Proteomics Guided Discovery of Flavopeptins: Anti-proliferative Aldehydes Synthesized by a Reductase Domain-Containing Non-ribosomal Peptide Synthetase

Due to the importance of proteases in regulating cellular processes, the development of protease inhibitors has garnered great attention. Peptide-based aldehydes are a class of compounds that exhibit inhibitory activities against various proteases and proteasomes in the context of anti-proliferative treatments for cancer and other diseases. More than a dozen peptide-based natural products containing aldehydes have been discovered such as chymostatin, leupeptin, and fellutamide; however, the biosynthetic origin of the aldehyde functionality has yet to be elucidated. Herein we describe the discovery of a new group of lipopeptide aldehydes, the flavopeptins, and the corresponding biosynthetic pathway arising from an orphan gene cluster in <i>Streptomyces</i> sp. NRRL-F6652, a close relative of <i>Streptomyces flavogriseus</i> ATCC 33331. This research was initiated using a proteomics approach that screens for expressed enzymes involved in secondary metabolism in microorganisms. Flavopeptins are synthesized through a non-ribosomal peptide synthetase containing a terminal NAD­(P)­H-dependent reductase domain likely for the reductive release of the peptide with a C-terminal aldehyde. Solid-phase peptide synthesis of several flavopeptin species and derivatives enabled structural verification and subsequent screening of biological activity. Flavopeptins exhibit sub-micromolar inhibition activities against cysteine proteases such as papain and calpain as well as the human 20S proteasome. They also show anti-proliferative activities against multiple myeloma and lymphoma cell lines

Proteomics Guided Discovery of Flavopeptins: Anti-proliferative Aldehydes Synthesized by a Reductase Domain-Containing Non-ribosomal Peptide Synthetase

Due to the importance of proteases in regulating cellular processes, the development of protease inhibitors has garnered great attention. Peptide-based aldehydes are a class of compounds that exhibit inhibitory activities against various proteases and proteasomes in the context of anti-proliferative treatments for cancer and other diseases. More than a dozen peptide-based natural products containing aldehydes have been discovered such as chymostatin, leupeptin, and fellutamide; however, the biosynthetic origin of the aldehyde functionality has yet to be elucidated. Herein we describe the discovery of a new group of lipopeptide aldehydes, the flavopeptins, and the corresponding biosynthetic pathway arising from an orphan gene cluster in <i>Streptomyces</i> sp. NRRL-F6652, a close relative of <i>Streptomyces flavogriseus</i> ATCC 33331. This research was initiated using a proteomics approach that screens for expressed enzymes involved in secondary metabolism in microorganisms. Flavopeptins are synthesized through a non-ribosomal peptide synthetase containing a terminal NAD­(P)­H-dependent reductase domain likely for the reductive release of the peptide with a C-terminal aldehyde. Solid-phase peptide synthesis of several flavopeptin species and derivatives enabled structural verification and subsequent screening of biological activity. Flavopeptins exhibit sub-micromolar inhibition activities against cysteine proteases such as papain and calpain as well as the human 20S proteasome. They also show anti-proliferative activities against multiple myeloma and lymphoma cell lines

<i>In Vitro</i> Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis

Genome sequencing has revealed that a far greater number of natural product biosynthetic pathways exist than there are known natural products. To access these molecules directly and deterministically, a new generation of heterologous expression methods is needed. Cell-free protein synthesis has not previously been used to study nonribosomal peptide biosynthesis, and provides a tunable platform with advantages over conventional methods for protein expression. Here, we demonstrate the use of cell-free protein synthesis to biosynthesize a cyclic dipeptide with correct absolute stereochemistry. From a single-pot reaction, we measured the expression of two nonribosomal peptide synthetases larger than 100 kDa, and detected high-level production of a diketopiperazine. Using quantitative LC–MS and synthetically prepared standard, we observed production of this metabolite at levels higher than previously reported from cell-based recombinant expression, approximately 12 mg/L. Overall, this work represents a first step to apply cell-free protein synthesis to discover and characterize new natural products

