Nodulisporic Acid Research Articles

Nodulisporic acid produces direct activation and positive allosteric modulation of AVR-14B, a glutamate-gated chloride channel from adult Brugia malayi

Glutamate-gated chloride channels (GluCls) are unique to invertebrates and are targeted by macrocyclic lactones. In this study, we cloned an AVR-14B GluCl subunit from adult Brugia malayi, a causative agent of lymphatic filariasis in humans. To elucidate this channel's pharmacological properties, we used Xenopus laevis oocytes for expression and performed two-electrode voltage-clamp electrophysiology. The receptor was gated by the natural ligand L-glutamate (effective concentration, 50% [EC50] = 0.4 mM) and ivermectin (IVM; EC50 = 1.8 nM). We also characterized the effects of nodulisporic acid (NA) on Bma-AVR-14B and NA-produced dual effects on the receptor as an agonist and a type II positive allosteric modulator. Here we report characterization of the complex activity of NA on a nematode GluCl. Bma-AVR-14B demonstrated some unique pharmacological characteristics. IVM did not produce potentiation of L-glutamate-mediated responses but instead, reduced the channel's sensitivity for the ligand. Further electrophysiological exploration showed that IVM (at a moderate concentration of 0.1 nM) functioned as an inhibitor of both agonist and positive allosteric modulatory effects of NA. This suggests that IVM and NA share a complex interaction. The pharmacological properties of Bma-AVR-14B indicate that the channel is an important target of IVM and NA. In addition, the unique electrophysiological characteristics of Bma-AVR-14B could explain the observed variation in drug sensitivities of various nematode parasites. We have also shown the inhibitory effects of IVM and NA on adult worm motility using Worminator. RNA interference (RNAi) knockdown suggests that AVR-14 plays a role in influencing locomotion in B. malayi.

New Methods and Strategies in the Synthesis of Terpenoid Natural Products.

Indoloterpenoids of the paxilline type belong to a large family of secondary metabolites that exhibit unique molecular architectures and a diverse set of biological activities. More than 100 congeners identified to date share a common structural motif that contains an indole moiety fused to a rearranged diterpenoid fragment. The representative physiological and cellular effects attributed to this family of natural products include neurological and insecticidal activities, modulation of lipid balance, and inhibition of mitosis. The uniting polycyclic motif combined with the diversity of individual structural features of paxilline indoloterpenoids and the broad scope of their biological activities have fascinated organic chemists for the past four decades and have led to the development of numerous syntheses. In this Account, we describe our contributions to this field and how they in turn shape new directions that are developing in our laboratory.We begin with the discussion of our strategy for the synthesis of the shared indoloterpenoid core. To address stereochemical challenges encountered in earlier reports, we planned to leverage a suitably substituted cyclopentanone in a polycyclization to form the desired trans-decalin motif. This polycyclization relied on a radical-polar crossover cascade initiated by hydrogen atom transfer. The original process exhibited poor diastereoselectivity, but we discovered an efficient solution to this problem that took advantage of intramolecular tethering effects, culminating in short synthesis of emindole SB. During these studies, we also identified indium-mediated alkenylation of silyl enol ethers with alkynes as a suitable method for the synthesis of highly substituted β,γ-unsaturated ketones that was critical to achieving brevity of our route. We subsequently developed a catalytic version of this transformation that allowed for a formal bimolecular ene reaction that exhibited unusual and potentially useful selectivity in construction of quaternary centers.To test the scope and limitations of our approach to paxilline indoloterpenoids and identify potential improvements, we developed a synthesis of the more complex congener nodulisporic acid C. The convergent assembly of this natural product was enabled by identification of new elements of stereocontrol in the radical-polar crossover polycyclization en route to the polycyclic terpenoid motif and development of a highly diastereoselective enyne cycloisomerization to access the indenopyran motif and a ketone arylation protocol to unite the two complex fragments.In subsequent studies, we expanded the radical-polar crossover cascade underlying our approach to paxilline indoloterpenoids to a bimolecular setting, which allowed for annulation of two unsaturated carbonyl components to produce functionalized cyclohexanes. This transformation is particularly well suited for installation of fully substituted carbons and can be complementary to the venerable Diels-Alder reaction. The utility of the new annulation was tested in the synthesis of forskolin, allowing for rapid construction of the complex polycyclic motif in this densely functionalized labdane diterpenoid.Over the past five years, our initial forays into the synthesis of paxilline indoloterpenoids have grown into a program that incorporates development of new synthetic methods and pursues artificial assembly of terpenoid natural products from several different families. We are encouraged by the increasing diversity of structural motifs made accessible by application of this chemistry and continue to discover new aspects of the underlying reactivity.

