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Purity: ≥98%
VPS34-IN2 (PIK-III; Vps34-PIK-III) is a novel, potent and selective inhibitor of VPS34 (IC50 = 18 nM) with the ability to modulate autophagy in Vivo. PIK-III blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo. In contrast to related kinases like PI(3)K, PIK-III binds to a particular hydrophobic pocket. The acute inhibition of autophagy and de novo lipidation of LC3 by PIK-III results in the stabilization of autophagy substrates.
| Targets |
Vps34 (IC50 = 18 nM); PI(3)Kδ (IC50 = 1.2 μM); PI(3)Kγ (IC50 = 3.04 μM); PI(3)Kα (IC50 = 3.96 μM)
VPS34 (IC₅₀ = 15 nM)[1] PI3Kδ (IC₅₀ = 820 nM)[1] TAK1 (10-fold less selective than VPS34, IC₅₀ not specified)[1] >280 other kinases including PI3Kα, β, γ (IC₅₀ >>100-fold selectivity over VPS34)[1] |
|---|---|
| ln Vitro |
VPS34 enzymatic function is essential for LC3 lipidation in mammalian cells and PIK-III is a robust inhibitor of autophagy and LC3 lipidation in mammalian cells. In H4 cells, both in basal conditions and when autophagy is induced using the mTOR inhibitor AZD8055, PIK-III reduces the formation of autolysosomes and boosts the cytosolic signal of LC3. PIK-III prevents the removal of mitochondria in a mitophagy model induced by CCCP. In H4 and PSN1 cells, treatment with PIK-III causes LC3-I levels to rise. In Panc10.05 cells, PIK-III raises the levels of LC3-II concurrently with LC3-I, indicating a cell type-specific response[1].
VPS34-IN2 inhibited VPS34 enzymatic activity with an IC₅₀ of 15 nM in a biochemical assay.[1] In a cellular GFP-FYVE reporter assay, which monitors PtdIns(3)P levels as a surrogate for VPS34 activity, VPS34-IN2 showed an IC₅₀ of 25 nM, indicating potent on-target cellular activity.[1] Treatment of DLD1 cells with VPS34-IN2 for 24 hours led to accumulation of autophagy substrates p62, NCOA4, NBR1, NDP52, and FTH1, similar to the known inhibitor PIK-III.[1] In DLD1 cells, treatment with VPS34-IN2 increased the levels of both lipidated (LC3-II) and non-lipidated (LC3-I) forms of LC3, consistent with inhibition of autophagic flux.[1] |
| ln Vivo |
The single-dose, fast-absorbing Vps34-PIK-III (10 mg/kg; valve) has a strong valve biological utilization (F% = 47) and a modest mean systemic clearance (30 mL/min/kg, or around 33% of hepatic blood flow) [1].
In RKO colon cancer tumor-bearing nude mice, oral administration of VPS34-IN2 at 50 mg/kg twice daily for 7 days resulted in a time-dependent accumulation of LC3-II in tumor samples, indicating inhibition of autophagy in vivo.[1] No reduction in tumor volume was observed during this 7-day treatment period.[1] |
| Enzyme Assay |
The biochemical inhibition of VPS34 was measured using a luminescence-based ATP detection assay. The assay determines the IC₅₀ value by measuring the depletion of ATP upon kinase activity.[1]
Selectivity profiling against a panel of over 280 kinases (including lipid and protein kinases) was performed to establish the selectivity profile of the compound.[1] |
| Cell Assay |
To determine whether inhibition of VPS34 function impacts autophagy，LC3 and known autophagy substrates such as damaged mitochondria or the autophagy cargo receptor p62 are monitored. H4 cells expressing mCherry-GFP-LC3 are treated overnight with the listed substances, fixed, stained with Hoechst 33342, and imaged by automated acquisition. HeLa cells expressing GFP-Parkin are treated with PIK-III for 12 hours, then added CCCP for another 12 hours. The cells are then fixed, stained for endogenous Tom20, and imaged.
A GFP-FYVE reporter assay was used to assess cellular VPS34 inhibition. Cells expressing the reporter were treated with compounds, and the redistribution or intensity of the GFP-FYVE signal, which binds to PtdIns(3)P, was measured to generate IC₅₀ values.[1] For autophagy substrate analysis, DLD1 cells were treated with compounds for 24 hours. Cell lysates were then analyzed by immunoblotting to detect proteins such as p62, NCOA4, and LC3.[1] |
| Animal Protocol |
Animal/Disease Models: C57BL/6 mice[1].
