E1(IC50: < 10 μM)
Ubiquitin-activating enzyme (E1). It is the first reported cell-permeable inhibitor of the ubiquitin E1 enzyme. In vitro, it inhibits ubiquitin loading onto E1 with an IC50 of <10 μM. At 50 μM, it achieves at least 95% inhibition [1].

PYR-41 not only prevents ubiquitylation but also raises total sumoylation in cells. PYR-41 reduces nuclear factor-κB activation mediated by cytokines. Additionally, PYR-41 stops IκBα from being ubiquitylated downstream and from being broken down by proteases. Moreover, PYR-41 stimulates this tumor suppressor's transcriptional activity and prevents p53 from being degraded[1]. The accumulation of ubiquitinated proteins is promoted by PYR-41 (50 μM).After 4 hours, Z138 cells exhibit a concentration-dependent (10-50 μM) decrease in DUB activity due to PYR-41. Potent inhibition of USP5 DUB activity is observed even at the lowest concentration (10 μM) of PYR-41. Potently (10-50 μM), PYR-41 inhibits the activity of multiple DUBs, which have been identified as USP9x, USP5, USP14, UCH37, and UCH-L3. When DTT and PYR-41 are administered together to Z138 cells, the accumulation of ubiquitinated proteins is totally eliminated[2].

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PYR-41 directly inhibits the ubiquitin E1 enzyme by blocking thioester bond formation between E1 and ubiquitin. It does not inhibit E2 enzymes (UbcH5B, Ube2G2) or caspases. It shows partial inhibition of HECT domain E3s (Nedd4, E6-AP) in vitro at concentrations that nearly completely inhibit E1 [1].
PYR-41 inhibits ubiquitin-mediated proteasomal degradation in vitro. It blocks cyclin E degradation in S100 cytosol, which is reversed by adding exogenous E1. It also inhibits E6-stimulated p53 degradation in rabbit reticulocyte lysates [1].
PYR-41 decreases the level of E1~Ub thioesters in cells with an IC50 between 10-25 μM. It blocks the accumulation of ubiquitin conjugates induced by proteasome inhibitor ALLN [1].
PYR-41 unexpectedly increases total sumoylation in cells, an effect also observed in temperature-sensitive E1 mutant cells at the restrictive temperature, suggesting inhibition of E1 is sufficient to increase sumoylation [1].
PYR-41 inhibits ligand-induced EGFR ubiquitylation, prolongs EGFR phosphorylation, and delays receptor down-regulation [1].
PYR-41 inhibits cytokine-induced NF-κB activation. It blocks IL-1α-induced TRAF6 ubiquitylation, delays IκBα phosphorylation, and prevents IκBα degradation following TNF-α stimulation [1].
PYR-41 increases p53 protein levels and transcriptional activity. At 20 μM, it induces p53 reporter activity comparable to Adriamycin. It also increases levels of Hdm2 and p21 (WAF-1) [1].
PYR-41 differentially kills transformed cells, particularly those expressing wild-type p53. E1A-transformed RPE cells are killed within 20 hours in a dose-dependent manner, while untransformed RPE cells are relatively resistant. p53-expressing transformed MEFs (C8) show substantially greater dose-dependent cell death compared to p53-deficient MEFs (A9) [1].

For 15 minutes, 32P-ubiquitin is incubated at room temperature in 1× reaction buffer (50 mM Tris (pH 7.4), 0.2 mM ATP, and 0.5 mM MgCl2) with rabbit or mouse E1 (apper 250 ng). Before conducting incubations and reactions, the His-tagged mouse E1 is bound to TALON cobalt affinity resin in certain experiments. Beads in 1× reaction buffer are mixed with mouse E1 and 32P-ubiquitin, and they are then incubated similarly to E1 reactions. Samples are resolved by SDS-PAGE after being heated in nonreducing SDS-PAGE sample buffer. Storm PhosphoImager visualizes thioesters that have ubiquitin.

