Figure 1.
Structure of albitiazolium and photoactivable analogs.
(A) The clinical antimalarial candidate albitiazolium, (B) the bifunctional bis-thiazolium derivative UA1936 and (C) the pharmacologically inactive bifunctional derivative UA2050 are depicted. The albitiazolium pharmacophore consists of two cationic thiazolium heads linked by a hydrophobic flexible spacer. The bifunctional compounds UA1936 and UA2050 incorporate a phenyl azido group as photoactivable moiety and a benzy azido group as “clickable” function.
Figure 2.
In vitro antimalarial activity of albitiazolium and related bifunctional analogs.
The compounds were added at various concentrations to the 3D7 P. falciparum strain and incubated for 48 h. [3H]hypoxanthine was added at 48 h to monitor parasite viability. Parasitemia is expressed as a percentage of the control cultures without drug. Half maximal inhibitory concentrations (IC50) are the concentrations needed to inhibit P. falciparum growth by 50%. The results are expressed as means ± SEM of three independent experiments conducted in triplicate. Albitiazolium (□), UA1936 (◊) and UA2050 (○).
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.
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.
Table 1.
Parasite proteins identified in interaction with the UA1936 bifunctional compound.
Figure 5.
Protein interaction networks were built in Cytoscape 2.5.1 using the PlasmoID plugin. Three clusters of UA1936-interacting proteins were found based on the following biological process terms: “phospholipid metabolism” depicted in pink, “vesicle-mediated transport” in blue and “transport” in violet. A fourth cluster of unknown or unrelated functions was created and named “miscellaneous”, as depicted in orange. For each protein (node), the PlasmoDB ID accession number is indicated in a colored rectangle followed by the putative name of the gene. Proteins in dark-colored rectangles were found in the three replicated experiments and proteins in light-colored rectangles were found in two replicates. The proteins depicted in ellipses are interacting proteins identified in the PlamsoMap interactome analysis with their respective likelihood scores. The colors on the edges in the network represent: Codes for (black), Interacts with (blue), and Participates in (orange). The p values for the gene ontology terms found to be significantly enriched are presented for genes having those annotations in the biological process.
Table 2.
Parasite proteins identified in UA1936 treated parasites under competition with albitiazolium.