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Triangularia mangenotti was analyzed for the production of secondary metabolites, resulting in the isolation of known zopfinol (1) and its new derivatives zopfinol B–C (2–4), the 10-membered lactones 7-O-acetylmultiplolide A (5) and 8-O-acetylmultiplolide A (6), together with sordarin (7), sordarin B (8), and hypoxysordarin (9). The absolute configuration of 1 was elucidated by the synthesis of MPTA-esters. Compound 1 showed antimicrobial activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus and the fungus Mucor hiemalis. While 4 was weakly antibacterial, 3 showed stronger antibiotic activity against the Gram-positive bacteria and weak antifungal activity against M. hiemalis and Rhodotorula glutinis. We furthermore observed the cytotoxicity of 1, 3 and 4 against the mammalian cell lines KB3.1 and L929. Moreover, the new genus Pseudorhypophila is introduced herein to accommodate Triangularia mangenotii together with several species of Zopfiella—Z. marina, Z. pilifera, and Z. submersa. These taxa formed a well-supported monophyletic clade in the recently introduced family Navicularisporaceae, located far from the type species of the respective original genera, in a phylogram based on the combined dataset sequences of the internal transcribed spacer region (ITS), the nuclear rDNA large subunit (LSU), and fragments of the ribosomal polymerase II subunit 2 (rpb2) and β-tubulin (tub2) genes. Zopfiella submersa is synonymized with P. marina due to the phylogenetic and morphological similarity. The isolation of zopfinols 1–4 and sordarins 7–9 confirms the potential of this fungal order as producers of bioactive compounds and suggests these compounds as potential chemotaxonomic markers.

To explore the chemical and biological diversities of diterpenoids from the fungus Talaromyces adpressus , a previously unknown biosynthetic gene cluster (BGC, tdn ) for sordarin (a well-known fungal antibiotics) was discovered by leveraging the genome mining method. Heterologous expressions of key genes of tdn in Aspergillus oryzae , led to the determination of one new diterpenoid, cycloaraneosene-9-ol-8-one ( 4 ), and three known diterpenoids, cycloaraneosene ( 1 ), cycloaraneosene-9-ol ( 2 ), cycloaraneosene-8,9-diol ( 3 ). The structures of 1 – 4 was elucidated well via detailed analysis of 1D and 2D NMR, GCMS, HRESIMS, IR data, and comparison with reported data. Structurally, compounds 1 – 4 were belonging to fusicoccane diterpenoids with a classical tricyclic 5/8/5 ring system, which are participated in the biosynthesis of sordarin. Compound 4 maybe a key precursor for a Baeyer–Villiger like reaction with C8–C9 bond cleavage in the biosynthetic pathway of sordarin. Moreover, all isolates were evaluated for their bioactivities, compounds 3 , and 4 exhibited inhibitory activities against the human cancer cell lines with IC 50 values ranging from 7.8 to 32.4 µ M. 3 and 4 promote cell apoptosis of HCT-116 and HepG2 cells, and suppress cell migration of HepG2 cells. As well, 3 and 4 also decrease gene expression of cell proliferation related molecules BCL-2 and cyclin D1, while increase expression of cell apoptosis related gene BAX. Targets predication and molecular docking indicate that compound 4 exhibits stronger affinity for DBL, suggesting its excellent binding potential. This finding will be enriched the structures and bioactivities of diterpenoids with a tricyclic 5/8/5 ring system, most importantly, will provide new strategies for the synthetic biological research of sordarins. Graphical Abstract

An 11.7‐Å‐resolution cryo‐EM map of the yeast 80S·eEF2 complex in the presence of the antibiotic sordarin was interpreted in molecular terms, revealing large conformational changes within eEF2 and the 80S ribosome, including a rearrangement of the functionally important ribosomal intersubunit bridges. Sordarin positions domain III of eEF2 so that it can interact with the sarcin–ricin loop of 25S rRNA and protein rpS23 (S12p). This particular conformation explains the inhibitory action of sordarin and suggests that eEF2 is stalled on the 80S ribosome in a conformation that has similarities with the GTPase activation state. A ratchet‐like subunit rearrangement (RSR) occurs in the 80S·eEF2·sordarin complex that, in contrast to Escherichia coli 70S ribosomes, is also present in vacant 80S ribosomes. A model is suggested, according to which the RSR is part of a mechanism for moving the tRNAs during the translocation reaction.

Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the msh3 locus of Schizosaccharomyces pombe impaired the function of the nearby located dph3 gene. Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. We constructed dph3 and msh3 strains with mutated ATG start codons ( ATGmut ), which allowed investigating drug sensitivity without potential interference by marker insertions. The dph3 - ATGmut and a dph3::loxP - ura4 - loxM gene disruption strain, but not msh3 - ATGmut , turned out to be sensitive to hydroxyurea and methyl methanesulfonate, likewise the strains with cassettes integrated at the msh3 locus. The fungicide sordarin, which inhibits diphthamide modified eEF2 of Saccharomyces cerevisiae , barely affected survival of wild type and msh3Δ S. pombe cells, while the dph3Δ mutant was sensitive. The msh3 - ATG mutation, but not dph3Δ or the dph3 - ATG mutation caused a defect in mating-type switching, indicating that the ura4 marker at the dph3 locus did not interfere with Msh3 function. We conclude that Dph3 is required for cellular resistance to the fungicide sordarin and to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. This is likely mediated by efficient translation of proteins in response to DNA damage and replication stress.

Diphtheria toxin (DT) inhibits eukaryotic translation elongation factor 2 (eEF2) by ADP-ribosylation in a fashion that requires diphthamide, a modified histidine residue on eEF2. In budding yeast, diphthamide formation involves seven genes, DPH1-DPH7. In an effort to further study diphthamide synthesis and interrelation among the Dph proteins, we found, by expression in E. coli and co-immune precipitation in yeast, that Dph1 and Dph2 interact and that they form a complex with Dph3. Protein-protein interaction mapping shows that Dph1-Dph3 complex formation can be dissected by progressive DPH1 gene truncations. This identifies N- and C-terminal domains on Dph1 that are crucial for diphthamide synthesis, DT action and cytotoxicity of sordarin, another microbial eEF2 inhibitor. Intriguingly, dph1 truncation mutants are sensitive to overexpression of DPH5, the gene necessary to synthesize diphthine from the first diphthamide pathway intermediate produced by Dph1-Dph3. This is in stark contrast to dph6 mutants, which also lack the ability to form diphthamide but are resistant to growth inhibition by excess Dph5 levels. As judged from site-specific mutagenesis, the amidation reaction itself relies on a conserved ATP binding domain in Dph6 that, when altered, blocks diphthamide formation and confers resistance to eEF2 inhibition by sordarin.

Using a sordarin derivative, an antifungal drug, it was possible to determine the structure of a eukaryotic ribosome·EF2 complex at 17.5 Å resolution by three‐dimensional (3D) cryo‐electron microscopy. EF2 is directly visible in the 3D map and the overall arrangement of the complex from Saccharomyces cerevisiae corresponds to that previously seen in Escherichia coli . However, pronounced differences were found in two prominent regions. First, in the yeast system the interaction between the elongation factor and the stalk region of the large subunit is much more extensive. Secondly, domain IV of EF2 contains additional mass that appears to interact with the head of the 40S subunit and the region of the main bridge of the 60S subunit. The shape and position of domain IV of EF2 suggest that it might interact directly with P‐site‐bound tRNA.

Using a sordarin derivative, an antifungal drug, it was possible to determine the structure of a eukaryotic ribosome EF2 complex at 17.5 Aa resolution by three-dimensional (3D) cryo- electron microscopy. EF2 is directly visible in the 3D map and the overall arrangement of the complex from Saccharomyces cerevisiae corresponds to that previously seen in Escherichia coli. However, pronounced differences were found in two prominent regions. First, in the yeast system the interaction between the elongation factor and the stalk region of the large subunit is much more extensive. Secondly, domain IV of EF2 contains additional mass that appears to interact with the head of the 40S subunit and the region of the main bridge of the 60S subunit. The shape and position of domain IV of EF2 suggest that it might interact directly with P-site-bound tRNA.

BMS-353645, also known as sordarin, was of interest based on its activity against pathogenic fungi. The objective of these studies was to provide high quality starting substrate for chemical modification aimed at further improving biological activity, with particular interest in the inhibition of Aspergillus. In the work presented here, Design of Experiments, or DOE, was successfully combined with traditional approaches to significantly improve sordarin yields in fermentation flasks. Overall, yields were increased 25-fold from <100 μg/g to as high as 2,609 μg/g in flasks through the use of various medium and conduction changes supplemented with DOE. The improved process was then successfully scaled to pilot plant tanks with the best batch producing 2,389 μg/g sordarin at the 250-l scale.

The increasing importance of invasive fungal infection as a cause of severe morbidity and mortality in immunocompromised patients has fuelled the development of new antifungal agents, including new triazole agents and echinocandins. Failure to diagnose fungal infection adequately has hampered the evaluation of these drugs in clinical trials. Consequently, the evidence base behind many prophylactic and empirical treatment strategies has been poor. Recent advances have improved diagnostic criteria and opened the way for more rational usage of these expensive preparations. The proven efficacy and improved safety profile of newer agents is a major advantage. Furthermore, better understanding of the immune response may allow novel strategies of adjunctive therapy and immunomodulation.