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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.

Subjecting the Australian marine-derived fungus Aspergillus noonimiae CMB-M0339 to cultivation profiling using an innovative miniaturized 24-well plate format (MATRIX) enabled access to new examples of the rare class of 2,6-diketopiperazines, noonazines A–C (1–3), along with the known analogue coelomycin (4), as well as a new azaphilone, noonaphilone A (5). Structures were assigned to 1–5 on the basis of a detailed spectroscopic analysis, and in the case of 1–2, an X-ray crystallographic analysis. Plausible biosynthetic pathways are proposed for 1–4, involving oxidative Schiff base coupling/dimerization of a putative Phe precursor. Of note, 2 incorporates a rare meta-Tyr motif, typically only reported in a limited array of Streptomyces metabolites. Similarly, a plausible biosynthetic pathway is proposed for 5, highlighting a single point for stereo-divergence that allows for the biosynthesis of alternate antipodes, for example, the 7R noonaphilone A (5) versus the 7S deflectin 1a (6).

Mucosal vaccines are highly attractive due to high patient compliance and their suitability for mass immunizations. However, all currently licensed mucosal vaccines are composed of attenuated/inactive whole microbes, which are associated with a variety of safety concerns. In contrast, modern subunit vaccines use minimal pathogenic components (antigens) that are safe but typically poorly immunogenic when delivered via mucosal administration. In this study, we demonstrated the utility of various functional polymer-based nanostructures as vaccine carriers. A Group A Streptococcus (GAS)-derived peptide antigen (PJ8) was selected in light of the recent global spread of invasive GAS infection. The vaccine candidates were prepared by either conjugation or physical mixing of PJ8 with rod-, sphere-, worm-, and tadpole-shaped polymeric nanoparticles. The roles of nanoparticle shape and antigen conjugation in vaccine immunogenicity were demonstrated through the comparison of three distinct immunization pathways (subcutaneous, intranasal, and oral). No additional adjuvant or carrier was required to induce bactericidal immune responses even upon oral vaccine administration.

The Australian roadside soil-derived fungus Penicillium shearii CMB-STF067 was prioritized for chemical investigation based on an SDA cultivation extract exhibiting both antibacterial properties and natural products with unprecedented molecular formulae (GNPS). Subsequent miniaturized 24-well plate cultivation profiling (MATRIX) identified red rice as optimal for the production of the target chemistry, with scaled-up cultivation, extraction and fractionation yielding four new xanthone–anthraquinone heterodimers, jugiones A–D (1–4), whose structures were assigned by detailed spectroscopic analysis and biosynthetic considerations. Of note, where 1–2 and 4 were active against the Gram-positive bacteria vancomycin-resistant Enterococcus faecalis (IC50 2.6–3.9 μM) and multiple-drug-resistant clinical isolates of Staphylococcus aureus (IC50 1.8–6.4 μM), and inactive against the Gram-negative bacteria Escherichia coli (IC50 > 30 μM), the closely related analog 3 exhibited no antibacterial properties (IC50 > 30 μM). Furthermore, where 1 was cytotoxic to human carcinoma (IC50 9.0–9.8 μM) and fungal (IC50 4.1 μM) cells, 2 and 4 displayed no such cytotoxicity (IC50 > 30 μM), revealing an informative structure activity relationship (SAR). We also extended the SAR study to other known compounds of this heterodimer class, which showed that the modification of ring G can reduce or eliminate the cytotoxicity while retaining the antibacterial activity.

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.

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.

Abstract Chemical cues produced by late-stage embryos of the cane toad (Rhinella marina) attract older conspecific larvae, which are highly cannibalistic and can consume an entire clutch. To clarify the molecular basis of this attraction response, we presented captive tadpoles with components present in toad eggs. As previously reported, attractivity arises from the distinctive toxins (bufadienolides) produced by cane toads, with some toxins (e.g., bufagenins) much stronger attractants than others (e.g., bufotoxins). Extracts of frozen toad parotoid glands (rich in bufagenins) were more attractive than were fresh MeOH extracts of the parotoid secretion (rich in bufotoxins), and purified marinobufagin was more effective than marinobufotoxin. Cardenolide aglycones (e.g., digitoxigenin) were active attractors, whereas C-3 glycosides (e.g., digoxin, oubain) were far less effective. A structure–activity relationship study revealed that tadpole attractant potency strongly correlated with Na+/K+ ATPase inhibitory activity, suggesting that tadpoles monitor and rapidly react to perturbations to Na+/K+ ATPase activity.

