Binding assays

For PI3K assays, 40 ml of reaction buffer (20 mM Tris/4 mM MgCl2/10 mM NaCl, pH 7.4) containing 50 mM D-myo-phosphatidylinositol 4,5-bisphosphate substrate and the desired PI3K isoform were aliquoted to 96-well plates; kinase concentrations were 250-500 ng/well, such that linear kinetics were achieved over 90 min. The compound to be tested was then added as 2.5 ml of a DMSO stock to final concentration range of 100 mM to 1 nM. Reactions were initiated by addition of 10 ml of ATP to a final concentration of 3 mM, and after 90 min, 50 ml of Kinase-Glo reagent added to quantify residual ATP levels; luminosity was measured using an Ultra 384 instrument. Control reactions omitting either test compound or substrate were also performed. IC50 values were derived from experimental data by nonlinear curve fitting.

Cell lines

Human umbilical vein EC

Preparation method

The solubility of this compound in DMSO is >3.5mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

10 μM, 24-72 h

Applications

In HUVECs, TG100-115 (10 μM) inhibited the VEGF-induced increase of total level of VE-cadherin. TG100-115 inhibited VEGF mediated phosphorylation of mTOR and p70S6 kinase. TG100-115 (125 nM to 10 μM) inhibited FGF-stimulated phosphorylation of Akt.

Animal models

Sprague–Dawley rats, Rodent and porcine myocardial ischemia (MI) models

Dosage form

intravenous injection, 5 mg/kg

Application

Pretreatment with TG100-115 (5 mg/kg) blocked both the edema and leukocytic infiltrate. In a rodent model of MI, TG100-115 (i.v. bolus 60 min after reperfusion) routinely reduced infarct size by ≥40%, with maximal efficacy reached by a dose of 0.5 mg/kg. In a porcine MI model, TG100-115 (0.5 mg/kg i.v. bolus 30 min after reperfusion) developed smaller infarcts.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

TG100-115 is an inhibitor of PI3Kγ and PI3Kδ with IC50 values of 83 nM and 235 nM, respectively. TG100-115 shows no obvious activity against PI3Kα and PI3Kβ [1].

TG100-115 remarkably reduced eosinophil accumulation, BALF levels of classic Th2 cytokine IL-13, perivascular and peribronchial leukocyte accumulations, and bronchiolar mucin accumulation in a murine model. Besides, TG100-115 showed to reduce airway hyper-responsiveness (AHR) by approximately 50% in asthmatic mice. Moreover, TG100-115 has been reported to reduce both smoke-and LPS-induced neutrophil and LPS-induced classic Th1 cytokine TNFα accumulations in mice [2].

References:
[1] Doukas J1, Wrasidlo W, Noronha G, Dneprovskaia E, Fine R, Weis S, Hood J, Demaria A, Soll R, Cheresh D. hosphoinositide 3-kinase gamma/delta inhibition limits infarct size after myocardial ischemia/reperfusion injury. Proc Natl Acad Sci U S A. 2006 Dec 26;103(52):19866-71.
[2] Doukas J1, Eide L, Stebbins K, Racanelli-Layton A, Dellamary L, Martin M, Dneprovskaia E, Noronha G, Soll R, Wrasidlo W, Acevedo LM, Cheresh DA. Aerosolized phosphoinositide 3-kinase gamma/delta inhibitor TG100-115 [3-[2,4-diamino-6-(3-hydroxyphenyl)pteridin-7-yl]phenol] as a therapeutic candidate for asthma and chronic obstructive pulmonary disease. J Pharmacol Exp Ther. 2009 Mar;328(3):758-65.