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Purity: ≥98%
PIK-93, a phenylthiazole analog, is a novel, potent, and selective PI4K (PI4KIIIβ) inhibitor with antiviral effects (antienterovirus activity). It inhibits PI4K (PI4KIIIβ) with an IC50 of 19 nM and PI3Kα with an IC50 of 39 NM.
| Targets |
PI4KIIIβ (IC50 = 19 nM); PI4KIIIα (IC50 = 1.1 μM); p110γ (IC50 = 16 nM); p110α (IC50 = 39 nM); p110δ (IC50 = 120 nM); p110β (IC50 = 590 nM); PI3KC2β (IC50 = 140 nM); PI3KC2α (IC50 = 16 μM); hsVPS34 (IC50 = 320 nM); DNA-PK (IC50 = 64 nM); ATM (IC50 = 490 nM); mTORC1 (IC50 = 1.38 μM); ATR (IC50 = 17 μM)
PI4KIIβ (IC50 = 19 nM) [1]; PI3Kα and PI3Kγ (isoform-selective; complete inhibition of PI3Kγ-dependent Akt phosphorylation in cells at 0.5-1 μM) [2] |
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| ln Vitro |
PIK-93 inhibits PI3Kγ and PI4KIIIβ, with IC50 values of 16 nM and 19 nM, respectively. PIK-93 also inhibits other members of PI3Ks, including PI3Kα, β, and δ, with IC50 values of 39 nM, 0.59 μM, and 0.12 μM, respectively. Even at a concentration of 10 M, PIK-93 has no discernible inhibitory effect on a panel of other kinases[1]. PIK-93 (0.5 M-1 M) decreases the consolidation and stability of the leading edge formed after treatment with uniform f-Met-Leu-Phe (fMLP) in differentiated HL60 (dHL60) cells. The fMLP-dependent accumulation of total F-actin is altered by PIK-93, but not in terms of quantity. PIK-93 increases cell turning frequency while decreasing the chemotactic index in fMLP gradients[2]. In COS-7 cells, PIK-93 (250 nM) successfully prevents the buildup of FL-Cer and the CERT-PH domain in the Golgi. The same concentration of PIK-93 also significantly reduces the ability of endogenous ceramide to transform into sphingomyelin when labeled with [3H]serine. These facts suggest that PI4KIIIβ plays a critical role in the regulation of spingomyelin synthesis as well as ceramide transport between the ER and Golgi[3]. PIK-93 (300 nM) inhibits net Ca2+ entry and carbachol-induced translocation of TRPC6 to the plasma membrane in T6.11 cells[4]. According to a recent study, PIK-93 has anti-enterovirus properties because it inhibits the replication of the poliovirus (PV) and the hepatitis C virus (HCV), with EC50 values of 0.14 µM and 1.9 µM, respectively[5].
In dHL60 cells, treatment with PIK-93 (0.5-1 μM) completely inhibits fMLP-stimulated phosphorylation of Akt, a downstream effector of PIP3 [2]. PIK-93 impairs the consolidation and stability of the leading edge in response to uniform fMLP, causing formation of multiple transient pseudopods instead of a single stable front [2]. In chemotaxis assays using an fMLP gradient, PIK-93 substantially reduces the chemotactic index (CI) and triples the turning frequency of dHL60 cells, resulting in jerky trajectories with less persistent orientation toward the attractant source [2]. Despite these effects, PIK-93 does not significantly reduce total F-actin accumulation in response to fMLP [2]. |
| ln Vivo |
N/A
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| Enzyme Assay |
Standard TLC lipid kinase activity assays are used to calculate IC50 values. In order to carry out kinase reactions, a reaction mixture containing kinase, PIK-93 (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 µg/mL) must be prepared. A final concentration of 10 or 100 µM is used to start the reactions, and they are then allowed to continue for 20 min at room temperature. The starting material for the reactions is ATP containing 10 µCi of γ-32P-ATP. The addition of 105 µL 1N HCl and 160 l of CHCl3:MeOH (1:1) then ends the reactions in preparation for TLC analysis. The organic phase is transferred to a new tube using a gel loading pipette tip that has been CHCl3-precoated after the biphasic mixture has been vortexed and briefly centrifuged. In a 65:35 solution of n-propanol:1M acetic acid, this extract is spotted on TLC plates and developed for 3–4 hours. Following drying, the TLC plates are shown to a phosphorimager screen and quantitated. As a two-fold dilution from the highest concentration of 100 μM, kinase activity is typically assessed at 10–12 concentrations of PIK–93.
