Efficient mining of myxobacterial metabolite profiles enabled by liquid chromatography-electrospray ionisation-time-of-flight mass spectrometry and compound-based principal component analysis

Bacteria producing secondary metabolites are an important source of natural products with highly diverse structures and biological activities. Developing methods to efficiently mine procaryotic secondary metabolomes for the presence of potentially novel natural products is therefore of considerable interest. Modern mass spectrometry-coupled liquid chromatography can effectively capture microbial metabolic diversity with ever improving sensitivity and accuracy. In addition, computational and statistical tools increasingly enable the targeted analysis and exploration of information-rich LC-MS datasets.In this article, we describe the use of such techniques for the characterization of myxobacterial secondary metabolomes. Using accurate mass data from high-resolution ESI-TOF measurements, target screening has facilitated the rapid identification of known myxobacterial metabolites in extracts from nine Myxococcus species. Furthermore, principal component analysis (PCA), implementing an advanced compound-based bucketing approach, readily revealed the presence of further compounds which contribute to variation among the metabolite profiles under investigation. The generation of molecular formulae for putative novel compounds with high confidence due to evaluation of both exact mass position and isotopic pattern, is exemplified as an important key for de-replication and prioritization of candidates for further characterization.     

Genome-Guided Discovery of the First Myxobacterial Biarylitide Myxarylin Reveals Distinct C–N Biaryl Crosslinking in RiPP Biosynthesis

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse group of natural products. They feature a wide range of intriguing post-translational modifications, as exemplified by the biarylitides. These are a family of cyclic tripeptides found in Planomonospora, carrying a biaryl linkage between two aromatic amino acids. Recent genomic analyses revealed that the minimal biosynthetic prerequisite of biarylitide biosynthesis consists of only one ribosomally synthesized pentapeptide precursor as the substrate and a modifying cytochrome-P450-dependent enzyme. In silico analyses revealed that minimal biarylitide RiPP clusters are widespread among natural product producers across phylogenetic borders, including myxobacteria. We report here the genome-guided discovery of the first myxobacterial biarylitide MeYLH, termed Myxarylin, from Pyxidicoccus fallax An d48. Myxarylin was found to be an N-methylated tripeptide that surprisingly exhibits a C–N biaryl crosslink. In contrast to Myxarylin, previously isolated biarylitides are N-acetylated tripeptides that feature a C–C biaryl crosslink. Furthermore, the formation of Myxarylin was confirmed by the heterologous expression of the identified biosynthetic genes in Myxococcus xanthus DK1622. These findings expand the structural and biosynthetic scope of biarylitide-type RiPPs and emphasize the distinct biochemistry found in the myxobacterial realm.     

Structure and Biosynthesis of Crocagins: Polycyclic Posttranslationally Modified Ribosomal Peptides from Chondromyces crocatus

Secondary metabolome mining efforts in the myxobacterial multiproducer of natural products, Chondromyces crocatus Cm c5, resulted in the isolation and structure elucidation of crocagins, which are novel polycyclic peptides containing a tetrahydropyrrolo[2,3-b]indole core. The gene cluster was identified through an approach combining genome analysis, targeted gene inactivation in the producer, and in vitro experiments. Based on our findings, we developed a biosynthetic scheme for crocagin biosynthesis. These natural products are formed from the three C-terminal amino acids of a precursor peptide and thus belong to a novel class of ribosomally synthesized and post-translationally modified peptides (RiPPs). We demonstrate that crocagin A binds to the carbon storage regulator protein CsrA, thereby inhibiting the ability of CsrA to bind to its cognate RNA target.     

Discovery and Total Synthesis of Natural Cystobactamid Derivatives with Superior Activity against Gram‐Negative Pathogens

Antibiotic discovery and development is challenging as chemical scaffolds of synthetic origin often lack the required pharmaceutical properties, and the discovery of novel ones from natural sources is tedious. Herein, we report the discovery of new cystobactamids with a significantly improved antibacterial profile in a detailed screening of myxobacterial producer strains. Some of these new derivatives display antibacterial activities in the low‐μg mL⁻¹ range against Gram‐negative pathogens, including clinical isolates of Klebsiella oxytoca, Pseudomonas aeruginosa, and fluoroquinolone‐resistant Enterobacteriaceae, which were not observed for previously reported cystobactamids. Our findings provide structure–activity relationships and show how pathogen resistance can be overcome by natural scaffold diversity. The most promising derivative 861‐2 was prepared by total synthesis, enabling further chemical optimization of this privileged scaffold.     

