Australian Marine and Terrestrial Streptomyces-Derived Surugamides, and Synthetic Analogs, and Their Ability to Inhibit Dirofilaria immitis (Heartworm) Motility.

A bioassay-guided chemical investigation of a bacterium, Streptomyces sp. CMB-MRB032, isolated from sheep feces collected near Bathurst, Victoria, Australia, yielded the known polyketide antimycins A4a (1) and A2a (2) as potent inhibitors of Dirofilaria immitis (heartworm) microfilaria (mf) motility (EC50 0.0013-0.0021 µg/mL), along with the octapeptide surugamide A (3) and the new N-methylated analog surugamide K (4). With biological data suggesting surugamides may also exhibit activity against D. immitis, a GNPS molecular network analysis of a library of microbes sourced from geographically diverse Australian ecosystems identified a further five taxonomically and chemically distinct surugamide producers. Scaled-up cultivation of one such producer, Streptomyces sp. CMB-M0112 isolated from a marine sediment collected at Shorncliff, Qld, Australia, yielded 3 along with the new acyl-surugamides A1-A4 (5-8). Solid-phase peptide synthesis provided additional synthetic analogs, surugamides S1-S3 (9-11), while derivatization of 3 returned the semi-synthetic surugamide S4 (12) and acyl-surugamides AS1-AS3 (13-15). The natural acyl-surugamide A3 (7) and semi-synthetic acyl-surugamide AS3 (15) were shown to selectively inhibit D. immitis mf motility (EC50 3.3-3.4 µg/mL), however, unlike antimycins 1 and 2, were inactive against the gastrointestinal nematode Haemonchus contortus L1-L3 larvae (EC50 > 25 µg/mL) and were not cytotoxic to mammalian cells (human colorectal carcinoma SW620, IC50 > 30 µg/mL). A structure-activity relationship (SAR) study on the surugamides 3-15 revealed that selective acylation of the Lys3-ε-NH2 correlates with anthelmintic activity.

Natural products as anthelmintics: safeguarding animal health.

Covering literature to December 2022This review provides a comprehensive account of all natural products (500 compounds, including 17 semi-synthetic derivatives) described in the primary literature up to December 2022, reported to be capable of inhibiting the egg hatching, motility, larval development and/or the survival of helminths (i.e., nematodes, flukes and tapeworms). These parasitic worms infect and compromise the health and welfare, productivity and lives of commercial livestock (i.e., sheep, cattle, horses, pigs, poultry and fish), companion animals (i.e., dogs and cats) and other high value, endangered and/or exotic animals. Attention is given to chemical structures, as well as source organisms and anthelmintic properties, including the nature of bioassay target species, in vivo animal hosts, and measures of potency.

Miniaturized Cultivation Profiling Returns Indolo-Sesquiterpene Michael Adducts from the Australian Soil-Derived Aspergillus terreus CMB-SWF012.

An integrated program of chemical profiling (GNPS) coupled with an expanded format 24-well-plate miniaturized cultivation profiling (MATRIX) utilizing traditional as well as grain/pulse and cereal media permitted rapid prioritization of Aspergillus terreus CMB-SWF012 as a source of unprecedented natural products. Scaled-up cultivation on rice and PDA yielded the rare tripeptides asterripeptides A-C (1-3), new indolo-sesquiterpene Michael adducts terreusides A and B (4 and 5), and known precursors asterresin A (6) and (+)-giluterrin (7). Structures for 1-7 were assigned by detailed spectroscopic and chemical analysis and biosynthetic considerations.

Glyclauxins A-E: Dimeric Oxaphenalenone Aminoglycosides from an Australian Wasp Nest-Derived Fungus Talaromyces sp. CMB-MW102.

