Preclinical evaluation of strasseriolides A-D, potent antiplasmodial macrolides isolated from Strasseria geniculata CF-247,251.

BACKGROUND: Malaria is a global health problem for which novel therapeutic compounds are needed. To this end, a recently published novel family of antiplasmodial macrolides, strasseriolides A-D, was herein subjected to in vivo efficacy studies and preclinical evaluation in order to identify the most promising candidate(s) for further development. METHODS: Preclinical evaluation of strasseriolides A-D was performed by MTT-based cytotoxicity assay in THLE-2 (CRL-2706) liver cells, cardiotoxicity screening using the FluxOR™ potassium assay in hERG expressed HEK cells, LC-MS-based analysis of drug-drug interaction involving CYP3A4, CYP2D6 and CYP2C9 isoforms inhibition and metabolic stability assays in human liver microsomes. Mice in vivo toxicity studies were also accomplished by i.v. administration of the compounds (vehicle: 0.5% HPMC, 0.5% Tween 80, 0.5% Benzyl alcohol) in mice at 25 mg/kg dosage. Plasma were prepared from mice blood samples obtained at different time points (over a 24-h period), and analysed by LC-MS to quantify compounds. The most promising compounds, strasseriolides C and D, were subjected to a preliminary in vivo efficacy study in which transgenic GFP-luciferase expressing Plasmodium berghei strain ANKA-infected Swiss Webster female mice (n = 4-5) were treated 48 h post-infection with an i.p. dosage of strasseriolide C at 50 mg/kg and strasseriolide D at 22 mg/kg for four days after which luciferase activity was quantified on day 5 in an IVIS® Lumina II imager. RESULTS: Strasseriolides A-D showed no cytotoxicity, no carditoxicity and no drug-drug interaction problems in vitro with varying intrinsic clearance (CLint). Only strasseriolide B was highly toxic to mice in vivo (even at 1 mg/kg i.v. dosage) and, therefore, discontinued in further in vivo studies. Strasseriolide D showed statistically significant activity in vivo giving rise to lower parasitaemia levels (70% lower) compared to the controls treated with vehicle. CONCLUSIONS: Animal efficacy and preclinical evaluation of the recently discovered potent antiplasmodial macrolides, strasseriolides A-D, led to the identification of strasseriolide D as the most promising compound for further development. Future studies dealing on structure optimization, formulation and establishment of optimal in vivo dosage explorations of this novel compound class could enhance their clinical potency and allow for progress to later stages of the developmental pipeline.

Diketopiperazines and other bioactive compounds from bacterial symbionts of marine sponges.

Humanity faces great challenges, such as the rise of bacterial antibiotic resistance and cancer incidence. Thus, the discovery of novel therapeutics from underexplored environments, such as marine habitats, is fundamental. In this study, twelve strains from the phylum Firmicutes and thirty-four strains from the phylum Proteobacteria, isolated from marine sponges of the Erylus genus, collected in Portuguese waters, were tested for bioactivities and the secondary metabolites were characterised. Bioactivity screenings comprised antimicrobial, anti-fungal, anti-parasitic and anti-cancer assays. Selected bioactive extracts were further analysed for already described molecules through high performance liquid chromatography and mass spectrometry. Several bioactivities were observed against the fungus Aspergillusfumigatus, the bacteria (methicillin-resistant Staphylococcus aureus and Escherichia coli), the human liver cancer cell line HepG2 and the parasite Trypanosoma cruzi. Medium scale-up volume extracts confirmed anti-fungal activity by strains Proteus mirabilis #118_13 and Proteus sp. (JX006497) strain #118_20. Anti-parasitic activity was also confirmed in Enterococcus faecalis strain #118_3. Moreover, P. mirabilis #118_13 showed bioactivity in human melanoma cell line A2058 and the human hepatocellular carcinoma cell line HepG2. The dereplication of bioactive extracts showed the existence of a variety of secondary metabolites, with some unidentifiable molecules. This work shows that bacterial communities of sponges are indeed good candidates for drug discovery and, as far as we know, we describe anti-parasitic activity of a strain of E. faecalis and the presence of diketopiperazines in Proteus genus for the first time.

