Stonikacidin A, an Antimicrobial 4-Bromopyrrole Alkaloid Containing L-Idonic Acid Core from the Northwestern Pacific Marine Sponge Lissodendoryx papillosa.

Stonikacidin A (1), the first representative of a new class of 4-bromopyrrole alkaloids containing an aldonic acid core, was isolated from the marine sponge Lissodendoryx papillosa. The compound is named in honor of Prof. Valentin A. Stonik, who is one of the outstanding investigators in the field of marine natural chemistry. The structure of 1 was determined using NMR, MS analysis, and chemical correlations. The L-idonic acid core was established by the comparison of GC, NMR, MS, and optical rotation data of methyl-pentaacetyl-aldonates obtained from the hydrolysis products of 1 and standard hexoses. The L-form of the idonic acid residue in 1 was confirmed by GC analysis of pentaacetate of (S)-2-butyl ester of the hydrolysis product from 1 and compared with corresponding derivatives of L- and D-idonic acids. The biosynthetic pathway for stonikacidin A (1) was proposed. The alkaloid 1 inhibited the growth of Staphylococcus aureus and Escherichia coli test strains, as well as affected the formation of S. aureus and E. coli biofilms. Compound 1 inhibited the activity of sortase A. Molecular docking data showed that stonikacidin A (1) can bind with sortase A due to the interactions between its bromine atoms and some amino acid residues of the enzyme.

Rhizochalinin Exhibits Anticancer Activity and Synergizes with EGFR Inhibitors in Glioblastoma In Vitro Models.

Rhizochalinin (Rhiz) is a recently discovered cytotoxic sphingolipid synthesized from the marine natural compound rhizochalin. Previously, Rhiz demonstrated high in vitro and in vivo efficacy in various cancer models. Here, we report Rhiz to be highly active in human glioblastoma cell lines as well as in patient-derived glioma-stem like neurosphere models. Rhiz counteracted glioblastoma cell proliferation by inducing apoptosis, G2/M-phase cell cycle arrest, and inhibition of autophagy. Proteomic profiling followed by bioinformatic analysis suggested suppression of the Akt pathway as one of the major biological effects of Rhiz. Suppression of Akt as well as IGF-1R and MEK1/2 kinase was confirmed in Rhiz-treated GBM cells. In addition, Rhiz pretreatment resulted in a more pronounced inhibitory effect of γ-irradiation on the growth of patient-derived glioma-spheres, an effect to which the Akt inhibition may also contribute decisively. In contrast, EGFR upregulation, observed in all GBM neurospheres under Rhiz treatment, was postulated to be a possible sign of incipient resistance. In line with this, combinational therapy with EGFR-targeted tyrosine kinase inhibitors synergistically increased the efficacy of Rhiz resulting in dramatic inhibition of GBM cell viability as well as a significant reduction of neurosphere size in the case of combination with lapatinib. Preliminary in vitro data generated using a parallel artificial membrane permeability (PAMPA) assay suggested that Rhiz cannot cross the blood brain barrier and therefore alternative drug delivery methods should be used in the further in vivo studies. In conclusion, Rhiz is a promising new candidate for the treatment of human glioblastoma, which should be further developed in combination with EGFR inhibitors.

Assimiloside A, a Glycolipid with Immunomodulatory Activity from the Northwestern Pacific Marine Sponge Hymeniacidon assimilis.

Assimiloside A (1), an unprecedented marine glycolipid containing a γ-lactone of 4R,16,26R-trihydroxy C28 fatty acid as an aglycon and a trisaccharide carbohydrate moiety, was isolated from the marine sponge Hymeniacidon assimilis. Its structure was elucidated by NMR spectroscopy, mass spectrometry, chemical transformations, and ECD spectroscopy combined with time-dependent density functional theory calculations. Assimiloside A at nontoxic concentrations of 0.01-0.1 µM was shown to present lysosomal activity stimulation and intracellular reactive oxygen species level elevation in RAW 264.7 murine macrophages.

Toporosides A and B, Cyclopentenyl-Containing ω-Glycosylated Fatty Acid Amides, and Toporosides C and D from the Northwestern Pacific Marine Sponge Stelodoryx toporoki.

Toporosides A-D (1-4), new ω-glycosylated fatty acid amides, were isolated from the sponge Stelodoryx toporoki. The structures of these compounds, including absolute configurations of stereogenic centers, were established using analysis of 1D and 2D NMR, ECD, and HR mass spectra as well as chemical transformations. Toporosides A (1) and B (2) are the first lipids containing a cyclopentenyl α,ß-unsaturated carbonyl moiety in the polymethylene chain. Toporoside C (3) is likely a precursor, which undergoes intramolecular aldol condensation to produce 1 and 2. Toporosides A, C, and D showed protective effects against TNF-α-induced injury in H9c2 cardiomyocytes.

