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Biological Activity
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Description
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AS-604850 is a selective, ATP-competitive PI3Kγ inhibitor with IC50 of 250 nM. |
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Targets
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PI3Kγ |
PI3Kα |
PI3Kβ |
PI3Kδ |
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IC50 |
250 nM |
4.5 μM |
> 20 μM |
> 20 μM [1] |
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In Vitro
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AS-604850 is ATP-competitive PI3Kγ inhibitor with Ki values of 0.18 μM. AS-604850 is isoform selective inhibitor of PI3Kγ with over 30-fold selectivity for PI3Kγ and β, and 18-fold selectivity over PI3Kα. (PI3Kα: IC50 = 4.5 μM, PI3Kγ and β: IC50 > 20μM) AS-604850 is capable of inhibiting C5a-mediated PKB phosphorylation in RAW264 mouse macrophages with an IC50 with 10 μM. AS-604850 blocks MCP-1–mediated chemotaxis in Pik3cg +/+ monocytes in a concentration- dependent manner, with an IC50 of 21 mM, but dosn’t affect chemotaxis in Pik3cg-/- cells, indicating that AS-604850 acts through PI3Kγ. [1] AS-604850 diminishes glycochenodeoxycholate (GCDC) induced Akt-phosphorylation and apoptosis in rat hepatocytes. AS-604850 diminishes bile salt-induced apoptosis in HepG2 Ntcp and Huh7-Ntcp cells. [2] AS604850 causes a concentration-dependent suppression of chemotactic responses of EoL-1 cells and blood eosinophils to platelet-activating factor (PAF). [3] |
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In Vivo
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AS-604850 reduces RANTES-induced peritoneal neutrophil recruitment with a ED50 of 42.4 mg/kg. In the thioglycollate-induced peritonitis model, oral administration of 10 mg/kg AS-604850 results in a 31% reduction of neutrophil recruitment. [1] |
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Clinical Trials
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Features
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Protocol
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Kinase Assay
[1]
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| In vitro PI3Kγ Kinase Assay |
Human PI3Kγ (100 ng) is incubated at RT with kinase buffer (10 mM MgCl2, 1 mM β-glycerophosphate, 1 mM DTT, 0.1 mM Na3VO4, 0.1% Na Cholate and 15 M ATP/100 nCi γ[33]ATP, final concentrations) and lipid vesicles containing 18 M PtdIns and 250 M of PtdSer (final concentrations), in the presence of AS-252424 or DMSO. Kinase reaction is stopped by adding 250 g of Neomycin-coated Scintillation Proximity Assay (SPA) beads and preceded. |
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Cell Assay
[2]
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| Cell Lines |
HepG2 Ntcp and Huh7-Ntcp cells |
| Concentrations |
2.5 μM |
| Incubation Time |
2 - 4 hours |
| Methods |
Hepatocyte cultures are treated with diluent (DMSO), 25 μM TLC, 250 μM TCDC, 50 μM GCDC, or 50 ng/ml Fas for 2–4 hours HepG2-Ntcp and Huh7-Ntcp cells are treated with DMSO, 20 μM TLC, 75 μM TCDC or GCDC, 200 μM etoposide or 200 ng/ml TNFa plus 28 ng/ml actinomycin D for 2–4 hours. The number of apoptotic cells is determined morphologically using fluorescent staining and expressed as % of cells. Apoptosis is confirmed in human cell lines by determination of caspase-3/-7 activity and in rat hepatocytes by detection of the 17 kDa proteolytic cleavage fragment of caspase-3 by immunoblotting; equal protein loading is monitored by immunoblotting for actin. |
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Animal Study
[1]
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| Animal Models |
RANTES (0.5 mg/kg in 200 ml saline) or thioglycollate (40 ml/kg) are intraperitoneally injected to C3H mice to induce peritonitis mouse models |
| Formulation |
AS-604850 is dissolved in vehicle (0.5% carboxymethylcellulose/0.25% Tween-20) and adjusted the solution to 10 mL/kg of body weight. |
| Doses |
0, 1, 3, 10 or 30 mg/kg for RANTES, 10 mg/kg for thioglycollate |
| Administration |
Oral administration 30 or 15 minutes before injection of RANTES or thioglycollate |
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References |
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[1] Camps M, et al, Nat Med, 2005, 11(9), 936-943.
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[2] Hohenester S, et al, J Hepatol, 2010, 53(5), 918-926.
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[3] Hasan AM, et al, Int Immunopharmacol, 2010, 10(9), 1017-1021.
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