A 2020 combinatorial-biosynthesis study reconstituted five fungal decalin-containing diterpenoid pyrone (DDP) pathways and four designed extended pathways in Aspergillus oryzae, producing a 22-compound library whose members show striking structure-dependent bioactivities: selective anti-HIV activity (compound 20, EC50 8.5 nM), Drosophila paralysis (subglutinol A, compound 5), inhibition of the insect IMD innate-immune pathway (compound 21, IC50 0.27 µM), and delayed amyloid-β42 (Aβ42) nucleation through apparent covalent trapping of Lys16. The study left three mechanistic questions open: (1) why two chemically unrelated tailoring enzymes, the berberine-bridge-enzyme-type oxidase DpmaF and the cytochrome P450 DpfgJ, strictly recognize only one C-8 stereoisomer of their substrates while other DDP tailoring enzymes accept both epimers — an observation established only in whole-cell reconstitutions; (2) the in vivo molecular targets and modes of action behind the library's headline bioactivities; (3) the full scope, kinetics, structural details, and functional consequences of the covalent adduct formed between the highly oxidized γ-pyrone of compounds 13/21 and lysine residues of full-length Aβ42, previously supported only by model chemistry with a protected lysine analog and surrogate-peptide MS/MS. No resolution of any of the three cores has been reported: no biochemical or structural characterization of any DDP tailoring enzyme exists, no target identification for compounds 20 or 21 or for the insect paralytic action of subglutinol A has appeared, and a 2025 review by the original Aβ co-authors still describes the DDP–lysine adduct with the 2020 model chemistry.

Background

Decalin-containing diterpenoid pyrones (DDPs) are fungal hybrid natural products in which a geranylgeranyl-derived decalin (a fused bicyclic C20 terpene) is joined to a polyketide-derived pyrone ring (an α-pyrone in the core intermediate; the highly oxidized form of the same ring in compounds 13 and 21 is designated the γ-pyrone). Family members include the subglutinols (immunosuppressive and insect-toxic metabolites of Metarhizium and Fusarium), the higginsianins (cytotoxins of Colletotrichum), and viridoxin-related compounds. Their biosynthesis is modular: a non-reducing polyketide synthase (NR-PKS, a multidomain enzyme that assembles and cyclizes the pyrone), a geranylgeranyl diphosphate synthase (GGPPS), and a prenyltransferase build a prenylated pyrone, which a flavin-dependent epoxidase and a membrane terpene cyclase convert into the trans-decalin intermediate shared by all DDP pathways. Downstream 'tailoring' enzymes then diversify the scaffold: short-chain dehydrogenase/reductases (SDRs) invert the configuration at carbon C-8, flavin monooxygenases (FMOs) and berberine-bridge-enzyme-type (BBE) oxidases (a flavin-dependent oxidase class named for the plant enzyme that builds the berberine bridge in alkaloid biosynthesis) install tetrahydrofuran rings and enones at the C5 unit, methyltransferases (MTs) methylate the pyrone, and cytochrome P450 monooxygenases perform staged oxidations of the pyrone's methyl groups.

Because total chemical synthesis of DDPs is elaborate (about twenty steps), Tsukada and co-workers (Nature Communications 11:1830, 2020) instead reconstructed the five native DDP pathways of Arthrinium, Metarhizium, Colletotrichum, Macrophomina and Fusarium, plus four designed non-natural 'extended' pathways, in the heterologous host Aspergillus oryzae (a fungus widely used to express fungal biosynthetic gene clusters because transferred genes are introduced stepwise and products accumulate in culture). This combinatorial biosynthesis — mixing and matching tailoring genes across species — delivered 22 DDPs, 15 of them new, at preparative titres, enabling a biological-activity screen of a structurally focused library.