Rosenbergiella bacteria have been previously isolated predominantly from floral nectar and identified in metagenomic screenings as associated with bees. Here, we isolated three Rosenbergiella strains from the robust Australian stingless bee Tetragonula carbonaria sharing over 99.4% sequence similarity with Rosenbergiella strains isolated from floral nectar. The three Rosenbergiella strains (D21B, D08K, D15G) from T. carbonaria exhibited near-identical 16S rDNA. The genome of strain D21B was sequenced; its draft genome contains 3,294,717 bp, with a GC content of 47.38%. Genome annotation revealed 3236 protein-coding genes. The genome of D21B differs sufficiently from the closest related strain, Rosenbergiella epipactidis 2.1A, to constitute a new species. In contrast to R. epipactidis 2.1A, strain D21B produces the volatile 2-phenylethanol. The D21B genome contains a polyketide/non-ribosomal peptide gene cluster not present in any other Rosenbergiella draft genomes. Moreover, the Rosenbergiella strains isolated from T. carbonaria grew in a minimal medium without thiamine, but R. epipactidis 2.1A was thiamine-dependent. Strain D21B was named R. meliponini D21B, reflecting its origin from stingless bees. Rosenbergiella strains may contribute to the fitness of T. carbonaria.

Intranasal vaccine administration can overcome the disadvantages of injectable vaccines and present greater efficiency for mass immunization. However, the development of intranasal vaccines is challenged by poor mucosal immunogenicity of antigens and the limited availability of mucosal adjuvants. Here, we examined a number of self-adjuvanting liposomal systems for intranasal delivery of lipopeptide vaccine against group A Streptococcus (GAS). Among them, two liposome formulations bearing lipidated cell-penetrating peptide KALA and a new lipidated chitosan derivative (oleoyl-quaternized chitosan, OTMC) stimulated high systemic antibody titers in outbred mice. The antibodies were fully functional and were able to kill GAS bacteria. Importantly, OTMC was far more effective at stimulating antibody production than the classical immune-stimulating trimethyl chitosan formulation. In a simple physical mixture, OTMC also enhanced the immune responses of the tested vaccine, without the need for a liposome delivery system. The adjuvanting capacity of OTMC was further confirmed by its ability to stimulate cytokine production by dendritic cells. Thus, we discovered a new immune stimulant with promising properties for mucosal vaccine development.

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.

Analytical scale chemical/cultivation profiling prioritized the Australian marine-derived fungus Aspergillus noonimiae CMB-M0339. Subsequent investigation permitted isolation of noonindoles A–F (5–10) and detection of eight minor analogues (i–viii) as new examples of a rare class of indole diterpene (IDT) amino acid conjugate, indicative of an acyl amino acid transferase capable of incorporating a diverse range of amino acid residues. Structures for 5–10 were assigned by detailed spectroscopic and X-ray crystallographic analysis. The metabolites 5–14 exhibited no antibacterial properties against G-ve and G+ve bacteria or the fungus Candida albicans, with the exception of 5 which exhibited moderate antifungal activity.

Peptide-based subunit vaccines include only minimal antigenic determinants, and, therefore, are less likely to induce allergic immune responses and adverse effects compared to traditional vaccines. However, peptides are weakly immunogenic and susceptible to enzymatic degradation when administered on their own. Hence, we designed polyelectrolyte complex (PEC)-based delivery systems to protect peptide antigens from degradation and improve immunogenicity. Lipopeptide (LCP-1) bearing J8 B-cell epitope derived from Group A Streptococcus (GAS) M-protein was selected as the model peptide antigen. In the pilot study, LCP-1 incorporated in alginate/cross-linked polyarginine-J8-based PEC induced high J8-specific IgG antibody titres. The PEC system was then further modified to improve its immune stimulating capability. Of the formulations tested, PEC-4, bearing LCP-1, alginate and cross-linked polylysine, induced the highest antibody titres in BALB/c mice following subcutaneous immunisation. The antibodies produced were more opsonic than those induced by mice immunised with other PECs, and as opsonic as those induced by antigen adjuvanted with powerful complete Freund’s adjuvant.

