Ubiquitin-activating enzyme E1 (UBA1).

IC50 for E1 enzyme inhibition = approximately 20 µM in a cell-free enzymatic assay.

In 5 of 8 leukemia and myeloma cell lines, less than 10 μM of PYZD-4409 (10–40 μM; 72 hours; myeloma, leukemia, and solid tumor cell lines, primary AML cells, and normal hematopoietic cells) results in cell death, or LD50. Solid tumor cell lines, on the other hand, have LD50s of roughly 15 to 20 μM, making them less susceptible. Compared to normal hematopoietic cells, PYZD-4409 primarily causes cytotoxicity against malignant cells [1]. PYZD-4409 (50 μM; 4 hours; K562 leukemia cells) treatment prevents ubiquitin from conjugating with the E2 enzyme cdc34 in an E1-dependent manner [1]. Grp78 and Hsp70 mRNA and protein levels were dramatically enhanced by PYZD-4409 (0-25 μM; 24 hours; K562 leukemia cells). Furthermore, phospho-JNK and phospho-p38 mitogen-activated protein kinases—which are linked to ER stress and the unfolded protein response—are also elevated by PYZD-4409 [1].

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Inhibition of E1 Enzyme Activity: PYZD-4409 inhibited the ATP-dependent activation of ubiquitin and the subsequent transfer of activated ubiquitin from the E1 enzyme to the E2 enzyme (UBE2E2) in gel-based assays. It also inhibited the E1-mediated loading of ubiquitin onto the E2 enzyme Cdc34 in K562 leukemia cells after 4 hours of treatment. The estimated IC50 for E1 inhibition was 20 µM in a cell-free assay. A structurally related control compound, PYZDmut, showed no inhibitory effect on E1 activity. [1]
Cytotoxicity in Cell Lines: PYZD-4409 induced cell death in a panel of leukemia, multiple myeloma, and solid tumor cell lines, as measured by Alamar Blue assay after 72 hours of treatment. Myeloma cell lines (LP1, KMS11, U266) were particularly sensitive, with LD50 values of 3 µM or less. Five out of eight leukemia and myeloma cell lines had an LD50 of less than 10 µM. Solid tumor cell lines were less sensitive, with LD50 values of approximately 15-20 µM. The control compound PYZDmut was not cytotoxic at concentrations up to 50 µM. Cell death was confirmed by Trypan Blue staining. [1]
Effect on Primary AML Cells vs. Normal Cells: In clonogenic growth assays, treatment with PYZD-4409 (10 µM for 24 hours) inhibited the colony formation of primary acute myeloid leukemia (AML) cells. In contrast, it did not reduce the clonogenic growth of normal hematopoietic cells (peripheral blood stem cells, PBSCs) from healthy donors under the same conditions. [1]
Impact on Short-lived Proteins and ER Stress:
Treatment with PYZD-4409 (50 µM) increased the protein levels of short half-life proteins regulated by ubiquitination, such as cyclin D3 (in K562 cells at 4 hours) and p53 (in HCT116 cells at 2 hours). [1]
PYZD-4409 treatment (10-25 µM for 24 hours) increased mRNA levels of ER stress markers GRP78 and HSP70 in K562 cells. It also increased protein levels of ER stress markers, including phospho-JNK, phospho-p38 MAPK (at 50 µM for 2.5 hours in K562 cells), as well as phospho-PERK, ATF4, and CHOP (at 10 µM for 24 hours in MDAY-D2 cells). The control compound PYZDmut did not induce these changes. [1]
Overexpression of BI-1, a protein that protects against ER stress, in HT1080 cells significantly inhibited cell death induced by increasing concentrations of PYZD-4409, suggesting ER stress is functionally important for its cytotoxic mechanism. [1]

PYZD-4409 (10 mg/kg; i.p.; once everyday every other day for 16 days; male severe combination immunodeficient mice) decreases tumor weight and volume without adverse effects [1].

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Efficacy in a Murine Leukemia Model: In a subcutaneous xenograft model, SCID mice bearing MDAY-D2 murine leukemia cells were treated with PYZD-4409 (10 mg/kg) or vehicle control via intraperitoneal injection every other day for 8 days. At day 16 post-tumor inoculation, treatment with PYZD-4409 significantly delayed tumor growth and reduced final tumor weight compared to the control group, without causing overt toxicity. However, increases in ER stress markers (phospho-PERK, Grp78, CHOP) were not detected in the tumors from treated mice by immunoblotting. [1]

