Jugiones A-D: Antibacterial Xanthone-Anthraquinone Heterodimers from Australian Soil-Derived Penicillium shearii CMB-STF067.

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.

Activity Relationship of Poly(ethylenimine)-Based Liposomes as Group A Streptococcus Vaccine Delivery Systems.

Untreated group A Streptococcus (GAS) can lead to a range of life-threatening diseases, including rheumatic heart disease. To date, no therapeutic or prophylactic vaccines are commercially available to treat or prevent GAS infection. Development of a peptide-based subunit vaccine offers a promising solution, negating the safety issues of live-attenuated or inactive vaccines. Subunit vaccines administer small peptide fragments (antigens), which are typically poorly immunogenic. Therefore, these peptide antigens require formulation with an immune stimulant and/or vaccine delivery platform to improve their immunogenicity. We investigated polyelectrolyte complexes (PECs) and polymer-coated liposomes as self-adjuvanting delivery vehicles for a GAS B cell peptide epitope conjugated to a universal T-helper epitope and a synthetic toll-like receptor 2-targeting moiety lipid core peptide-1 (LCP-1). A structure-activity relationship of cationic PEC vaccines containing different external PEI-coatings (poly(ethylenimine); 10 kDa PEI, 25 kDa PEI, and a synthetic mannose-functionalized 25 kDa PEI) formed vaccines PEC-1, PEC-2, and PEC-3, respectively. All three PEC vaccines induced J8-specific systemic immunoglobulin G (IgG) antibodies when administered intranasally to female BALB/c mice without the use of additional adjuvants. Interestingly, PEC-3 induced the highest antibody titers among all tested vaccines, with the ability to effectively opsonize two clinically isolated GAS strains. A comparative study of PEC-2 and PEC-3 with liposome-based delivery systems was performed subcutaneously. LCP-1 was incorporated into a liposome formulation (DPPC, DPPG and cholesterol), and the liposomes were externally coated with PEI (25 kDa; Lip-2) or mannosylated PEI (25 kDa; Lip-3). All liposome vaccines induced stronger humoral immune responses compared to their PEC counterparts. Notably, sera of mice immunized with Lip-2 and Lip-3 produced significantly higher opsonic activity against clinically isolated GAS strains compared to the positive control, P25-J8 emulsified with the commercial adjuvant, complete Freund's adjuvant (CFA). This study highlights the capability of a PEI-liposome system to act as a self-adjuvanting vehicle for the delivery of GAS peptide antigens and protection against GAS infection.

Noonindoles G-L: Indole Diterpene Glycosides from the Australian Marine-Derived Fungus Aspergillus noonimiae CMB-M0339.

Fungal indole diterpenes (IDTs) occupy a valuable region of bioactive natural product chemical space, displaying potent and selective inhibition of therapeutically important ion channels and with potential application in the treatment of glaucoma, cancer, and neurodegenerative diseases, as well as insecticides and antivirals. We have employed an integrated workflow of analytical scale chemical profiling using GNPS (Global Natural Products Social molecular networking) and cultivation profiling (also known as "MATRIX" miniaturized microbioreactor) to detect, prioritize, optimize the production, isolate, characterize, and identify a new series of indole diterpenes, noonindoles G-L (7-12), from an Australian marine-derived fungus, Aspergillus noonimiae CMB-M0339. The first reported examples of IDT glycosides, the molecular structures for 7-12, were assigned on the basis of detailed spectroscopic analysis and biosynthetic considerations.

Phoslactomycins Revisited: Polyketide Tetrahydrofurans and Lactones from an Australian Wasp Nest-Derived Streptomyces sp. CMB-MW079.