The natural product indole-3-carbinol (I3C) and its major digestive product 3,3′-diindolylmethane (DIM) have shown clinical promise in multiple forms of cancer including breast cancer. In this study, we explored the calcium channel activity of DIM, its synthetic derivative 3,3′-Diindolylmethanone (DIM-one) and related I3C and DIM-one analogs. For the first time, DIM, DIM-one and analog IX were identified as selective blockers for T-type CaV3.3 (IC50s DIM 2.09 µM; DIM-one 9.07 µM) while compound IX inhibited both CaV3.2 (6.68 µM) and CaV3.3 (IC50 = 3.05 µM) using a FLIPR cell-based assay to measure inhibition of T-type calcium channel window current. Further characterization of DIM by electrophysiology revealed it inhibited inward Ca2+ current through CaV3.1 (IC50 = 8.32 µM) and CaV3.3 (IC50 = 9.63 µM), while IX partially blocked CaV3.2 and CaV3.3 inward Ca2+ current. In contrast, DIM-one preferentially blocked CaV3.1 inward Ca2+ current (IC50 = 1.53 µM). The anti-proliferative activities of these compounds revealed that oxidation of the methylene group of DIM shifted the selectivity of DIMs from breast cancer cell line MCF-7 to colon cancer cell line HT-29.

Adjuvants and delivery systems are essential components of vaccines to increase immunogenicity against target antigens, particularly for peptide epitopes (poor immunogens). Emulsions, nanoparticles, and liposomes are commonly used as a delivery system for peptide-based vaccines. A Poly(hydrophobic amino acids) delivery system was previously conjugated to Group A Streptococcus (GAS)-derived peptide epitopes, allowing the conjugates to self-assemble into nanoparticles with self adjuvanting ability. Their hydrophobic amino acid tail also serves as an anchoring moiety for the peptide epitope, enabling it to be integrated into the liposome bilayer, to further boost the immunological responses. Polyleucine-based conjugates were anchored to cationic liposomes using the film hydration method and administered to mice subcutaneously. The polyleucine-peptide conjugate, its liposomal formulation, and simple liposomal encapsulation of GAS peptide epitope induced mucosal (saliva IgG) and systemic (serum IgG, IgG1 and IgG2c) immunity in mice. Polyleucine acted as a potent liposome anchoring portion, which stimulated the production of highly opsonic antibodies. The absence of polyleucine in the liposomal formulation (encapsulated GAS peptide) induced high levels of antibody titers, but with poor opsonic ability against GAS bacteria. However, the liposomal formulation of the conjugated vaccine was no more effective than conjugates alone self-assembled into nanoparticles.

A library of fungi previously recovered from the gastrointestinal tract (GIT) of several fresh, commercially sourced Australian mullet fish was re-profiled for production of a rare class of phenylpropanoid piperazine alkaloids (chrysosporazines) using an integrated platform of; (i) miniaturized 24-well plate cultivation profiling (MATRIX), (ii) UPLC-DAD and UPLC-QTOF-MS/MS (GNPS) chemical profiling, and; (iii) precursor directed biosynthesis to manipulate in situ biosynthetic performance and outputs; to detect two new fungal producers of chrysosporazines. Chemical analysis of an optimized PDA solid phase cultivation of Aspergillus sp. CMB-F661 yielded the new regioisomeric chrysosporazine T (1) and U (2), while precursor directed cultivation amplified production and yielded the very minor new natural products azachrysosporazine T1 (3) and U1 (4), and the new unnatural analogues neochrysosporazine R (5) and S (6). Likewise, chemical analysis of an optimized M1 solid phase cultivation of Spiromastix sp. CMB-F455 lead to the GNPS detection of multiple chrysosporazines and brasiliamides, and the isolation and structure elucidation of chrysosporazine D (7) and brasiliamide A (8). Access to new chrysosporazine regioisomers facilitated structure activity relationship investigations to better define the chrysosporazine P-glycoprotein (P-gp) inhibitory pharmacophore, which is exceptionally potent at reversing doxorubrin resistance in P-gp over expressing colon carcinoma cells (SW600 Ad300).

