Unique assembly of carbonylpyridinium and chromene reveals mitochondrial thiol starvation under ferroptosis and novel ferroptosis inducer

Abstract

To reveal the delicate function of mitochondria, spatiotemporally precise detection tools remain highly desirable. However, current probes with positively charged warheads for targeting mitochondria diffuse out of the mitochondria as the potential of the mitochondrial membrane changes, which directly influences the accuracy of the detection. Herein, we assembled carbonylpyridinium and chromene to afford the probe CM-Mit. Following the ultrafast response to thiol and the dissociation of carbonylpyridinium, the formation of o-quinone methide from CM-Mit was proposed to label proteins, thus avoiding diffusion out of the mitochondria. Therefore, the accurate spatiotemporal detection of thiol in mitochondria was realized. With this excellent probe, ferroptosis inducers were proved to stimulate thiol starvation in mitochondria for the first time in cancer cells. Moreover, CM-Mit was used to screen a compound library developed in-house and the stemona alkaloid analog SA-11 was shown to induce ferroptosis in various cancer cell lines, including a drug-resistant one.

Fig. 1. Previous work: (a) thiol-mediated nucleophilic attack on unsaturated α,β-ketone and (b) self-immolation to release the fluorophore. (c) Design strategy of this work.

Fig. 2. (a) Absorption changes of CM-Mit (5 μM, DMSO : PBS, 1 : 9, v/v) after adding 200 μM Cys, Hcy, or GSH. (b) Fluorescence intensity changes of probe CM-Mit (5 μM, DMSO : PBS, 1 : 9, v/v) with the addition of 200 μM of various amino acid after 5 min; (λ_ex = 475 nm, slit: 5 nm/5 nm). (c) Fluorescence spectral changes of CM-Mit (5 μM, DMSO : PBS, 1 : 9, v/v) as the concentration of Cys (0–200 μM) increases. (d) The kinetic curve of CM-Mit (5 μM) in the mixed solvent of DMSO and PBS (1 : 9, v/v) without or with 200 μM Cys, 20 μM Hcy, and 1 mM GSH at 578 nm (λ_ex = 475 nm, slit: 5 nm/5 nm).

Fig. 3. The working curve of CM-Mit (5 μM) in the presence of different concentrations of Cys. Each point represents the average and standard deviation of three independent repeated experiments.

Fig. 4. (a) Calculated UV-vis absorption spectra of CM-Mit and the product after the reaction of CM-Mit and thiol. (b) The simplified structure of CM-Mit (S-CM-Mit). (c) S-CM-Mit and the product after the response in the ground state (the Franck–Condon state) and the ICT state. The insets show the dihedral angles (α and β) along with bond 3.

Fig. 5. Confocal fluorescence images of HeLa cells co-stained with CM-Mit and a tracker. (a–c) Cells pretreated with CM-Mit (10 μM) for 20 min and subsequently Mito-Tracker Green (1 μM) at 37 °C for 20 min. (a and d) Images from CM-Mit, λ_ex = 475 nm and λ_em = 563–593 nm. (b) Fluorescence images from Mito-Tracker Green, λ_ex = 488 nm and λ_em = 500–540 nm. (d) Overlay of the orange and green channels. (e) Line profile: intensity profile of the white line in image overlap. Scale bars: 5 μm.

Fig. 6. Fluorescence images of CM-Mit responding to thiols in living HeLa cells along with reaction time by confocal fluorescence imaging. (a) Images of Mito-Tracker Green; (b) images of CM-Mit. (c) Merged images of (a) and (b). (d) Time-dependent Pearson's coefficient. The HeLa cells were first incubated with CM-Mit for 20 min, followed by incubation with Mito-Tracker Green for 20 min. Fluorescence images were captured from the green channel of 500–540 nm with excitation at 488 nm (first row) and orange channel of 563–593 nm (second row) with excitation at 475 nm.

Fig. 7. Imaging of endogenous thiols in live HeLa cells treated with ferroptosis inducer. (Above) Cells were pretreated without other reagents. (Middle and below) Cells were pretreated with ferroptosis inducer, erastin (15 μM) and RSL3 (0.2 μM). All of the above were cultured at 37 °C for 24 h, followed by exchange into PBS (pH = 7.4) with 10 μM CM-Mit and incubation for another 15 min at 37 °C. Data are represented as mean ± SD (n = 3).

Fig. 8. Screening of stemona alkaloids and their derivatives for controlling the thiol level. (a) HeLa cells were pretreated with ferroptosis inducer erastin (5 μM), SA-11 and its analogs (15 μM) in a 5% carbon dioxide incubator at 37 °C for 24 h, and the cells were incubated with 10 μM CM-Mit for 15 min. The mean intensity from Fig. S9, where the control group is defined as 1.0. Data are represented as mean ± SD (n = 3); (b) the structure of SA-11.

Fig. 9. SA-11 induces cancer cell death through ferroptosis. (a) Ability of apoptosis inhibitor z-VAD or necroptosis inhibitor Nec-1 to protect cells against SA-11 (30 μmol) treatment after 24 hours in ES-2 cells. For (b) and (c) effect of ferroptosis Fer-1 (2 μmol) and NAC (1 mmol) on the viability of ES-2 cells after treated with SA-11 (20 μM, 25 μM, 30 μM), erastin (10 μM) and erastin (10 μM) combined with SA-11 (20 μmol) for 24 hours. Data are represented as mean ± SD (n = 3).

Fig. 10. Effect of NAC on viability of (a) HCT-116 and (b) HCT116P53^−/− after incubation with different concentration of SA-11. Data are represented as mean ± SD (n = 3).