Antiparasitic Activity of Bromotyrosine Alkaloids and New Analogues Isolated from the Fijian Marine Sponge Aplysinella rhax.

Ten bromotyrosine alkaloids were isolated and characterised from the marine sponge Aplysinella rhax (de Laubenfels 1954) collected from the Fiji Islands, which included one new bromotyrosine analogue, psammaplin P and two other analogues, psammaplin O and 3-bromo-2-hydroxy-5-(methoxycarbonyl)benzoic acid, which have not been previously reported from natural sources. HR-ESI-MS, 1D and 2D NMR spectroscopic methods were used in the elucidation of the compounds. Bisaprasin, a biphenylic dimer of psammaplin A, showed moderate activity with IC50 at 19±5 and 29±6 µM against Trypanzoma cruzi Tulahuen C4, and the lethal human malaria species Plasmodium falciparum clone 3D7, respectively, while psammaplins A and D exhibited low activity against both parasites. This is the first report of the antimalarial and antitrypanosomal activity of the psammaplin-type compounds. Additionally, the biosynthesis hypotheses of three natural products were proposed.

Signalling and Bioactive Metabolites from Streptomyces sp. RK44.

Streptomyces remains one of the prolific sources of structural diversity, and a reservoir to mine for novel natural products. Continued screening for new Streptomyces strains in our laboratory led to the isolation of Streptomyces sp. RK44 from the underexplored areas of Kintampo waterfalls, Ghana, Africa. Preliminary screening of the metabolites from this strain resulted in the characterization of a new 2-alkyl-4-hydroxymethylfuran carboxamide (AHFA) 1 together with five known compounds, cyclo-(L-Pro-Gly) 2, cyclo-(L-Pro-L-Phe) 3, cyclo-(L-Pro-L-Val) 4, cyclo-(L-Leu-Hyp) 5, and deferoxamine E 6. AHFA 1, a methylenomycin (MMF) homolog, exhibited anti-proliferative activity (EC50 = 89.6 µM) against melanoma A2058 cell lines. This activity, albeit weak is the first report amongst MMFs. Furthermore, the putative biosynthetic gene cluster (ahfa) was identified for the biosynthesis of AHFA 1. DFO-E 6 displayed potent anti-plasmodial activity (IC50 = 1.08µM) against P. falciparum 3D7. High-resolution electrospray ionization mass spectrometry (HR ESIMS) and molecular network assisted the targeted-isolation process, and tentatively identified six AHFA analogues, 7-12 and six siderophores 13-18.

MDN-0185, an Antiplasmodial Polycyclic Xanthone Isolated from Micromonospora sp. CA-256353.

A potent antiplasmodial polycyclic xanthone, MDN-0185 (1), was isolated from an unidentified species of the genus Micromonospora. The planar structure of 1 was established as a seven-ring polycyclic xanthone with partial structures very similar to two known natural products, namely, xantholipin and Sch 54445. Using ROESY correlations, the relative stereochemistry of the two independent stereoclusters of compound 1 could be determined. Mosher analysis and comparison of the specific rotation of compound 1 with that of xantholipin allowed the determination of its absolute configuration. Compound 1 exhibited an IC50 of 9 nM against Plasmodium falciparum 3D7 parasites.

Establishment of a screening platform based on human coronavirus OC43 for the identification of microbial natural products with antiviral activity.

