My personal notes from the 2nd European Conference for Natural Products.
Monday
Manuela Toisin: Chemical probes for the capture and functionalisation of polyketide intermediates
"Stealing" intermediates with non-hydrolysable ACP mimics. This works in vivo and in vitro. Capture can be combined with functional groups on the probes to then do downstream "click" chemistry or similar modifications to generate new motifs.
Martin Grininger: Engineering FAS for directed polyketide synthesis
Using the close relationship of PKSs & FASs to utilise the insights on FAS pathways. Work on chain length control for iterative FASs & PKSs. Built up kinetic models. Chain length control seems to depend on binding affinity to AT (elongation happens) or MPT (exit of iteration happens) domains.
Florian Seebeck: Mechanisms of ergothioneine biosynthesis
Ergothioneine, a thiohistidine produced by bacteria and some fungi. Uptake transporters in all eukaryotes, though. Might be an antioxidant, as ergothioneine-transport-defective nematodes show oxidative DNA damage. Ergothioneine also occurs in some secondary metabolite products. Ovothiol is a similar thiohistidine. Erwinia tasmaniensis uses ovothiol to protect itself from the H2O2 oxidative burst during plant infection. EgtD is a histidine methyltransferase that, despite similar catalytic efficiency per step, prefers to methylate histidine all the way to 3-methyl-histidine. Me-His and especially 2-Me-His have a much higher affinity to active site, and thus prevent the exessive methylation of all histidine in the cell. Also, 3-Me-His has a higher affinity to the active site than His, thus serving as feedback inhibitor. EgtB is a 3-His facial triade non-heme iron enzyme that synthesises sulfoxide moieties in a 4 electron oxidation, a very unique mode of action. Conservative mutation can convert EgtB into a deoxidase, raising questions about current conceptions of how deoxidases work.
Tobias Gulder: Novel (Bio-)synthetic Strategies to Polycyclic Natural Products
Talking about polycyclic tetramate macrolactams (PTMs). Very unusual iterative PKS/NRPS module? Multiple reductive tailoring enzymes perform a hydride induced cyclisation cascade. Heterologously expressed enzymes can perform the synthesis in vitro with a similar yield as full synthesis, but with simpler experimental setup.
Markus Nett: Biosynthetic pathways from predatory bacteria
Pyxidicoccus fallax produces RNA synthesis inhibiting macrolide antibiotic, apparently very specific against Gram+ive prey.
Herpetosiphon aurantiacus, a filamentous predatory bacterium. Contains many secondary metabolites. Most gene clusters are cryptic, though, and no genetic tools exist.
Daniel Petras: The biosynthesis of albicidins
Albicidin is a PKS-NRPS hybrid gyrase inhibitor. Contains a large number of unusual AAs. Used Pieter Dorrestein's clustering tool (GNPS) to analyse LC-MS/MS data.
Martin Schäfer: Structure-function relationships of the DNA gyrase inhibitor simocyclinone
Aminocoumarin cluster in Streptomyces antibioticus Tü6040. Is there a sequence for Tü6040 available? ~72 kb cluster. Literature pathway for Simocyclinone D8 is likely wrong, SimC7, while important for antibiotic activity of the product, seems to be a ketoreductase, not a dehydratase.
Roderich Süssmuth: Peptide Antibiotics
Honeybees are important for cross-pollination, and thus important for agriculture. Paenibacillus larvae is a highly successful bee larvae pathogen. P. larvae genome encodes for 4 NRPS clusters. Paenilamicin, an 11-monomer modular PKS/NRPS hybrid, has some antibacterial & antifungal activity.
Engineering on the cyclodepsipeptides like enniatins show that substrate specificity & chain length control is dependent on specific modules in the iterative NRPS cluster.
Xinyu Liu: Hapalindole alkaloid biosynthesis: a treasure trove of novel enzymatic transformations
Stignematalean cyanobacteria, large regiochemical diversity in a single producer organism. Identification of two representative gene clusters gives insights into biosynthesis. Identified a new type of halogenases, the carrier protein dependent NHI halogenases. Both narrow & broad spectrum halogenases were identified. Enzymes are evolvable, interesting for synthetic biology.
Tohru Dairi: A novel enzyme capping N-terminus of various peptides with amidino-PheGly derivatives
Pheganomycin biosynthesis incooperates a non-proteinogenic N-terminus and a very variable C-terminus. So both ribosomal and NRPS synthesis would not be able to produce this compound. Enzymes for the production of the precursor dihydro-phenylglycine were found. Also, a RiPP prepeptide ORF and a peptidase were found. But how are these compounds linked? Cluster seems to contain an NRPS domain (antiSMASH predicts Valin specificity), but a knockout shows no pheganomycin deficiency. Instead, a peptide ligase was observed. The peptide ligase needs an amidino group on the N-terminal substrate, and can accept a wide variety of C-terminal peptides. Crystal structure analysis shows a large binding pocket and a long cleft around the catalytic Arg residue.
Kenan Bozhüyük & Florian Fleischhacker: Reprogramming nonribosomal peptide synthetases from Xenorhabdus and Photorhabdus.
Exchange ATC units, not TCA modules. C-A linker region highly conserved, cut
before WNATE motif to combine units. Less yield loss compared to module
exchange.
Till Schäberle: Corallopyronin A - two in one sweep
Observed new kind of shift domain, similar to DH domain but with different residues in the active site.