Reconsideration of the spectroscopic data for penipacids A–E, first reported in 2013 as the acyclic amidines 1–5 from the South China deep sea sediment-derived fungus Penicillium paneum SD-44, prompted a total synthesis structure revision as the hydrazones 6–10. This revision strongly supported the proposition that penipacids A–B (6–7) were artifact Schiff base adducts of the cryptic (undetected) natural product N-aminoanthranilic acid (11) with diacetone alcohol, induced by excessive exposure to acetone and methanol under acidic handling conditions. Likewise, the revised structures for penipacids C–D (8–9) and E (10) raise the possibility that they may also be artifact Schiff base adducts of 11 and the media constituents pyruvic acid and furfural, respectively. A review of the natural products literature revealed other Schiff base (hydrazone) natural products that might also be viewed as Schiff base adduct artifacts of 11. Having raised the prospect that 11 is an undetected and reactive cryptic natural product, we went on to establish that 11 is not cytotoxic to a range of bacterial, fungal or mammalian (human) cell types. Instead, when added as a supplement to microbial cultivations, 11 can act as a chemical cue/transcriptional regulator, activating and/or enhancing the yield of biosynthetic gene clusters encoding for other natural product chemical defenses. This study demonstrates the value of challenging the structure and artifact status of natural products, as a window into the hidden world of cryptic and highly reactive natural products.

The knowledge that natural products (NPs) are potent and selective modulators of important biomacromolecules (e.g., DNA and proteins) has inspired some of the world’s most successful pharmaceuticals and agrochemicals. Notwithstanding these successes and despite a growing number of reports on naturally occurring pairs of enantiomers, this area of NP science still remains largely unexplored, consistent with the adage “If you don’t seek, you don’t find”. Statistically, a rapidly growing number of enantiomeric NPs have been reported in the last several years. The current review provides a comprehensive overview of recent records on natural enantiomers, with the aim of advancing awareness and providing a better understanding of the chemical diversity and biogenetic context, as well as the biological properties and therapeutic (drug discovery) potential, of enantiomeric NPs.

The ethyl acetate extract of an ISP-2 agar cultivation of the wasp nest-associated fungus Penicillium sp. CMB-MD14 exhibited promising antibacterial activity against vancomycin-resistant enterococci (VRE), with a bioassay guided chemical investigation yielding the new meroterpene, oxandrastin A (1), the first andrastin-like metabolite with an extra oxygenation at C-2. A culture media optimisation strategy informed a scaled-up rice cultivation that yielded 1, together with three new oxandrastins B–D (2–4), two known andrastins C (5) and F (6), and a new meroterpene of the austalide family, isoaustalide F (7). Structures of 1–7 were assigned based on detailed spectroscopic analysis and chemical interconversion. A GNPS molecular networking analysis of the rice cultivation extract detected the known austalides B (8), H (9), and H acid (10), tentatively identified based on molecular formulae and co-clustering with 7. That the anti-VRE properties of the CMB-MD14 extract were exclusively attributed to 1 (IC50 6.0 µM, MIC99 13.9 µM), highlights the importance of the 2-OAc and 3-OAc moieties to the oxandrastin anti-VRE pharmacophore.

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.

