IRE1α (inositol-requiring enzyme 1 alpha). It is a Type I ATP-competitive inhibitor that binds to the kinase domain's ATP-binding site, stabilizing an active (DFG-in) conformation. [1]

APY29 modifies IRE1α's oligomeric state and RNase activity in distinct ways. Using the same binding site, APY29 affects RNase activity in opposing ways. To varied degrees, APY29 influences the oligomerization of IRE1α. The dose-dependent action of APY29 on the activation of endogenous IRE1α RNase caused by ER stress is opposite [1].

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APY29 is an ATP-competitive inhibitor that binds to the kinase domain of IRE1α. Co-crystal structures of yeast IRE1 bound with APY29 show that the kinase catalytic domain is in an active conformation, which is typically adopted by protein kinases when bound to ATP and other type I inhibitors. [1]
APY29 inhibits IRE1α autophosphorylation in a dose-dependent manner. In an in vitro kinase assay using a recombinant human IRE1α protein (IRE1α, residues 469-977), APY29 demonstrated an IC50 for inhibiting autophosphorylation. (Quantitative IC50 value not provided in text/figures). [1]
Unlike its effect on kinase activity, APY29 activates the RNase domain of IRE1α. In an in vitro RNase assay using a FRET-quenched XBP1 RNA mini-substrate, APY29 increased the cleavage activity of a dephosphorylated, low-activity form of IRE1α (dP-IRE1α) in a dose-dependent manner, restoring activity to ~60% of the level of phosphorylated IRE1α. The EC50 for this activation was determined. (Quantitative EC50 value not provided in text/figures). [1]
APY29 promotes the oligomerization of IRE1α. In cross-linking experiments with increasing concentrations of IRE1α, the presence of a saturating concentration of APY29 (200 μM) enhanced the ratio of oligomeric (dimers and higher-order species) to monomeric IRE1α compared to DMSO control. [1]
In competition experiments, increasing concentrations of APY29 progressively reversed the inhibition of IRE1α RNase activity caused by a fixed concentration of the type II inhibitor, compound 3. This suggests that APY29 and compound 3 exert their opposing effects on RNase activity through the same ATP-binding site. [1]
Biochemical footprinting experiments using isotope-coded affinity tag (ICAT) reagents showed that APY29 binds to the ATP-binding site of IRE1α. It shielded Cys645 (located in the kinase hinge region) from alkylation, and slowed the alkylation rate of Cys715 (located in the activation loop). This pattern is consistent with APY29 stabilizing a DFG-in conformation of the activation loop. [1]
Molecular docking studies predicted that APY29 binds favorably to a DFG-in conformation of the human IRE1α ATP-binding site, forming hydrogen bonds with the kinase hinge region via its pyrazole ring and occupying the adenine pocket with its pyrimidine moiety. No favorable poses were obtained for APY29 bound to a DFG-out conformation model. [1]

XBP1 mRNA Splicing Assay in INS-1 Cells: Rat insulinoma INS-1 cells were pre-treated for 1 hour with varying concentrations of APY29 or DMSO control. Endoplasmic reticulum (ER) stress was then induced by treating the cells with 6 nM thapsigargin (Tg) for 4 hours. Total RNA was extracted, reverse transcribed, and XBP1 cDNA was amplified by PCR. The products were resolved on agarose gels, and the ratio of spliced XBP1 (XBP1s) to total XBP1 (spliced + unspliced) was quantified. APY29 demonstrated dose-dependent opposing effects on ER stress-induced XBP1 mRNA splicing. (Specific EC50/IC50 values not provided). [1]
IRE1α Phosphorylation Assay in INS-1 Cells: INS-1 cells were pre-treated with compounds, followed by ER stress induction with Tg. Cell extracts were analyzed by immunoblotting using anti-total IRE1α and anti-phospho IRE1α antibodies. The results showed that APY29 inhibits IRE1α autophosphorylation in cells. [1]

[1]. Divergent allosteric control of the IRE1α endoribonuclease using kinase inhibitors. Nat Chem Biol. 2012 Dec;8(12):982-9.

APY29 is a predicted type I kinase inhibitor that stabilizes the active (DFG-in) conformation of the IRE1α kinase domain. [1]
By stabilizing this active kinase conformation, APY29 acts as an allosteric activator of the adjacent RNase domain. This demonstrates that ligand occupancy of the kinase ATP-binding site alone can trigger RNase activation, even without upstream ER stress or autophosphorylation. [1]
The divergent effects of APY29 (type I) and compound 3 (type II) on IRE1α's RNase activity—activation vs. inhibition—highlight the potential for pharmacologically controlling this master UPR regulator in both directions by targeting its kinase domain with different classes of ATP-competitive inhibitors. [1]

C17H16N8

332.371

332.149

1216665-49-4

42627755

White to yellow solid powder

1.6±0.1 g/cm3

782.3±70.0 °C at 760 mmHg

426.9±35.7 °C

0.0±2.7 mmHg at 25°C

1.861

1.66

4

6

5

25

458

0

C1CC1C2=CC(=NN2)NC3=NC(=NC=C3)NC4=CC5=C(C=C4)N=CN5

WJNBSTLIALIIEW-UHFFFAOYSA-N

InChI=1S/C17H16N8/c1-2-10(1)13-8-16(25-24-13)22-15-5-6-18-17(23-15)21-11-3-4-12-14(7-11)20-9-19-12/h3-10H,1-2H2,(H,19,20)(H3,18,21,22,23,24,25)

2-N-(3H-benzimidazol-5-yl)-4-N-(5-cyclopropyl-1H-pyrazol-3-yl)pyrimidine-2,4-diamine

APY29 APY 29 APY-29

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 : ~53.33 mg/mL (~160.46 mM)
H2O : ~5 mg/mL (~15.04 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (15.04 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 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 (15.04 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: ≥ 5 mg/mL (15.04 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 50.0 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.)