E1 Enzymatic Activity Assay (IC50 Determination): To determine the IC50, recombinant His-tagged human E1 (1 µM) was incubated with His-tagged human E2 enzyme UbcH5A (10 µM), ubiquitin (20 µM), and ATP (1 mM) in a reaction buffer (50 mM Tris-HCl, pH 7, 5 mM MgCl2) with increasing concentrations of PYZD-4409 in a 96-well plate. An inorganic pyrophosphate detection reagent was added immediately, and fluorescence signals (excitation 530 nm, emission 567 nm) were read over 30 minutes. The inorganic pyrophosphate resulting from ATP hydrolysis during E1-catalyzed ubiquitin activation was quantified to estimate enzyme activity. The IC50 was determined to be approximately 20 µM. [1]
E1-Ubiquitin Conjugate Formation Assay: GST-tagged human E1 (0.5 µM) and fluorescein-labeled ubiquitin (1 µM) were incubated in reaction buffer (50 mM Tris-HCl, pH 7, 5 mM MgCl2, 250 µM ATP) with or without increasing concentrations of PYZD-4409 or the control compound PYZDmut for 30 minutes at 37°C. The products were then fractionated by SDS-PAGE under non-reducing conditions. Formation of E1-Ub conjugates was assessed by visualizing the fluorescein signals using a gel imager. [1]
E2 Loading Assay: GST-tagged human E1 (1 µM), His-tagged human E2 (UbcH5A, 5 µM), ubiquitin (20 µM), and ATP (1 mM) were incubated with or without increasing concentrations of PYZD-4409 or PYZDmut for 30 minutes at 30°C. The reactions were fractionated by SDS-PAGE, followed by immunoblotting with an anti-His antibody and fluorescent dye-labeled secondary antibody. Fluorescent signals corresponding to E2-ubiquitin conjugates were detected using an infrared imaging system. [1]
Cellular E1 Activity Assay (E2 Loading): To assess E1 inhibition in cells, K562 cells were treated with PYZD-4409 (50 µM) for 4 hours. Cell lysates were then prepared and heated in either non-reducing or reducing SDS-PAGE sample buffer. Samples were fractionated by SDS-PAGE and immunoblotted with antibodies against the E2 protein cdc34 to detect the formation of E2-ubiquitin conjugates, which is dependent on E1 activity. [1]

Cytotoxicity assay[1]
Cell Types: Myeloma, leukemia and solid tumor cell lines, primary AML cells and normal hematopoietic cells
Tested Concentrations: 10 μM, 20 μM, 30 μM, 40 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Induction of cell death Five of eight leukemia and myeloma cell lines had LD50s below 10 μM. In contrast, solid tumor cell lines are less sensitive, with LD50s of approximately 15 to 20 μM.

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Western Blot Analysis[1]
Cell Types: K562 Leukemia Cells
Tested Concentrations: 50 μM
Incubation Duration: 4 hrs (hours)
Experimental Results: Blocks E1-dependent conjugation of ubiquitin to the E2 enzyme cdc34.

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RT-PCR[1]
Cell Types: K562 Cell
Tested Concentrations: 0 μM, 10 μM, 25 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: The mRNA and protein levels of Grp78 and Hsp70 were Dramatically increased.

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Cell Viability Assay (Alamar Blue/MTS): Cells were seeded in 96-well plates and treated with increasing concentrations of PYZD-4409 for 72 hours. After incubation, Alamar Blue or MTS reagent was added according to the manufacturer's instructions, and fluorescence or absorbance was measured to determine cell viability relative to control. For trypan blue exclusion, aliquots of cells were mixed with the dye and non-stained (viable) cells were counted. For Annexin V/PI staining, cells were stained with Annexin V-FITC and PI and analyzed by flow cytometry. [1]
Colony Formation Assay: 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 methylcellulose-based medium (MethoCult GF H4434) containing cytokines. Colonies were counted after 7 days of incubation for AML cells and after 2 weeks for normal hematopoietic cells. [1]
Western Blot Analysis: Cells treated with PYZD-4409, control compound PYZDmut, or DMSO were lysed in SDS sample buffer. Protein concentrations were determined, and lysates were supplemented with β-mercaptoethanol (for reducing conditions), heated, and fractionated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes and hybridized with primary antibodies against targets of interest (e.g., E1, ubiquitin, cyclin D3, p53, GRP78, phospho-JNK, phospho-p38, phospho-PERK, ATF4, CHOP). Signals were visualized using an enhanced chemiluminescence kit. [1]
Real-Time Quantitative RT-PCR: K562 cells treated with PYZD-4409 at indicated concentrations for 24 hours were harvested, and total RNA was isolated. cDNA was synthesized, and real-time PCR was performed using primers for GRP78, HSP70, and 18S rRNA. Relative mRNA expression was determined using the ΔΔCT normalization method with 18S rRNA as the housekeeping gene. [1]
BI-1 Overexpression Protection Assay: HT1080 cells stably overexpressing BI-1 (HT1080-BI-1) or containing empty vector (HT1080-neo) were seeded overnight in 96-well plates. The next day, cells were treated with increasing concentrations of PYZD-4409 for 24 hours. Cell viability was then determined by the Alamar Blue assay. [1]