To determine the selectivity profile of PIK-93, in vitro kinase assays were performed using purified recombinant PI3K family members (15 enzymes) and other kinases. IC50 values were measured by incubating the kinase with varying concentrations of inhibitor and ATP, then quantifying product formation [1]. PIK-93 was found to potently inhibit PI4KIIβ with an IC50 of 19 nM in a lipid kinase assay using purified enzyme [1]. Crystal structures of PIK-93 bound to human p110γ were determined at 2.5-2.6 Å resolution. Recombinant human p110γ (residues 144-1102 with a C-terminal His6 tag) was purified from baculovirus-infected Sf9 cells. Crystals were grown at 17°C using sitting-drop vapor diffusion and soaked with inhibitor. Diffraction data were collected at a synchrotron radiation facility. The structure revealed hydrogen bonds between PIK-93 and Val882 (backbone amide and carbonyl) and Asp964 (side chain) [1]. |
| Cell Assay |
For actin staining, dHL60 cells are preincubated in suspension with PIK-93 or vehicle for 40 min, centrifuged for 5 min at 2000 rpm at room temperature in a J6-B centrifuge, resuspended in mHBSS containing the appropriate agent at the same concentration, and then allowed to adhere to fibronectin-covered coverslips for 3 min. Rhodamine-phalloidin (10 units/mL) is used to stain cells that have been fixed in 3.7% PFA for 15 minutes.
dHL60 cells were differentiated with DMSO and then preincubated in suspension with PIK-93 (0.5-1 μM, from a stock in DMSO, final DMSO 1.0%) for 40 minutes at room temperature. Cells were then centrifuged, resuspended in medium containing the same inhibitor concentration, allowed to adhere to fibronectin-coated coverslips, and stimulated with 100 nM fMLP for 3 minutes. After stimulation, cells were fixed with 3.7% paraformaldehyde and stained with rhodamine-phalloidin to visualize F-actin distribution [2]. For live-cell imaging of polarity, dHL60 cells expressing YFP-actin were preincubated with PIK-93 (1 μM) for 40 minutes, then plated on fibronectin-coated coverslips and stimulated with uniform 100 nM fMLP. Time-lapse images were acquired at 1-minute intervals using an inverted microscope with a cooled CCD camera [2]. Chemotaxis assays were performed using a micropipette containing fMLP to create a gradient. Cells preincubated with PIK-93 (0.5 μM) were placed on fibronectin-coated coverslips, and migration trajectories were tracked for 25 minutes. The chemotactic index (displacement in correct direction divided by path length) and turning frequency (deviations ≥60° between successive 2-minute vectors) were calculated [2]. To assess Akt phosphorylation, dHL60 cells were preincubated with PIK-93 (0.5-1 μM) for 40 minutes, then stimulated with 100 nM fMLP for 1 minute. Cell lysates were prepared and subjected to SDS-PAGE and Western blotting using anti-phospho-Akt (Thr308) antibody [2]. |
| Animal Protocol |
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| References |
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| Additional Infomation |
PIK-93 is one of the most polar inhibitors in the PI3K inhibitor panel with a calculated logP (CLogP) of 1.69 [1].
X-ray crystallography revealed that PIK-93 binds to p110γ in the ATP-binding pocket, making three hydrogen bonds: to the backbone amide and carbonyl of Val882 and to the side chain of Asp964. Unlike selective inhibitors such as PIK-39, PIK-93 adopts a flat conformation and projects into a deeper affinity pocket [1]. PIK-93 is a potent inhibitor of PI4KIIβ (IC50=19 nM) and represents the first potent synthetic PI4-kinase inhibitor, used to dissect the role of PI4-K isoforms in calcium signaling [1]. In neutrophil chemotaxis, PIK-93 (along with PIK-90) demonstrates that PI3Kγ is required for maintaining stable polarity by promoting both frontness (pseudopod stability) and backness (RhoA-dependent actomyosin contraction) via Cdc42 activation [2]. |
| Molecular Formula |
C14H16N3O4S2CL
|
|---|---|
| Molecular Weight |
389.87754
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| Exact Mass |
389.027
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| Elemental Analysis |
C, 43.13; H, 4.14; Cl, 9.09; N, 10.78; O, 16.41; S, 16.45
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| CAS # |
593960-11-3
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| Related CAS # |
593960-11-3;
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| PubChem CID |
6852167
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| Appearance |
White to off-white solid powder
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| Density |
1.48 g/cm3
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| Boiling Point |
611.8ºC at 760 mmHg
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| Flash Point |
323.8ºC
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| LogP |
4.122
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
24
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| Complexity |
543
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC(NC1=NC(C)=C(S1)C2=CC=C(C(S(=O)(NCCO)=O)=C2)Cl)=O
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| InChi Key |
JFVNFXCESCXMBC-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C14H16ClN3O4S2/c1-8-13(23-14(17-8)18-9(2)20)10-3-4-11(15)12(7-10)24(21,22)16-5-6-19/h3-4,7,16,19H,5-6H2,1-2H3,(H,17,18,20)
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| Chemical Name |
N-(5-(4-chloro-3-(N-(2-hydroxyethyl)sulfamoyl)phenyl)-4-methylthiazol-2-yl)acetamide
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| Synonyms |
PIK-93; PIK93; PIK 93
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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: ~78 mg/mL (200.1 mM)
Water: <1 mg/mL Ethanol: <1 mg/mL |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 4.55 mg/mL (11.67 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 45.5 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 (6.41 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 (6.41 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5%Propylene glycol:30mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5649 mL | 12.8245 mL | 25.6489 mL | |
| 5 mM | 0.5130 mL | 2.5649 mL | 5.1298 mL | |
| 10 mM | 0.2565 mL | 1.2824 mL | 2.5649 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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