Studies on the Synthesis of Indothiazinone and Its Derivatives via Direct 3-Acylation of Indole

Indothiazinone is a natural 3-acylindole alkaloid, isolated from a culture of myxobacterial strain. It was found to possess antibacterial activity against yeast and filamentous fungi. Indothiazinone is also structurally related with a mammalian endogenous aryl hydrocarbon receptor ligand, (2-(1′H-indole-3′-carbonyl)thiazol-4-carboxylic acid methyl ester (ITE). In this article, the synthesis of indothiazinone has been disclosed for the first time. Key feature includes direct and selective 3-acylation of indole in the presence of Lewis acid. In addition, an efficient preparation of N-substituted indothiazinone derivatives has been demonstrated.     

Syntheses of cystothiazole A and its stereoisomers: importance of stereochemistry for antifungal activity

The enantiocontrolled total syntheses of all the stereoisomers of a myxobacterial antibiotic, cystothiazole A, are described. The natural syn stereochemistry at the C4-C5 position was controlled by the asymmetric Evans aldol process, whereas the anti relationship was introduced by a modified Evans aldol methodology. Starting with a known aldehyde, the common substrate of the aldol reactions, cystothiazole A and its three stereoisomers were synthesized in 9 steps. All three stereoisomers did not show antifungal activity even at a dosage 2500-fold that of cystothiazole A.     

Heterologous expression and genetic engineering of the tubulysin biosynthetic gene cluster using Red/ET recombineering and inactivation mutagenesis

Although the tubulysin (tub) biosynthetic gene cluster has been located in two myxobacterial strains, it appears in both cases to be incomplete as obvious candidates for acyl transfer and oxidation functions are lacking. Here, we report the engineering of?a heterologous expression system for the tub biosynthetic pathway from strain Cystobacter sp. SBCb004. The entire tub core cluster was reconstituted from two cosmids using Red/ET recombineering and heterologous expression achieved in strains Pseudomonas putida and Myxococcus xanthus. Availability of the heterologous expression system and the natural producer strain SBCb004 provided a platform for the functional investigation of various biosynthetic genes by targeted inactivation. In addition, BLAST analysis of SBCb004 genome data was?used to identify multiple candidate monooxygenases, whose involvement in tubulysin assembly was evaluated using a combination of knockout mutagenesis and heterologous expression.     

Evolution of polyketides: post-PKS processing in the formation of spiroketals

It is now well recognized that natural products have directly or indirectly contributed to the discovery and development of as much as 75% of our current treatments for cancer and infectious disease as well as other indications. It cannot be overemphasized that new sources of chemical diversity are essential to the discovery of the next generation of chemotherapeutic agents. Characterization of the polyketide gene clusters responsible for the production of the spirangienes A and B provided detailed information regarding the biochemistry of myxobacterial secondary metabolism as well as significant insights into the evolution of post-PKS enzymes. The implications of these findings, coupled with additional examples, suggest that putative PKS products represent a new source of chemical diversity with chemotherapeutic potential and are thus worthy of further investigation.     

Utilization of alternate substrates by the first three modules of the epothilone synthetase assembly line

The epothilones, a family of macrolactone natural products produced by the myxobacterial species Sorangium cellulosum, are of current clinical interest as antitumor agents. Inspection of the structure of the epothilones suggests a hybrid polyketide/nonribosomal peptide biosynthetic origin, and the recent sequencing of the epothilone biosynthetic gene cluster has validated this proposal. Here we have examined unnatural substrates with the first two enzymes of the biosynthetic pathway, EpoA and EpoB, to investigate the enzymatic construction of alternate heterocyclic structures and the subsequent elongation of these products by the third enzyme of the pathway, EpoC. The epothilone biosynthetic machinery can utilize serine to install an oxazole in place of a thiazole in the epothilone structure and will tolerate functionalized donor groups from the EpoA-ACP domain to produce epothilone fragments modified at the C21 position. These studies with the early enzymes of the epothilone biosynthesis cluster suggest that combinatorial biosynthesis may be a viable means for producing a variety of epothilone analogues that incorporate diversity into the heterocycle starter unit.     

Configurational assignment of rhizopodin, an actin-binding macrolide from the myxobacterium Myxococcus stipitatus

The relative and absolute stereochemistry of the 38-membered myxobacterial polyketide rhizopodin, a potent actin-binding macrolide, was determined by J-based configurational analysis in combination with molecular modeling and chemical derivatization.     

A concise total synthesis of melithiazole C

A short and convergent synthesis of the myxobacterial antibiotic melithiazole C is described featuring a highly E-selective cross-metathesis as the key step.     

