Pyxipyrrolones: Structure Elucidation and Biosynthesis of Cytotoxic Myxobacterial Metabolites

In the search for new secondary metabolites from myxobacteria, a strain from the genus Pyxidicoccus was investigated. This led to the identification of a new class of natural products showing structural novelty and interesting biological activity. Isolation and structure elucidation of two analogues led to the identification of pyxipyrrolone A and B, harboring the novel 3‐methylene‐2,3,4,5,6,7,8,9‐octahydro‐1H‐benzo[e]isoindol‐1‐one scaffold. Mosher's ester analysis combined with NMR studies allowed the determination of all stereocenters but one. Genome sequencing of the producer strain led to the identification of a putative biosynthetic gene cluster for the pyxipyrrolones. The compounds showed activity against several cancer cell lines (μm range) with pyxipyrrolone B having 2‐ to 11‐fold higher activity than A, although they differ only by one methylene group.     

Expanding the Myxochelin Natural Product Family by Nicotinic Acid Containing Congeners

Myxobacteria represent a viable source of chemically diverse and biologically active secondary metabolites. The myxochelins are a well-studied family of catecholate-type siderophores produced by various myxobacterial strains. Here, we report the discovery, isolation, and structure elucidation of three new myxochelins N1–N3 from the terrestrial myxobacterium Corallococcus sp. MCy9049, featuring an unusual nicotinic acid moiety. Precursor-directed biosynthesis (PDB) experiments and total synthesis were performed in order to confirm structures, improve access to pure compounds for bioactivity testing, and to devise a biosynthesis proposal. The combined evaluation of metabolome and genome data covering myxobacteria supports the notion that the new myxochelin congeners reported here are in fact frequent side products of the known myxochelin A biosynthetic pathway in myxobacteria.     

Recent Advances in Discovery of Lead Structures from Microbial Natural Products: Genomics- and Metabolomics-Guided Acceleration

Natural products (NPs) are evolutionarily optimized as drug-like molecules and remain the most consistently successful source of drugs and drug leads. They offer major opportunities for finding novel lead structures that are active against a broad spectrum of assay targets, particularly those from secondary metabolites of microbial origin. Due to traditional discovery approaches’ limitations relying on untargeted screening methods, there is a growing trend to employ unconventional secondary metabolomics techniques. Aided by the more in-depth understanding of different biosynthetic pathways and the technological advancement in analytical instrumentation, the development of new methodologies provides an alternative that can accelerate discoveries of new lead-structures of natural origin. This present mini-review briefly discusses selected examples regarding advancements in bioinformatics and genomics (focusing on genome mining and metagenomics approaches), as well as bioanalytics (mass-spectrometry) towards the microbial NPs-based drug discovery and development. The selected recent discoveries from 2015 to 2020 are featured herein.     

Genomics-Driven Discovery of a Novel Glutarimide Antibiotic from Burkholderia gladioli Reveals an Unusual Polyketide Synthase Chain Release Mechanism

A gene cluster encoding a cryptic trans-acyl transferase polyketide synthase (PKS) was identified in the genomes of Burkholderia gladioli BCC0238 and BCC1622, both isolated from the lungs of cystic fibrosis patients. Bioinfomatics analyses indicated the PKS assembles a novel member of the glutarimide class of antibiotics, hitherto only isolated from Streptomyces species. Screening of a range of growth parameters led to the identification of gladiostatin, the metabolic product of the PKS. NMR spectroscopic analysis revealed that gladiostatin, which has promising activity against several human cancer cell lines and inhibits tumor cell migration, contains an unusual 2-acyl-4-hydroxy-3-methylbutenolide in addition to the glutarimide pharmacophore. An AfsA-like domain at the C-terminus of the PKS was shown to catalyze condensation of 3-ketothioesters with dihydroxyacetone phosphate, thus indicating it plays a key role in polyketide chain release and butenolide formation.     

