Enhanced Production and Anticancer Properties of Photoactivated Perylenequinones

Abstract

Hypocrellins and hypomycins are naturally occurring fungal perylenequinones with potential photodynamic activity against cancer and microbial diseases. This project pursued three lines of research. First, the production of perylenequinones was enhanced by investigating the effect of culture medium and light exposure on their biosynthesis. Solid-fermentation cultures on rice medium allowed for enhanced production of hypocrellins as compared to Cheerios or oatmeal medium. Alternatively, increased production of hypomycins, which are structurally related to the hypocrellins, was observed on oatmeal medium. In both cases, light exposure was an essential factor for the enhanced biosynthesis. In addition, this led to the discovery of two new perylenequinones, ent-shiraiachrome A (5) and hypomycin E (8), which were elucidated based on spectroscopic data. Finally, the photocytotoxic effects of both classes of compounds were evaluated against human skin melanoma, with EC₅₀ values at nanomolar levels for hypocrellins and micromolar levels for hypomycins. In contrast, both classes of compounds showed reduced dark toxicity (EC₅₀ values >100 μM), demonstrating promising phototherapeutic indices.

Figure 1.

Panels A and B show the relative percentages of 5 and 1, respectively, across cultures grown on rice, Cheerios, and oatmeal media under 12:12 h light:dark cycles, continuous LED, or in darkness. The relative percentages were measured by LC-HRESIMS in three biological replicates and multiplied by the extract weight, and then normalized according to the extract with highest abundance. Panels C and D show the amounts isolated of 5 and 1, respectively, from cultures shown in panels A and B and grown on rice, Cheerios, and oatmeal media under either 12:12 h light:dark cycles or LED conditions.

Figure 2.

The atropisomerization process (A) and the tautomerism process (B) of hypocrellins.

Figure 3.

Panels A-C show the relative percentages of 6–8, respectively, across cultures grown on rice, Cheerios, and oatmeal media under 12:12 h light:dark cycles, continuous LED, or in darkness. The relative percentages were measured by LC-HRMS in three biological replicates and multiplied by the extract weight, and then normalized according to the extract with highest abundance.

Figure 4.

Experimental ECD spectrum of 6 compared with calculated ECD spectra of [M(R), 1S, 2R, 14R, 15S, 17S]- configuration and its enantiomer (left frame), and [M(R), 1R, 2S, 14S, 15R, 17R]- configuration and its enantiomer (right frame).

Figure 5.

Experimental VCD (left frame) and IR (right frame) spectra observed for hypomycin A (6) compared with calculated VCD and IR spectra of [M(R), 1S, 2R, 14R, 15S, 17S]-configuration (0.65 similarity factor to 6) and [M(R), 1R, 2S, 14S, 15R, 17R]- configuration (0.11 similarity factor to 6). Specdis software was utilized to calculate the similarity factors.

Figure 6.

Key HMBC (A) and NOESY (B) correlations for hypomycin E (8).

Figure 7.

Panel A: photocytotoxic activity plot for compounds 1, 3, and 5–8 against SK-MEL-28 melanoma cells without (black) or with visible- (blue) or red- (red) light irradiation. Panel B: phototherapeutic index (PI) activity plot for compounds 1, 3, 5–8 against SK-MEL-28 melanoma cells treated with visible- (blue) or red- light (red).