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For E1 inhibition assays, rabbit or mouse E1 (~250 ng) was incubated with ³²P-ubiquitin in reaction buffer [50 mmol/L Tris (pH 7.4), 0.2 mmol/L ATP, 0.5 mmol/L MgCl2] at room temperature for 15 minutes. In some experiments, His-tagged mouse E1 was immobilized on TALON cobalt affinity resin before incubation. Samples were heated in nonreducing SDS-PAGE sample buffer and resolved by SDS-PAGE. Thioesters with ubiquitin were visualized by Phosphorimager. To test reversibility, immobilized E1 was treated with PYR-41 for 15 minutes, then washed thoroughly before the thioester assay [1].
For E2 assays, GST-UbcH5B (~900 ng) was bound to glutathione Sepharose beads. Mouse E1 and ³²P-ubiquitin were added in reaction buffer and incubated as for E1 reactions [1].
For competition experiments, immobilized E1 (2 pmol) was pretreated with PYR-41 together with reduced glutathione (GSH) at 10⁴-10⁵ fold excess relative to E1 for 15 minutes. Beads were washed, and Ub~E1 thioester formation was assessed [1].
For in vitro degradation assays, ³⁵S-labeled cyclin E was added to S100 cytosol (50 μg) pre-incubated with DMSO or PYR-41 (50 μmol/L) for 30 minutes. Aliquots were taken over 90 minutes and analyzed by SDS-PAGE and autoradiography. For rescue experiments, recombinant E1 (250 ng, 50 nmol/L final) was added 30 minutes after PYR-41 treatment [1].
For p53 degradation, ³⁵S-labeled p53 and HPV-16 E6 were incubated in rabbit reticulocyte lysates with or without PYR-41 for up to 3 hours and analyzed by SDS-PAGE and autoradiography [1].

For cellular E1 thioester analysis, RPE cells were treated with indicated compounds (50 μmol/L PYR-41, 20 μmol/L HL98C, or 10 mmol/L iodoacetamide) for 30 minutes. Cell lysates were prepared in urea-containing buffer, heated in nonreducing or reducing sample buffer, and immunoblotted with anti-E1 antibody [1].
For ubiquitin conjugate accumulation, RPE cells were exposed to PYR-41 (50 μmol/L) or DMSO for 30 minutes, then treated with or without ALLN (50 μmol/L) for 90 minutes. Lysates were immunoblotted with anti-ubiquitin antibody [1].
For sumoylation studies, HEK293 cells with tetracycline-inducible Myc-SUMO-1 were induced with doxycycline (2 μg/mL) for 20 hours, then treated with PYR-41 (0-50 μmol/L) for 4 hours. Lysates were immunoblotted with anti-Myc antibody [1].
For EGFR studies, HeLa cells were pretreated with PYR-41 (50 μmol/L) for 15 minutes, then stimulated with EGF (100 ng/mL) for indicated times. Cell lysates were immunoprecipitated with anti-EGFR and immunoblotted for ubiquitin, or directly immunoblotted for phosphorylated EGFR and total EGFR [1].
For NF-κB luciferase assay, HeLa cells transfected with NF-κB response element-driven luciferase reporter were pretreated with PYR-41 for 30 minutes, then stimulated with IL-1α (1 or 10 ng/mL) for 2 hours. Luciferase activity was measured [1].
For TRAF6 ubiquitylation, HeLa cells were treated with PYR-41 (50 μmol/L) for 15 minutes, stimulated with IL-1α (10 ng/mL) for 5 minutes, immunoprecipitated with anti-TRAF6, and immunoblotted for ubiquitin [1].
For IκBα analysis, HeLa cells were pretreated with PYR-41 for 10 minutes, stimulated with IL-1α (10 ng/mL) for indicated times, and lysates immunoblotted for IκBα and phospho-IκBα. 2B4 T-cells were pretreated with DMSO, PYR-41 (50 μmol/L), or ALLN (50 μmol/L) for 15 minutes, then stimulated with TNF-α [1].
For p53 activity, U2OS-pG13 cells (with p53 response element-driven luciferase) were treated with DMSO, Adriamycin (1 μg/mL), or PYR-41 (10-50 μmol/L) for 20 hours, then luciferase activity measured. U2OS cells were treated with Adriamycin (1 μg/mL), MG132 (50 μmol/L), or PYR-41 (10-50 μmol/L) for 6 hours and immunoblotted for p53, Hdm2, and p21 [1].
For cell viability assays, RPE and RPE-E1A cells were incubated with PYR-41 (0-50 μmol/L) for 20 hours, and viability assessed by trypan blue exclusion. C8 (p53+/+) and A9 (p53-/-) transformed MEFs were treated with PYR-41 (0-50 μmol/L) for up to 24 hours, and viability assessed [1].

