Nemiralisib hydrochloride  (Synonyms: GSK2269557)

Inhibition of PI3Kinase enzymatic activity is determined using a homogeneous time resolved fluorescence (HTRF) kit assay format. Reactions are performed in assay buffer containing 50 mM HEPES, pH 7.0, 150 mM NaCl, 10 mM MgCl₂, <1 % cholate (w/v), <1 % CHAPS (w/v), 0.05 % sodium azide (w/v) and 1 mM DTT. Enzymes are preincubated with compound, serially diluted 4-fold in 100 % DMSO, for 15 mins prior to reaction initiation upon addition of substrate solution containing ATP at K_m for the specific isoform tested (PI3Kα at 250 μM, PI3Kβ at 400 μM, PI3Kδ at 80 μM and PI3Kγ at 15 μM), PIP2 at either 5 μM (PI3Kδ) or 8 μM (PI3Kα, PI3Kβ and PI3Kγ) and 10 nM biotin-PIP3. Assays are quenched after 60 mins by addition of a quench/detection solution prepared in 50 mM HEPES pH 7.0, 150 mM NaCl, <1 % cholate, <1 % Tween 20, 30 mM EDTA, 40 mM potassium fluoride and 1 mM DTT containing 16.5 nM GRP-1 PH domain, 8.3 nM Streptavidin-APC and 2 nM Europium-anti-GST, and are left for a further 60 mins in the dark to equilibrate prior to reading using a BMG RubyStar plate reader. Ratio data are normalised to high (no compound) and low (no enzyme) controls prior to fitting using a logistical four parameter equation to determine IC₅₀^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Rats^[1]
In vivo pharmacokinetics is tested in Sprague Dawley male rats. Compounds (e.g., GSK2269557) are administered discretely by the oral or intravenous routes, at a dose level of 3 and 1 mg/kg respectively (n=2 rats/route). Compounds (e.g., GSK2269557) are formulated as a solution in DMSO:PEG200:water (5:45:50 v/v/v) at a dose volume of 6 (oral) and 2 (intravenous) mL/kg. All animals are serially bled from the tail vein and blood samples collected over a time-course of 0-7 h are submitted to LC-MS/MS analysis for the quantification of the parent compound. The main pharmacokinetic parameters are estimated by non-compartmental analysis.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Down K et al. Optimization of Novel Indazoles as Highly Potent and Selective Inhibitors of Phosphoinositide 3-Kinase δ for the Treatment of Respiratory Disease.  [Content Brief]