Chemical analysis of cultures of a Queensland mud dauber wasp nest-derived fungus, Talaromyces sp. CMB-MW102, yielded the known dimeric oxaphenalenone duclauxin (1) along with a family of new 1-deoxy-d-glucosamine adducts, glyclauxins A-E (2-6). Despite 1D NMR spectra of 2-6 being compromised by broadening of selected resonances, structures inclusive of absolute configuration were assigned on the basis of detailed spectroscopic analysis and biogenetic considerations, as well as biomimetic semisynthesis and chemical interconversion. For example, exposure of duclauxin (1) to synthetic 1-deoxy-d-glucosamine yielded glyclauxin B (3), while on handling and storage, glyclauxins C (4) and D (5) (bearing a 7-OMe moiety) proved chemically labile and underwent quantitative transformation to glyclauxins B (3) and A (2), respectively. These latter observations on chemical reactivity and stability informed a proposed biogenetic relationship linking all known members of the extended duclauxin family. Notwithstanding their potential status as artifacts, the detection of glyclauxins B (3) and A (2) in a fresh CMB-MW102 culture extract confirmed their natural product status.

Case Studies in Molecular Network-Guided Marine Biodiscovery.

In reviewing a selection of recent case studies from our laboratory, we revealed some lessons learned and benefits accrued from the application of mass spectrometry (MS/MS) molecular networking in the field of marine sponge natural products. Molecular networking proved pivotal to our discovery of many new natural products and even new classes of natural product, some of which were opaque to alternate dereplication and prioritization strategies. Case studies included the discovery of: (i) trachycladindoles, an exceptionally rare class of bioactive indole alkaloid previously only known from a single southern Australia sample of Trachycladus laevispirulifer; (ii) dysidealactams, an unprecedented class of sesquiterpene glycinyl-lactam and glycinyl-imide from a Dysidea sp., a sponge genera often discounted as having been exhaustively studied; (iii) cacolides, an unprecedented family of sesterterpene α-methyl-γ-hydroxybutenolides from a Cacospongia sp., all too easily mischaracterized and deprioritized during dereplication as a well-known class of sponge sesterterpene tetronic acids; and (iv) thorectandrins, a new class of indole alkaloid which revealed unexpected insights into the chemical and biological properties of the aplysinopsins, one of the earliest and more extensively reported class of sponge natural products.

Talarolides Revisited: Cyclic Heptapeptides from an Australian Marine Tunicate-Associated Fungus, Talaromyces sp. CMB-TU011.

Application of a miniaturized 24-well plate system for cultivation profiling (MATRIX) permitted optimization of the cultivation conditions for the marine-derived fungus Talaromyces sp. CMB-TU011, facilitating access to the rare cycloheptapeptide talarolide A (1) along with three new analogues, B-D (2-4). Detailed spectroscopic analysis supported by Marfey's analysis methodology was refined to resolve N-Me-l-Ala from N-Me-d-Ala, l-allo-Ile from l-Ile and l-Leu, and partial and total syntheses of 2, and permitted unambiguous assignment of structures for 1 (revised) and 2-4. Consideration of diagnostic ROESY correlations for the hydroxamates 1 and 3-4, and a calculated solution structure for 1, revealed how cross-ring H-bonding to the hydroxamate moiety influences (defines/stabilizes) the cyclic peptide conformation. Such knowledge draws attention to the prospect that hydroxamates may be used as molecular bridges to access new cyclic peptide conformations, offering the prospect of new biological properties, including enhanced oral bioavailability.

Glenthmycins A-M: Macrocyclic Spirotetronate Polyketide Antibacterials from the Australian Pasture Plant-Derived Streptomyces sp. CMB-PB041.

