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 4
Flow chart for the UA1936-target fishing approach using photocrosslinking and click chemistry.
Free parasites were obtained by saponin treatment of P. falciparum-infected red blood cells (IRBC) and incubated at 37°C with 100 µM UA1936 or 100 µM UA2050 for 1 h in HEPES-buffered RPMI 1640 medium. Control experiments were also conducted without compound. In competitive experiments, free parasites were first incubated with 100 µM albitiazolium for 30 min and then with 100 µM of UA1936. Parasites were then irradiated at 254 nm for 2.5 min. After centrifugation and wash, parasites were lysed and 10 mg of parasite proteins were used for click chemistry reactions with the alkyne agarose resin. After stringent washes, the resin-bound proteins were digested with trypsin overnight. The peptides were analyzed by mass spectrometry in a LTQ-Orbitrap VELOS mass spectrometer. After spectral data analysis, the identified parasite proteins were clustered based on gene ontology annotations using different bioinformatics packages.