tumor proliferation was determined by Ki-67 IHC staining [1]
.
- **GDH1 Activity in Tumors:** Resected tumor samples were analyzed for GDH1 protein expression by Western blotting and GDH enzyme activity using the GDH activity assay [1]
.

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Toxicity Study: R162 was administered to mice at 30 mg/kg/day for 30 days by intraperitoneal injection. Vehicle control groups received 50% DMSO in PBS. After 30 days, histopathological analysis of tissues and complete blood counts were performed to assess toxicity [1]
.
- Xenograft Study: Nude mice (athymic nu/nu, female, 4-6 weeks old) were subcutaneously injected with 1×10⁷ H1299 cells. One day after injection, mice were divided into two groups (n=8/group) and treated with either R162 (20 mg/kg/day) or control DMSO for 35 days by daily intraperitoneal injection. Tumor growth was recorded by measurement of two perpendicular diameters and tumor size was calculated using the formula 4π/3 × (width/2)² × (length/2). Tumors were harvested and weighed at the experimental endpoint. Tumor proliferation was determined by Ki-67 IHC staining [1]
.
- GDH1 Activity in Tumors: Resected tumor samples were analyzed for GDH1 protein expression by Western blotting and GDH enzyme activity using the GDH activity assay [1]
.

Cell Permeability: R162 was identified as a purpurin analog with improved cell permeability due to its allyl group, making it more suitable for cellular and in vivo studies compared to the parent compound purpurin [1]
.
- Detailed pharmacokinetic parameters (half-life, bioavailability, etc.) were not described in the provided text [1]
.

In Vitro Selectivity: R162 did not affect cell viability of non-malignant proliferating human cells including HaCaT (keratinocytes), MRC-5 (fetal lung fibroblasts), and HFF (foreskin fibroblasts), suggesting selectivity for cancer cells [1]
.
- Primary Cell Toxicity: R162 did not affect cell viability of peripheral blood mononucleocytes from healthy human donors, while effectively reducing viability of primary leukemia cells from patients [1]
.
- In Vivo Toxicity: Chronic R162 treatment (30 mg/kg/day for 30 days) did not result in significant histopathological changes in tissues, nor altered complete blood counts or hematopoietic properties, suggesting minimal toxicity in vivo [1]
.
- Therapeutic Window: The compound showed promising efficacy in inhibiting tumor growth at 20 mg/kg/day with no apparent toxicity at 30 mg/kg/day, suggesting a favorable therapeutic window [1]
.

[1]. Glutamate dehydrogenase 1 signals through antioxidant glutathione peroxidase 1 to regulate redox homeostasis and tumor growth. Cancer Cell. 2015 Feb 9;27(2):257-70.

2-Allyl-1-hydroxy-9,10-anthraquinone is a monohydroxyanthraquinone compound in which the hydrogen atoms at positions 1 and 2 of 9,10-anthraquinone are replaced by hydroxyl and allyl groups, respectively. It is an EC 1.4.1.3 {glutamate dehydrogenase [NAD(P)(+)]} inhibitor.

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Chemical Identity and Source: R162 is a purpurin analog identified from a library of 2,000 FDA-approved small molecule compounds. It was developed as a more cell-permeable derivative of purpurin by addition of an allyl group [1]
.
- Discovery Strategy: R162 was identified through a series of screening assays designed to identify GDH1-selective inhibitors. The initial lead compound purpurin showed dramatic inhibitory effects on GDH1 enzyme activity in vitro but was not cell permeable. Structure-activity relationship studies led to the identification of the purpurin analog R162 as a potent GDH1 inhibitor with improved cell permeability [1]
.
- Mechanism of Action: R162 directly binds to GDH1 and inhibits its enzyme activity. This leads to decreased intracellular α-KG and fumarate levels, resulting in reduced GPx activity, elevated ROS, and ultimately inhibited cancer cell proliferation and tumor growth. The effects are consistent with those observed in GDH1 knockdown cells [1]
.
- Selectivity Profile: Unlike EGCG (epigallocatechin gallate), a previously reported GDH1 inhibitor that also inhibits other NADPH-dependent enzymes such as 6-phosphogluconate dehydrogenase (6PGD) and fumarate hydratase (FH), R162 specifically inhibits GDH1 without affecting these other enzymes, demonstrating improved selectivity [1]
.
- Clinical Potential: R162 demonstrated promising efficacy in inhibiting cancer cell proliferation and tumor growth with minimal toxicity in vitro and in vivo. It also effectively reduced viability of primary leukemia cells from patients. These findings provide proof-of-principle that GDH1 is a promising therapeutic target and R162 is a potential lead compound for treating human cancers that rely heavily on glutamine metabolism [1]
.
- Dosing in Animal Studies: For toxicity studies: 30 mg/kg/day IP for 30 days. For efficacy studies: 20 mg/kg/day IP for 35 days [1]
.

C17H12O3

264.28

264.078

64302-87-0

4412951

Light yellow to yellow solid powder

1.3±0.1 g/cm3

459.0±34.0 °C at 760 mmHg

245.5±22.2 °C

0.0±1.2 mmHg at 25°C

1.659

4.99

1

3

2

20

428

0

C=CCC1=C(C2=C(C=C1)C(=O)C3=CC=CC=C3C2=O)O

IMUBGIOLZQTIGI-UHFFFAOYSA-N

InChI=1S/C17H12O3/c1-2-5-10-8-9-13-14(15(10)18)17(20)12-7-4-3-6-11(12)16(13)19/h2-4,6-9,18H,1,5H2

1-hydroxy-2-prop-2-enylanthracene-9,10-dione

R162 R-162 R 162

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 : ~10 mg/mL (~37.84 mM)
Ethanol : ~1 mg/mL (~3.78 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.)