Myco-metabolites as biological control agents against the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae)

Fungi are a promising source for bioactive secondary metabolites against various agricultural pests. Soil samples were collected from the rhizosphere of various plants at El-Khatatba, Egypt, in May 2016. Sixty-two fungal isolates were locally isolated and screened against Tetranychus urticae Koch (Acari: Tetranychidae). Four fungal strains, showed potent control activities, were morphologically identified. Laboratory evaluation of the crude extracts of the selected strains, Aspergillus melleus, A. terreus, Emericella nidulans, and Chaetomium globosum, exhibited LC50 values of 10.27, 33.05, 14.68, and 22.40 mg/ml against females of T. urticae, respectively. Correspondent LC50 values that exhibited on eggs were 8.81, 23.17, 11.66, and 11.05 mg/ml. Consequently, secondary metabolites of A. melleus were separated by liquid chromatography/mass spectrometry. Compounds separated from the active fraction were identified as mellamide, ochratoxin C, nodulisporic acid, 7-Oxocurvularin, and 6-(4′-hydroxy-2′-methyl phenoxy)-(−)-(3R)-mellein. Obtained secondary metabolites are promising sources for biopesticides to be used in bio-rationale control of T. urticae.

Nodulisporic acid E biosynthesis: in vivo characterisation of NodD1, an indole-diterpene prenyltransferase that acts on an emindole SB derived indole-diterpene scaffold.

Prenylation of aromatic compounds is a key tailoring reaction in biosynthesis of bioactive indole-diterpenes. Here, we identify NodD1 as the enzyme responsible for the bisprenylation of nodulisporic acid F. This prenyltransferase showed a preference for its natural indole-diterpene substrate whereas other related enzymes were not able to catalyse this conversion.

Synthesis of (–)-Nodulisporic Acid C

Synthesis of (–)-Nodulisporic Acid C

Twelve-Step Asymmetric Synthesis of (-)-Nodulisporic Acid C.

A short, enantioselective synthesis of (-)-nodulisporic acid C is described. The route features two highly diastereoselective polycyclizations en route to the terpenoid core and the indenopyran fragment and a highly convergent assembly of a challenging indole moiety. Application of this chemistry allows for a 12-step synthesis of the target indoloterpenoid from commercially available material.

Draft Genome Sequence of the Filamentous Fungus Hypoxylon pulicicidum ATCC 74245.

ABSTRACTHypoxylon pulicicidum strain MF5954 (ATCC 74245) (formerly classified as Nodulisporium sp.) is a filamentous fungal species known for its production of the secondary metabolite nodulisporic acid A. We present here the 41.5-Mb draft genome sequence for this organism.

Heterologous Biosynthesis of Nodulisporic Acid F.

Nodulisporic acids comprise a group of valuable indole diterpenes that exhibit potent insecticidal activities. We report the identification of a gene cluster in the genome of the filamentous fungus Hypoxylon pulicicidum (Nodulisporium sp.) that contains genes responsible for the biosynthesis of nodulisporic acids. Using Penicillium paxilli as a heterologous host, and through pathway reconstitution experiments, we identified the function of four genes involved in the biosynthesis of the nodulisporic acid core compound, nodulisporic acid F (NAF). Two of these genes (nodM and nodW) are especially significant as they encode enzymes with previously unreported functionality: nodM encodes a 3-geranylgeranylindole epoxidase capable of catalyzing only a single epoxidation step to prime formation of the distinctive ring structure of nodulisporic acids, and nodW encodes the first reported gene product capable of introducing a carboxylic acid moiety to an indole diterpene core structure that acts as a reactive handle for further modification. Here, we present the enzymatic basis for the biosynthetic branch point that gives rise to nodulisporic acids.

Characterization of the secondary metabolites from endophytic fungiNodulisporiumsp. isolated from the medicinal plantMikania laevigata(Asteraceae) by reversed-phase high-performance liquid chromatography coupled with mass spectrometric multistage