Chemical investigation of Australian pasture plant-derived Streptomyces sp. CMB-PB041, supported by miniaturized cultivation profiling and molecular network analysis, led to the isolation and characterization of 13 new macrocyclic spirotetronates, glenthmycins A-M (1-13), with structures assigned by detailed spectroscopic analysis, chemical degradation and derivatization, and mechanistic and biosynthetic considerations. Hydrolysis of glenthmycin B (2) yielded the aglycone 14, whose structure and absolute configuration were secured by X-ray analysis, along with the unexpected amino sugar residues glenthose lactams A (15) and B (16), with Mosher analysis of 15 facilitating assignment of absolute configurations of the amino sugar. While the glenthmycins proved to be acid stable, treatment of isomeric glenthmycins (i.e., 3, 6, and 8) with base catalyzed rapid intramolecular trans-esterification to regio-isomeric mixtures (i.e., 3 + 6 + 8). Exposure of 5 to base achieved the same intramolecular trans-esterification and was instrumental in detecting and tentatively identifying two additional minor co-metabolites, glenthmycins N (19) and O (20). A structure-activity relationship analysis carried out on 1-13 and the semisynthetic analogues 14 and 21-26 revealed a promising Gram +ve antibacterial pharmacophore, effective against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE), but with no detectable cytotoxicity to eukaryotic cells (i.e., fungal and human carcinoma). Of particular note, the semisynthetic analogue glenthmycin K 9-valerate (26) was unique among glenthmycins in potently inhibiting growth of the full panel of Gram +ve pathogens (IC50 0.2-1.6 µM). We conclude with an observation that any future evaluation of the antibacterial potential of glenthmycins and related macrocyclic spirotetronates may do well to include important soil-derived Gram +ve pathogens, such as Bacillus anthrax, Clostridium botulinum, and Rhodococcus equi, the causative agents of anthrax, botulism, and livestock pneumonia.

Glenthenamines A-F: Enamine Pyranonaphthoquinones from the Australian Pasture Plant Derived Streptomyces sp. CMB-PB042.

Chemical investigations into solid phase cultivations of an Australian sheep station pasture plant derived Streptomyces sp. CMB-PB042 yielded the rare enamine naphthopyranoquinones BE-54238A (1) and BE-54238B (2), together with four new analogues, glenthenamines B-D (4-6) and F (8), and two handling artifacts, glenthenamines A (3) and E (7). Single-crystal X-ray analyses of 1 and 2 resolved configurational ambiguities in the scientific literature, while detailed spectroscopic analysis and biosynthetic considerations assigned structures inclusive of absolute configuration to 3-8. We propose a plausible sequence of biosynthetic transformations linking structural and configurational features of 1-8 and apply a novel Schiff base "fishing" approach to detect a key deoxyaminosugar precursor. These enamine naphthopyranoquinones disclose a new P-gp inhibitory pharmacophore capable of reversing doxorubicin resistance in P-gp overexpressing colon carcinoma cells.

Molecular Networking and Cultivation Profiling Reveals Diverse Natural Product Classes from an Australian Soil-Derived Fungus Aspergillus sp. CMB-MRF324.

This study showcases the application of an integrated workflow of molecular networking chemical profiling (GNPS), together with miniaturized microbioreactor cultivation profiling (MATRIX) to successfully detect, dereplicate, prioritize, optimize the production, isolate, characterize, and identify a diverse selection of new chemically labile natural products from the Queensland sheep pasture soil-derived fungus Aspergillus sp. CMB-MRF324. More specifically, we report the new tryptamine enamino tripeptide aspergillamides E-F (7-8), dihydroquinoline-2-one aflaquinolones H-I (11-12), and prenylated phenylbutyrolactone aspulvinone Y (14), along with an array of known co-metabolites, including asterriquinones SU5228 (9) and CT5 (10), terrecyclic acid A (13), and aspulvinones N-CR (15), B (16), D (17), and H (18). Structure elucidation was achieved by a combination of detailed spectroscopic and chemical analysis, biosynthetic considerations, and in the case of 11, an X-ray crystallographic analysis.

GluClR-mediated inhibitory postsynaptic currents reveal targets for ivermectin and potential mechanisms of ivermectin resistance.