IMPORTANCE: The COVID-19 pandemic has revealed the lack of effective treatments against betacoronaviruses and the urgent need for new broad-spectrum antivirals. Natural products are a valuable source of bioactive compounds with pharmaceutical potential that may lead to the discovery of new antiviral agents. Specifically, compared to conventional synthetic molecules, microbial natural extracts possess a unique and vast chemical diversity and are amenable to large-scale production. The implementation of a high-throughput screening platform using the betacoronavirus OC43 in a human cell line infection model has provided proof of concept of the approach and has allowed for the rapid and efficient evaluation of 1,280 microbial extracts. The identification of several active compounds validates the potential of the platform for the search for new compounds with antiviral capacity.

Antiparasitic Meroterpenoids Isolated from Memnoniella dichroa CF-080171.

Memnoniella is a fungal genus from which a wide range of diverse biologically active compounds have been isolated. A Memnoniella dichroa CF-080171 extract was identified to exhibit potent activity against Plasmodium falciparum 3D7 and Trypanosoma cruzi Tulahuen whole parasites in a high-throughput screening (HTS) campaign of microbial extracts from the Fundación MEDINA's collection. Bioassay-guided isolation of the active metabolites from this extract afforded eight new meroterpenoids of varying potencies, namely, memnobotrins C-E (1-3), a glycosylated isobenzofuranone (4), a tricyclic isobenzofuranone (5), a tetracyclic benzopyrane (6), a tetracyclic isobenzofuranone (7), and a pentacyclic isobenzofuranone (8). The structures of the isolated compounds were established by (+)-ESI-TOF high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Compounds 1, 2, and 4 exhibited potent antiparasitic activity against P. falciparum 3D7 (EC50 0.04-0.243 µM) and T. cruzi Tulahuen (EC50 0.266-1.37 µM) parasites, as well as cytotoxic activity against HepG2 tumoral liver cells (EC50 1.20-4.84 µM). The remaining compounds (3, 5-8) showed moderate or no activity against the above-mentioned parasites and cells.

Antiparasitic Activities of Compounds Isolated from Aspergillus fumigatus Strain Discovered in Northcentral Nigeria.

In this study, we explored a fungal strain UIAU-3F identified as Aspergillus fumigatus isolated from soil samples collected from the River Oyun in Kwara State, Nigeria. In order to explore its chemical diversity, the fungal strain UIAU-3F was cultured in three different fermentation media, which resulted in different chemical profiles, evidenced by LC-ESI-MS-based metabolomics and multivariate analysis. The methanolic extract afforded two known compounds, fumitremorgin C (1) and pseurotin D (2). The in vitro antiparasitic assays of 1 against Trypanosoma cruzi and Plasmodium falciparum showed moderate activity with IC50 values of 9.6 µM and 2.3 µM, respectively, while 2 displayed IC50 values > 50 µM. Molecular docking analysis was performed on major protein targets to better understand the potential mechanism of the antitrypanosomal and antiplasmodial activities of the two known compounds.

Strasseriolides display in vitro and in vivo activity against trypanosomal parasites and cause morphological and size defects in Trypanosoma cruzi.

Neglected diseases caused by kinetoplastid parasites are a health burden in tropical and subtropical countries. The need to create safe and effective medicines to improve treatment remains a priority. Microbial natural products are a source of chemical diversity that provides a valuable approach for identifying new drug candidates. We recently reported the discovery and bioassay-guided isolation of a novel family of macrolides with antiplasmodial activity. The novel family of four potent antimalarial macrolides, strasseriolides A-D, was isolated from cultures of Strasseria geniculata CF-247251, a fungal strain obtained from plant tissues. In the present study, we analyze these strasseriolides for activity against kinetoplastid protozoan parasites, namely, Trypanosoma brucei brucei, Leishmania donovani and Trypanosoma cruzi. Compounds exhibited mostly low activities against T. b. brucei, yet notable growth inhibition and selectivity were observed for strasseriolides C and D in the clinically relevant intracellular T. cruzi and L. donovani amastigotes with EC50 values in the low micromolar range. Compound C is fast-acting and active against both intracellular and trypomastigote forms of T. cruzi. While cell cycle defects were not identified, prominent morphological changes were visualized by differential interference contrast microscopy and smaller and rounded parasites were visualized upon exposure to strasseriolide C. Moreover, compound C lowers parasitaemia in vivo in acute models of infection of Chagas disease. Hence, strasseriolide C is a novel natural product active against different forms of T. cruzi in vitro and in vivo. The study provides an avenue for blocking infection of new cells, a strategy that could additionally contribute to avoid treatment failure.

