Phomoxanthone A Targets ATP Synthase

Abstract

Phomoxanthone A is a naturally occurring molecule and a powerful anti-cancer agent, although its mechanism of action is unknown. To facilitate the determination of its biological target(s), we used affinity-based labelling using a phomoxanthone A probe. Labelled proteins were pulled down, subjected to chemoproteomics analysis using LC-MS/MS and ATP synthase was identified as a likely target. Mitochondrial ATP synthase was validated in cultured cells lysates and in live intact cells. Our studies show sixty percent inhibition of ATP synthase by 260 μM phomoxanthone A.

Keywords: anticancer; biconjugation; microscopy; natural product; photoaffinity labelling.

Figure 1.

Photoaffinity labelling studies of anticancer agent phomoxanthone A.

Figure 2.

Live cell protein labelling with PAL-1. (A) Structures of PAL-1 and 5, the compounds used to probe the selective protein labelling abilities of PAL-1. (B) Experimental process for bioconjugating and tagging PAL-1 and 5. (C) SDS-PAGE separation and visualization by in-gel fluorescence scanning.

Figure 3.

Volcano plots depicting hits derived from phomoxanthone A proteomics.

Figure 4.

(A) Relative rates of ATP synthase activity after treatment with oligomycin (2 mM) or phomoxanthone A (260 μM) in comparison to the control (DMSO). ****For both phomoxanthone A and oligomycin A, p≤0.0001. (B) Effect of phomoxanthone on ATP synthase activity. Each data point is the mean of triplicate measurements. The error bars represent calculated standard deviations.

Figure 5.

Phomoxanthone A decreases NADH lifetime in HeLa cell studies. (A) Representative HeLa cells treated with DMSO or 5μM or 50 SymbolM of phomoxanthone (left to right) showing autofluorescence corresponding to primarily to NADH/NAD+ redox levels. (B) Phasor representation of raw lifetimes values of each image pixel showing heterogeneity. The distinct regions on the phasor plots are highlighted by colored circles indicating distinct lifetimes (red lifetime center= 1.57 ns, orange lifetime center= 1.39 ns, yellow lifetime center= 1.24 ns, green lifetime center= 1.11 ns, blue lifetime center= 1 ns). The pixels underlying these circles are false colored and overlaid on grayscale cell images. The cells have been treated with DMSO or 5 mM or 50 mM of phomoxanthone (left to right, respectively) where the asterisks represented significant differences * = p ≤0.05, **=p≤0.01, ***=p≤0.001, ****=p≤0.0001. Error bars denote standard deviation. (D) Decrease in lifetime (%) at 5 μM and 50 μM phomoxanthone A when compared to the control experiment. (E and F) Decrease in autofluorescence of HeLa cells treated with 5 mM of phomoxanthone after 24 hours when compared to the control (DMSO treatment), and a similar study showing the immediate decrease. Note that decrease in the lifetime is proportional to the dose of phomoxanthone

Scheme 1.

Photoaffinity label optimization on chromenone model system.

Scheme 2.

(A) Photoaffinity labelling of phomoxanthone A and (B) Comparison of cytotoxicity of 1 and PAL-1 in Jurkat cells.