Leveraging the Aggregated Protein Dye YAT2150 for Malaria Chemotherapy

Abstract

Background/Objectives: YAT2150 is a first-in-class antiplasmodial compound that has been recently proposed as a new interesting drug for malaria therapy. Methods/Results: The fluorescence of YAT2150 rapidly increases upon its entry into Plasmodium, a property that can be of use for the design of highly sensitive diagnostic approaches. YAT2150 blocks the in vitro development of the ookinete stage of Plasmodium and, when added to an infected blood meal, inhibits oocyst formation in the mosquito. Thus, the compound could possibly contribute to future transmission-blocking antimalarial strategies. Cell influx/efflux studies in Caco-2 cells suggest that YAT2150 is internalized by endocytosis and also through the OATP2B1 transporter, whereas its main export route would be via OSTα. YAT2150 has an overall favorable drug metabolism and pharmacokinetics profile, and its moderate cytotoxicity can be significantly reduced upon encapsulation in immunoliposomes, which leads to a dramatic increase in the drug selectivity index to values close to 1000. Although YAT2150 binds amyloid-forming peptides, its in vitro fluorescence emission is stronger upon association with peptides that form amorphous aggregates, suggesting that regions enriched in unstructured proteins are the preferential binding sites of the drug inside Plasmodium cells. The reduction of protein aggregation in the parasite after YAT2150 treatment, which has been suggested to be directly related to the drug's mode of action, is also observed following treatment with quinoline antimalarials like chloroquine and primaquine. Conclusions: Altogether, the data presented here indicate that YAT2150 can represent the spearhead of a new family of compounds for malaria diagnosis and therapy due to its presumed novel mode of action based on the interaction with functional protein aggregates in the pathogen.

Keywords: Plasmodium falciparum; YAT2150; malaria; protein aggregation.

Figure 1

Chemical structure of YAT2150.

Figure 2

YAT2150 staining of clinical samples of P. falciparum and P. ovale infections. The merge panels refer to fluorescence images only. Arrowheads indicate the Plasmodium-infected red blood cells present in the microscope fields shown.

Figure 3

Transmission blocking assays. (A,B) Ex vivo P. berghei ookinete maturation assay. (A) Scheme of the experimental protocol. (B) Effect of DONE3TCl and YAT2150 on ookinete development. Mean values ± standard deviations are indicated (n = 3). **: p < 0.01 (one-way ANOVA, Dunnett’s post-hoc test). (C,D) Membrane feeding assay to test the effect of YAT2150 on P. berghei oocyst production. (C) Scheme of the experimental protocol. A total of 10 µM YAT2150 was added to blood infected with P. berghei and immediately fed to A. gambiae mosquitoes. Control feeding was the same blood mixed with DMSO at the same concentration as in the YAT2150-containing sample. The illustrative GFP-oocyst image is from Lantero et al. [33]. (D) Effect of YAT2150 on oocyst development in three independent experiments (Exp 1 to 3). Percentages in the graph indicate the prevalence of infection (% of infected mosquitoes). ****: p < 0.0001 (Mann–Whitney non-parametric test).

Figure 4

YAT2150 (A) influx and (B) efflux study in Caco-2 cells in FBS-free medium. Representative microscopy images of YAT2150 fluorescence from three to five independent experiments are shown. Each panel corresponds to a single inhibitor blocking a particular pathway (in parenthesis). The bar graphs show the quantification of YAT2150 accumulation as reported in Materials and Methods using a fluorescence imaging system and normalized to YAT2150 without inhibitors (YAT2150, red line). The negative control sample (control) corresponds to the fluorescence in cells cultured for the same time in the absence of YAT2150 (no image of this condition is provided because fluorescence was almost negligible). Results are the mean ± SEM from three to five independent experiments. Statistical significance relative to YAT2150 influx and efflux was determined by Student’s t-test: ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5

Characterization of the fast-acting activity of YAT2150. (A) Stage arrest assay of 3D7 P. falciparum synchronized at schizont stage, treated for up to 72 h with the IC₈₀ of YAT2150 (200 nM). At the indicated times after treatment start, blood smears stained with Giemsa were prepared, and parasite population was noted for at least 100 pRBCs. Bars show the percentages of asexual blood stages present at each time; *: indicates the presence of only pyknotic and dead parasites of which the population could not be annotated (n = 2 independent experiments). (B) Parasite-killing profile of 3D7 P. falciparum parasites treated for 24 and 48 h with 10 times the IC₅₀ of YAT2150 or with fast- (in green, chloroquine and artesunate), moderate- (in orange, pyrimethamine), and slow-acting (in red, atovaquone) antimalarials. (C) Representative images of Giemsa-stained pRBCs in the stage arrest assay of panel (A).

Figure 6

ThT analysis in P. falciparum asexual blood stage cultures of the effect on protein aggregation of four clinically used antimalarial drugs. ThT fluorescence assay of P. falciparum culture extracts normalized to have equal protein content, either non-treated or treated for 4 h with artemisinin, atovaquone, chloroquine, or primaquine at their respective in vitro IC₅₀ (10.8 nM, 1 nM, 7 nM, and 3 µM, respectively, as determined in our experimental setting). The p-values refer to the fluorescence intensity measured at the maximum emission wavelength.

Figure 7

Interaction of YAT2150 with aggregative peptides. (A–C) Relative fluorescence emission intensity of solutions of non-disaggregated KDLLF, KVVNI, and LYWIYY peptides treated with YAT2150 and ThT. Peptides (12.5 µM each) were incubated in the presence of (A) 25 µM ThT or (B) 10 µM YAT2150 before proceeding to measuring fluorescence emission. The insets in panel (A) show representative TEM images of the aggregates formed by LYWIYY and KDLLF. (C) Fluorescence emission spectra of peptide-free 10 µM YAT2150 solutions in PBS (dashed line) and DMSO (solid line). (D) Transmission electron microscopy analysis of the effect of 100 nM YAT2150 on the aggregation of LYWIYY at 24 h and 48 h after undergoing a disaggregation process. Size bars: 1 µm (upper panels, 30,000×), 250 nm (lower panels, 120,000×).