Chemical analysis of an M1 agar plate cultivation of a marine fish-gut-derived fungus, Chrysosporium sp. CMB-F214, revealed the known chrysosporazines A–D (11–14) in addition to a suite of very minor aza analogues 1–6. A microbioreactor (MATRIX) cultivation profiling analysis failed to deliver cultivation conditions that significantly improved the yields of 1–6; however, it did reveal that M2 agar cultivation produced the new natural product 15. A precursor-directed biosynthesis strategy adopting supplementation of a CMB-F214 M1 solid agar culture with sodium nicotinate enhanced production of otherwise inaccessible azachrysposorazines A1 (1), A2 (2), B1 (3), C1 (4), C2 (5) and D1 (6), in addition to four new chrysosporazines; chrysosporazines N–P (7–9) and spirochrysosporazine A (10). Structures inclusive of absolute configurations were assigned to 1–15 based on detailed spectroscopic and chemical analyses, and biosynthetic considerations. Non-cytotoxic to human carcinoma cells, azachrysosporazies 1–5 were capable of reversing doxorubicin resistance in P-glycoprotein (P-gp)-overexpressing human colon carcinoma cells (SW620 Ad300), with optimum activity exhibited by the C-2′ substituted analogues 3–5.

Peptide-based vaccine development represents a highly promising strategy for preventing Group A Streptococcus (GAS) infection. However, these vaccines need to be administered with the help of a delivery system and/or immune adjuvant. Cell-penetrating peptides (CPPs) have been used as a powerful tool for delivering various therapeutic agents, including peptides, as they can overcome the permeability barrier of cell membranes. Here, we used CPPs to deliver our lead lipopeptide-based vaccine (LCP-1). CPPs were anchored through a spacer to LCP-1-bearing multilamellar and unilamellar liposomes and administered to Swiss outbred mice. Tat47–57 conjugated to two palmitic acids via a (Gly)6 spacer (to form a liposome-anchoring moiety) was the most efficient system for triggering immune responses when combined with multilamellar liposomes bearing LCP-1. The immunostimulatory potential of a variety of other CPPs was examined following intranasal administration in mice. Among them, LCP-1/liposomes/Tat47–57 and LCP-1/liposomes/KALA induced the highest antibody titers. The antibodies produced showed high opsonic activity against clinically isolated GAS strains D3840 and GC2 203. The use of the CPP-liposome delivery system is a promising strategy for liposome-based GAS vaccine development.

Further investigation into a fish gut-derived fungus Evlachovaea sp. CMB-F563, previously reported to produce the unprecedented Schiff base prolinimines A–B (1–2), revealed a new cryptic natural product, N-amino-l-proline methyl ester (5)—only the second reported natural occurrence of an N-amino-proline, and the first from a microbial source. To enable these investigations, we developed a highly sensitive analytical derivitization methodology, using 2,4-dinitrobenzaldehyde (2,4-DNB) to cause a rapid in situ transformation of 5 to the Schiff base 9, with the latter more readily detectable by UHPLC-DAD (400 nm) and HPLC-MS analyses. Moreover, we demonstrate that during cultivation 5 is retained in fungal mycelia, and it is only when solvent extraction disrupts mycelia that 5 is released to come in contact with the furans 7–8 (which are themselves produced by thermal transformation of carbohydrates during media autoclaving prior to fungal inoculation). Significantly, on contact, 5 undergoes a spontaneous condensation with 7–8 to yield the Schiff base prolinimines 1–2, respectively. Observations made during this study prompted us to reflect on what it is to be a natural product (i.e., 5), versus an artifact (i.e., 1–2), versus a media component (i.e., 7–8).

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.

Group A Streptococcus (GAS) and GAS-associated infections are a global challenge, with no licensed GAS vaccine on the market. The GAS M protein is a critical virulence factor in the fight against GAS infection, and it has been a primary target for GAS vaccine development. Measuring functional opsonic antibodies against GAS is an important component in the clinical development path for effective vaccines. In this study, we compared the opsonic activity of two synthetic, self-adjuvanting subunit vaccines containing either the J8- or 88/30-epitope in Swiss outbred mice using intranasal administration. Following primary immunization and three boosts, sera were assessed for IgG activity using ELISA, and opsonization activity against seven randomly selected clinical isolates of GAS was measured. Vaccine constructs containing the conservative J8-epitope showed significant opsonic activity against six out of the seven GAS clinical isolates, while the vaccine containing the variable 88/30-epitope did not show any significant opsonic activity.

