Activity-Independent Discovery of Secondary Metabolites Using Chemical Elicitation and Cheminformatic Inference

Abstract

Most existing antibiotics were discovered through screens of environmental microbes, particularly the streptomycetes, for the capacity to prevent the growth of pathogenic bacteria. This “activity-guided screening” method has been largely abandoned because it repeatedly rediscovers those compounds that are highly expressed during laboratory culture. Most of these metabolites have already been biochemically characterized. However, the sequencing of streptomycete genomes has revealed a large number of “cryptic” secondary metabolic genes that are either poorly expressed in the laboratory or that have biological activities that cannot be discovered through standard activity-guided screens. Methods that reveal these uncharacterized compounds, particularly methods that are not biased in favor of the highly expressed metabolites, would provide direct access to a large number of potentially useful biologically active small molecules. To address this need, we have devised a discovery method in which a chemical elicitor called Cl-ARC is used to elevate the expression of cryptic biosynthetic genes. We show that the resulting change in product yield permits the direct discovery of secondary metabolites without requiring knowledge of their biological activity. We used this approach to identify three rare secondary metabolites and find that two of them target eukaryotic cells and not bacterial cells. In parallel, we report the first paired use of cheminformatic inference and chemical genetic epistasis in yeast to identify the target. In this way, we demonstrate that oxohygrolidin, one of the eukaryote-active compounds we identified through activity-independent screening, targets the V1 ATPase in yeast and human cells and secondarily HSP90.

Antibiotic dialogues: induction of silent biosynthetic gene clusters by exogenous small molecules

“…A derivative of ARC2, called Cl-ARC (Fig. 1, 14), has recently been applied to 50 additional Streptomyces, resulting in at least one induced compound in a majority of the strains for a total of 216 cryptic metabolites (Pimentel-Elardo et al 2015).…”

An epigenetic modifier induces production of (10′ S )-verruculide B, an inhibitor of protein tyrosine phosphatases by Phoma sp. nov. LG0217, a fungal endophyte of Parkinsonia microphylla

“…2 However, when cultured utilizing traditional fermentation techniques, fungi often express only a subset of biosynthetic genes which encode for their secondary metabolite production. 3 Realization of this has recently led to the discovery of strategies to activate these silent biosynthetic gene clusters in fungi including the use of epigenetic modifiers to activate silent biosynthetic pathways, 4 and simulation of natural gene cluster activating conditions by biotic (co-cultivation), 5 and abiotic (elicitation by chemical and physical means) 6 methods. Among these, incorporation of small-molecule modifiers into fungal culture media to manipulate epigenetic regulation of gene transcription has received considerable attention primarily focusing on inhibitors of histone deacetylase (HDAC), 7 DNA methyltransferase (DNMT) 8 and the proteasome.…”

Symbiosis-inspired approaches to antibiotic discovery

“…In a subsequent study, an analog of ARC2, Cl-ARC, was synthesized on the basis of structure-activity relationship towards secondary metabolite induction. 219 Among the compounds induced via addition of Cl-ARC were the antibiotics oxohygrolidin, 9-methylstreptimidone, and dynactin. Taken together, this work demonstrates how knowledge obtained using a model system can lead to identification of a trigger, in this case ARC, which can coax production of secondary metabolites from orphan BGCs.…”