Background: The endophytic fungi are an excellent source of secondary metabolites as a natural product with antimicrobial, insecticidal, cytotoxic, antioxidant, and anticancer activities. No studies on the chemical compounds of the endophytes of Mikania laevigata have been described in the literature. Objective: The objective of the study is to investigate by reversed phase high-performance liquid chromatography coupled with mass spectrometric multistage (RP-HPLC-MS/MS) the secondary metabolites of the endophytic fungi Nodulisporium sp., isolated from the medicinal plant M. laevigata (Asteraceae). Materials and Methods: The mycelia biomass strains of Nodulisporium sp. were accumulated and separated from the aqueous medium by filtration; the filtrate was subjected to a liquid–liquid partition with ethyl acetate (EtOAc) resulting in the crude EtOAc extract. This extract was analyzed by RP-HPLC-MS/MS. Results: The total mass chromatogram of the EtOAc crude strain Nodulisporium sp. extract showed compounds eluted between 13 and 20 min. The peak 1 (Rt 13.8 min), peak 2 (Rt. 14.8 min), peak 3 (Rt. 15.7 min) and peak 4 (Rt. 19.6 min) were characterized as dechlorogriseofulvin (C17H18O6), griseofulvin (C17H17O6Cl), cytochalasin D (C30H37NO6) and nodulisporic acid B2 (C43H57NO7), respectively. Conclusion: The present work provides the first scientific report on constituents of endophytic fungi Nodulisporium sp. isolated from the M. laevigata (Asteraceae). Abbreviations used: EtOAc: Ethyl acetate; RP-HPLC-MS/MS: Reversed-Phase High-Performance Liquid Chromatography coupled with mass spectrometric multistage; CCMB: Culture collection of microorganisms from Bahia; Rt: Retention time.

ChemInform Abstract: Total Synthesis of (—)‐Nodulisporic Acid D.

A convergent total synthesis of the architecturally complex indole diterpenoid (−)-nodulisporic acid D has been achieved. Key synthetic transformations include vicinal difunctionalization of an advanced α,β-unsaturated aldehyde to form the E,F-trans-fused 5,6-ring system of the eastern hemisphere and a cascade cross-coupling/indolization protocol leading to the CDE multisubstituted indole core.

Synthesis of (–)-Nodulisporic Acid D

Key words nodulisporic acid D - diterpenoids - indoles - cross-coupling - Horner–Wadsworth–Emmons reaction

Total Synthesis of (-)-Nodulisporic Acid D.

A convergent total synthesis of the architecturally complex indole diterpenoid (-)-nodulisporic acid D has been achieved. Key synthetic transformations include vicinal difunctionalization of an advanced α,β-unsaturated aldehyde to form the E,F-trans-fused 5,6-ring system of the eastern hemisphere and a cascade cross-coupling/indolization protocol leading to the CDE multisubstituted indole core.

Hypoxylon pulicicidum sp. nov. (Ascomycota, Xylariales), a pantropical insecticide-producing endophyte.

BackgroundNodulisporic acids (NAs) are indole diterpene fungal metabolites exhibiting potent systemic efficacy against blood-feeding arthropods, e.g., bedbugs, fleas and ticks, via binding to arthropod specific glutamate-gated chloride channels. Intensive medicinal chemistry efforts employing a nodulisporic acid A template have led to the development of N-tert-butyl nodulisporamide as a product candidate for a once monthly treatment of fleas and ticks on companion animals. The source of the NAs is a monophyletic lineage of asexual endophytic fungal strains that is widely distributed in the tropics, tentatively identified as a Nodulisporium species and hypothesized to be the asexual state of a Hypoxylon species.Methods and ResultsInferences from GenBank sequences indicated that multiple researchers have encountered similar Nodulisporium endophytes in tropical plants and in air samples. Ascomata-derived cultures from a wood-inhabiting fungus, from Martinique and closely resembling Hypoxylon investiens, belonged to the same monophyletic clade as the NAs-producing endophytes. The hypothesis that the Martinique Hypoxylon collections were the sexual state of the NAs-producing endophytes was tested by mass spectrometric analysis of NAs, multi-gene phylogenetic analysis, and phenotypic comparisons of the conidial states. We established that the Martinique Hypoxylon strains produced an ample spectrum of NAs and were conspecific with the pantropical Nodulisporium endophytes, yet were distinct from H. investiens. A new species, H. pulicicidum, is proposed to accommodate this widespread organism.Conclusions and SignificanceKnowledge of the life cycle of H. pulicicidum will facilitate an understanding of the role of insecticidal compounds produced by the fungus, the significance of its infections in living plants and how it colonizes dead wood. The case of H. pulicicidum exemplifies how life cycle studies can consolidate disparate observations of a fungal organism, whether from environmental sequences, vegetative mycelia or field specimens, resulting in holistic species concepts critical to the assessment of the dimensions of fungal diversity.

