A stock solution of sapanisertib was serially diluted to yield final concentrations ranging from 1 to 500 nM in the assay. Synchronous ring-stage PKG (PF3D7_1436600) (12 (link)), PI4Kβ (PF3D7_0509800), and PI3K (PF3D7_0515300) cKD parasite lines, as well as a control cell line expressing an aptamer-regulatable fluorescent protein, were maintained in the presence of high aTc (500 nM) or low aTc (2 nM in the case of PKG and no aTc in the case of PI4Kβ and PI3K) and distributed into 384-well polystyrene microplates (Corning). Compounds were transferred to the parasite-containing plates using the Janus platform (PerkinElmer). DMSO and dihydroartemisinin treatment (500 nM) served as reference controls. Luminescence was measured after 72 hours using the Renilla-Glo Luciferase Assay System (Promega E2750) and the GloMax Discover Multimode Microplate Reader (Promega), and IC50 values were obtained from corrected dose-response curves using Graph-Pad Prism.
Protocol detail
Sapanisertib Dose-Response Assay in Malaria Parasites
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384-well polystyrene microplates
Janus platform
Renilla-Glo Luciferase Assay System
GloMax Discover Multimode Microplate Reader
Assay
Cell line
Dihydroartemisinin
Dmso
Luminescence
Parasite
Pi3k
Polystyrene
Prism
Promega
Protein
Renilla luciferase
Sapanisertib
Corresponding Organization : South African Medical Research Council
Other organizations : Columbia University Irving Medical Center, Pennsylvania State University, Massachusetts Institute of Technology, GlaxoSmithKline (Spain), University of Pretoria, University of the Witwatersrand, National Health Laboratory Service, University of California, San Diego
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Variable analysis
independent variables
- Concentration of sapanisertib in the assay, ranging from 1 to 500 nM
- Luminescence measured after 72 hours using the Renilla-Glo Luciferase Assay System
- IC50 values obtained from corrected dose-response curves
- Synchronous ring-stage PKG (PF3D7_1436600), PI4Kβ (PF3D7_0509800), and PI3K (PF3D7_0515300) cKD parasite lines, as well as a control cell line expressing an aptamer-regulatable fluorescent protein
- Presence of high aTc (500 nM) or low aTc (2 nM in the case of PKG and no aTc in the case of PI4Kβ and PI3K)
- DMSO treatment as a reference control
- Dihydroartemisinin treatment (500 nM) as a reference control
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