CNX-1351 is a potent, isoform-selective, and targeted covalent inhibitor of PI3Kα.

PI3Kα:6.8 nM

Biochemistry assay: The motor domains of KSP (amino acids 1–360) is expressed as in Escherichia coli BL21(DE3) as COOH-terminal 6-his fusion proteins. Bacterial pellets are lysed in a microfluidizer with a lysis buffer [50 mM Tris-HCl; 50 mM KCl, 10 mM imidazole, 2 mM MgCl2, 8 mM β-mercaptoethanol, 0.1 mM ATP (pH 7.4)], and proteins are purified using Ni-NTA agarose affinity chromatography, with an elution buffer consisting of 50 mM PIPES, 10% sucrose, 300 mM imidazole, 50 mM KCl, 2 mM MgCl2, mM β-mercaptoethanol, and 0.1 mM ATP (pH 6.8). Steady-state measurements of ATPase activity are performed with a pyruvate kinase–lactate dehydrogenase detection system that coupled the appearance of ADP with oxidation of NADH. Absorbance changes are monitored at 340 nm. All biochemical experiments are performed in PEM25 buffer [25 mM Pipes/KOH (pH 6.8), 2 mM MgCl2, 1 mM EGTA] supplemented with 10 μM SB 743921 for experiments involving microtubules. Rates of ADP release are measured in a stopped-flow apparatus; the decrease in fluorescence of MANT-ATP is monitored. Rates of Pi release are measured in a stopped-flow apparatus, using bacterial phosphate binding protein modified with 7-diethylamino-3-((((2 maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) dye. Ki estimates of KSP inhibitors are extracted from the dose–response curves, with explicit correction for enzyme concentration. Tubulin polymerization by measuring changes in absorbance at 340 nm is monitored. The assay is performed in 100-μL volumes in 96-well half-area microtiter plates, using a microplate reader with the incubation temperature set at 37 °C.