Ubiquitin-activating enzyme UBA1 (E1). It inhibits the ATP-dependent activation of ubiquitin by E1. [1]

In canine heart Purkinje fibers and guinea pig atrial tissue, A-935142 reduces action potential duration (APD90) [1]. A-935142 has concentration- and voltage-dependent effects on cardiac action potential shortening and hERG current amplitude enhancement. The linear component of the fully activated IV relationship exhibits an increase in slope conductance and outward and inward K(+) currents when A-935142 (60μM) is added. At two sample voltages (-10 mV: tau=100+/-17 ms vs. 164+/-24 ms, n=6, P<0.01; +30 mV: tau=16.7+/-1.8 ms vs. 18.9+/-1.8 ms, n=5, P<0.05), A-935142 significantly decreased the time constant τ of hERG channel activation. Additionally, it shifts the voltage dependence of hERG activation by 9 mV along the hyperpolarization direction [2].

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PYZD-4409 inhibits the E1 enzyme in cell-free assays with an estimated IC50 of 20 μM. [1]
PYZD-4409 blocks the E1-dependent conjugation of ubiquitin to the E2 enzyme cdc34 in K562 leukemia cells after 4 hours of treatment at 50 μM. [1]
In a panel of leukemia, myeloma, and solid tumor cell lines, PYZD-4409 induces cell death with varying sensitivity. Myeloma cell lines (LP1, KMS11, U266) are particularly sensitive, with LD50 values of 3 μM or less after 72 hours of treatment. Leukemia and solid tumor cell lines have higher LD50 values (e.g., approximately 15-20 μM for solid tumor lines). [1]
PYZD-4409 (10 μM, 24h) inhibits the clonogenic growth of primary acute myeloid leukemia (AML) cells but does not reduce the colony formation of normal hematopoietic peripheral blood stem cells (PBSCs). [1]
Treatment with PYZD-4409 (50 μM, 2-4h) increases the abundance of short half-life proteins regulated by ubiquitination, such as cyclin D3 (in K562 cells) and p53 (in HCT116 cells). [1]
PYZD-4409 induces endoplasmic reticulum (ER) stress. In K562 cells, it increases mRNA and protein levels of ER stress markers Grp78 and Hsp70 (at 10-25 μM for 24h), and increases levels of phospho-JNK and phospho-p38 (at 50 μM for 2.5h). In MDAY-D2 cells, it increases phospho-PERK, ATF4, and CHOP (at 10 μM for 24h). [1]
Overexpression of BI-1, an ER stress protective protein, in HT1080 cells inhibits cell death induced by PYZD-4409 (0-30 μM, 24h), suggesting ER stress is functionally important for its cytotoxic effect. [1]
The structurally related inactive control compound, PYZDmut, shows no effect on E1 activity or cytotoxicity. [1]

In a murine xenograft model, intraperitoneal administration of PYZD-4409 (10 mg/kg every other day for 8 days) to SCID mice bearing subcutaneous MDAY-D2 murine leukemia cells significantly delays tumor growth and reduces tumor weight compared to control treatment, without causing untoward toxicity. [1]

To determine the IC50, recombinant His-tagged human E1 enzyme (1 μM) was incubated with His-tagged human E2 enzyme (10 μM), ubiquitin (20 μM), and ATP (1 mM) in a reaction buffer containing 50 mM Tris-HCl (pH 7), 5 mM MgCl2, and increasing concentrations of PYZD-4409. The reaction was initiated, and inorganic pyrophosphate released from ATP hydrolysis during E1-catalyzed ubiquitin activation was immediately quantified using a fluorogenic pyrophosphate detection kit. Fluorescence signals were measured over 30 minutes in a microplate reader. [1]
For the E1-ubiquitin conjugate assay, GST-tagged human E1 (0.5 μM) and fluorescein-labeled ubiquitin (1 μM) were incubated with or without increasing concentrations of PYZD-4409 or the inactive control PYZDmut in reaction buffer for 30 minutes at 37°C. The products were then fractionated on 4-20% gradient SDS-PAGE under non-reducing conditions. Formation of the E1-Ub conjugate was assessed by visualizing the fluorescent signals using a gel imager. [1]
To assess the effect on E2 loading, His-tagged E1 (0.5 μM), His-tagged E2 (5 μM), and ubiquitin (5 μM) were incubated with increasing concentrations of PYZD-4409 or PYZDmut. The products were resolved on non-reducing 4-20% gradient SDS-PAGE followed by immunoblotting with an anti-His antibody. The fluorescent bands, corresponding to E2 and E2-ubiquitin conjugates, were detected using a fluorescent dye-labeled secondary antibody and a gel imager. [1]