Tuesday:
Keynote: David Sherman
Polyketide synthetases are modular systems, but despite all the hype, lego-isation of PKSs has not been viable so far. On the way to a better understanding of the biosynthesis, chemoenzymatic synthesis helped to work with individual modules. Cryo-EM, and individual PKS/NRPS modules are at the lower end of the size you can visualise with that, is also giving great new insights in the mode of action. PikAIII, different to the excised DEBS-KS-AT model, has a single reaction center in the dimer. Substrate controlled divergence seems to direkt the chain length in methymycin / pikromycin biosysnthesis. The pikromycin TE can be engineered to allow for unnatural products with different stereochemistry by a single AA replacement in the active site. Reasons still not known.
Christian Ducho: Structure-activity relationship studies on muraymycin nucleoside-peptide antibiotics
Full synthesis of Muramycin-related structures allows to play with a number of side-chains to find new inhibitors to membrane protein translocase MraY. Assay is in vitro, though, not convinced that bioavailability is given. An omega-functionalised fatty acid was proposed to help get the substance through lipid membranes. To check, a flouresescence-tagged version of the omega-functionalised fatty acid was combined with immobilised vesicles and seemed to penetrate the membrane of those vesicles. Still, not entirely convinced. There's a reason why nature targes cell wall biosynthesis pathways outside of the membrane, not inside.
Esther Schmitt: Cyclomarin A Kills Mycobacteria and Malaria Parasites Using Distinct Modes of Action
Malarial parasites Plasmodium vivax and Plasmodium falciparum have a complex life cycle. Most malarial drugs attack plasmodia in the blood stages, where they are easily accessible. This works well against P. falciparum, but not as well agains the "relapsing malaria" caused by P. vivax, where some parasites go dormant inside liver cells, only to resurface at some later point, often years later. Additionaly, the current gold standard treatment of Arteminisin, while having been used successfully > 800 Mio. times already, is beginning to fail in some tropical regions with plasmodia beginning to show resistance due to a point mutation.
Cyclomarin A works against Mycobacteria by deregulating the ClpC1 protease, thus also killing of dormant mycobacteria once they leave hibernation. While plasmodia have a Clp protease analog, the target of Cyclomarin A is the diadenosine triphosphate hydrolase PFAp3Ase. PFAp3Ase cleaves the signaling molecule Ap3A, which in turn seems to be a tumor suppressor in mammals. Function in plasmodia is unknown.
Challenge on investigating the dormant stage is that experiments need to be done in monkey primary hepatocytes, which only survive in cell culture for several days. On the other hand, forming dormant stages by the monkey-specific P. cynomolgi also takes several days, race against time.
Mark Brönstrup: Structure-based optimisation of anabaenopeptins as TAFI inhibitors
Pilot stody to explore NPs from cyanobacteria. Anabaenopeptin B is a good TAFI inhibitor, thus functioning as an antithromboical agent. However, a generic Astra Zeneca patent covers the function, without being anabaenopeptin-specific. Also, the oral bioavailability is not really good. Idea: generate analogs with better bioavailability not covered by patent. Crystal structure of TAFI with bound anabaenopeptin B gave insights into the mode of action, allowing to create simple but active derivatives. While current leads are based on full synthesis, the functional insights from the natural product was vital to the understanding the bioactivity.
Shu-Ming Li: Prenyltransferase genes in fungal genomes
Prenylated indole alkaloids in fungy are (di)peptide derivatives with prenyl moiety at different positions, and with different biological activities. The corresponding prenyltransferases cluster into different classes in a phylogenetic analysis.
Prenylation of the indole ring incresases the cytotoxicity of cyclic dipeptides.
Jennifer Herrmann: Antibacterial disciformycins from myxobacteria as novel RNA polymerase inhibitors
Disciformicins inhibit S. aureus RNA polymerase subunits RpoB and RpoC, and show no cross-resistance to other known RNA polymerase inhibitors.
Peter Spieth: New strategies to bypass plant-based bioinsecticide production
The Neem tree (Azadirachta indica) contains endophytes that seems to produce a wide variety of compounds with insecticidal activity.
Wednesday
Tilmann Weber: Tools for the genomics driven discovery and engineering of natural products
antiSMASH talk! Tilmann is not only talking about antiSMASH, but also presenting the class IV lanthipeptide work on streptocollin, and our CRISPR-Cas9 toolset.
Olga Genilloud: Accessing previously uncultured bacteria with the Diffusion Sandwich System
Goal: Access as of yet uncultured bacteria to find new natural products. Reformulated culturing media, supplements of signalling molecules, diffusion chamber systems.
Diffusion Sandwich Systems are simpler to build than iChip. Basically a metal plate with holes. Holes contain plugs of agar or rubber gum containing 1-10 viable cells. Then buried in soil for 1-3 months. Growing cells are then "domesticated", Pilot study increased culturability of viable cells from 0.06% using the standard dilution approach up to ~8% using the sandwich system.
Chambers Hughes: Reactivity-guided isolation of biologically-active natural products
Ideally, you want to identify irreversible inhibitors of targets, substances that covalently bind their targets. But screening for this by target or biosynthesis is hard, as they are very diverse. However, they are often electrophilic, and that can be used for the screening. Use Thiol-based probes to scan for this, that contains a nice UV tag, a Br tag for MS, and a stereochemical reference. Alternative probe with a Cl tag and UV tag works very specifically on epoxide-containing products.
Max Crüsemann: Novel secondary metabolites from Salinispora through mass spectrometry-guidede genome mining approaches
We're drowning in genomic data, and methods to bridge MS data to genomic data is desirable. Pieter Dorrestein's Global natural products social (GNPS) molecular networking.
Wilfried Schwab: Activity-based profiling of physiologic aglycone library
Natural products from plants seem to be annoying to deal with.
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