Metabolic Investigation and Auxiliary Enzyme Modelization of the Pyrrocidine Pathway Allow Rationalization of Paracyclophane-Decahydrofluorene Formation.

Fungal paracyclophane-decahydrofluorene-containing natural products are complex polycyclic metabolites derived from similar hybrid PKS-NRPS pathways. Herein we studied the biosynthesis of pyrrocidines, one representative of this family, by gene inactivation in the producer Sarocladium zeae coupled to thorough metabolic analysis and molecular modeling of key enzymes. We characterized nine pyrrocidines and analogues as well as in mutants a variety of accumulating metabolites with new structures including rare cis-decalin, cytochalasan, and fused 6/15/5 macrocycles. This diversity highlights the extraordinary plasticity of the pyrrocidine biosynthetic gene cluster. From accumulating metabolites, we delineated the scenario of pyrrocidine biosynthesis. The ring A of the decahydrofluorene is installed by PrcB, a membrane-bound cyclizing isomerase, on a PKS-NRPS-derived pyrrolidone precursor. Docking experiments in PrcB allowed us to characterize the active site suggesting a mechanism triggered by arginine-mediated deprotonation at the terminal methyl of the substrate. Next, two integral membrane proteins, PrcD and PrcE, each predicted as a four-helix bundle, perform hydroxylation of the pyrrolidone ring and paracyclophane formation, respectively. Modelization of PrcE highlights a topological homology with vitamin K oxido-reductase and the presence of a disulfide bond. Our results suggest a previously unsuspected coupling mechanism via a transient loss of aromaticity of tyrosine residue to form the strained paracyclophane motif. Finally, the lipocalin-like protein PrcX drives the exo-cycloaddition yielding ring B and C of the decahydrofluorene to afford pyrrocidine A, which is transformed by a reductase PrcI to form pyrrocidine B. These insights will greatly facilitate the microbial production of pyrrocidine analogues by synthetic biology.

Identification of novel Zika virus NS3 protease inhibitors with different inhibition modes by integrative experimental and computational approaches.

Zika virus (ZIKV) infection is associated with severe neurological disorders and congenital malformation. Despite efforts to eradicate the disease, there is still neither vaccine nor approved drugs to treat ZIKV infection. The NS2B-NS3 protease is a validated drug target since it is essential to polyprotein virus maturation. In the present study, we describe an experimental screening of 2,320 compounds from the chemical library of the Muséum National d'Histoire Naturelle of Paris on ZIKV NS2B-NS3 protease. A total of 96 hits were identified with 90% or more of inhibitory activity at 10 µM. Amongst the most active compounds, five were analyzed for their inhibitory mechanisms by kinetics assays and computational approaches such as molecular docking. 2-(3-methoxyphenoxy) benzoic acid (compound 945) show characteristics of a competitive inhibition (Ki = 0.49 µM) that was corroborated by its molecular docking at the active site of the NS2B-NS3 protease. Taxifolin (compound 2292) behaves as an allosteric inhibitor whereas 3,8,9-trihydroxy-2-methyl-1H-phenalen-1-one (compound 128), harmol (compound 368) and anthrapurpurin (compound 1499) show uncompetitive inhibitions. These new NS2B-NS3 protease inhibitors are valuable hits to further hit-to-lead optimization.

Use of 2,6-diaminopurine as a potent suppressor of UGA premature stop codons in cystic fibrosis.

Nonsense mutations are responsible for around 10% of cases of genetic diseases, including cystic fibrosis. 2,6-diaminopurine (DAP) has recently been shown to promote efficient readthrough of UGA premature stop codons. In this study, we show that DAP can correct a nonsense mutation in the Cftr gene in vivo in a new CF mouse model, in utero, and through breastfeeding, thanks, notably, to adequate pharmacokinetic properties. DAP turns out to be very stable in plasma and is distributed throughout the body. The ability of DAP to correct various endogenous UGA nonsense mutations in the CFTR gene and to restore its function in mice, in organoids derived from murine or patient cells, and in cells from patients with cystic fibrosis reveals the potential of such readthrough-stimulating molecules in developing a therapeutic approach. The fact that correction by DAP of certain nonsense mutations reaches a clinically relevant level, as judged from previous studies, makes the use of this compound all the more attractive.

2,6-Diaminopurine as a highly potent corrector of UGA nonsense mutations.