Peptide subunit vaccines hold great potential compared to traditional vaccines. However, peptides alone are poorly immunogenic. Therefore, it is of great importance that a vaccine delivery platform and/or adjuvant that enhances the immunogenicity of peptide antigens is developed. Here, we report the development of two different systems for the delivery of lipopeptide subunit vaccine (LCP-1) against group A streptococcus: polymer-coated liposomes and polyelectrolyte complexes (PECs). First, LCP-1-loaded and alginate/trimethyl chitosan (TMC)-coated liposomes (Lip-1) and LCP-1/alginate/TMC PECs (PEC-1) were examined for their ability to trigger required immune responses in outbred Swiss mice; PEC-1 induced stronger humoral immune responses than Lip-1. To further assess the adjuvanting effect of anionic polymers in PECs, a series of PECs (PEC-1 to PEC-5) were prepared by mixing LCP-1 with different anionic polymers, namely alginate, chondroitin sulfate, dextran, hyaluronic acid, and heparin, then coated with TMC. All produced PECs had similar particle sizes (around 200 nm) and surface charges (around + 30 mV). Notably, PEC-5, which contained heparin, induced higher antigen-specific systemic IgG and mucosal IgA titers than all other PECs. PEC systems, especially when containing heparin and TMC, could function as a promising platform for peptide-based subunit vaccine delivery for intranasal administration.

Concern about finding new antibiotics against drug-resistant pathogens is increasing every year. Antarctic bacteria have been proposed as an unexplored source of bioactive metabolites; however, most biosynthetic gene clusters (BGCs) producing secondary metabolites remain silent under common culture conditions. Our work aimed to characterize elicitation conditions for the production of antibacterial secondary metabolites from 34 Antarctic bacterial strains based on MS/MS metabolomics and genome mining approaches. Bacterial strains were cultivated under different nutrient and elicitation conditions, including the addition of lipopolysaccharide (LPS), sodium nitroprusside (SNP), and coculture. Metabolomes were obtained by HPLC-QTOF-MS/MS and analyzed through molecular networking. Antibacterial activity was determined, and seven strains were selected for genome sequencing and analysis. Biosynthesis pathways were activated by all the elicitation treatments, which varies among strains and dependents of culture media. Increased antibacterial activity was observed for a few strains and addition of LPS was related with inhibition of Gram-negative pathogens. Antibiotic BGCs were found for all selected strains and the expressions of putative actinomycin, carotenoids, and bacillibactin were characterized by comparison of genomic and metabolomic data. This work established the use of promising new elicitors for bioprospection of Antarctic bacteria and highlights the importance of new “-omics” comparative approaches for drug discovery.

Group A Streptococcus (GAS)-associated rheumatic heart disease is a leading cause of death caused by GAS infection. While antibiotics can treat the infection in most cases, growing antibiotic resistance, late medical intervention, and recurrent infection are major obstacles to the effective treatment of GAS-associated diseases. As GAS infection typically originates from the bacterial colonization of mucosal tissue in the throat, an oral vaccine that can generate both systemic and mucosal immune responses would solve problems associated with traditional medical interventions. Moreover, orally delivered vaccines are more easily administered and less expensive for mass immunization. In this study, the B-cell epitope J8, derived from GAS M protein, and universal T-helper Pan HLA-DR-binding epitope peptide (PADRE), were conjugated to poly (methyl acrylate) (PMA) to form a self-assembled nanoparticle vaccine candidate (PMA-P-J8). Strong systemic and mucosal immune responses were induced upon single oral immunization of mice with the conjugate. The antibodies generated were opsonic against GAS clinical isolates as measured after boost immunization. Thus, we developed a simple conjugate as an effective, adjuvant-free oral peptide-based vaccine.

