Tanja Grkovic

Elicitation of secondary metabolism in actinomycetes

Genomic sequence data have revealed the presence of a large fraction of putatively silent biosynthetic gene clusters in the genomes of actinomycetes that encode for secondary metabolites, which are not detected under standard fermentation conditions. This review focuses on the effects of biological (co-cultivation), chemical, as well as molecular elicitation on secondary metabolism in actinomycetes. Our review covers the literature until June 2014 and exemplifies the diversity of natural products that have been recovered by such approaches from the phylum Actinobacteria.

Production of Induced Secondary Metabolites by a Co-Culture of Sponge-Associated Actinomycetes, Actinokineospora sp. EG49 and Nocardiopsis sp. RV163

Two sponge-derived actinomycetes, Actinokineospora sp. EG49 and Nocardiopsis sp. RV163, were grown in co-culture and the presence of induced metabolites monitored by 1H NMR. Ten known compounds, including angucycline, diketopiperazine and β-carboline derivatives 1–10, were isolated from the EtOAc extracts of Actinokineospora sp. EG49 and Nocardiopsis sp. RV163. Co-cultivation of Actinokineospora sp. EG49 and Nocardiopsis sp. RV163 induced the biosynthesis of three natural products that were not detected in the single culture of either microorganism, namely N-(2-hydroxyphenyl)-acetamide (11), 1,6-dihydroxyphenazine (12) and 5a,6,11a,12-tetrahydro-5a,11a-dimethyl[1,4]benzoxazino[3,2-b][1,4]benzoxazine (13a). When tested for biological activity against a range of bacteria and parasites, only the phenazine 12 was active against Bacillus sp. P25, Trypanosoma brucei and interestingly, against Actinokineospora sp. EG49. These findings highlight the co-cultivation approach as an effective strategy to access the bioactive secondary metabolites hidden in the genomes of marine actinomycetes.

Dereplication strategies for targeted isolation of new antitrypanosomal actinosporins A and B from a marine sponge associated-Actinokineospora sp. EG49.

High resolution Fourier transform mass spectrometry (HRFTMS) and nuclear magnetic resonance (NMR) spectroscopy were employed as complementary metabolomic tools to dereplicate the chemical profile of the new and antitrypanosomally active sponge-associated bacterium Actinokineospora sp. EG49 extract. Principal Component (PCA), hierarchical clustering (HCA), and orthogonal partial least square-discriminant analysis (OPLS-DA) were used to evaluate the HRFTMS and NMR data of crude extracts from four different fermentation approaches. Statistical analysis identified the best culture one-strain-many-compounds (OSMAC) condition and extraction procedure, which was used for the isolation of novel bioactive metabolites. As a result, two new O-glycosylated angucyclines, named actinosporins A (1) and B (2), were isolated from the broth culture of Actinokineospora sp. strain EG49, which was cultivated from the Red Sea sponge Spheciospongia vagabunda. The structures of actinosporins A and B were determined by 1D- and 2D-NMR techniques, as well as high resolution tandem mass spectrometry. Testing for antiparasitic properties showed that actinosporin A exhibited activity against Trypanosoma brucei brucei with an IC50 value of 15 µM; however no activity was detected against Leishmania major and Plasmodium falciparum, therefore suggesting its selectivity against the parasite Trypanosoma brucei brucei; the causative agent of sleeping sickness.

Anti-staphylococcal activity of C-methyl flavanones from propolis of Australian stingless bees (Tetragonula carbonaria) and fruit resins of Corymbia torelliana (Myrtaceae).

Propolis of Australian stingless bees (Tetragonula carbonaria, Meliponini) originating from Corymbia torelliana (Myrtaceae) fruit resins was tested for its antimicrobial activities as well as its flavonoid contents. This study aimed at the isolation, structural elucidation and antibacterial testing of flavanones of C. torelliana fruit resins that are incorporated into stingless bee propolis. Flavanones of this study were elucidated by spectroscopic and spectrometric methods including UV, 1D and 2D NMR, EI-MS, ESI-MS and HR-MS. The results indicated known C-methylated flavanones namely, 1 (2S)-cryptostrobin, its regioisomer 2 (2S)- stroboponin, 3 (2S)- cryptostrobin 7-methyl ether, and 6 (2S)- desmethoxymatteucinol, and known flavanones 4 (2S)- pinostrobin and 5 (2S)- pinocembrin as markers for C. torelliana fruit resins and one propolis type. Ethanolic preparations of propolis were shown to be active against Staphylococcus aureus (ATCC 25923) and to a lesser extent against Pseudomonas aeruginosa (ATCC 27853). C. torelliana flavanones inhibited the growth of S. aureus therefore contributing to the antibacterial effects observed for Australian stingless bee propolis extracts.

Cryptocaryols A-H, α-pyrone-containing 1,3-polyols from Cryptocarya sp. implicated in stabilizing the tumor suppressor Pdcd4.

