A Chemical Proteomics Approach for the Search of Pharmacological Targets of the Antimalarial Clinical Candidate Albitiazolium in Plasmodium falciparum Using Photocrosslinking and Click Chemistry
Figure 3
Labeling and detection of UA1936-bound parasite proteins.
(A) In-gel fluorescence of UA1936-bound parasite proteins. Saponin isolated parasites were incubated with 100 µM UA1936 and irradiated or not at 254 nm for 2.5 min, parasite proteins were clicked to 10 µM Alexa 647 alkyne. Then 50 µg protein of each sample were separated by SDS-PAGE and imaged in a LiCor OdysseyFc infrared imaging system under an infrared camera. The gel was subsequently stained with Coomassie blue and scanned. (B) and (C). Fluorescence microscopy of UA1936 and its localization in P. falciparum-infected red blood cells. P. falciparum-infected red blood cells (asynchronous cultures) were cultured with or without 100 µM UA1936 for 1 h and then fixed with 4% paraformaldehyde. In-cell click chemistry was performed with 2.5 µM Alexa 488 alkyne (green) or 2.5 TAMRA alkyne (red) for 30 min. For immunodetection, P. falciparum-infected red blood cells were incubated with specific antibodies against the endoplasmic reticulum markers ERC and BiP, against the cis-Golgi marker ERD2, against the food vacuole membrane marker CRT and against the apicoplast marker ACP. Samples were mounted with Vectashield mounting medium with DAPI (blue) and observed under a Zeiss Axioimager epifluorescence microscope.