Pipecolisporin, a Novel Cyclic Peptide with Antimalarial and Antitrypanosome Activities from a Wheat Endophytic Nigrospora oryzae.

A novel cyclic antimalarial and antitrypanosome hexapeptide, pipecolisporin (1), was isolated from cultures of Nigrospora oryzae CF-298113, a fungal endophyte isolated from roots of Triticum sp. collected in a traditional agricultural land of Montefrío, Granada, Spain. The structure of this compound, including its absolute configuration, was elucidated by HRMS, 1-D and 2-D NMR spectroscopy, and Marfey's analysis. This metabolite displayed interesting activity against Plasmodium falciparum and Trypanosoma cruzi, with IC50 values in the micromolar range, and no significant cytotoxicity against the human cancer cell lines A549, A2058, MCF7, MIA PaCa-2, and HepG2.

Cytotoxycity and antiplasmodial activity of phenolic derivatives from Albizia zygia (DC.) J.F. Macbr. (Mimosaceae).

BACKGROUND: The proliferation and resistance of microorganisms area serious threat against humankind and the search for new therapeutics is needed. The present report describes the antiplasmodial and anticancer activities of samples isolated from the methanol extract of Albizia zygia (Mimosaseae). MATERIAL: The plant extract was prepared by maceration in methanol. Standard chromatographic, HPLC and spectroscopic methods were used to isolate and identify six compounds (1-6). The acetylated derivatives (7-10) were prepared by modifying 2-O-ß-D-glucopyranosyl-4-hydroxyphenylacetic acid and quercetin 3-O-α-L-rhamnopyranoside, previously isolated from A. zygia (Mimosaceae). A two-fold serial micro-dilution method was used to determine the IC50s against five tumor cell lines and Plasmodium falciparum. RESULTS: In general, compounds showed moderate activity against the human pancreatic carcinoma cell line MiaPaca-2 (10 < IC50 < 20 µM) and weak activity against other tumor cell lines such as lung (A-549), hepatocarcinoma (HepG2) and human breast adenocarcinoma (MCF-7and A2058) (IC50 > 20 µM). Additionally, the two semi-synthetic derivatives of quercetin 3-O-α-L-rhamnopyranoside exhibited significant activity against P. falciparum with IC50 of 7.47 ± 0.25 µM for compound 9 and 6.77 ± 0.25 µM for compound 10, higher than that of their natural precursor (IC50 25.1 ± 0.25 µM). CONCLUSION: The results of this study clearly suggest that, the appropriate introduction of acetyl groups into some flavonoids could lead to more useful derivatives for the development of an antiplasmodial agent.

Strasseriolides A-D, A Family of Antiplasmodial Macrolides Isolated from the Fungus Strasseria geniculata CF-247251.

A novel family of four potent antimalarial macrolides, strasseriolides A-D (1-4), has been isolated from cultures of Strasseria geniculata CF-247251, a fungal strain obtained from plant tissues. The structures of these compounds, including their absolute configurations, were elucidated by HRMS, NMR spectroscopy, and X-ray single-crystal diffraction. The four compounds gave respective IC50 values of 9.810, 0.013, 0.123, and 0.128 µM against Plasmodium falciparum 3D7 parasites with no significant cytotoxicity against the HepG2 cell line.

Bioactivities and Extract Dereplication of Actinomycetales Isolated From Marine Sponges.