Ascription of poorly defined taxa to taxonomic entities using molecular phylogenies: a case study on Nodulisporium sp. producers of nodulisporic acid

Ascription of poorly defined taxa to taxonomic entities using molecular phylogenies: a case study on <I>Nodulisporium</I> sp. producers of nodulisporic acid

Discovery of the Development Candidate N-tert-Butyl Nodulisporamide: A Safe and Efficacious Once Monthly Oral Agent for the Control of Fleas and Ticks on Companion Animals

Nodulisporic acid A (1) is a structurally complex fungal metabolite that exhibits systemic efficacy against fleas via modulation of an invertebrate specific glutamate-gated ion channel. In order to identify a nodulisporamide suitable for monthly oral dosing in dogs, a library of 335 nodulisporamides was examined in an artificial flea feeding system for intrinsic systemic potency as well as in a mouse/bedbug assay for systemic efficacy and safety. A cohort of 66 nodulisporamides were selected for evaluation in a dog/flea model; pharmacokinetic analysis correlated plasma levels with flea efficacy. These efforts resulted in the identification of the development candidate N-tert-butyl nodulisporamide (3) as a potent and efficacious once monthly oral agent for the control of fleas and ticks on dogs and cats which was directly compared to the topical agents fipronil and imidacloprid, with favorable results obtained. Multidose studies over 3 months confirmed the in vivo ectoparasiticidal efficacy and established that 3 lacked overt mammalian toxicity. Tissue distribution studies in mice using [(14)C]-labeled 3 indicate that adipose beds serve as ligand depots, contributing to the long terminal half-lives of these compounds.

The role of tryptophan as a biosynthetic precursor of indole-diterpenoid fungal metabolites: Continuing a debate

Studies in the 1980s and 1990s on the origin of the indole moiety in fungal indole-diterpenoids using 14C-labelled tryptophan consistently showed autoradiographic evidence but gave low % incorporation of the probe. Recent studies on a member of the group (nodulisporic acid A), using more specific 13C methodology, demonstrated a role of the tryptophan biosynthetic pathway but, in failing to show involvement of end-product, concluded that the indole was derived from indole-3-glycerol phosphate and suggested that the previous 14C data arose via metabolic scrambling of label. In considering the protocol for the 13C studies, there is concern that the fungal material was starved of an exogenous nitrogen source and thus could have degraded added labelled tryptophan. Consequently, synthesis of the serine necessary for anabolic formation of tryptophan may have been constrained. It is suggested that 13C studies on appropriate fungi early in the idiophase of submerged or surface fermentation should be made before the biosynthesis of indole-diterpenoids can become clearer.

The tryptophan synthetase gene TRP1 of Nodulisporium sp.: molecular characterization and its relation to nodulisporic acid A production

Nodulisporic acid A (NAA), an insecticidal indole diterpene, is produced by the fungus Nodulisporium sp. Since indole-3-glycerolphosphate is the precursor of the indole moiety of NAA, it is suggested that the activity of tryptophan synthetase may play a role in NAA biosynthesis. To investigate this hypothesis, the tryptophan synthetase gene TRP1 of Nodulisporium sp. was cloned and characterized. The gene consists of three introns of 146, 68, and 57 bp. The four exons encode a protein of 712 amino acids, the sequence of which is highly homologous to that of other fungal tryptophan synthetase proteins. The transcription initiation site was mapped 66 bp upstream to the ATG, and the polyA tail attachment site is 169 bp downstream to the translation stop codon. Replacement of the N-terminal half of the gene with a hygromycin selection marker yielded mutants with the tryptophan auxotroph/hygromycin-resistance (trp(-)/hyr) phenotype. The TRP1 mutants required a high concentration of tryptophan supplement in solid medium (10 mM) to sustain minimal growth and failed to produce NAA in the production medium (FFL-CAM) supplemented with high concentrations of tryptophan.

Indole Diterpene Synthetic Studies. Total Synthesis of (+)‐Nodulisporic Acid F and Construction of the Heptacyclic Cores of (+)‐Nodulisporic Acids A and B and (‐)‐Nodulisporic Acid D.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Indole Diterpene Synthetic Studies. Total Synthesis of (+)-Nodulisporic Acid F and Construction of the Heptacyclic Cores of (+)-Nodulisporic Acids A and B and (−)-Nodulisporic Acid D

A first-generation strategy for construction of (+)-nodulisporic acids A (1) and B (2) is described. The strategy entails union of the eastern and western hemisphere subtargets via the indole synthesis protocol developed in our laboratory. Subsequent elaboration of rings E and F, however, revealed the considerable acid instability of the C(24) hydroxyl, thereby preventing further advancement. Nonetheless, preparation of the heptacyclic core of (+)-nodulisporic acids A and B, the total synthesis of (+)-nodulisporic acid F, the simplest member of the nodulisporic acid family, and elaboration of the heptacyclic core of (-)-nodulisporic acid D were achieved.

Indole Diterpenoid Synthetic Studies. The Total Synthesis of (+)‐Nodulisporic Acid F.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.