A high-throughput cell-based reporter assay designed to identify small-molecule stabilizers of the tumor suppressor Pdcd4 was used to screen extracts in the NCI Natural Products Repository. Bioassay-guided fractionation of an extract from a Papua New Guinea collection of the tropical tree Cryptocarya sp. provided a series of new 5,6-dihydro-α-pyrone-containing 1,3-polyols (1-8), named cryptocaryols A-H. Their structures were assigned from a combination of NMR, MS, and CD studies in conjunction with NMR database comparisons. Compounds 1-8 were found to rescue Pdcd4 from TPA-induced degradation with EC50 concentrations that ranged from 1.3 to 4.9 μM.

NMR Fingerprints of the Drug‐like Natural‐Product Space Identify Iotrochotazine A: A Chemical Probe to Study Parkinson’s Disease

The NMR spectrum of a mixture of small molecules is a fingerprint of all of its components. Herein, we present an NMR fingerprint method that takes advantage of the fact that fractions contain simplified NMR profiles, with minimal signal overlap, to allow the identification of unique spectral patterns. The approach is exemplified in the identification of a novel natural product, iotrochotazine A (1), sourced from an Australian marine sponge Iotrochota sp. Compound 1 was used as a chemical probe in a phenotypic assay panel based on human olfactory neurosphere-derived cells (hONS) from idiopathic Parkinson’s disease patients. Compound 1 at 1 μm was not cytotoxic but specifically affected the morphology and cellular distribution of lysosomes and early endosomes.

Enantiomeric Discorhabdin Alkaloids and Establishment of Their Absolute Configurations Using Theoretical Calculations of Electronic Circular Dichroism Spectra

Enantiomeric pairs of the cytotoxic pyrroloiminoquinone marine alkaloids discorhabdins B (2), G*/I (3), L (4), and W (5) have been isolated from Latrunculia species sponges collected at different locations around the coast of New Zealand. The absolute configuration of all compounds was secured by comparison of observed data with the results of time-dependent density functional theory (TDDFT) calculations of electronic circular dichroism (ECD) spectra. Enantiomeric discorhabdins exhibit equipotent antiproliferative biological activity.

Potential of marine natural products against drug-resistant fungal, viral, and parasitic infections

Antibiotics have revolutionised medicine in many aspects, and their discovery is considered a turning point in human history. However, the most serious consequence of the use of antibiotics is the concomitant development of resistance against them. The marine environment has proven to be a very rich source of diverse natural products with significant antibacterial, antifungal, antiviral, antiparasitic, antitumour, anti-inflammatory, antioxidant, and immunomodulatory activities. Many marine natural products (MNPs)-for example, neoechinulin B-have been found to be promising drug candidates to alleviate the mortality and morbidity rates caused by drug-resistant infections, and several MNP-based anti-infectives have already entered phase 1, 2, and 3 clinical trials, with six approved for usage by the US Food and Drug Administration and one by the EU. In this Review, we discuss the diversity of marine natural products that have shown in-vivo efficacy or in-vitro potential against drug-resistant infections of fungal, viral, and parasitic origin, and describe their mechanism of action. We highlight the drug-like physicochemical properties of the reported natural products that have bioactivity against drug-resistant pathogens in order to assess their drug potential. Difficulty in isolation and purification procedures, toxicity associated with the active compound, ecological impacts on natural environment, and insufficient investments by pharmaceutical companies are some of the clear reasons behind market failures and a poor pipeline of MNPs available to date. However, the diverse abundance of natural products in the marine environment could serve as a ray of light for the therapy of drug-resistant infections. Development of resistance-resistant antibiotics could be achieved via the coordinated networking of clinicians, microbiologists, natural product chemists, and pharmacologists together with pharmaceutical venture capitalist companies.

Isolation and characterization of diastereomers of discorhabdins H and K and assignment of absolute configuration to discorhabdins D, N, Q, S, T, and U.

Investigations of four different sponge populations of Latrunculia species collected in New Zealand waters has led to the characterization of a new diastereomer of discorhabdin H, named discorhabdin H2, confirmation of the structure of discorhabdin K ((+)-7), and presentation of a new diastereomer, discorhabdin K2 ((-)-8). In each case the structures were established by extensive NMR and MS studies and the absolute configurations interrogated by electronic circular dichroism (ECD). Absolute configurations were assigned to the known metabolites discorhabdins H, D, 2-hydroxy-D, N, and Q by comparison of ECD spectra with those recorded for discorhabdin alkaloids of defined absolute configuration, while the configurations of discorhabdins S, T, and U were assigned by semisynthesis from (+)-(6S,8S)-discorhabdin B.

Two new antioxidant actinosporin analogues from the calcium alginate beads culture of sponge-associated Actinokineospora sp. strain EG49

Marine sponge-associated actinomycetes represent an exciting new resource for the identification of new and novel natural products . Previously, we have reported the isolation and structural elucidation of actinosporins A (1) and B (2) from Actinokineospora sp. strain EG49 isolated from the marine sponge Spheciospongia vagabunda. Herein, by employing different fermentation conditions on the same microorganism, we report on the isolation and antioxidant activity of structurally related metabolites, actinosporins C (3) and D (4). The antioxidant potential of actinosporins C and D was demonstrated using the ferric reducing antioxidant power (FRAP) assay. Additionally, at 1.25 μM, actinosporins C and D showed a significant antioxidant and protective capacity from the genomic damage induced by hydrogen peroxide in the human promyelocytic (HL-60) cell line.