Kinase experiment:

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[33P]ATP, final concentrations) and lipid vesicles containing 18 M PtdIns and 250 M of PtdSer (final concentrations), in the presence of inhibitors or DMSO. Kinase reaction is stopped by adding 250 g of Neomycin-coated Scintillation Proximity Assay (SPA) bead and proceeded.

Cell experiment:

A total of 5×105 BDC2.5 splenocytes and 50 μg/mL BDC2.5-peptide are incubated in vitro in a 96-well round-bottom plate for 48 h. Then the cultures are pulsed with 1 μCi of tritiated thymidine [3H] to determine cell proliferation.

Animal experiment:

In this study, rats are bred for one week to affirm body weight and then randomLy divided into four experimental groups: (a) control group (rats are given vehicle only); (b) BLM group (rats are induced with BLM); (c) BLM + 25 mg/kg AS605240 group (rats are induced with BLM and then administrated with 25 mg/kg AS605240); (d) BLM + 50 mg/kg AS605240 group (the same protocol as the former group except a different dose of 50 mg/kg AS605240). In addition, five rats are given 50 mg/kg AS605240 only to detect whether AS605240 has any side effect simultaneously as the previous four groups. Rats in (c), (d) and AS605240-given-only group are administered orally 25, 50 and 50 mg/kg AS605240 by gavage while rats in control group and BLM group are given only equivalent saline at day-1 (the day rats are given BLM is marked as day-0). The same dosage is maintained once everyday for 28 days.

AS-605240 is a selective PI3K γ inhibitor with an IC50 of 8 nM. [1]

PI3Ks are a family of enzymes, which phosphorylate the 3’- OH position of the inositol ring of phosphoinositides.They have been divided into three classes on the basis of structural features and in vitro lipid substrate specificity. The three class-Ia PI3-kinases (p110 α / β / δ ) and the sole class-Ib PI 3-kinase (p110 γ ) couple growth factor receptors and G-protein-coupled receptors, respectively, to a wide range of downstream pathways. Signal transduction via the PI3K/Akt pathway is essential for regulating cellular responses, including proliferation, survival, migration, motility and tumorigenesis, in a variety of cell types. The key selectivity features were identified by co-crystallization with PI3Kγ, which revealed that thiazolidinedione nitrogen makes a salt – bridge interaction with the side chain of Lys-833, and also forms the link to Val-882.[1,2]

AS-605240 is a cell permeable inhibitor for PI3Ks and inhibits p110 α/β/γ/δ in vitro?with IC50 values of 0.06, 0.27, 0.008 0.3 μΜ, respectively. AS-605240 was tested its ability to inhibit C5a-mediated PKB phosphorylation in RAW264 mouse macrophages and AS-605240 showed an IC50 of 0.09 mM. [1]

As in many in?ammatory processes, neutrophils are predominant in the initial in?ux of leukocytes, followed by monocytes-macrophages and lymphocytes. in vivo efficacy in blocking leukocyte migration assays were conducted, AS-605240 was tested in two different mouse models of peritonitis, including RANTES-induced or thioglycollate, the outcome showed ED50 of 9.1 mg/kg and 10 mg/kg respectively. AS-605240 also show that it is an orally active, selective PI3Kγ inhibitor suppresses joint inflammation and damage in a mouse model of collagen-induced arthritis.[1]

References:
[1]. Camps M, Rückle T, Ji H, et al. Blockade of PI3Kγ suppresses joint inflammation and damage in mouse models of rheumatoid arthritis[J]. Nature medicine, 2005, 11(9): 936-943.
[2]. Marone R, Cmiljanovic V, Giese B, et al. Targeting phosphoinositide 3-kinase—moving towards therapy[J]. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 2008, 1784(1): 159-185.