Nonsense mutations cause about 10% of genetic disease cases, and no treatments are available. Nonsense mutations can be corrected by molecules with nonsense mutation readthrough activity. An extract of the mushroom Lepista inversa has recently shown high-efficiency correction of UGA and UAA nonsense mutations. One active constituent of this extract is 2,6-diaminopurine (DAP). In Calu-6 cancer cells, in which TP53 gene has a UGA nonsense mutation, DAP treatment increases p53 level. It also decreases the growth of tumors arising from Calu-6 cells injected into immunodeficient nude mice. DAP acts by interfering with the activity of a tRNA-specific 2'-O-methyltransferase (FTSJ1) responsible for cytosine 34 modification in tRNATrp. Low-toxicity and high-efficiency UGA nonsense mutation correction make DAP a good candidate for the development of treatments for genetic diseases caused by nonsense mutations.

Optimized approach for the identification of highly efficient correctors of nonsense mutations in human diseases.

About 10% of patients with a genetic disease carry a nonsense mutation causing their pathology. A strategy for correcting nonsense mutations is premature termination codon (PTC) readthrough, i.e. incorporation of an amino acid at the PTC position during translation. PTC-readthrough-activating molecules appear as promising therapeutic tools for these patients. Unfortunately, the molecules shown to induce PTC readthrough show low efficacy, probably because the mRNAs carrying a nonsense mutation are scarce, as they are also substrates of the quality control mechanism called nonsense-mediated mRNA decay (NMD). The screening systems previously developed to identify readthrough-promoting molecules used cDNA constructs encoding mRNAs immune to NMD. As the molecules identified were not selected for the ability to correct nonsense mutations on NMD-prone PTC-mRNAs, they could be unsuitable for the context of nonsense-mutation-linked human pathologies. Here, a screening system based on an NMD-prone mRNA is described. It should be suitable for identifying molecules capable of efficiently rescuing the expression of human genes harboring a nonsense mutation. This system should favor the discovery of candidate drugs for treating genetic diseases caused by nonsense mutations. One hit selected with this screening system is presented and validated on cells from three cystic fibrosis patients.

A Reactive Eremophilane and Its Antibacterial 2(1H)-Naphthalenone Rearrangement Product, Witnesses of a Microbial Chemical Warfare.

Two sesquiterpenes, 4-epi-microsphaeropsisin (1) and a dihydrofurano-2(1H)-naphthalenone (variabilone, 2) which represents a new skeleton, were isolated from endophytic fungus Paraconiothyrium variabile. Reactivity studies showed that eremophilane 1 is a precursor of 2 through acid-promoted methyl 1,2-migration and aromatization. An electrophilic intermediate of this transformation was intercepted by N-acetylcysteamine, a biomimetic nucleophile. Only compound 2 was antibacterial against endophytic bacterium Bacillus subtilis (coisolated with P. variabile), suggesting a role in the microbial competition in plants.

Automated Detection of Natural Halogenated Compounds from LC-MS Profiles-Application to the Isolation of Bioactive Chlorinated Compounds from Marine-Derived Fungi.

A collection of culture extracts obtained from several marine-derived fungal strains collected on the French Atlantic coast was investigated by high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) in order to prospect for halogenated compounds and to identify potentially new ones. To achieve a fast, automated, and efficient data analysis, a bioinformatics tool named MeHaloCoA (Marine Halogenated Compound Analysis) was developed and included into R. After extraction of all the peaks from the metabolic fingerprints and their associated mass spectra, a mathematical filter based on mass isotopic profiles allowed the selective detection of halogenated (Cl and Br) molecules. Integrating MeHaloCoA into a dereplication approach allowed the identification of known and new halogenated compounds in a competitive amount of time. Subsequent targeted purification led to the isolation of several chlorinated metabolites, including two new natural products with bioactive potential, griseophenone I and chlorogriseofulvin, from a marine-derived Penicillium canescens strain.

Direct biosynthetic cyclization of a distorted paracyclophane highlighted by double isotopic labelling of L-tyrosine.

The biosynthesis of pyrrocidines, fungal PK-NRP compounds featuring a strained [9]paracyclophane, was investigated in Acremonium zeae. We used a synthetic L-tyrosine probe, labelled with oxygen 18 as a reporter of phenol reactivity and carbon 13 as a tracer of incorporation of this exogenous precursor. The ((18)O)phenolic oxygen was incorporated, suggesting that phenol behaves as a nucleophile during the formation of the bent aryl ether.

The fungal leaf endophyte Paraconiothyrium variabile specifically metabolizes the host-plant metabolome for its own benefit.

Fungal endophytes live inside plant tissues and some have been found to provide benefits to their host. Nevertheless, their ecological impact is not adequately understood. Considering the fact that endophytes are continuously interacting with their hosts, it is conceivable that both partners have substantial influence on each other's metabolic processes. In this context, we have investigated the action of the endophytic fungus Paraconiothyrium variabile, isolated from the leaves of Cephalotaxus harringtonia, on the secondary metabolome of the host-plant. The alteration of the leaf compounds by the fungus was monitored through metabolomic approaches followed by structural characterization of the altered products. Out of more than a thousand molecules present in the crude extract of the plant leaf, we have observed a specific biotransformation of glycosylated flavonoids by the endophyte. In all cases it led to the production of the corresponding aglycone via deglycosylation. The deglycosylated flavonoids turned out to display significant beneficial effects on the hyphal growth of germinated spores. Our finding, along with the known allelopathic role of flavonoids, illustrates the chemical cooperation underlying the mutualistic relationship between the plant and the endophyte.

