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APY29

Alias: APY29 APY 29 APY-29
Cat No.:V7033 Purity: ≥98%
APY29 is an ATP competitive inhibitor, a specific allosteric modulator of IRE1α, which inhibits the autonomous phosphorylation of IRE1α by binding to the ATP binding pocket, with IC50 of 280 nM.
APY29
APY29 Chemical Structure CAS No.: 1216665-49-4
Product category: New1
This product is for research use only, not for human use. We do not sell to patients.
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Product Description
APY29 is an ATP competitive inhibitor, a specific allosteric modulator of IRE1α, which inhibits the autonomous phosphorylation of IRE1α by binding to the ATP binding pocket, with IC50 of 280 nM. APY29 can also allosterically activate the adjacent RNase domain of IRE1α.
Biological Activity I Assay Protocols (From Reference)
ln Vitro
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].
References

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

These protocols are for reference only. InvivoChem does not independently validate these methods.
Physicochemical Properties
Molecular Formula
C17H16N8
Molecular Weight
332.371
Exact Mass
332.149
CAS #
1216665-49-4
PubChem CID
42627755
Appearance
White to yellow solid powder
Density
1.6±0.1 g/cm3
Boiling Point
782.3±70.0 °C at 760 mmHg
Flash Point
426.9±35.7 °C
Vapour Pressure
0.0±2.7 mmHg at 25°C
Index of Refraction
1.861
LogP
1.66
Hydrogen Bond Donor Count
4
Hydrogen Bond Acceptor Count
6
Rotatable Bond Count
5
Heavy Atom Count
25
Complexity
458
Defined Atom Stereocenter Count
0
InChi Key
WJNBSTLIALIIEW-UHFFFAOYSA-N
InChi Code
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)
Chemical Name
2-N-(3H-benzimidazol-5-yl)-4-N-(5-cyclopropyl-1H-pyrazol-3-yl)pyrimidine-2,4-diamine
Synonyms
APY29 APY 29 APY-29
HS Tariff Code
2934.99.9001
Storage

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Shipping Condition
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
Solubility Data
Solubility (In Vitro)
DMSO : ~53.33 mg/mL (~160.46 mM)
H2O : ~5 mg/mL (~15.04 mM)
Solubility (In Vivo)
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.

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.


 (Please use freshly prepared in vivo formulations for optimal results.)
Preparing Stock Solutions 1 mg 5 mg 10 mg
1 mM 3.0087 mL 15.0435 mL 30.0870 mL
5 mM 0.6017 mL 3.0087 mL 6.0174 mL
10 mM 0.3009 mL 1.5043 mL 3.0087 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.

Calculator

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An example of molarity calculation using the molarity calculator is shown below:
What is the mass of compound required to make a 10 mM stock solution in 5 ml of DMSO given that the molecular weight of the compound is 350.26 g/mol?
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  • The answer of 17.513 mg appears in the Mass box. In a similar way, you may calculate the volume and concentration.

Dilution Calculator allows you to calculate how to dilute a stock solution of known concentrations. For example, you may Enter C1, C2 & V2 to calculate V1, as detailed below:

What volume of a given 10 mM stock solution is required to make 25 ml of a 25 μM solution?
Using the equation C1V1 = C2V2, where C1=10 mM, C2=25 μM, V2=25 ml and V1 is the unknown:
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  • The answer of 62.5 μL (0.1 ml) appears in the Volume (Start) box
g/mol

Molecular Weight Calculator allows you to calculate the molar mass and elemental composition of a compound, as detailed below:

Note: Chemical formula is case sensitive: C12H18N3O4  c12h18n3o4
Instructions to calculate molar mass (molecular weight) of a chemical compound:
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Definitions of molecular mass, molecular weight, molar mass and molar weight:
  • Molecular mass (or molecular weight) is the mass of one molecule of a substance and is expressed in the unified atomic mass units (u). (1 u is equal to 1/12 the mass of one atom of carbon-12)
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Reconstitution Calculator allows you to calculate the volume of solvent required to reconstitute your vial.