Molecular network analysis of Streptomyces sp. CMB-MW079 detected rare phosphorylated natural products. Miniaturized cultivation profiling (MATRIX) established optimal conditions for the production, isolation, and identification of the polyketide δ-lactone phoslactomycin E (1) and new ester homologues, phoslactomycins J and K (2 and 3), as well as unprecedented heterocyclic analogues, the tetrahydrofuran cyclolactomycins A-D (4-7) and γ-lactone isocyclolactomycins A-C (8-10). We propose a biogenetic relationship linking these cometabolites with the known lactomycins A-C which were tentatively identified as minor cometabolites.

Diindolylmethane Derivatives: New Selective Blockers for T-Type Calcium Channels.

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.

Chrysosporazines Revisited: Regioisomeric Phenylpropanoid Piperazine P-Glycoprotein Inhibitors from Australian Marine Fish-Derived Fungi.

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).

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.

Neochrysosporazines: Precursor-Directed Biosynthesis Defines a Marine-Derived Fungal Natural Product P-Glycoprotein Inhibitory Pharmacophore.

Upregulation of ATP binding cassette (ABC) transporter efflux pumps (i.e. P-glycoprotein, P-gp) can impart multidrug resistance, rendering many chemotherapeutics ineffective and seriously limiting treatment regimes. While ABC transporters remain an attractive target for therapeutic intervention, the development of clinically useful small-molecule inhibitors has proved challenging. In this report, we describe the structure-activity relationship (SAR) analysis of a newly discovered P-gp inhibitory pharmacophore, phenylpropanoid piperazine chrysosporazines, produced by co-isolated marine-derived fungi. In the absence of any total syntheses, we apply an innovative precursor-directed biosynthesis strategy that successfully repurposed fungal biosynthetic output, allowing us to isolate, characterize, and identify the structurally diverse neochrysosporazines A-Q. SAR analysis utilizing all known (and new) neochrysosporazines, chrysosporazines, and azachrysosporazines, plus semi-synthetic analogues, established the key structure requirements for the P-gp inhibitory pharmacophore, and, in addition, identified non-essential sites that allow for the design of affinity and other conjugated probes.

Precursor-Directed Biosynthesis Mediated Amplification of Minor Aza Phenylpropanoid Piperazines in an Australian Marine Fish-Gut-Derived Fungus, Chrysosporium sp. CMB-F214.

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.

Poly(hydrophobic amino acid)-Based Self-Adjuvanting Nanoparticles for Group A Streptococcus Vaccine Delivery.

Peptide antigens have been widely used in the development of vaccines, especially for those against autoimmunity-inducing pathogens and cancers. However, peptide-based vaccines require adjuvant and/or a delivery system to stimulate desired immune responses. Here, we explored the potential of self-adjuvanting poly(hydrophobic amino acids) (pHAAs) to deliver peptide-based vaccine against Group A Streptococcus (GAS). We designed and synthesized self-assembled nanoparticles with a variety of conjugates bearing a peptide antigen (J8-PADRE) and polymerized hydrophobic amino acids to evaluate the effects of structural arrangement and pHAAs properties on a system's ability to induce humoral immune responses. Immunogenicity of the developed conjugates was also compared to commercially available human adjuvants. We found that a linear conjugate bearing J8-PADRE and 15 copies of leucine induced equally effective, or greater, immune responses than commercial adjuvants. Our fully defined, adjuvant-free, single molecule-based vaccine induced the production of antibodies capable of killing GAS 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.

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).

A tetrapeptide class of biased analgesics from an Australian fungus targets the µ-opioid receptor.