Genome Mining Reveals Two Missing CrtP and AldH Enzymes in the C30 Carotenoid Biosynthesis Pathway in Planococcus faecalis AJ003T

Planococcus faecalis AJ003^T produces glycosyl-4,4′-diaponeurosporen-4′-ol-4-oic acid as its main carotenoid. Five carotenoid pathway genes were presumed to be present in the genome of P. faecalis AJ003^T; however, 4,4-diaponeurosporene oxidase (CrtP) was non-functional, and a gene encoding aldehyde dehydrogenase (AldH) was not identified. In the present study, a genome mining approach identified two missing enzymes, CrtP2 and AldH2454, in the glycosyl-4,4′-diaponeurosporen-4′-ol-4-oic acid biosynthetic pathway. Moreover, CrtP2 and AldH enzymes were functional in heterologous Escherichia coli and generated two carotenoid aldehydes (4,4′-diapolycopene-dial and 4,4′-diaponeurosporene-4-al) and two carotenoid carboxylic acids (4,4′-diaponeurosporenoic acid and 4,4′-diapolycopenoic acid). Furthermore, the genes encoding CrtP2 and AldH2454 were located at a distance the carotenoid gene cluster of P. faecalis.     

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.     

Biosynthesis of the Apoptolidins in Nocardiopsis sp. FU 40

The apoptolidins are 20/21-membered macrolides produced by Nocardiopsis sp. FU40. Several members of this family are potent and remarkably selective inducers of apoptosis in cancer cell lines, likely via a distinct mitochondria associated target. To investigate the biosynthesis of this natural product, the complete genome of the apoptolidin producer Nocardiopsis sp. FU40 was sequenced and a 116 kb region was identified containing a putative apoptolidin biosynthetic gene cluster. The apoptolidin gene cluster comprises a type I polyketide synthase, with 13 homologating modules, apparently initiated in an unprecedented fashion via transfer from a methoxymalonyl-acyl carrier protein loading module. Spanning approximately 39 open reading frames, the gene cluster was cloned into a series of overlapping cosmids and functionally validated by targeted gene disruption experiments in the producing organism. Disruption of putative PKS and P₄₅₀ genes delineated the roles of these genes in apoptolidin biosynthesis and chemical complementation studies demonstrated intact biosynthesis peripheral to the disrupted genes. This work provides insight into details of the biosynthesis of this biologically significant natural product and provides a basis for future mutasynthetic methods for the generation of non-natural apoptolidins.     

Secure and Sustainable Sourcing of Plant Tissues for the Exhaustive Exploration of Their Chemodiversity

The main challenge of plant chemical diversity exploration is how to develop tools to study exhaustively plant tissues. Their sustainable sourcing is a limitation as bioguided strategies and dereplication need quite large amounts of plant material. We examine if alternative solutions could overcome these difficulties by obtaining a secure, sustainable, and scalable source of tissues able to biosynthesize an array of metabolites. As this approach would be as independent of the botanical origin as possible, we chose eight plant species from different families. We applied a four steps culture establishment procedure, monitoring targeted compounds through mass spectrometry-based analytical methods. We also characterized the capacities of leaf explants in culture to produce diverse secondary metabolites. In vitro cultures were successfully established for six species with leaf explants still producing a diversity of compounds after the culture establishment procedure. Furthermore, explants from leaves of axenic plantlets were also analyzed. The detection of marker compounds was confirmed after six days in culture for all tested species. Our results show that the first stage of this approach aiming at easing exploration of plant chemodiversity was completed, and leaf tissues could offer an interesting alternative providing a constant source of natural compounds.     

Modern Approaches in the Discovery and Development of Plant-Based Natural Products and Their Analogues as Potential Therapeutic Agents

Natural products represents an important source of new lead compounds in drug discovery research. Several drugs currently used as therapeutic agents have been developed from natural sources; plant sources are specifically important. In the past few decades, pharmaceutical companies demonstrated insignificant attention towards natural product drug discovery, mainly due to its intrinsic complexity. Recently, technological advancements greatly helped to address the challenges and resulted in the revived scientific interest in drug discovery from natural sources. This review provides a comprehensive overview of various approaches used in the selection, authentication, extraction/isolation, biological screening, and analogue development through the application of modern drug-development principles of plant-based natural products. Main focus is given to the bioactivity-guided fractionation approach along with associated challenges and major advancements. A brief outline of historical development in natural product drug discovery and a snapshot of the prominent natural drugs developed in the last few decades are also presented. The researcher’s opinions indicated that an integrated interdisciplinary approach utilizing technological advances is necessary for the successful development of natural products. These involve the application of efficient selection method, well-designed extraction/isolation procedure, advanced structure elucidation techniques, and bioassays with a high-throughput capacity to establish druggability and patentability of phyto-compounds. A number of modern approaches including molecular modeling, virtual screening, natural product library, and database mining are being used for improving natural product drug discovery research. Renewed scientific interest and recent research trends in natural product drug discovery clearly indicated that natural products will play important role in the future development of new therapeutic drugs and it is also anticipated that efficient application of new approaches will further improve the drug discovery campaign.     