Glutamate-gated chloride channel receptors (GluClRs) mediate inhibitory neurotransmission at invertebrate synapses and are primary targets of parasites that impact drastically on agriculture and human health. Ivermectin (IVM) is a broad-spectrum pesticide that binds and potentiates GluClR activity. Resistance to IVM is a major economic and health concern, but the molecular and synaptic mechanisms of resistance are ill-defined. Here we focus on GluClRs of the agricultural endoparasite, Haemonchus contortus. We demonstrate that IVM potentiates inhibitory input by inducing a tonic current that plateaus over 15 minutes and by enhancing post-synaptic current peak amplitude and decay times. We further demonstrate that IVM greatly enhances the active durations of single receptors. These effects are greatly attenuated when endogenous IVM-insensitive subunits are incorporated into GluClRs, suggesting a mechanism of IVM resistance that does not affect glutamate sensitivity. We discovered functional groups of IVM that contribute to tuning its potency at different isoforms and show that the dominant mode of access of IVM is via the cell membrane to the receptor.

Lincolnenins A-D: Isomeric Bactericidal Bianthracenes from Streptomyces lincolnensis.

Cultivation profiling followed by chemical analysis of Streptomyces lincolnensis yielded four new isomeric bianthracenes, lincolnenins A-D (1-4), with relative stereostructures assigned on the basis of detailed spectroscopic analysis. Lincolnenins A (1) and B (2) exhibit restricted rotation about alternate bianthracene 9-9' and 9-8' bridges, respectively, and exist as single atropisomers, whereas C (3) and D (4) are thermally interconvertible atropisomers sharing a common 8-8' bianthracene bridge. Absolute configurations were assigned to 1-4 on the basis of diagnostic ROESY correlations and ECD calculations, whereas acid-mediated dehydration of 1 led to formation and revision of the absolute configuration of the biosynthetically related known Streptomyces antibiotic, setomimycin (5). Lincolnenin A (1) exhibited significant bactericidal activity against multiple susceptible and drug-resistant Gram-positive pathogens (MIC99 < 2.0 µM), including Mycobacterium tuberculosis H37Ra (MIC99 = 0.9 µM).

Amaurones A-K: Polyketides from the Fish Gut-Derived Fungus Amauroascus sp. CMB-F713.

Using a molecular networking guided strategy, chemical analysis of the Australian mullet fish gastrointestinal tract-derived fungus Amauroascus sp. CMB-F713 yielded a family of polyketide pyrones, amaurones A-I (1-9), featuring an unprecedented carbon skeleton. Structures were assigned to 1-9 by detailed spectroscopic analysis (including X-ray analysis of 1), biosynthetic considerations, and chemical interconversions. For example, the orthoacetate 5 was unstable when stored dry at room temperature, transforming to the monoacetates 2 and 3, while mild heating (40 °C) prompted quantitative conversion of 3 to 2, via an intramolecular trans-acetylation. Likewise, during handling, the monoacetate 1 was prone to intramolecular trans-acetylation, leading to an equilibrium mixture with the isomeric monoacetate amaurone J (10), confirmed when partial hydrolysis of the diacetate 2 yielded the monoacetates 1 and 10 and the triol amaurone K (11).

Methods in Microbial Biodiscovery.

This review presents an account of the microbial biodiscovery methodology developed and applied in our laboratory at The University of Queensland, Institute for Molecular Bioscience, with examples drawn from our experiences studying natural products produced by Australian marine-derived (and terrestrial) fungi and bacteria.

New from Old: Thorectandrin Alkaloids in a Southern Australian Marine Sponge, Thorectandra choanoides (CMB-01889).

