Protein disulfide isomerase (PDI) – IC₅₀ = 0.17 ± 0.01 μM in insulin turbidity assay [1]

3-Mmethyltoxoflavin has an IC50 of 170 nM, making it a strong inhibitor of protein disulfide isomerase (PDI). 3. Human glioblastoma cell lines are susceptible to the toxicity of methyltoxin. The most cytotoxic PDI inhibitor, according to the screening results, is 3-methyltoxin. The induction of Nrf2 antioxidant response, endoplasmic reticulum stress response, and autophagy was demonstrated by 3-methyltoxoflavin using bru-seq (bromouridine labeling and sequencing) of nascent RNA. More precisely, 3-Mmethyltoxoflavin suppresses PDI target genes like TXNIP and EGR1 while upregulating heme oxygenase 1 and SLC7A11 transcription and protein expression. Remarkably, a combination of autophagy and ferroptosis rather than apoptosis or necrosis drives the process of 3-methyltoxin-induced cell death [1].

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35G8 (1,3,6-trimethylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione) inhibited recombinant human PDI activity with an IC₅₀ of 0.17 ± 0.01 μM in the insulin turbidity assay. [1]

In a thermal shift assay, 35G8 destabilized PDI in a dose-dependent manner: melting temperature (Tₘ) shifts of -1.43°C at 1 μM, -2.94°C at 10 μM, and -3.64°C at 100 μM, indicating binding to PDI at a unique site. [1]

In the cellular thermal shift assay (CETSA), 35G8 also destabilized PDI in U87MG cells, with little effect on the related molecular chaperone GRP78, but appeared to stabilize GSTO1. [1]

In the drug affinity responsive target stability (DARTS) assay using U87MG cell lysates, 35G8 (100 μM) protected PDI from pronase degradation, similar to PACMA31. [1]

35G8 induced reactive oxygen species (ROS) formation in U87MG cells. At 5 μM, significant ROS induction was observed as early as 4 hours post-treatment, and at 24 hours, 5 μM 35G8 achieved maximal ROS induction comparable to 100 μM hydrogen peroxide. [1]

35G8 demonstrated potent cytotoxicity in a panel of human glioblastoma cell lines: IC₅₀ values were 1.1 ± 0.2 μM in U87MG, and under 10 μM in U118MG, A172 and NU04 cells. [1]

N-acetyl-cysteine (NAC) did not significantly affect the cytotoxicity of 35G8 in U87MG cells. [1]

Pretreatment with the caspase inhibitor Z-VAD-FMK (to block apoptosis) or necrostatin-1 (to block necroptosis) did not protect U87MG cells from 35G8-induced cell death. [1]

In the presence of the iron chelator deferoxamine (DFO), the IC₅₀ of 35G8 in U87MG cells increased from 2.2 ± 0.7 μM (without DFO) to 5.8 ± 1.0 μM (with DFO), indicating that ferroptosis is involved. [1]

35G8 (2 μM, 24 h treatment) upregulated protein expression of heme oxygenase 1 (HMOX1), SLC7A11, GRP78, and DDIT3 (CHOP) in U87MG cells, and increased cleaved LC3B expression, while ATG3, ATG5, ATG7 and beclin 1 levels did not change. [1]

Bromouridine labeling and sequencing (Bru-seq) of nascent RNA in U87MG cells treated with 1 μM 35G8 for 4 hours showed that 498 genes were upregulated ≥2-fold and 238 genes were downregulated ≥2-fold. Upregulated genes included HMOX1 (19-fold), SLC7A11 (63-fold), AKR1C1 (59-fold), CHAC1 (46-fold), TRIB3 (23-fold), TMEM74 (28-fold), and IRS2 (12-fold). Downregulated genes included TXNIP (-7.40-fold), EGR1 (-5.65-fold), and ITGA3 (-3.89-fold). [1]

