Bioactive secondary metabolites from fungi with special emphasis on anti-infectives

Plenary talk, HRH-Hub conference, Ogbomosho, Nigeria, 291 Oct. 2025 Marc Stadler, Dept. Microbial Drugs, HZI Braunschweig, Germany; [email protected] Bioactive secondary metabolites from fungi with special emphasis on anti-infectives 2 Strong competition in tropical rainforests Image by Thomas Læssøe (Khao Yai NP, Thailand, 2007) Amauroderma sp. (Basidiomycota, Ganodermataceae) Squamotubera leratii (Ascomycota, Xylariaceae) Sei Fungal metabolites as drugs & pesticides Pharma indications Antibacterial (Penicillins, Cephalosporins) & antimycotic (Caspofungin, Micafungin) antibiotics A Immunosuppressive agents (e.g. Cyclosporin, Mycophenolic acid) A CNS-active drugs (e.g. Ergotamine) A Cardiovascular drugs (e.g. cholesterin-lowering Statins) A Agro indications Fungicides (e,g. Strobilurins) B Antiparasitic agents (e.g. emodepsin) A Fungal metabolites continue to be of great value as lead structures for development of new drugs & pesticides A: From Ascomycota B: From Basidiomycota A „novel“ class of antibiotics Clitopilus prunulus (Entolomataceae)Pleuromutilin Retapamulin (Altabax®/Altargo®) (semisynthetic derivative of pleuromutilin) First discovered from cultures of „Pleurotus“ spp. in 1951 Chemotaxonomic marker metabolite for the genus Clitopilus! Highly efficient against Gram-positive bacteria (inhibitor of protein synthesis) Only In 2007, Retapamulin was approved for treatment of skin infections (further derivatives are in development) First systemic pleuromutilin type antibiotic was approved by the US FDA in August of 2019 ! Antimycotic agents on the market/ under development Images from Mapook et al. Fungal Dviers 116 (2022) Many of them are derived from bacteria or fungi (as in case of the antibacterials) Ibrexafungerp, a semisynthetic emfumafungin Images from Mapook et al. Fungal Dviers 116 (2022) • First newly FDA-approved antimycotic in ages • (1,3)- β -D-glucan synthase inhibitor • Indications: e.g. invasive candidiasis, invasive aspergillosis First drug ever approved that is derived from a fungal endopyhte! Impressions from field work in Thailand Post-MC10 mycological foray (Mushroom Reseach Centre, Chiang Mai Prov., Aug 2014) Post IMC10 Foray, Mushroom Research Centre, Chiang Mai Prov., Thailand (2014) New pleurotins from Hohenbuehelia grisea H. grisea (Thailand) Birthe Sandargo Frank Surup B. Sandargo et al. (2018) J. Nat. Prod. 81:286-291 Benjarong Thongbai Metabolites of the “Orange Pingpong Bat Fungus” O O CH3 H3C CH3 CH3 CH3 O CH3 O O H3C Strobilurin G 10 9 pterulinic acid O HO O Cl 8 2,3-dihydro-1-benzoxepin derivative O H2N OCl 7 Favolon H3CO HO CH3 H3C CH3 H CH3 H H O O OH CH3 H3C HO O O O H3C Clara Chepkirui Metabolites of the “Orange Pingpong Bat Fungus” O O CH3 H3C CH3 CH3 CH3 O CH3 O O H3C Strobilurin G 10 9 pterulinic acid O HO O Cl 8 2,3-dihydro-1-benzoxepin derivative O H2N OCl 7 Favolon H3CO HO CH3 H3C CH3 H CH3 H H O O OH CH3 H3C HO O O O H3C Clara Chepkirui Chepkirui et al. (2016) Phytochemistry 132:95-101. Favolone – promising lead for a new antimycotic agent? Favolascha represents the dangerous Neomycota Due to global warming & globalisation, these fungi now invade temperate climates and are a big threat for our ecosystems! Data from Anke et al. (J. Antibiotics 1995) Compounds Antifungal activity MIC MIC (µg/ml) Cytotoxicity-L929 IC 50 (µg/mL) Candida tenuis - MUCL29892 Mucor plumbeusMUCL49355 Mouse fibroblastL929 HeLa- (KB3.1) 1 ≤ 9.37 ≤ 18.75 15.00 5.50 2 ≤ 4.68 ≤ 9.37 18.00 6.00 3 ≤ 4.68 ≤ 9.37 0.28 0.51 4 ≤ 4.68 ≤ 9.37 1.10 5.50 Favolone ≤ 2.34 ≤ 2.34 - - Nystatin c ≤ 2.34 ≤ 4.68 - - Epothilone B c - - - 0.00022 Epothilone A c - - 0.0038 - Methanol c - - - - 1-4: New Strobilurins [a] No antibacterial activities were observed for all the compounds against E. coli (DSM498) and B. subtilis (DSM10) at concentration ≥ 300µg/mL. [b] Nystatinantifungal reference. Epothilone A and B cytotoxicity test references. Methanolnegative control, - no activity. Chepkirui et al. (2016) Phytochemistry 132:95-101. The Favolaschia species that invades Europe is different from F. calocera Favolaschia claudopus has already been found in our neighbour countries (B, NL) and will soon reach Germany from New Zealand ! Name der PräsentationSeite 20 | New terpenoids from a tropical genus of Mycenaceae Name der PräsentationSeite 21 | S. Pfütze et al. (2023) J. Nat Prof./Molecules New terpenoids from a tropical genus of Mycenaceae From solid state culture after 7 months of growth From shake cultures Heimiomyces: cultured and studied on its secondary metabolites for the first time Frank Surup Sebastian Pfütze Even German forests harbour many basidiomycetes that are yet untapped for secondary metabolites Name der PräsentationSeite 23 | Name der PräsentationSeite 24 | New carbon skeleton; antiviral activities Significant antivral activity (HCV) Several new acorane sesquiterpenoids were obtained concurrently Kakamega rainforest (February 2023) • collected in RhinelandPalatinate from a dead trunk of Pseudotsuga menziesii in 2013 • most prolific producer of inhouse strain collection (previous screening) • In-depth investigation on secondary metabolism Page 32 | Disputation New melleolide-type meroterpenoids from A. ostoyae Armillaria ostoyae Frank SurupSebastian Pfütze Rita Toshe Esther Schulzke Esteban Charria Artit Khonsanit Tony New melleolide-type meroterpenoids from A. ostoyae Page 33 | Disputation Known melleolide-type meroterpenoids from A. ostoyae Aldehydes O O OH OHO HO OH 30 (melleolide D) Cl OH H O O OH OHHO HO OH 31 (melleolide E) H H O O OH OHO HO OH Cl OH 38 (10-hydroxy-5'-methoxy6'-chloroarmillane) H H O O OH OHO HO OH 28 (melleolide B) H H O O OH OHO HO OH Cl 32 (melleolide I) H H O O OH OHHO HO OH 33 (melledonol) OH H O O OH OHO HO OH 29 (melleolide C) OH H O O OH OHO 36 (A52a) Cl HO H H O O OH OHO HO O 35 (10-oxo-melleolide B) H H O O OH OHO HO 34 (10-dehydroxy-melleolide B) H H O O OH OHO HO 37 (5'-methoxy-6'- chloroarmillane) H H Cl OH Alcohols Page 34 | Disputation → together with the new derivatives, ~40% of all melleolides described in literature were isolated only from cultures of A. ostoyae A. ostoyae − metabolomics Esteban Charria Girón HPLC-UV/Vis chromatograms (210 nm) of different extracts Page 35 | Disputation • assessment of melleolide production in A. ostoyae under different cultivation conditions (three different media: rice, Q6 ½, YM6.3) • analysis of extracts by tandem mass spectrometry (timsTOF) • at first principal component analysis (PCA) to obtain overall picture of metabolomes observed in each extract rice Q6 ½ YM6.3 rice Q6 ½ myc Q6 ½ sup YM6.3 myc YM6.3 sup PCA scores plot A. ostoyae − metabolomics Page 36 | Disputation • tandem mass spectrometry analysis (timsTOF) of isolated pure compounds and evaluation of their fragmentation patterns • distinctive patterns observed → characteristic neutral losses corresponding to loss of orsellinic acid (OA) moiety • identification of chlorination and O-methylation in OA, as well as presence of hydroxylations, fatty acid side chains, dimerisation etc. Reference MS/MS spectrum Esteban Charria Girón A. ostoyae − metabolomics Page 37 | Disputation • for feature based molecular network (FBMN) analyses UHPLC-DAD-IMMS/MS data pre-processed with MetaboScape software • detected features organized into 156 molecular families (MFs), with 22 MFs belonging to compound class of “melleolides” • grouping of new “melleolides” by hierarchical clustering to show effect of cultivation conditions on metabolome in a heatmap Esteban Charria Girón Paradigm shift in agrochemistry ? • In the past decades, the Big Agro companies have essentally given • up their internal R&D activities on natural products and biocontrol. • Unfortunately, this has now led to serious problems regarding the development of innovative products that can help to satisfy the need of their customers …increasing resistances against strobilurins; problematic use of azoles in both, agrochemistry and antimycotic therapy Fungi and their secondary metabolites could fill some innovation gaps because mycologists have made substantial progress in the past years! Nematode antagonists can yield interesting chemistry Helaly et al. (2018) J. Nat Prod. Metabolites of new nematode antagonistic fungus that represents a new genus Remy B. Teponno Soleiman Helaly Species of a new genus of root endophytes attack nematodes and thereby protect the host plants! 6 New metabolites from Polydomus Compound [M + H] ion at m/z Formula (Ion) 4804.49 C44H62N5O9 5818.52 C45H64N5O9 13 788.50 C44H61N5O8 7430.25 C25H35NO5 8448.23 C24H33NO7 9462.26 C25H35NO7 10 446.27 C25H35NO6 11 476.27 C26H37NO7 12 502.28 C28H49NO7 61132.65 C60H89N7O14 Exact Mass: 1131,65 Sum Formula: C60H89N7O14 Exact mass: 461.56 Sum Formula: C25H35NO7 Exact Mass: 475,26 Sum Formula: C26H37NO7 911 Table 1. Compounds isolated from Polydomus Wennrich et al., submited Frank Surup JP Wennrich A new endophyte genus from Algeria Characterized by phylogenetic methods and new secondary metabolites Sarah Raouia Noumeur Close to pathogens like Madurella and also thermophilic ! Xylariales HQ Genomes Project Genome sequencing of 12 representatives of Hypoxylaceae and the ex-epitype strain of Xylaria hypoxylon High genome quality (between 16 and 88 contigs) with N50 ranging from 1,165,420 bp to 5,039,066 bp based on third generation techniques (Oxford nanopore/ONP) and PACBIO) polished with Illumina Gene prediction and annotation using Augustus and Genemark Between 8,988 and 11,762 genes per genome Eric Kuhnert Collaboration with Russell Cox and J. Kalinowsi in the course of DFG SPP 1991 „Taxon-omics“ Genome data provided by CEBITEC Bielefeld and DSMZ Wibberg et al. (2021) Fungal Divers. 1st in Class phylogenomic study on an ascomycete family using 3rd generation genome data Hypomontagnella spongiphila, the first fungal species that was erected based on phylogenomic comparison data 51 Several BGC analogues putatively encoding for metabolites that were never before found in Xylariales detected The majority of BGC detected could not be associated with any known class of fungal metabolites Name der PräsentationSeite 52 | 50 further high quality genomes generated (ONP) & annotated Examples with BGC counts see below DFG SPP1991, project Hypoxylomics (2022-2025) Hypoxylon texense genome: 10 contigs, nine of which represent the chromosome and the tenth the mitochondrion! . Zeng et al. JOF 9:726 (2023) Haoxuan Zeng New genus justified by chemotaxonomic evidence & molecular phylogeny Christopher Lambert Sporothriolides = Genus-specific marker metabolites with selective antifungal effects Preliminary data: Sporothriolide is a selective antifungal agent! Surprisingly, no cytotoxicity and no antibacterial effects were observed, despite the presence of an exo-methylene keto moiety ! Biosynthesis of sporothriolides and sporochartins Several key enzymes identified by heterologous expression Tian et al. (2020) Chem. Sci. 11:12477-12484 Alkyl citrates and their biosynthesis Feeding experiments Tian et al. (2020) Chem. Sci. 11:12477-12484 Transcriptome studies (and concurrent HPLC analysis)