A yeast-based in vivo bioassay to screen for class I phosphatidylinositol 3-kinase specific inhibitors.
The phosphatidylinositol 3-kinase (PI3K) pathway couples receptor-mediated signaling to essential cellular functions by generating the lipid second messenger phosphatidylinositol-3,4,5-trisphosphate. This pathway is implicated in multiple aspects of oncogenesis. A low-cost bioassay that readily measures PI3K inhibition in vivo would serve as a valuable tool for research in this field. Using heterologous expression, we have previously reconstituted the PI3K pathway in the model organism Saccharomyces cerevisiae. On the basis of the fact that the overproduction of PI3K is toxic in yeast, we tested the ability of commercial PI3K inhibitors to rescue cell growth. All compounds tested counteracted the PI3K-induced toxicity. Among them, 15e and PI-103 were the most active. Strategies to raise the intracellular drug concentration, specifically the use of 0.003% sodium dodecyl sulfate and the elimination of the Snq2 detoxification pump, optimized the bioassay by enhancing its sensitivity. The humanized yeast-based assay was then tested on a pilot scale for high-throughput screening (HTS) purposes using a collection of natural products of microbial origin. From 9600 extracts tested, 0.6% led to a recovery of yeast growth reproducibly, selectively, and in a dose-dependent manner. Cumulatively, we show that the developed PI3K inhibition bioassay is robust and applicable to large-scale HTS.
Dpto. Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Plaza de Ramón y Cajal, Madrid, Spain.
Teresa
T
Fernández-Acero
Isabel
I
Rodríguez-Escudero
Francisca
F
Vicente
Maria Cândida
MC
Monteiro
José R
JR
Tormo
Juan
J
Cantizani
María
M
Molina
Víctor J
VJ
Cid
D064413:3-Phosphoinositide-Dependent Protein Kinases; D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D005663:Furans / Q000494:pharmacology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D047428:Protein Kinase Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D012967:Sodium Dodecyl Sulfate
D018528:ATP-Binding Cassette Transporters; D005663:Furans; C522973:PI103; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D011743:Pyrimidines; C101797:SNQ2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012967:Sodium Dodecyl Sulfate; D064413:3-Phosphoinositide-Dependent Protein Kinases; D017346:Protein Serine-Threonine Kinases