An Australian marine tunicate-derived fungus, Talaromyces sp. CMB-TU011 was subjected to a program of analytical microbioreactor (MATRIX) cultivations, supported by UHPLC-QTOF profiling, to reveal conditions for producing a new class of extensively N-methylated 11-12 residue linear peptides, talaropeptides A-D (2-5). The structures for 2-5, inclusive of absolute configurations, were determined by a combination of detailed spectroscopic and chemical (e.g., C3 and C18 Marfey's) analyses. We report on the biological properties of 2-5, including plasma stability, as well as antibacterial, antifungal and cell cytotoxicity. The talaropeptide mega non-ribosomal peptide synthetase (NRPS) is described, as second only in size to that for the fungus-derived immunosuppressant cyclosporine (an 11-residue extensively N-methylated cyclic peptide).

Streptomyces are Gram-positive bacteria that occupy diverse ecological niches including host-associations with animals and plants. Members of this genus are known for their overwhelming repertoire of natural products, which has been exploited for almost a century as a source of medicines and agrochemicals. Notwithstanding intense scientific and commercial interest in Streptomyces natural products, surprisingly little is known of the intra- and/or inter-species ecological roles played by these metabolites. In this report we describe the chemical structures, biological properties, and biosynthetic relationships between natural products produced by Streptomyces isolated from internal tissues of predatory Conus snails, collected from the Great Barrier Reef, Australia. Using chromatographic, spectroscopic and bioassays methodology, we demonstrate that Streptomyces isolated from five different Conus species produce identical chemical and antifungal profiles – comprising a suite of polycyclic tetramic acid macrolactams (PTMs). To investigate possible ecological (and evolutionary) relationships we used genome analyses to reveal a close taxonomic relationship with other sponge-derived and free-living PTM producing Streptomyces (i.e., Streptomyces albus). In-depth phylogenomic analysis of PTM biosynthetic gene clusters indicated PTM structure diversity was governed by a small repertoire of genetic elements, including discrete gene acquisition events involving dehydrogenases. Overall, our study shows a Streptomyces-Conus ecological relationship that is concomitant with specific PTM chemical profiles. We provide an evolutionary framework to explain this relationship, driven by anti-fungal properties that protect Conus snails from fungal pathogens.

ATP binding cassette (ABC) transporters, such as P-gp, BCRP and MRP1, can increase efflux of clinical chemotherapeutic agents and lead to multi-drug resistance (MDR) in cancer cells. While the discovery and development of clinically useful inhibitors has proved elusive to date, this molecular target nevertheless remains a promising strategy for addressing and potentially overcoming MDR. In a search for new classes of inhibitor, we used fluorescent accumulation and efflux assays supported by cell flow cytometry and MDR reversal assays, against a panel of sensitive and MDR human cancer cell lines, to evaluate the marine sponge co-metabolites 1–12 as inhibitors of P-gp, BCRP or MRP1 initiated MDR. These studies identified and characterized lamellarin O (11) as a selective inhibitor of BCRP mediated drug efflux. A structure–activity relationship analysis inclusive of the natural products 1–12 and the synthetic analogues 13–19, supported by in silico docking studies, revealed key structural requirements for the lamellarin O (11) BCRP inhibitory pharmacophore.

The heronapyrroles A–C have first been isolated from a marine-derived Streptomyces sp. (CMB-0423) in 2010. Structurally, these natural products feature an unusual nitropyrrole system to which a partially oxidized farnesyl chain is attached. The varying degree of oxidation of the sesquiterpenyl subunit in heronapyrroles A–C provoked the hypothesis that there might exist other hitherto unidentified metabolites. On biosynthetic grounds a mono-tetrahydrofuran-diol named heronapyrrole D appeared a possible candidate. We here describe a short asymmetric synthesis of heronapyrrole D, its detection in cultivations of CMB-0423 and finally the evaluation of its antibacterial activity. We thus demonstrate that biosynthetic considerations and the joint effort of synthetic and natural product chemists can result in the identification of new members of a rare class of natural products.