In the beginning of the twenty-first century, humanity faces great challenges regarding diseases and health-related quality of life. A drastic rise in bacterial antibiotic resistance, in the number of cancer patients, in the obesity epidemics and in chronic diseases due to life expectation extension are some of these challenges. The discovery of novel therapeutics is fundamental and it may come from underexplored environments, like marine habitats, and microbial origin. Actinobacteria are well-known as treasure chests for the discovery of novel natural compounds. In this study, eighteen Actinomycetales isolated from marine sponges of three Erylus genera collected in Portuguese waters were tested for bioactivities with the main goal of isolating and characterizing the responsible bioactive metabolites. The screening comprehended antimicrobial, anti-fungal, anti-parasitic, anti-cancer and anti-obesity properties. Fermentations of the selected strains were prepared using ten different culturing media. Several bioactivities against the fungus Aspergillus fumigatus, the bacteria Staphylococcus aureus methicillin-resistant (MRSA) and the human liver cancer cell line HepG2 were obtained in small volume cultures. Screening in higher volumes showed consistent anti-fungal activity by strain Dermacoccus sp. #91-17 and Micrococcus luteus Berg02-26. Gordonia sp. Berg02-22.2 showed anti-parasitic (Trypanosoma cruzi) and anti-cancer activity against several cell lines (melanoma A2058, liver HepG2, colon HT29, breast MCF7 and pancreatic MiaPaca). For the anti-obesity assay, Microbacterium foliorum #91-29 and #91-40 induced lipid reduction on the larvae of zebrafish (Danio rerio). Dereplication of the extracts from several bacteria showed the existence of a variety of secondary metabolites, with some undiscovered molecules. This work showed that Actinomycetales are indeed good candidates for drug discovery.

Antiprotozoan sesterterpenes and triterpenes isolated from two Ghanaian mushrooms.

Bioassay-guided compound isolation led to the discovery of two new scalarane sesterterpenes (1 and 2) and two new triterpenes (3 and 4) from two mushroom species, Pleurotus ostreatus (edible) and Scleroderma areolatum, collected from Ghana. Their structures, including absolute stereochemistry, were established by spectroscopic methods, particularly (+)-ESI-TOF mass spectrometry and 1D and 2D NMR. The four compounds exhibited IC50 values of 1.65-7.63 µM against Plasmodium falciparum 3D7 and 5.04-13.65 µM against Trypanosoma cruzi Tulahuen C4 parasites and were also non-cytotoxic against HepG2 tumoral human liver cells. This is the first report describing the isolation of sesterterpenes belonging to the scalarane structural class from a terrestrial source.

High-Throughput Screening Platform for the Discovery of New Immunomodulator Molecules from Natural Product Extract Libraries.

It is widely accepted that central nervous system inflammation and systemic inflammation play a significant role in the progression of chronic neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, neurotropic viral infections, stroke, paraneoplastic disorders, traumatic brain injury, and multiple sclerosis. Therefore, it seems reasonable to propose that the use of anti-inflammatory drugs might diminish the cumulative effects of inflammation. Indeed, some epidemiological studies suggest that sustained use of anti-inflammatory drugs may prevent or slow down the progression of neurodegenerative diseases. However, the anti-inflammatory drugs and biologics used clinically have the disadvantage of causing side effects and a high cost of treatment. Alternatively, natural products offer great potential for the identification and development of bioactive lead compounds into drugs for treating inflammatory diseases with an improved safety profile. In this work, we present a validated high-throughput screening approach in 96-well plate format for the discovery of new molecules with anti-inflammatory/immunomodulatory activity. The in vitro models are based on the quantitation of nitrite levels in RAW264.7 murine macrophages and interleukin-8 in Caco-2 cells. We have used this platform in a pilot project to screen a subset of 5976 noncytotoxic crude microbial extracts from the MEDINA microbial natural product collection. To our knowledge, this is the first report on an high-throughput screening of microbial natural product extracts for the discovery of immunomodulators.