PDI activity was measured using an insulin turbidity assay based on PDI-catalyzed reduction of insulin. Recombinant human PDI protein (0.4 μM) was pre-incubated with test compounds at 37°C for 1 hour in sodium phosphate buffer (100 mM sodium phosphate, 2 mM EDTA, 8 μM DTT, pH 7.0). Then a mixture of sodium phosphate buffer, DTT (500 μM), and bovine insulin (130 μM) was added. The reduction reaction was catalyzed by PDI at room temperature, and the resulting aggregation of reduced insulin B chains was measured at 620 nm. PDI activity (%) was calculated as: [(ODT80(PDI+DTT+compound) - ODT0(PDI+DTT+compound)) - (ODT80(DTT) - ODT0(DTT))] / [(ODT80(PDI+DTT) - ODT0(PDI+DTT)) - (ODT80(DTT) - ODT0(DTT))] × 100. IC₅₀ for 35G8 was determined as 0.17 ± 0.01 μM. [1]

Thermal shift assay: Purified PDI (0.3 mg/mL in 100 mM NaPO₄, pH 7.0) was mixed with protein dye (1,8-ANS, 0.3 mM) and test compound solutions. Silicone oil was added to prevent evaporation. The plate was heated from 25°C to 90°C at 1°C/min in a ThermoFluor instrument. Fluorescence emission was detected by CCD camera. DMSO (2% in buffer) was used as control. Melting temperature shifts (ΔTₘ) were calculated. 35G8 caused negative thermal shifts: -1.43°C at 1 μM, -2.94°C at 10 μM, and -3.64°C at 100 μM. [1]

Cellular thermal shift assay (CETSA): U87MG cells (2×10⁶ per 100 mm dish) were treated with 0.5, 1.0, or 2.0 μM 35G8 or DMSO for 2 hours at 37°C. Cells were trypsinized, washed, suspended in DPBS, split into aliquots, heated at indicated temperatures for 3 minutes, then flash-frozen twice and centrifuged. Supernatants were analyzed by Western blot for PDI, GRP78, GSTO1, and actin. [1]

Drug affinity responsive target stability (DARTS): U87MG cell lysates (5 mg/mL protein) were incubated with 100 μM 35G8, 100 μM PACMA31, or DMSO for 30 minutes with shaking at room temperature. Pronase was added at concentrations of 0, 1:1000 (0.005 μg/μL), 1:500 (0.01 μg/μL), or 1:250 (0.02 μg/μL) for 30 minutes. Digestion was stopped with protease inhibitor cocktail. Samples were analyzed by Western blot for PDI, GRP78, and GSTO1. [1]

Redox cycling assay: In a 384-well plate, HBSS buffer, catalase (100 U), H₂O₂ (100 μM), DTT (500 μM), 10 μM 35G8 with or without DTT and catalase were added to a final volume of 60 μL. After 30 minutes at room temperature, phenol red-HRP detection reagent was added (final concentration: 100 μg/mL phenol red, 60 μg/mL HRP). After 1 hour, 10 μL of 1 N NaOH was added and absorbance measured at 610 nm. [1]

Cell growth inhibition was assessed by MTT assay. Cells (U87MG, U118MG, A172, NU04, or HCT116) were seeded in 96-well plates at 7000–10000 cells/well. After overnight incubation, cells were treated with indicated compounds for 72 hours at 37°C in 5% CO₂. Then 20 μL of MTT solution (3 mg/mL) was added, incubated for 4 hours, supernatant removed, 100 μL DMSO added, plates shaken for 15 minutes, and absorbance measured at 570 nm. Cell growth inhibition (%) = [1 - (At - Ab)/(Ac - Ab)] × 100. For 35G8, IC₅₀ in U87MG was 1.1 ± 0.2 μM. For combination studies, NAC was added simultaneously with 35G8; Z-VAD-FMK and necrostatin-1 were added 1 hour prior to 35G8. [1]

Ferroptosis rescue assay: U87MG cells were seeded at 5000 cells/well in 96-well plates. Deferoxamine was added in a five-point, three-fold dilution series from 400 μM. 35G8 was added immediately after in a five-point, three-fold dilution series from 100 μM. Cells were incubated for 12 hours, then MTT assay performed. The IC₅₀ of 35G8 increased from 2.2 ± 0.7 μM (without DFO) to 5.8 ± 1.0 μM (with DFO). [1]