For cell viability assays, leukemia, myeloma, and solid tumor cell lines were seeded in 96-well plates and treated with increasing concentrations of PYZD-4409 for 72 hours. Cell growth and viability were then measured using the Alamar Blue assay according to the manufacturer's instructions. Cell death was confirmed by Trypan Blue dye exclusion assay, where aliquots of cells were mixed with the dye and non-stained (viable) cells were counted. [1]
For clonogenic growth assays, primary human AML cells or normal PBSCs were treated with PYZD-4409 (10 μM) or buffer control for 24 hours. After treatment, cells were washed and plated in duplicate in MethoCult GF H4434 methylcellulose medium. AML colonies were counted after 7 days of incubation, while normal hematopoietic colonies were counted after 2 weeks. [1]
For Western blot analysis, cells (e.g., K562, HCT116, MDAY-D2) were treated with PYZD-4409 or control at specified concentrations and times (e.g., 2-24 hours). Cells were then lysed in SDS sample buffer, and protein concentrations were determined. Equal amounts of protein were fractionated by SDS-PAGE, transferred to nitrocellulose membranes, and hybridized with specific primary antibodies (e.g., anti-cyclin D3, anti-p53, anti-GRP78, anti-phospho-JNK). Signals were visualized using an enhanced chemiluminescence kit. [1]
To measure E1 activity in cells, K562 cells were treated with PYZD-4409 (50 μM) or PYZDmut for 4 hours. Cell lysates were heated in either non-reducing or reducing SDS-PAGE sample buffer, fractionated, and immunoblotted with antibodies against the E2 protein cdc34 to assess E1-mediated loading of ubiquitin onto cdc34. [1]
For real-time RT-PCR, K562 cells were treated with PYZD-4409 (10 and 25 μM) for 24 hours. Total cellular RNA was isolated, and the expression of GRP78 and HSP70 mRNA was measured relative to 18S rRNA using real-time quantitative RT-PCR with the ΔΔCT normalization method. [1]
For the cell death protection assay, HT1080 cells stably overexpressing BI-1 (HT1080-BI-1) or empty vector (HT1080-neo) were seeded in 96-well plates overnight. The next day, cells were treated with increasing concentrations of PYZD-4409 (0-30 μM) for 24 hours. Cell viability was determined by the Alamar Blue assay. [1]

Male SCID mice were injected subcutaneously with 1 × 10^5 MDAY-D2 murine leukemia cells. The next day, animals were randomly assigned to treatment groups. Mice received intraperitoneal injections of PYZD-4409 at 10 mg/kg in saline, or saline buffer control alone. The treatment was administered once every other day over a total of 8 days. Tumor growth was monitored at least every other day using external calipers after tumors became palpable. Sixteen days after tumor inoculation, mice were euthanized by CO2 asphyxiation, and tumors were excised and weighed. [1]

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Male SCID mice were injected subcutaneously with 1 × 10^5 MDAY-D2 murine leukemia cells. The next day, animals were randomly assigned to treatment groups. Mice received intraperitoneal injections of PYZD-4409 at 10 mg/kg in saline, or saline buffer control alone. The treatment was administered once every other day over a total of 8 days. Tumor growth was monitored at least every other day using external calipers after tumors became palpable. Sixteen days after tumor inoculation, mice were euthanized by CO2 asphyxiation, and tumors were excised and weighed. [1]

In the mouse xenograft model, intraperitoneal administration of PYZD-4409 at 10 mg/kg every other day for 8 days delayed tumor growth without causing untoward toxicity. [1]
In cell-based assays, PYZD-4409 showed preferential cytotoxicity, inhibiting the clonogenic growth of primary AML cells at 10 μM without affecting normal hematopoietic PBSCs. [1]

[1]. Absorption, disposition, metabolism and excretion of [14C]mizagliflozin, a novel selective SGLT1 inhibitor, in rats. Xenobiotica. 2019 Apr;49(4):463-473.

[2]. Characterization of A-935142, a hERG enhancer, in the presence and absence of standard hERG blockers. Life Sci. 2012 Apr 20;90(15-16):607-11.

PYZD-4409 is a novel 3,5-dioxopyrazolidine compound identified from a screen of a focused chemical library based on the pyrazolidine pharmacophore. Its chemical name is 1-(3-chloro-4-fluorophenyl)-4-[(5-nitro-2-furyl)methylene]-3,5-pyrazolidinedione. It has a molecular weight of 352 and a CAS number of 423148-78-1. The compound is structurally similar to another E1 inhibitor, PYR-41. [1]
The study uses PYZD-4409 as a chemical probe to demonstrate that inhibition of the E1 enzyme induces cell death in malignant cells through a mechanism involving ER stress and an unfolded protein response, highlighting E1 as a potential therapeutic target for hematologic malignancies like leukemia and multiple myeloma. [1]

C18H19F3N2O2

352.350875139236

352.139

1031335-85-9

25125452

White to off-white solid powder

4.5

2

6

4

25

456

0

FC(C1=CC(C2C=CC(=CC=2)C2CCC(CC(=O)O)CC2)=NN1)(F)F

YPHFQSYEKIEMNC-UHFFFAOYSA-N

InChI=1S/C18H19F3N2O2/c19-18(20,21)16-10-15(22-23-16)14-7-5-13(6-8-14)12-3-1-11(2-4-12)9-17(24)25/h5-8,10-12H,1-4,9H2,(H,22,23)(H,24,25)

2-[4-[4-[5-(trifluoromethyl)-1H-pyrazol-3-yl]phenyl]cyclohexyl]acetic acid

A-935142 A935142 A 935142

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.)