Doses: 10 mg/kg; 2 mg/kg Route of Administration: oral; intravenous (iv) (iv)injection; individual Experimental Results: 1.19 pharmacokinetic/PK/PK parameters of Vps34-PIK-III in C57BL/6 mice [1]. IV (2 mg/kg) PO (10 mg/kg) Tmax (h) 0.7 Cmax (nM) 2994 AUCinf (nM·h) 2855 6725 t1/2 (h) 1.2 CL (mL/min/kg) 30 Vdss ( Liter/kg)1.5F(%)47% The pharmacokinetic profile was determined in C57BL/6 mice. VPS34-IN2 was administered as a single intravenous dose (2 mg/kg) or a single oral dose (10 mg/kg). Blood samples were collected at various time points to determine PK parameters.[1] For the in vivo efficacy/pharmacodynamics study, RKO tumor-bearing nude mice were treated orally with VPS34-IN2 at a dose of 50 mg/kg, twice daily (BID), for 7 days. Tumor samples were collected at the end of the treatment for immunoblot analysis of LC3-II levels.[1] |
| ADME/Pharmacokinetics |
In C57BL/6 mice, the mean systemic clearance (CL) was 30 mL/min/kg and the steady-state volume of distribution (Vss) was 1.5 L/kg after a single intravenous injection (2 mg/kg). [1]
The terminal half-life (t₁/₂) after intravenous administration was 1.2 hours. [1] After a single oral administration (10 mg/kg), the peak plasma concentration (Cmax) was 2994 nM and the time to peak concentration (Tmax) was 0.7 hours. [1] The oral bioavailability (F) was calculated to be 47%. [1] |
| References |
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| Additional Infomation |
VPS34-IN2 (compound 19) was developed through medicinal chemistry optimization of lead compounds screened in high-throughput screening, aiming to achieve high efficiency and selectivity for VPS34, making it superior to other PI3K subtypes and multiple kinases. [1]
The X-ray co-crystal structure of VPS34 and VPS34-IN2 confirmed its binding mode. The compound binds to the ATP binding site, where the aminopyrimidine moiety interacts with the hinge region (Ile685), and the cyclopropyl group occupies the hydrophobic pocket specific to VPS34, thus conferring selectivity. [1] VPS34-IN2 has been described as the first publicly disclosed selective VPS34 inhibitor with high oral bioavailability and has been shown to inhibit autophagy in vivo, making it an important chemical tool for studying autophagy biology. [1] |
| Molecular Formula |
C17H17N7
|
|---|---|
| Molecular Weight |
319.3638
|
| Exact Mass |
319.155
|
| Elemental Analysis |
C, 63.93; H, 5.37; N, 30.70
|
| CAS # |
1383716-40-2
|
| Related CAS # |
1383716-40-2
|
| PubChem CID |
67983123
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| Appearance |
White to off-white solid powder
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| LogP |
3.261
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| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
7
|
| Rotatable Bond Count |
5
|
| Heavy Atom Count |
24
|
| Complexity |
396
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
N1C(N([H])[H])=NC([H])=C(C2C([H])=C([H])N=C(N([H])C3C([H])=C([H])N=C([H])C=3[H])N=2)C=1C([H])([H])C1([H])C([H])([H])C1([H])[H]
|
| InChi Key |
XXSDLQLNIVFIJI-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H17N7/c18-16-21-10-13(15(23-16)9-11-1-2-11)14-5-8-20-17(24-14)22-12-3-6-19-7-4-12/h3-8,10-11H,1-2,9H2,(H2,18,21,23)(H,19,20,22,24)
|
| Chemical Name |
4-(cyclopropylmethyl)-5-[2-(pyridin-4-ylamino)pyrimidin-4-yl]pyrimidin-2-amine
|
| Synonyms |
Vps34 PIK-III; Vps34-PIK III; VPS34-IN2; VPS34-IN 2; Vps34-PIK-III; PIK-III; PIK III; PIKIII; VPS34-IN-2
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO: 31~63 mg/mL (97~197.3 mM)
Ethanol: ~63 mg/mL (~197.3 mM) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.83 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.83 mM) (saturation unknown) 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. 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 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (7.83 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1313 mL | 15.6563 mL | 31.3126 mL | |
| 5 mM | 0.6263 mL | 3.1313 mL | 6.2625 mL | |
| 10 mM | 0.3131 mL | 1.5656 mL | 3.1313 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.
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.
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