Fig. 1

Development of YZSA-based probes to study heme biology and identify ring-stage inhibitors for the most pathogenic parasite, P. falciparum. a The unique reaction between Yingzhaosu A and heme. b Design I for FRET-based heme-reactive probes. c Absorption spectra of X-1a, X-2a, X-2b and the BODIPY fluorophore in PBS (5 μM). d PL spectra of X-1a, X-2a, X-2b and the BODIPY fluorophore in PBS (5 μM). e PL intensity of three probes incubated with/without heme under different conditions for 2 h. The different conditions were as follows: 1, X-1a; 2, X-1a+GSH; 3, X-1a+SA; 4, X-2a; 5, X-2a+GSH; 6, X-2a+SA; 7, X-2b; 8, X-2b+GSH; 9, X-2b+SA; 10, hemin+SA+X-1a; 11, hemin+SA+GSH+X-1a; 12, hemin+X-1a; 13, hemin+GSH+X-1a; 14, hemin+SA+X-2a; 15, hemin+SA+GSH+X-2a; 16, hemin+X-2a; 17, hemin+GSH+X-2a; 18, hemin+SA+X-2b; 19, hemin+SA+GSH+X-2b; 20, hemin+X-2b; and 21, hemin+GSH+X-2b. SA sodium ascorbate. Results are shown as the mean ± SD with n = 3 biological replicates. f Time-dependent PL intensity of probe X-2b treated with hemin and SA. Results are shown as the mean ± SD with n = 3 biological replicates. g PL intensity of probe X-2b treated with different concentrations of hemin in the presence of SA. The fluorescence intensity of the probe steadily increased as the concentration of heme increased from 0 to 25 μM. Higher concentrations of heme (50 and 100 μM) did not further increase the fluorescence intensity but instead caused it to decrease greatly, which was ascribed to the quenching effect of heme. Results are shown as the mean ± SD with n = 3 biological replicates. h Confocal images of P. falciparum (3D7) treated with X-1a, X-2a, or X-2b (10 μM) for 3 h. BF bright field, FL fluorescence. i Confocal images of P. falciparum (3D7) treated with X-2b, Hoechst and lysosome tracker red (LTR). j The ratio of probe-responsive iRBCs treated with DFO or ALLN for 1 h and subsequently coincubated with X-2b for 3 h. The ratio of probe-responsive iRBCs denotes the number of probe-imaged RBCs among the examined 100,000 RBCs. Trophozoite-stage parasites were used in the assay. Trophozoite-stage parasites were obtained by allowing the sorbitol-treated parasites to grow further for 16 h. DFO a chelator of ferrous iron, ALL, a cysteine protease inhibitor. Results are shown as the mean ± SD with n = 5 biological replicates. k Comparing the growth patterns of 3D7 and ART-resistant P. falciparum by measuring the ratio of X-2b-imaged RBCs during one life cycle. iRBC infected red blood cell, RBC red blood cell, ALA the precursor of heme biosynthesis, SA the inhibitor of heme biosynthesis. Results are shown as the mean ± SD with n = 3 biological replicates. l The high-throughput screening results for a library of 100 compounds. The ratio of probe-responsive iRBCs in the presence of the screened compound was normalized to that of the control group without the addition of screened compounds. m Ring-stage survival of DHA- and 96-treated 0–3 hpi rings. Results are shown as the mean ± SD with n = 5 biological replicates