Natural aristolactams and aporphine alkaloids as inhibitors of CDK1/cyclin B and DYRK1A.

In an effort to find potent inhibitors of the protein kinases DYRK1A and CDK1/Cyclin B, a systematic in vitro evaluation of 2,500 plant extracts from New Caledonia and French Guyana was performed. Some extracts were found to strongly inhibit the activity of these kinases. Four aristolactams and one lignan were purified from the ethyl acetate extracts of Oxandra asbeckii and Goniothalamus dumontetii, and eleven aporphine alkaloids were isolated from the alkaloid extracts of Siparuna pachyantha, S. decipiens, S. guianensis and S. poeppigii. Among these compounds, velutinam, aristolactam AIIIA and medioresinol showed submicromolar IC50 values on DYRK1A.

Identification of novel inhibitors of DNA methylation by screening of a chemical library.

In order to discover new inhibitors of the DNA methyltransferase 3A/3L complex, we used a medium-throughput nonradioactive screen on a random collection of 1120 small organic compounds. After a primary hit detection against DNA methylation activity of the murine Dnmt3A/3L catalytic complex, we further evaluated the EC50 of the 12 most potent hits as well as their cytotoxicity on DU145 prostate cancer cultured cells. Interestingly, most of the inhibitors showed low micromolar activities and little cytotoxicity. Dichlone, a small halogenated naphthoquinone, classically used as pesticide and fungicide, showed the lowest EC50 at 460 nM. We briefly assessed the selectivity of a subset of our new inhibitors against hDNMT1 and bacterial Dnmts, including M. SssI and EcoDam, and the protein lysine methyltransferase PKMT G9a and the mode of inhibition. Globally, the tested molecules showed a clear preference for the DNA methyltransferases, but poor selectivity among them. Two molecules including Dichlone efficiently reactivated YFP gene expression in a stable HEK293 cell line by promoter demethylation. Their efficacy was comparable to the DNMT inhibitor of reference 5-azacytidine.

Ciliate Nassula sp. grazing on a microcystin-producing cyanobacterium (Planktothrix agardhii): impact on cell growth and in the microcystin fractions.

The proliferation of microcystins (MCs)-producing cyanobacteria (MCs) can have detrimental effects on the food chain in aquatic environments. Until recently, few studies had focused on the fate of MCs in exposed organisms, such as primary consumers of cyanobacteria. In this study, we investigate the impact of an MC-producing strain of the cyanobacterium Planktothrix agardhii on the growth and physiology of a Nassula sp. ciliate isolated from a non-toxic cyanobacterial bloom. We show that this Nassula sp. strain was able to consume and grow while feeding exclusively on an MC-producing cyanobacterium over a prolonged period of time (8 months). In short-term exposure experiments (8 days), ciliates consuming an MC-producing cyanobacterial strain displayed slower growth rate and higher levels of antioxidant enzymes than ciliates feeding on two non-MC-producing strains. Three high-performance methods (LC/MS, LC/MS-MS and ELISA) were used to quantify the free and bound MCs in the culture medium and in the cells. We show that ciliate grazing led to a marked decrease in free MCs (methanol extractable) in cells, the MCs were therefore no longer found in the surrounding culture medium. These findings suggest that MCs may have undergone redistribution (free vs bound MCs) or chemical degradation within the ciliates.

Guaiane sesquiterpenes from Biscogniauxia nummularia featuring potent antigerminative activity.

Xylaranone, a previously unreported guaiane sesquiterpene along with the known terpenoid xylaranol B and the two mellein derivatives 3,5-dimethyl-8-methoxy-3,4-dihydroisocoumarin and 3,5-dimethyl-8-hydroxy-3,4-dihydroisocoumarin were isolated from Biscogniauxia nummularia. Pogostol was also isolated from this fungus, and in light of our spectroscopic data, its structure was revised and corrected. This fungus, which was isolated as an endophyte from the plum yew Cephalotaxus harringtonia, is also suspected of being a pathogen. Interestingly, we report here the potent antigerminative activity of xylaranone and xylaranol B against seeds of Raphanus sativus at concentrations comparable to glyphosate, a commonly used herbicide. This effect suggests a role for these metabolites in the latent fungal pathogenesis of B. nummularia.