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In vivo Formulation Calculator (Clear solution)
Step 1: Enter information below (Recommended: An additional animal to make allowance for loss during the experiment)
Step 2: Enter in vivo formulation (This is only a calculator, not the exact formulation for a specific product. Please contact us first if there is no in vivo formulation in the solubility section.)
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Calculation results

Working concentration mg/mL;

Method for preparing DMSO stock solution mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.

Method for preparing in vivo formulation:Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.

(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
             (2) Be sure to add the solvent(s) in order.

Biological Data
  • Interaction of ATP-competitive inhibitors with the bifunctional kinase/RNase, IRE1α (a) Proposed binding modes of type I and type II kinase inhibitors with the ATP-binding pocket of IRE1α. Left panel shows the contacts the type I inhibitor APY29 forms with yeast IRE1α (PDB code 3SDJ)18. The right panel shows the proposed contacts a type II inhibitor 1 forms with IRE1α based on the co-crystal structure of the same inhibitor bound to Src (PDB code 3EL8) (also see Supplementary Fig. 3). (b) XBP1 RNA minisubstrate assay used for screening IRE1α modulators. The recombinant human IRE1α—IRE1α*—used in the assay spans residues 469–977, which includes the cytosolic kinase and RNase domains. Cleavage of the 5’FAM-3’BHQ-labeled XBP1 minisubstrate by IRE1α* results in FRET-dequenching. [1].Wang L, et al. Divergent allosteric control of the IRE1α endoribonuclease using kinase inhibitors. Nat Chem Biol. 2012 Dec;8(12):982-9.
  • APY29 and 3 divergently modulate the RNase activity and oligomerization state of IRE1α* (a) Inhibition of IRE1α* autophosphorylation in vitro by APY29 and 3. Normalized autophosphorylation levels and IC50 values for both compounds are shown. (b) λ-PPase treatment of IRE1α* produces dephosphorylated IRE1α* (dP-IRE1α*). Immunoblots using anti-IRE1α and anti-phospho IRE1α antibodies are shown. (c) RNase activities of IRE1α* and dP-IRE1 α* under varying concentrations of APY29 or 3 per the assay of Figure 1b. EC50 values were determined by fitting normalized fluorescence intensities (mean ± SD, n = 3). (d) Urea PAGE of XBP1 mini-substrate cleavage by IRE1α* and dP-IRE1α* with and without 3 or APY29. (e) RNase competition assays between APY29 and 3. The red line shows IRE1α* RNase activity under fixed 3 and varying APY29 concentrations. The black line shows IRE1α* RNase activity under fixed APY29 and varying 3 concentrations. The blue line shows IRE1α* RNase activity under fixed STF-083010 and varying APY29 concentrations (mean ± SD, n = 3).[1].Wang L, et al. Divergent allosteric control of the IRE1α endoribonuclease using kinase inhibitors. Nat Chem Biol. 2012 Dec;8(12):982-9.
  • Characterization of 3's interaction with the ATP-binding site of IRE1α Results of the ICAT footprinting experiments with IRE1α*. Alkylation rates were measured in the presence of DMSO (black), APY29 (blue) (20 µM), or 3 (red) (20 µM) (mean ± SD, n = 3). (a) Alkylation rate of Cys572. (b) Alkylation rate of Cys645. (c) Alkylation rate of Cys715. (d) A molecular model of 3's interaction with the ATP-binding site of IRE1α (grey). IRE1α is in the DFG-out inactive conformation. The imidazopyrazine ring of 3 occupies the adenine pocket and the 3-trifluoromethylurea occupies the DFG-out pocket. No favorable poses for 3 bound to the DFG-in conformation of IRE1α could be determined.[1].Wang L, et al. Divergent allosteric control of the IRE1α endoribonuclease using kinase inhibitors. Nat Chem Biol. 2012 Dec;8(12):982-9.
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