Animal/Disease Models: Male severe combined immunodeficiency (SCID) mice with MDAY-D2 murine leukemia cells [1]
Doses: 10 mg/kg
Route of Administration: intraperitoneal (ip) injection; daily, every other day; for 16 days
Experimental Results: Delayed tumor growth , tumor weight was diminished without adverse toxicity.

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Murine Leukemia Xenograft Efficacy Study:** Male SCID mice were injected subcutaneously with 1 x 10^5 MDAY-D2 murine leukemia cells. The following day, treatment began. Mice received intraperitoneal injections of PYZD-4409 at a dose of 10 mg/kg in saline, or an equal volume of saline vehicle alone as a control. Treatment was administered once every other day for a total of 8 days. Tumor growth was monitored at least every other day using external calipers once tumors became palpable. Sixteen days after tumor inoculation, mice were euthanized by CO2 asphyxiation. Tumors were then excised and weighed. [1]

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Murine Leukemia Xenograft Efficacy Study: Male SCID mice were injected subcutaneously with 1 x 10^5 MDAY-D2 murine leukemia cells. The following day, treatment began. Mice received intraperitoneal injections of PYZD-4409 at a dose of 10 mg/kg in saline, or an equal volume of saline vehicle alone as a control. Treatment was administered once every other day for a total of 8 days. Tumor growth was monitored at least every other day using external calipers once tumors became palpable. Sixteen days after tumor inoculation, mice were euthanized by CO2 asphyxiation. Tumors were then excised and weighed. [1]

In Vivo Tolerability: In the murine efficacy study, intraperitoneal administration of PYZD-4409 at 10 mg/kg every other day for 8 days was described as being without untoward toxicity, as no overt signs of toxicity were reported. [1]
In Vitro Selectivity: PYZD-4409 demonstrated preferential cytotoxicity towards malignant cells. It inhibited the clonogenic growth of primary AML cells at 10 µM but did not reduce the colony formation of normal hematopoietic cells (PBSCs) from healthy donors. Additionally, it showed no inhibitory effect on unrelated enzymes such as α-Mannosidase II or Luciferase at concentrations up to 100 µM, and it did not inhibit the SUMO E1 enzyme, Uba2, at similar concentrations. [1]

[1]. The ubiquitin-activating enzyme E1 as a therapeutic target for the treatment of leukemia and multiple myeloma. Blood. 2010 Mar 18;115(11):2251-9.

Background and Mechanism: PYZD-4409 (1-(3-chloro-4-fluorophenyl)-4-[(5-nitro-2-furyl)methylene]-3,5-pyrazolidinedione) is a novel small-molecule inhibitor of the ubiquitin-activating enzyme E1 (UBA1). It was identified from a screen of a focused chemical library based on the pyrazolidine pharmacophore. By inhibiting E1, the compound blocks the first step in the ubiquitination cascade, preventing the activation and subsequent transfer of ubiquitin to E2 enzymes. This inhibition leads to the accumulation of ubiquitinated proteins and short half-life proteins (like p53 and cyclin D3), and ultimately induces cell death through a mechanism involving endoplasmic reticulum (ER) stress and the unfolded protein response. The study provides a proof-of-concept for targeting the E1 enzyme as a therapeutic strategy in hematologic malignancies like leukemia and multiple myeloma. [1]

351.006

423148-78-1

60111983

Light brown to black solid powder

3.305

1

6

2

24

595

0

FC1=CC=C(N2C(/C(C(N2)=O)=C\C3=CC=C([N+]([O-])=O)O3)=O)C=C1Cl

MSYMKEYWUWVZQY-TWGQIWQCSA-N

InChI=1S/C14H7ClFN3O5/c15-10-5-7(1-3-11(10)16)18-14(21)9(13(20)17-18)6-8-2-4-12(24-8)19(22)23/h1-6H,(H,17,20)/b9-6-

(4Z)-1-(3-chloro-4-fluorophenyl)-4-[(5-nitrofuran-2-yl)methylidene]pyrazolidine-3,5-dione

PYZD4409 PYZD-4409 PYZD 4409

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 : ≥ 35 mg/mL (~99.53 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.11 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (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 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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 (Please use freshly prepared in vivo formulations for optimal results.)