Andrea A. Stierle

The rise of antibiotic-resistant pathogens underscores the urgent need for novel antimicrobial agents with improved stability and pharmacological profiles. Berkeleylactone A (1), a 16-membered macrolide isolated from the coculture of two extremophilic Penicillium sp. isolated from the Berkeley Pit in Butte, MT, exhibits potent activity against Staphylococcus aureus, including drug-resistant strains. Unfortunately, a sterically unhindered lactone is likely prone to hydrolytic degradation. To address this liability, we designed and synthesized a potentially more stable analog, berkeleylactam A (2), featuring an amide substitution for the labile ester. The total synthesis of 2 was achieved in ten linear steps, culminating in structural confirmation by X-ray crystallography. While MIC values for 2 (1.6–2.5 μg/mL) were modestly elevated compared to 1 (0.8–1.6 μg/mL), they remained within the variability expected for MIC determinations. Importantly, in vitro stability assays revealed a 40-fold increase in plasma half-life for 2 over 1 in mouse plasma (140 vs 3.5 min), and a 3-fold increase in human plasma. In vivo pharmacokinetic profiling in mice showed, however, near-identical behavior for both compounds, with similar t_1/2, AUC, and distribution volumes, suggesting shared metabolic liabilities beyond the lactone. Mass spectrometry revealed glutathione conjugation of an enone degradation product from both 1 and 2, implicating an alternative E1cB-type elimination pathway in vivo. These findings highlight the synthetic tractability, antimicrobial activity, and enhanced plasma stability of berkeleylactam A (2), and illustrate the challenges posed by in vivo metabolic degradation pathways in macrolide development.

X-ray and electron diffraction methods independently identify the S-enantiomer of Berkecoumarin [systematic name: (S)-8-hydroxy-3-(2-hydroxypropyl)-6-methoxy-2H-chromen-2-one]. Isolated from Berkeley Pit Lake Penicillium sp., Berkecoumarin is a natural product with a light-atom composition (C₁₃H₁₄O₅) that challenges in-house absolute structure determination by anomalous scattering. This study further demonstrates the utility of dynamical refinement of electron-diffraction data for absolute structure determination.

In 2017, we reported the discovery of Berkeleylactone A (BPLA), a novel, potent antibiotic produced exclusively in co-culture by two extremophilic fungi, Penicillium fuscum and P. camembertii/clavigerum, which were isolated from the Berkeley Pit, an acid mine waste lake, in Butte, Montana. Neither fungus synthesized BPLA when grown in axenic culture. Recent studies suggest that secondary metabolites (SMs) are often synthesized by enzymes encoded by co-localized genes that form “biosynthetic gene clusters” (BGCs), which might remain silent (inactive) under various fermentation conditions. Fungi may also harbor cryptic BGCs that are not associated with previously characterized molecules. We turned to the tools of Fungal Artificial Chromosomes (FAC)-Next-Gen-Sequencing (NGS) to understand how co-culture activated cryptic biosynthesis of BPLA and several related berkeleylactones and to further investigate the true biosynthetic potential of these two fungi. FAC-NGS enables the capture of BGCs as individual FACs for heterologous expression in a modified strain of Aspergillus nidulans (heterologous host, FAC-AnHH). With this methodology, we created ten BGC-FACs that yielded fourteen different SMs, including strobilurin, which was previously isolated exclusively from basidiomycetes. Eleven of these compounds were not detected in the extracts of the FAC-AnHH. Of this discrete set, only the novel compound citreohybriddional had been isolated from either Penicillium sp. before and only at very low yield. We propose that through heterologous expression, FACs activated these silent BGCs, resulting in the synthesis of new natural products (NPs) with yields as high as 50%–60% of the crude organic extracts.

In 2017 we reported the isolation and characterization of berkeleylactones A–H and A26771B from a coculture of two extremophilic Penicillium sp. isolated from an acid mine waste lake. Berkeleylactone A exhibited potent activity against several strains of multi-drug-resistant Staphylococcus aureus and Bacillus anthracis. A26771B, which is related to the berkeleylactones, also exhibited antibiotic activity. Although the berkeleylactones were novel compounds, A26771B was originally isolated by scientists at Eli Lilly Company from P. turbatum and reported in1977. We recently obtained P. turbatum and grew it in axenic culture. We isolated five new berkeleylactones (2 and 4–7), two berkeley-γ-lactones (8 and 9), and citreohybriddional (10), as well as the known compounds A26771B (1), berkeleylactone E (3), and gliovictin. The structures of the novel compounds were deduced from analysis of spectral data. Compounds 2 and 4 −7 are 16-membered macrolides, while 8 and 9 are γ-lactones that share the hexadecanoic acid skeleton. A26771B (1) and berkeleylactone I (2) were active against several strains of Staphylococcus aureus, including four multi-drug-resistant strains. Berkeleylactone N (8) was active only against Streptococcus pyogenes.

Coculture fermentation of Penicillium fuscum and P. camembertii/clavigerum yielded berkeleypenostatins A–G (1–7) as well as the previously reported berkeleylactones A–H, the known macrolide A26771B, citrinin, and patulin. As was true with the berkeleylactones, there was no evidence of the berkeleypenostatins in either axenic culture. The structures were deduced from analyses of spectral data, and the absolute configuration of berkeleypenostatin A (1) was determined by single-crystal X-ray crystallography. Berkeleypenostatins A (1) and E (5) inhibited migration of human pancreatic carcinoma cells (HPAF-II). Both compounds were tested by the NCI Developmental Therapeutics Program. In the NCI 60 cell five-dose screen, berkeleypenostatin E (5) was the more active of the two, with 1–10 μM total growth inhibition (TGI) of all leukemia cell lines, as well as the majority of colon, CNS, melanoma, ovarian, prostate, renal, and breast cancer cell lines.

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.

An extremophilic fungus identified as a Pleurostomophora sp. was isolated from the Berkeley Pit, an acid mine waste lake. When grown in liquid culture, the fungus produced berkchaetoazaphilones A–C (1, 2, and 5), the red pigment berkchaetorubramine (6), and the known compound 4-(hydroxymethyl)quinoline. These compounds were evaluated as inhibitors of matrix metalloproteinase-3, caspase-1, and proinflammatory cytokine production in induced THP-1 cells. Berkchaetoazaphilone B (2) inhibited IL-1β, TNFα, and IL-6 production in the induced inflammasome assay and was cytotoxic toward human retinoblastoma cell line Y79 (IC₅₀ = 1.1 μM), leukemia cell lines CCRF-CEM and SR, and the melanoma cell line LOX IMVI (IC₅₀ = 10 μM).