Chemical analysis of a cultivation of an Australian Mugil mullet gastrointestinal tract (GIT) derived fungus, Scopulariopsis sp. CMB-F458, yielded the known lipodepsipeptides scopularides A (1) and B (2). A comparative global natural product social (GNPS) molecular networking analysis of ×63 co-isolated fungi, detected two additional fungi producing new scopularides, with Beauveria sp. CMB-F585 yielding scopularides C−G (3−7) and Scopulariopsis sp. CMB-F115 yielding scopularide H (8). Structures inclusive of absolute configurations were assigned by detailed spectroscopic and C3 Marfey’s analysis, together with X-ray analyses of 3 and 8, and biosynthetic considerations. Scopularides A−H (1−8) did not exhibit significant growth inhibitory activity against a selection of Gram positive (+ve) and negative (−ve) bacteria, a fungus, or a panel of three human carcinoma cell lines.

Chemical analysis of a southern Australian sponge, Leucetta sp., led to the discovery of a pair of solvolysis adducts, leucettazoles A1 (1a) and B1 (2a), as artifacts of an unprecedented family of 15-membered macrocyclic alkaloid dimers featuring a pair of imino bridged 2-aminoimidazoles, together with a putative monomeric precursor, leucettazine A (3). The dimeric alkaloids 1a and 2a, and monomer 3, were identified by detailed spectroscopic analysis, supported by chemical transformations, analytical mass spectrometry, and biosynthetic considerations. Global natural product social networking (GNPS) molecular analysis of crude sponge extracts and solvent partitions, supported by single ion extraction (SIE) and diagnostic MS/MS fragmentations, revealed the associated natural products, leucettazoles A (1) and B (2). This study highlights that the study of natural product artifacts can be useful, and can on occasion serve as a pathway to discover cryptic new classes of natural products.

T-type calcium channel (CaV3.x) blockers are receiving increasing attention as potential therapeutics for the treatment of pathophysiological disorders and diseases, including absence epilepsy, Parkinson’s disease (PD), hypertension, cardiovascular diseases, cancers, and pain. However, few clinically approved CaV3.x blockers are available, and selective pharmacological tools are needed to further unravel the roles of individual CaV3.x subtypes. In this work, through an efficient synthetic route to the marine fungal product pseudellone C, we obtained bisindole alkaloid analogs of pseudellone C with a modified tryptophan moiety and identified two CaV3.2 (2, IC50 = 18.24 µM; 3, IC50 = 6.59 µM) and CaV3.3 (2, IC50 = 7.71 µM; 3, IC50 = 3.81 µM) selective blockers using a FLIPR cell-based assay measuring CaV3.x window currents. Further characterization by whole-cell patch-clamp revealed a preferential block of CaV3.1 activated current (2, IC50 = 5.60 µM; 3, IC50 = 9.91 µM), suggesting their state-dependent block is subtype specific.

Chemical analysis of a marine sponge, Cacospongia sp. (CMB-03404), obtained during deep sea commercial fishing activities off the southern coast of Australia, yielded an unprecedented family of sesterterpene α-methyl-γ-hydroxybutenolides, cacolides A⁻L (1⁻12), together with biosynthetically related norsesterterpene carboxylic acids, cacolic acids A⁻C (13⁻15). Structures were assigned on the basis of detailed spectroscopic analysis with comparisons to known natural products and biosynthetic considerations. In addition to revealing new chemical diversity, this study provided a valuable platform for comparing and contrasting the capabilities of the traditional dereplication technologies of HPLC-DAD, HPLC-MS and NMR, with those of the emerging HPLC-MS/MS approach known as global natural products social molecular networking (GNPS), as applied to marine sponge sesterterpene tetronic acids.

This study describes an investigation into secondary metabolites that are produced by a marine red alga, Symphyocladia latiuscula, which was collected from coastal waters off Qingdao, China. A combination of normal, reversed phase, and gel chromatography was used to isolate six citric acid derived natural products, aconitates A–F (1–6), together with two known and ten new polybrominated phenols, symphyocladins C/D (7a/b), and symphyocladins H–Q (8a/b, 9a/b and 10–15), respectively. Structure elucidation was achieved by detailed spectroscopic (including X-ray crystallographic) analysis. We propose a plausible and convergent biosynthetic pathway involving a key quinone methide intermediate, linking aconitates and symphyocladins.