Thorectandra choanoides (CMB-01889) was prioritized as a source of promising new chemistry from a library of 960 southern Australian marine sponge extracts, using a global natural products social (GNPS) molecular networking approach. The sponge was collected at a depth of 45 m. Chemical fractionation followed by detailed spectroscopic analysis led to the discovery of a new tryptophan-derived alkaloid, thorectandrin A (1), with the GNPS cluster revealing a halo of related alkaloids 1a-1n. In considering biosynthetic origins, we propose that Thorectandrachoanoides (CMB-01889) produces four well-known alkaloids, 6-bromo-1',8-dihydroaplysinopsin (2), 6-bromoaplysinopsin (3), aplysinopsin (4), and 1',8-dihydroaplysinopsin (10), all of which are susceptible to processing by a putative indoleamine 2,3-dioxygenase-like (IDO) enzyme to 1a-1n. Where the 1',8-dihydroalkaloids 2 and 10 are fully transformed to stable ring-opened thorectandrins 1 and 1a-1b, and 1h-1j, respectively, the conjugated precursors 3 and 4 are transformed to highly reactive Michael acceptors that during extraction and handling undergo complete transformation to artifacts 1c-1g, and 1k-1n, respectively. Knowledge of the susceptibility of aplysinopsins as substrates for IDOs, and the relative reactivity of Michael acceptor transformation products, informs our understanding of the pharmaceutical potential of this vintage marine pharmacophore. For example, the cancer tissue specificity of IDOs could be exploited for an immunotherapeutic response, with aplysinopsins transforming in situ to Michael acceptor thorectandrins, which covalently bind and inhibit the enzyme.

Bhimamycin J, a Rare Benzo[f]isoindole-dione Alkaloid from the Marine-Derived Actinomycete Streptomyces sp. MS180069.

Chemical investigation on a Streptomyces sp. strain MS180069 isolated from a sediment sample collected from the South China Sea, yielded the new benzo[f]isoindole-dione alkaloid, bhimamycin J (1). The structure was determined by extensive spectroscopic analysis, including HRMS, 1D, 2D NMR, and X-ray diffraction techniques. A molecular docking study revealed 1 as a new molecular motif that binds with human angiotensin converting enzyme2 (ACE2), recently described as the cell surface receptor responsible for uptake of 2019-CoV-2. Using enzyme assays we confirm that 1 inhibits human ACE2 79.7 % at 25 µg/mL.

Dysidealactams and Dysidealactones: Sesquiterpene Glycinyl-Lactams, Imides, and Lactones from a Dysidea sp. Marine Sponge Collected in Southern Australia.

A GNPS molecular networking approach mapped a library of 960 southern Australian marine sponges and prioritized Dysidea sp. (CMB-01171) for chemical investigation. Although the published natural products literature on Australian Dysidea sponges extends back over half a century and suffers from the perception of being near exhausted, fractionation of Dysidea sp. (CMB-01171) led to the discovery of a family of 10 new biosynthetically and chemically related sesquiterpenes. Detailed spectroscopic analysis guided structure elucidation identified dysidealactams A-F (1-6), dysidealactones A and B (7 and 8), and two solvolysis artifacts, 9 and 10. The dysidealactams A-D (1-4) incorporate a rare glycinyl-lactam functionality, while dysidealactam E (5) is particularly noteworthy in incorporating an unprecedented glycinyl-imide moiety. In addition to expanding knowledge of Dysidea natural products, this study demonstrates the value of applying GNPS molecular networking to map chemical diversity and prioritize the selection of marine sponge extracts for more detailed chemical analysis.

Chrysosporazines F-M: P-Glycoprotein Inhibitory Phenylpropanoid Piperazines from an Australian Marine Fish Derived Fungus, Chrysosporium sp. CMB-F294.

Chemical analysis of the fungus Chrysosporium sp. CMB-F294 isolated from the gastrointestinal tract of a market-purchased specimen of Mugil mullet yielded eight new alkaloids, belonging to a rare class of phenylpropanoid piperazines. Chrysosporazines F-M (1-8) occur as an equilibrium mixture of acetamide rotamers and feature unprecedented carbocyclic and heterocyclic scaffolds. Structures inclusive of absolute configuration were assigned by detailed spectroscopic analysis, supported by biosynthetic considerations. Structure-activity relationship studies determined that selected chrysosporazines were promising noncytotoxic inhibitors of the multidrug resistance efflux pump P-glycoprotein (P-gp), capable of reversing doxorubicin resistance in P-gp-overexpressing human colon carcinoma cells (SW620 Ad300). Chrysosporazine F (1) was particularly noteworthy, with a 2.5 µM cotreatment inducing a doxorubicin gain in sensitivity (GS 14) > 2-fold that of the positive control verapamil (GS 6.1).