Three observations from that study still lack molecular-level explanation. First, comparing product profiles of transformants showed that two tailoring enzymes of unrelated classes — DpmaF (a BBE-type oxidase catalysing tetrahydrofuran-ring formation) and DpfgJ (a P450 catalysing three-step oxidation of a pyrone methyl to a carboxylic acid, plus C-26 hydroxylation) — strictly recognize the C-8 configuration of their substrates, whereas DpasF and DpchF (FMOs), DpmpI and DpfgI (MTs), and DpmpJ (a P450) accept both C-8 epimers. All of these selectivity assignments rest on which products appear or disappear in whole-cell reconstitutions; no enzyme has been purified, assayed in vitro with defined stereoisomeric substrates, or structurally characterized. Second, the screen revealed potent but mechanistically opaque bioactivities: compound 20 (an extended-pathway product) selectively inhibits HIV-1 in a MAGI assay (a single-round infection assay in reporter cells bearing a β-galactosidase gene under the HIV-1 promoter; EC50 8.5 nM, no cytotoxicity up to 10 µM); subglutinol A (compound 5) paralyses injected adult Drosophila and inhibits insect-cell lines at low nanomolar concentration; compound 21 inhibits the Drosophila immune deficiency (IMD) pathway — an innate-immune signalling cascade homologous to mammalian TNF receptor signalling — with an IC50 of 0.27 µM in an Attacin-luciferase reporter assay (luciferase driven by the promoter of the IMD-inducible antimicrobial-peptide gene Attacin). No molecular target is known for any of these phenotypes. Third, compounds 13 and 21, which carry a highly oxidized γ-pyrone (hydroxyl group plus methyl ester), delay the nucleation phase of Aβ42 aggregation (thioflavin-T assay, a fluorescence assay reporting amyloid fibril formation, and electron microscopy); mass spectrometry of Aβ42 treated with 21 showed adducts consistent with Michael-type addition of lysine to the γ-pyrone with loss of 32 Da, and MS/MS of a surrogate Aβ9-35 peptide implicated Lys16. The chemical mechanism, however, was established only with Nα-benzyl-protected lysine model chemistry (nucleophilic attack at C-24 of an ortho-ester form with methoxide elimination, giving C-21 epimeric adducts) — not on full-length Aβ42, and without kinetics, adduct structures, or any demonstration of functional consequences for Aβ-mediated toxicity in cellular or animal Alzheimer's disease models.

Neighbouring work frames but does not answer these questions. Subglutinol A has a separate pre-existing mammalian pharmacology — suppression of activated T cells via exacerbated mitochondrial damage, estrogen-receptor antagonism, and a medicinal-chemistry analog program — none of which addresses its insect paralytic action. The general strategy of lysine-targeting covalent inhibitors of amyloid aggregation has advanced with other chemotypes (e.g., a perilla-derived chalcone active in vitro and in vivo), and chemical synthesis can now supply several natural DDPs, but the DDP-specific mechanistic questions remain untouched.

Problem Statement

Provide a molecular-level mechanistic account of the three unexplained observation classes of the fungal DDP combinatorial-biosynthesis library of Tsukada et al. (2020), jointly converting its phenomenological findings into mechanism:

  1. Substrate-selectivity determinants of DDP tailoring enzymes. Determine the structural and catalytic features that make the BBE-type oxidase DpmaF and the cytochrome P450 DpfgJ strict for the C-8 configuration of their substrates while the FMOs DpasF and DpchF, the methyltransferases DpmpI and DpfgI, and the P450 DpmpJ are permissive — including whether the selectivity observed in whole-cell reconstitutions is reproduced by the isolated enzymes, and which active-site or remote features enforce or relax C-8 discrimination.

  2. Molecular targets and modes of action of the library's headline bioactivities. Identify, with causally validated evidence, the in vivo molecular target(s) and mode of action of (i) the selective anti-HIV activity of compound 20 (EC50 8.5 nM, no cytotoxicity to 10 µM in the MAGI assay); (ii) the paralytic/insecticidal action of subglutinol A (compound 5) in Drosophila; and (iii) the inhibition of the Drosophila IMD innate-immune pathway by compound 21 (IC50 0.27 µM), distinguishing direct pathway-component binding from upstream cellular effects.

  3. Covalent chemistry of the oxidized γ-pyrone with amyloid-β. Establish the full reaction of compounds 13/21 (and their γ-pyrone congeners) with full-length Aβ42: adduct sites and stoichiometry, reaction kinetics (rate and equilibrium constants for adduct formation and, where applicable, reversibility), a structural description of the adduct, and the functional consequences of adduct formation for Aβ42 aggregation and Aβ-mediated toxicity in cellular and in vivo Alzheimer's disease models.

The common objective is mechanistic closure of the DDP library study: each component explains at the molecular level one class of observation (biosynthetic selectivity, bioactivity phenotype, covalent amyloid modulation) that the 2020 study could only record. The three components are jointly posed: the structural and catalytic determinants of the C-8 discrimination shown by the DDP tailoring enzymes (component 1); the in vivo molecular targets and modes of action behind the headline bioactivities (component 2); and the adduct sites and stoichiometry, reaction kinetics, structural description, and functional consequences for Aβ42 aggregation and Aβ-mediated toxicity of the γ-pyrone–Aβ42 reaction (component 3).

References

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