ROS detection: U87MG cells were detached, resuspended, treated with 20 μM H₂DCFDA for 30 minutes at 37°C, washed, and placed in black-wall 384-well plates at 20,000 cells/well. After 30 minutes, cells were treated with compounds. Fluorescence was read at 493/523 nm at 4, 6, and 24 hours. 35G8 at 5 μM induced significant ROS at 4, 6, and 24 hours. [1]

Western blot: U87MG cells were treated with DMSO or 2 μM 35G8 for 1, 3, 6, 12, or 24 hours. Cells were lysed in buffer (25 mM Tris, 150 mM NaCl, 17 mM Triton X-100, 3.5 mM SDS, pH 7.4), sonicated, and centrifuged. Protein concentration was determined by BCA assay. 50 μg protein was loaded onto 10% or 12% acrylamide gels, electro-transferred to PVDF membranes, blocked, and probed with primary antibodies (P4HB, GRP78, GSTO1, HMOX1, CHAC1, CHOP, LC3B, SLC7A11, GAPDH) overnight at 4°C, then with secondary antibodies, and imaged. 35G8 (2 μM, 24 h) upregulated HMOX1, SLC7A11, GRP78, DDIT3, and cleaved LC3B. [1]

Bru-seq: U87MG cells were treated with DMSO or 1 μM 35G8 for 4 hours. Bromouridine (2 mM final) was added for the last 30 minutes to label nascent RNA. Total RNA was isolated, bromouridine-labeled RNA was isolated and sequenced. 498 genes upregulated ≥2-fold, 238 genes downregulated ≥2-fold. [1]

Using ADMET predictor (Version 7.0) qualitative model, 35G8 was predicted to have high likelihood of blood-brain barrier (BBB) permeation. AlogP is between -1.1 and 1.1. Polar surface area is less than 90 Ų (cutoff for predicted CNS penetration). Molecular weight is 207 Da. [1]

[1]. Discovery and Mechanistic Elucidation of a Class of Protein Disulfide Isomerase Inhibitors for the Treatment of Glioblastoma. ChemMedChem. 2018 Jan 22;13(2):164-177.

1,3,6-Trimethylpyrimidino[5,4-e][1,2,4]triazine-5,7-dione is a pyrimidinotriazine.

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35G8 (1,3,6-trimethylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione) is a novel nanomolar PDI inhibitor identified from a screen of 20,000 diverse compounds in HCT116 colon cancer cells. It was the most cytotoxic PDI inhibitor from the screen. 35G8 induces Nrf2 antioxidant response, ER stress response, and autophagy in glioblastoma cells. It upregulates HMOX1 and SLC7A11, and represses PDI target genes such as TXNIP and EGR1. 35G8-induced cell death does not proceed via apoptosis or necrosis, but by a mixture of autophagy and ferroptosis. The compound is expected to cross the blood-brain barrier, making it a potential therapeutic agent for glioblastoma. [1]

C8H9N5O2

207.193

207.076

32502-62-8

32502-62-8

460747

Light yellow to yellow solid powder

-0.7

0

3

0

15

448

0

CPXHNWKHOFNPDO-UHFFFAOYSA-N

InChI=1S/C8H9N5O2/c1-4-9-5-6(13(3)11-4)10-8(15)12(2)7(5)14/h1-3H3 S

1,3,6-Trimethyl-pyrimido[5,4-e]-1,2,4-triazine-5,7(1H,6H)-dione

3-Methyltoxoflavin 3 Methyltoxoflavin 3Methyltoxoflavin GNF-Pf-2272.

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 (~241.32 mM)

Solubility in Formulation 1: 5 mg/mL (24.13 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 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: 5 mg/mL (24.13 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (12.07 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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 (Please use freshly prepared in vivo formulations for optimal results.)