The Cytotoxic Natural Product Vioprolide A Targets Nucleolar Protein 14, Which Is Essential for Ribosome Biogenesis

Novel targets are needed for treatment of devastating diseases such as cancer. For decades, natural products have guided innovative therapies by addressing diverse pathways. Inspired by the potent cytotoxic bioactivity of myxobacterial vioprolides A–D, we performed in‐depth studies on their mode of action. Based on its prominent potency against human acute lymphoblastic leukemia (ALL) cells, we conducted thermal proteome profiling (TPP) and deciphered the target proteins of the most active derivative vioprolide A (VioA) in Jurkat cells. Nucleolar protein 14 (NOP14), which is essential in ribosome biogenesis, was confirmed as a specific target of VioA by a suite of proteomic and biological follow‐up experiments. Given its activity against ALL cells compared to healthy lymphocytes, VioA exhibits unique therapeutic potential for anticancer therapy through a novel mode of action.     

Production of the Bengamide Class of Marine Natural Products in Myxobacteria: Biosynthesis and Structure–Activity Relationships

The bengamides, sponge‐derived natural products that have been characterized as inhibitors of methionine aminopeptidases (MetAPs), have been intensively investigated as anticancer compounds. We embarked on a multidisciplinary project to supply bengamides by fermentation of the terrestrial myxobacterium M. virescens, decipher their biosynthesis, and optimize their properties as drug leads. The characterization of the biosynthetic pathway revealed that bacterial resistance to bengamides is conferred by Leu 154 of the myxobacterial MetAP protein, and enabled transfer of the entire gene cluster into the more suitable production host M. xanthus DK1622. A combination of semisynthesis of microbially derived bengamides and total synthesis resulted in an optimized derivative that combined high cellular potency in the nanomolar range with high metabolic stability, which translated to an improved half‐life in mice and antitumor efficacy in a melanoma mouse model.     

Chromatography-bioluminescence coupling reveals surprising bioactivity of inthomycin A

By employing a novel technique for the direct coupling of a bacterial bioassay with chromatography, we discovered a gyrA promotor active compound in myxobacterial extracts and elucidated the structure directly without any isolation step. As a result, we identified inthomycin A as the bioactive substance. Our method is based on a whole-cell bioluminescent reporter gene assay coupled with thin-layer chromatography for primary hit detection and with liquid chromatography (LC)/mass spectrometry or LC/NMR for dereplication and structure elucidation. Previously, inthomycin A has been isolated from Streptomycetes and was associated with the inhibition of cellulose biosynthesis and herbicidal activity. Thus, our findings support the basic principle that completely different microbial phyla are able to synthesize the same natural product. Moreover, our results indicate that inthomycin A affects bacterial DNA supercoiling, which reveals an unexpected bioactivity for this compound. These results can possibly promote further investigation of the biosynthesis as well as the biological activity of inthomycins and related natural products.

Stereochemical Determination of Tuscolid/Tuscorons and Total Synthesis of Tuscoron D and E: Insights into the Tuscolid/Tuscoron Rearrangement

The stereochemistry of the structurally unique myxobacterial polyketides tuscolid/tuscorons was determined by a combination of high‐field NMR studies, molecular modeling, and chemical derivatization and confirmed by a modular total synthesis of tuscorons D and E. Together with the discovery of three novel tuscorons, this study provides detailed insight into the chemically unprecedented tuscolid/tuscoron rearrangement cascade.     

Aetheramides A and B, potent HIV-inhibitory depsipeptides from a myxobacterium of the new genus "Aetherobacter"

Aetheramides are structurally distinctive cyclic peptides isolated from a novel myxobacterial genus proposed to be termed "Aetherobacter". The structures were solved by a combination of NMR analyses, quantum mechanical calculations, and chemical derivatizations. Aetheramides which contain a unique polyketide moiety and two amino acid residues potently inhibited HIV-1 infection with IC(50) values of ~0.015 μM. Furthermore aetheramides showed cytostatic activity against human colon carcinoma (HCT-116) cells with IC(50) values of 0.11 μM.     

Stereochemical determination of Archazolid A and B, highly potent vacuolar-type ATPase inhibitors from the Myxobacterium Archangium gephyra

[structure: see text] The relative and absolute stereochemistry of the structurally unique 24-membered myxobacterial macrolides archazolid A and B, highly potent vacuolar-type ATPase (V-ATPase) inhibitors in vitro and in vivo, was determined on the basis of a combination of extensive high-field NMR studies, including J-based configuration analysis, molecular modeling, and chemical methods.     

Pretubulysin,a Potent and Chemically Accessible Tubulysin Precursor from Angiococcus disciformis

Simplify, simplify, simplify! Pretubulysin (structure without the green substituents), a simplified tubulysin was prepared in the laboratory and also found in a natural myxobacterial source. This biosynthetic precursor of the tubulysins is not as active as tubulysins A and D but is still effective in picomolar concentrations against cancer cell lines.