New Aspercryptins, Lipopeptide Natural Products, Revealed by HDAC Inhibition in <i>Aspergillus nidulans</i>

Unlocking the biochemical stores of fungi is key for developing future pharmaceuticals. Through reduced expression of a critical histone deacetylase in <i>Aspergillus nidulans</i>, increases of up to 100-fold were observed in the levels of 15 new aspercryptins, recently described lipopeptides with two noncanonical amino acids derived from octanoic and dodecanoic acids. In addition to two NMR-verified structures, MS/MS networking helped uncover an additional 13 aspercryptins. The aspercryptins break the conventional structural orientation of lipopeptides and appear “backward” when compared to known compounds of this class. We have also confirmed the 14-gene aspercryptin biosynthetic gene cluster, which encodes two fatty acid synthases and several enzymes to convert saturated octanoic and dodecanoic acid to α-amino acids

Large-Scale Metabolomics Reveals a Complex Response of <i>Aspergillus nidulans</i> to Epigenetic Perturbation

The microbial world offers a rich source of bioactive compounds for those able to sift through it. Technologies capable of quantitatively detecting natural products while simultaneously identifying known compounds would expedite the search for new pharmaceutical leads. Prior efforts have targeted histone deacetylases in fungi to globally activate the production of new secondary metabolites, yet no study has directly assessed its effects with minimal bias at the metabolomic level. Using untargeted metabolomics, we monitored changes in >1000 small molecules secreted from the model fungus, <i>Aspergillus nidulans</i>, following genetic or chemical reductions in histone deacetylase activity (HDACi). Through quantitative, differential analyses, we found that nearly equal numbers of compounds were up- and down-regulated by >100 fold. We detected products from both known and unknown biosynthetic pathways and discovered that <i>A. nidulans</i> is capable of producing fellutamides, proteasome inhibitors whose expression was induced by ∼100 fold or greater upon HDACi. This work adds momentum to an “omics”-driven resurgence in natural products research, where direct detection replaces bioactivity as the primary screen for new pharmacophores

Elucidating the Rimosamide-Detoxin Natural Product Families and Their Biosynthesis Using Metabolite/Gene Cluster Correlations

As microbial genome sequencing becomes more widespread, the capacity of microorganisms to produce an immense number of metabolites has come into better view. Utilizing a metabolite/gene cluster correlation platform, the biosynthetic origins of a new family of natural products, the rimosamides, were discovered. The rimosamides were identified in <i>Streptomyces rimosus</i> and associated with their NRPS/PKS-type gene cluster based upon their high frequency of co-occurrence across 179 strains of actinobacteria. This also led to the discovery of the related detoxin gene cluster. The core of each of these families of natural products contains a depsipeptide bond at the point of bifurcation in their unusual branched structures, the origins of which are definitively assigned to nonlinear biosynthetic pathways <i>via</i> heterologous expression in <i>Streptomyces lividans</i>. The rimosamides were found to antagonize the antibiotic activity of blasticidin S against <i>Bacillus cereus</i>

Discovery of the Tyrobetaine Natural Products and Their Biosynthetic Gene Cluster <i>via</i> Metabologenomics

Natural products (NPs) are a rich source of medicines, but traditional discovery methods are often unsuccessful due to high rates of rediscovery. Genetic approaches for NP discovery are promising, but progress has been slow due to the difficulty of identifying unique biosynthetic gene clusters (BGCs) and poor gene expression. We previously developed the metabologenomics method, which combines genomic and metabolomic data to discover new NPs and their BGCs. Here, we utilize metabologenomics in combination with molecular networking to discover a novel class of NPs, the tyrobetaines: nonribosomal peptides with an unusual trimethylammonium tyrosine residue. The BGC for this unusual class of compounds was identified using metabologenomics and computational structure prediction data. Heterologous expression confirmed the BGC and suggests an unusual mechanism for trimethylammonium formation. Overall, the discovery of the tyrobetaines shows the great potential of metabologenomics combined with molecular networking and computational structure prediction for identifying interesting biosynthetic reactions and novel NPs