Cell lines

RPE cells, U2OS cells transfected with GFPu; RAW 264.7 cells

Preparation method

The solubility of this compound in DMSO is >18.6mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

RPE cells: 50 μmol/L; 30 min; 37℃RAW 264.7 cells: 5, 10, and 20 μM

Applications

In RPE cells, PYR-41 markedly reduced Ub~E1 thioesters with IC50 between 10 and 25 μmol/L. PYR-41 also blocked accumulation of ubiquitin conjugates in response to the proteasome inhibitor ALLN. In U2OS cells transfected with GFPu, PYR-41 inhibited both ubiquitylation and proteasomal degradation of GFPu. In RAW 264.7 cells stimulated by LPS, PYR-41 (10 and 20 μM) restored the expression levels of IκB to 89% and 95% of those in the non LPS-stimulated RAW 264.7 cells, respectively. PYR-41 also reduced TNF-α levels.

Animal models

Male C57BL/6 mice with sepsis induced by cecal ligation and puncture (CLP)

Dosage form

5 mg/kg; intravenous injection immediately after CLP

Application

In septic mice induced by CLP, PYR-41 significantly reduced serum levels of proinflammatory cytokines TNF-α, IL-1β, and IL-6 by 79%, 77%, and 89%, respectively. PYR-41 also reduced serum levels of organ injury markers AST, ALT, and LDH by 27%, 43%, and 52%, respectively. Treatment with PYR-41 improved the morphologic appearance of lung tissues and showed a 74% reduction in histology injury score.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

• Cell Death Dis 13.4 (2022): 1-12.PMID: 35365616

Ubiquitylation is catalyzed by the sequential action of ubiquitinactivating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and a ubiquitin protein ligase (E3). Ubiquitylation is essential to numerous cellular and developmental processes, including protein quality control, growth, apoptosis, antigen presentation, DNA repair, and signal transduction. PYR-41, 4[4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester, is a selective inhibitor of Ubiquitin-Activating Enzyme (E1).

In vitro: In addition to blocking ubiquitylation, PYR-41 was found to increase total sumoylation in cells. PYR-41 could attenuate cytokine-mediated nuclear factor-KBactivation. This correlated with inhibition of nonproteasomal ubiquitylation of TRAF6, which is important to IKBkinase activation. PYR-41 also prevented the downstream ubiquitylation and proteasomal degradation of IKBA. Moreover, PYR-41 has demonstrated effective UAE E1 inhibition as well as some off-target inhibition of the other ubiquitin regulatory enzymes and signal-transducing proteins, suggesting it is a nonspecific inhibitor [1].

In vivo: No animal in vivo data have been published so far.

Clinical trial: Up to now, PYR-41 is still in the preclinical development stage.

Reference:
[1] Yang Y1 Kitagaki J, Dai RM, Tsai YC, Lorick KL, Ludwig RL, Pierre SA, Jensen JP, Davydov IV, Oberoi P, Li CC, Kenten JH, Beutler JA, Vousden KH, Weissman AM. Inhibitors of ubiquitin-activating enzyme (E1), a new class of potential cancer therapeutics. Cancer Res. 2007 Oct 1;67(19):9472-81.