The compound shows differential cytotoxicity, preferentially killing transformed cells. In RPE-E1A transformed cells, PYR-41 induces dose-dependent cell death within 20 hours. Untransformed RPE cells show relative resistance, though some growth inhibition is observed at higher concentrations (20 μmol/L). The apoptotic nature of cell death was confirmed by PARP cleavage and DNA fragmentation. In transformed MEFs, p53-expressing cells (C8) show substantially greater susceptibility to PYR-41-induced death compared to p53-deficient cells (A9) [1].

[1]. Inhibitors of ubiquitin-activating enzyme (E1), a new class of potential cancer therapeutics. Cancer Res. 2007 Oct 1;67(19):9472-81.

[2]. Protein cross-linking as a novel mechanism of action of a ubiquitin-activating enzyme inhibitor with anti-tumor activity. Biochem Pharmacol. 2011 Aug 15;82(4):341-9.

PYR-41 is an ethyl ester formed by the condensation of the carboxyl group of 4-{4-[(5-nitrofuran-2-yl)methylene]-3,5-dioxopyrazol-1-yl}benzoic acid with ethanol. It is an irreversible, cell-membrane-crossing inhibitor of ubiquitin-activating enzyme E1. It is both an EC 6.2.1.45 (E1 ubiquitin-activating enzyme) inhibitor and an antitumor drug. It is an ethyl ester belonging to the furans, C-nitro compounds, pyrazolidine compounds, and benzoic acid esters.

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PYR-41 is the first described cell-permeable inhibitor of the ubiquitin-activating enzyme (E1). It was identified through a high-throughput screen for inhibitors of Hdm2 ubiquitin ligase activity, but was found to inhibit E1 instead. Its structure features a nitro-substituted furan ring that may be important for activity, and it likely functions by covalently modifying the active site cysteine of E1, as suggested by quenching of activity by excess free thiols (GSH) [1].
Inhibition of E1 blocks both proteasomal and non-proteasomal functions of ubiquitination. The compound reveals a reciprocal relationship between ubiquitination and sumoylation, as E1 inhibition leads to increased sumoylation in cells—a finding confirmed in temperature-sensitive E1 mutant cells [1].
PYR-41 has multiple potentially therapeutic effects: it inhibits NF-κB activation (at both upstream K63-linked ubiquitination of TRAF6 and downstream proteasomal degradation of IκBα), stabilizes and activates p53, increases p21 levels, and differentially kills transformed cells, particularly those with wild-type p53. These properties suggest that E1 inhibitors could have therapeutic potential in cancer, despite their broad effects on multiple cellular pathways [1].

C17H13N3O7

371.30102

371.075

C, 54.99; H, 3.53; N, 11.32; O, 30.16

418805-02-4

418805-02-4

5335621

Brown solid powder

1.5±0.1 g/cm3

1.652

2.87

1

7

5

27

664

0

O1C(=C([H])C([H])=C1/C(/[H])=C1/C(N([H])N(C/1=O)C1C([H])=C([H])C(C(=O)OC([H])([H])C([H])([H])[H])=C([H])C=1[H])=O)[N+](=O)[O-]

ARGIPZKQJGFSGQ-LCYFTJDESA-N

InChI=1S/C17H13N3O7/c1-2-26-17(23)10-3-5-11(6-4-10)19-16(22)13(15(21)18-19)9-12-7-8-14(27-12)20(24)25/h3-9H,2H2,1H3,(H,18,21)/b13-9-

ethyl 4-(4-((5-nitrofuran-2-yl)methylene)-3,5-dioxopyrazolidin-1-yl)benzoate

PYR41; PYR 41; PYR-41.

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 : 46~74 mg/mL ( 123.89~199.29 mM )

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.73 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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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Solubility in Formulation 2: 2% DMSO + 30% PEG300 + 5% Tween80 + 63% ddH2O: 2mg/ml

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