The Berkeleylactones, Antibiotic Macrolides from Fungal Coculture

Abstract

A carefully timed coculture fermentation of Penicillium fuscum and P. camembertii/clavigerum yielded eight new 16-membered-ring macrolides, berkeleylactones A-H (1, 4, 6-9, 12, 13), as well as the known antibiotic macrolide A26771B (5), patulin, and citrinin. There was no evidence of the production of the berkeleylactones or A26771B (5) by either fungus when grown as axenic cultures. The structures were deduced from analyses of spectral data, and the absolute configurations of compounds 1 and 9 were determined by single-crystal X-ray crystallography. Berkeleylactone A (1) exhibited the most potent antimicrobial activity of the macrolide series, with low micromolar activity (MIC = 1-2 μg/mL) against four MRSA strains, as well as Bacillus anthracis, Streptococcus pyogenes, Candida albicans, and Candida glabrata. Mode of action studies have shown that, unlike other macrolide antibiotics, berkeleylactone A (1) does not inhibit protein synthesis nor target the ribosome, which suggests a novel mode of action for its antibiotic activity.

Figure 1

Selected long-range correlations from the HMBC spectrum of berkeleylactone A (1).

Figure 2

X-ray crystal structure of berkeleylactone A (1).

Figure 3

Selected δΔ values of the (S)- and (R)-MTPA esters of berkeleylactone F (11) [δΔ = chemical shift of the (S)-MTPA ester minus the chemical shift of the (R)-MTPA ester in ppm].

Figure 4

X-ray crystal structure of berkeleylactone F triacetate (10).

Figure 5

The extension inhibition assay allows the direct monitoring of the formation of stalled ribosome complexes (SRCs) on mRNA. It can assess the ability of a specific antibiotic to stall the ribosome at a specific mRNA codon. In this case, the effect of berkeleylactone A (1, DNA76) was compared to the known macrolide antibiotics erythromycin, josamycin, and tylosin. Unlike the known antibiotics, compound 1 did not induce SRC formation.

Figure 6

Cell-free translation of GFP protein compared the effects of berkeleylactone A (1) and three known macrolide antibiotics—erythromycin, josamycin, and tylosin—on protein synthesis. The known macrolides were tested at 50 μM, and compound 1 was tested at 50 and 250 μM. Compound 1 did not inhibit protein synthesis, and its effect was comparable to that of the control (no antibiotic).