The Effects of Conspecific Alarm Cues on Larval Cane Toads (Rhinella marina).

Many aquatic organisms detect and avoid damage-released cues from conspecifics, but the chemical basis of such responses, and the effects of prolonged exposure to such cues, remain poorly understood. Injured tadpoles of the cane toad (Rhinella marina) produce chemical cues that induce avoidance by conspecific tadpoles; and chronic exposure to those cues decreases rates of tadpole survival and growth, and reduces body size at metamorphosis. Such effects suggest that we might be able to use the cane toads' alarm cue for biocontrol of invasive populations in Australia. In the present study, we examined behavioral and ecological effects of compounds that are present in cane toad tadpoles and thus, might trigger avoidance of crushed conspecifics. Four chemicals (L-Arg, L-Leu-L-Leu-OH, L-Leu-L-Ile-OH and suberic acid) induced behavioral avoidance in toad tadpoles at some (but not all) dosage levels, so we then exposed toad larvae to these chemicals over the entire period of larval development. Larval survival and size at metamorphosis were decreased by chronic exposure to crushed conspecifics (consistent with earlier studies), but not by exposure to any of the four chemicals. Indeed, L-Arg increased body size at metamorphosis. We conclude that the behavioral response to crushed conspecifics by cane toad tadpoles can be elicited by a variety of chemical cues, but that consistent exposure to these individual chemical cues does not affect tadpole viability or developmental trajectory. The optimal behavioral tactic of a tadpole may be to flee if it encounters even a single chemical cue likely to have come from an injured conspecific (indicative of predation risk), whereas the continuing presence of that single chemical (but no others) provides a less reliable signal of predation risk. Our data are consistent with results from studies on fish, that suggest a role for multiple chemicals in initiating alarm responses to damage-released cues.

Trivirensols: Selectively Bacteriostatic Sesquiterpene Trimers from the Australian Termite Nest-Derived Fungus Trichoderma virens CMB-TN16.

The termite nest-derived fungus Trichoderma virens CMB-TN16 cultivated on rice-based media produced seven new first-in-class trimeric sesquiterpenes, trivirensols A-G (11-17). Structures inclusive of absolute configurations were assigned by detailed spectroscopic analysis and biosynthetic considerations. Although trivirensols exhibit no cytotoxicity to mammalian carcinoma cells, selected examples are bacteriostatic against vancomycin-resistant Enterococcus faecalis (VRE). Structure-activity relationship (SAR) investigations combined with in situ chemical stability studies documented bacteriostatic activity for trivirensols A (11) and B (12) and the co-metabolite divirensols A (4), B (5), and G (10), all of which share a common terminal butenolide. Significantly, SAR studies also revealed bacteriostatic activity for trivirensols C (13) and G (17) and the co-metabolite divirensol C (6), all of which share a common hydrated butenolide terminal. Of note, when exposed to VRE cell cultures, the hydrated butenolides 6, 13, and 17 undergo rapid in situ dehydration to corresponding butenolides, suggesting hydrated butenolides are a pro-drug form of the butenolide VRE bacteriostatic pharmacophore.

Divirensols: Sesquiterpene Dimers from the Australian Termite Nest-Derived Fungus Trichoderma virens CMB-TN16.

A chemical investigation of the Australian termite nest-derived fungus Trichoderma virens CMB-TN16 yielded the known sesquiterpene gliocladic acid (1), together with two new acetylated analogues, 3-acetylgliocladic acid (2) and 14-acetylgliocladic acid (3), and seven new dimeric congeners, divirensols A-G (4-10). All metabolites were identified by detailed spectroscopic analysis, supported by biosynthetic considerations, and were assessed for antibacterial and cytotoxic properties. The divirensols are examples of an exceptionally rare class of dimeric sesquiterpene, likely linked via a highly convergent biosynthetic pathway. HPLC-DAD-MS analysis of the crude fungal extract detected ions attributed to putative monomeric biosynthetic precursors.