Constrained Cyclopeptides: Biaryl Formation through Pd‐Catalyzed C−H Activation in Peptides—Structural Control of the Cyclization vs. Cyclodimerization Outcome

A series of short tryptophan‐phenylalanine peptides containing an iodo substituent on the phenyl ring was subjected to Pd‐catalyzed CH activation reactions to give the corresponding aryl‐indole coupled products. Two types of adducts were generated: cyclomonomer and cyclodimeric peptides; no evidence of oligo‐ or polymerization products was detected. Contrary to standard peptide macrocyclizations, the factors controlling the fate of the reaction are the number of amino acids between the aromatic residues and the regiochemistry of the parent iodo derivative, independent of both the concentration and the cyclization mode. The method is general and allows access to novel biaryl peptidic topologies, which have been fully characterized.     

Unusual Acetylation‐Dependent Reaction Cascade in the Biosynthesis of the Pyrroloindole Drug Physostigmine

Physostigmine is a parasympathomimetic drug used to treat a variety of neurological disorders, including Alzheimer’s disease and glaucoma. Because of its potent biological activity and unique pyrroloindole skeleton, physostigmine has been the target of many organic syntheses. However, the biosynthesis of physostigmine has been relatively understudied. In this study, we identified a biosynthetic gene cluster for physostigmine by genome mining. The 8.5 kb gene cluster encodes eight proteins (PsmA–H), seven of which are required for the synthesis of physostigmine from 5‐hydroxytryptophan, as shown by in vitro total reconstitution. Further genetic and enzymatic studies enabled us to delineate the biosynthetic pathway for physostigmine. The pathway features an unusual reaction cascade consisting of highly coordinated methylation and acetylation/deacetylation reactions.     

Bioorthogonal Probes for the Study of MDM2‐p53 Inhibitors in Cells and Development of High‐Content Screening Assays for Drug Discovery

To study the behavior of MDM2‐p53 inhibitors in a disease‐relevant cellular model, we have developed and validated a set of bioorthogonal probes that can be fluorescently labeled in cells and used in high‐content screening assays. By using automated image analysis with single‐cell resolution, we could visualize the intracellular target binding of compounds by co‐localization and quantify target upregulation upon MDM2‐p53 inhibition in an osteosarcoma model. Additionally, we developed a high‐throughput assay to quantify target occupancy of non‐tagged MDM2‐p53 inhibitors by competition and to identify novel chemical matter. This approach could be expanded to other targets for lead discovery applications.     

In Silico Analysis of P450s and Their Role in Secondary Metabolism in the Bacterial Class Gammaproteobacteria

The impact of lifestyle on shaping the genome content of an organism is a well-known phenomenon and cytochrome P450 enzymes (CYPs/P450s), heme-thiolate proteins that are ubiquitously present in organisms, are no exception. Recent studies focusing on a few bacterial species such as Streptomyces, Mycobacterium, Cyanobacteria and Firmicutes revealed that the impact of lifestyle affected the P450 repertoire in these species. However, this phenomenon needs to be understood in other bacterial species. We therefore performed genome data mining, annotation, phylogenetic analysis of P450s and their role in secondary metabolism in the bacterial class Gammaproteobacteria. Genome-wide data mining for P450s in 1261 Gammaproteobacterial species belonging to 161 genera revealed that only 169 species belonging to 41 genera have P450s. A total of 277 P450s found in 169 species grouped into 84 P450 families and 105 P450 subfamilies, where 38 new P450 families were found. Only 18% of P450s were found to be involved in secondary metabolism in Gammaproteobacterial species, as observed in Firmicutes as well. The pathogenic or commensal lifestyle of Gammaproteobacterial species influences them to such an extent that they have the lowest number of P450s compared to other bacterial species, indicating the impact of lifestyle on shaping the P450 repertoire. This study is the first report on comprehensive analysis of P450s in Gammaproteobacteria.