An Australian estuarine isolate of Penicillium sp. MST-MF667 yielded 3 tetrapeptides named the bilaids with an unusual alternating LDLD chirality. Given their resemblance to known short peptide opioid agonists, we elucidated that they were weak (Ki low micromolar) µ-opioid agonists, which led to the design of bilorphin, a potent and selective µ-opioid receptor (MOPr) agonist (Ki 1.1 nM). In sharp contrast to all-natural product opioid peptides that efficaciously recruit ß-arrestin, bilorphin is G protein biased, weakly phosphorylating the MOPr and marginally recruiting ß-arrestin, with no receptor internalization. Importantly, bilorphin exhibits a similar G protein bias to oliceridine, a small nonpeptide with improved overdose safety. Molecular dynamics simulations of bilorphin and the strongly arrestin-biased endomorphin-2 with the MOPr indicate distinct receptor interactions and receptor conformations that could underlie their large differences in bias. Whereas bilorphin is systemically inactive, a glycosylated analog, bilactorphin, is orally active with similar in vivo potency to morphine. Bilorphin is both a unique molecular tool that enhances understanding of MOPr biased signaling and a promising lead in the development of next generation analgesics.

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.

Chrysosporazines A-E: P-Glycoprotein Inhibitory Piperazines from an Australian Marine Fish Gastrointestinal Tract-Derived Fungus, Chrysosporium sp. CMB-F214.

Chemical analysis of Chrysosporium sp. CMB-F214, yielded five new piperazines, chrysosporazines A-E (1-5), with structures assigned by spectroscopic and X-ray analyses and biosynthetic considerations. The chrysosporazines 2-5 exist as an equilibrium of major and minor N-acyl rotamers, while 1-3 incorporate an unprecedented hexahydro-6H-pyrazino[1,2-b]isoquinolin-6-one scaffold. The noncytotoxic chrysosporazines reverse doxorubicin drug resistance in P-glycoprotein overexpressing colon carcinoma cells (SW620 Ad300), with 2 delivering a comparable gain in sensitivity to the positive control, verapamil.

Cinerols, Nitrogenous Meroterpenoids from the Marine Sponge Dysidea cinerea.

Eleven new nitrogenous meroterpenoids, cinerols A-K (1-11), were isolated from the marine sponge Dysidea cinerea collected in the South China Sea, and their structures were determined by detailed spectroscopic analysis. Cinerols A (1) and B (2) feature a rare 5H-pyrrolo[1,2a]benzimidazole moiety, while cinerols C-G (3-7) are examples of rare meroterpene benzoxazoles. The cinerols are noncytotoxic to human melanoma A375 cells at the concentration of 32 µM; however, selected cinerols exhibit moderate inhibitory activity against one or more of protein-tyrosine phosphatase 1B, ATP-citrate lyase, and SH2 domain-containing phosphatase-1 with IC50 values of 2.8-27 µM.

Scopularides Revisited: Molecular Networking Guided Exploration of Lipodepsipeptides in Australian Marine Fish Gastrointestinal Tract-Derived Fungi.

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.

Acylpeptide hydrolase is a novel regulator of KRAS plasma membrane localization and function.

The primary site for KRAS signaling is the inner leaflet of the plasma membrane (PM). We previously reported that oxanthroquinone G01 (G01) inhibited KRAS PM localization and blocked KRAS signaling. In this study, we identified acylpeptide hydrolase (APEH) as a molecular target of G01. APEH formed a stable complex with biotinylated G01, and the enzymatic activity of APEH was inhibited by G01. APEH knockdown caused profound mislocalization of KRAS and reduced clustering of KRAS that remained PM localized. APEH knockdown also disrupted the PM localization of phosphatidylserine (PtdSer), a lipid critical for KRAS PM binding and clustering. The mislocalization of KRAS was fully rescued by ectopic expression of APEH in knockdown cells. APEH knockdown disrupted the endocytic recycling of epidermal growth factor receptor and transferrin receptor, suggesting that abrogation of recycling endosome function was mechanistically linked to the loss of KRAS and PtdSer from the PM. APEH knockdown abrogated RAS-RAF-MAPK signaling in cells expressing the constitutively active (oncogenic) mutant of KRAS (KRASG12V), and selectively inhibited the proliferation of KRAS-transformed pancreatic cancer cells. Taken together, these results identify APEH as a novel drug target for a potential anti-KRAS therapeutic.