New Guanidine Alkaloids Batzelladines O and P from the Marine Sponge Monanchora pulchra Induce Apoptosis and Autophagy in Prostate Cancer Cells.

Two new guanidine alkaloids, batzelladines O (1) and P (2), were isolated from the deep-water marine sponge Monanchora pulchra. The structures of these metabolites were determined by NMR spectroscopy, mass spectrometry, and ECD. The isolated compounds exhibited cytotoxic activity in human prostate cancer cells PC3, PC3-DR, and 22Rv1 at low micromolar concentrations and inhibited colony formation and survival of the cancer cells. Batzelladines O (1) and P (2) induced apoptosis, which was detected by Western blotting as caspase-3 and PARP cleavage. Additionally, induction of pro-survival autophagy indicated as upregulation of LC3B-II and suppression of mTOR was observed in the treated cells. In line with this, the combination with autophagy inhibitor 3-methyladenine synergistically increased the cytotoxic activity of batzelladines O (1) and P (2). Both compounds were equally active in docetaxel-sensitive and docetaxel-resistant prostate cancer cells, despite exhibiting a slight p-glycoprotein substrate-like activity. In combination with docetaxel, an additive effect was observed. In conclusion, the isolated new guanidine alkaloids are promising drug candidates for the treatment of taxane-resistant prostate cancer.

Oceanalin B, a Hybrid α,ω-Bifunctionalized Sphingoid Tetrahydroisoquinoline ß-Glycoside from the Marine Sponge Oceanapia sp.

Oceanalin B (1), an α,ω-bipolar natural product belonging to a rare family of sphingoid tetrahydoisoquinoline ß-glycosides, was isolated from the EtOH extract of the lyophilized marine sponge Oceanapia sp. as the second member of the series after oceanalin A (2) from the same animal. The compounds are of particular interest due to their biogenetically unexpected structures as well as their biological activities. The structure and absolute stereochemistry of 1 as a α,ω-bifunctionalized sphingoid tetrahydroisoquinoline ß-glycoside was elucidated using NMR, CD and MS spectral analysis and chemical degradation. Oceanalin B exhibited in vitro antifungal activity against Candidaglabrata with a MIC of 25 µg/mL.

Application of Oxidative and Reductive Transformations in the Structure Determination of Marine Natural Products.

This review highlights the application of oxidative and reductive chemical transformations in the structure determination of complex marine natural products, including their absolute configurations. Workability of the Baeyer-Villiger reaction, ozonolysis, periodate oxidation, hydrogenolysis, and reductive amination, as well as other related chemical transformations, are discussed.

Gracilosulfates A-G, Monosulfated Polyoxygenated Steroids from the Marine Sponge Haliclona gracilis.

Seven new polyoxygenated steroids belonging to a new structural group of sponge steroids, gracilosulfates A-G (1-7), possessing 3ß-O-sulfonato, 5ß,6ß epoxy (or 5(6)-dehydro), and 4ß,23-dihydroxy substitution patterns as a common structural motif, were isolated from the marine sponge Haliclona gracilis. Their structures were determined by NMR and MS methods. The compounds 1, 2, 4, 6, and 7 inhibited the expression of prostate-specific antigen (PSA) in 22Rv1 tumor cells.

Leptogorgins A-C, Humulane Sesquiterpenoids from the Vietnamese Gorgonian Leptogorgia sp.

Leptogorgins A-C (1-3), new humulane sesquiterpenoids, and leptogorgoid A (4), a new dihydroxyketosteroid, were isolated from the gorgonian Leptogorgia sp. collected from the South China Sea. The structures were established using MS and NMR data. The absolute configuration of 1 was confirmed by a modification of Mosher's method. Configurations of double bonds followed from NMR data, including NOE correlations. This is the first report of humulane-type sesquiterpenoids from marine invertebrates. Sesquiterpenoids leptogorgins A (1) and B (2) exhibited a moderate cytotoxicity and some selectivity against human drug-resistant prostate cancer cells 22Rv1.

Cyclobutastellettolides A and B, C19 Norterpenoids from a Stelletta sp. Marine Sponge.

Two novel C19 terpenoids (1, 2) with an unprecedented carbon skeleton (A) were isolated from a Stelletta sp. sponge collected from Vietnamese waters. Their structures and absolute configurations were established by extensive NMR, MS, and ECD analyses together with quantum chemical modeling and biogenetic considerations. The probable pathways of biogenesis of 1 and 2 from isomalabaricane triterpenoids are discussed. Compounds 1 and 2 significantly increase the production of reactive oxygen species in murine peritoneal macrophages.

Guitarrins A-E and Aluminumguitarrin A: 5-Azaindoles from the Northwestern Pacific Marine Sponge Guitarra fimbriata.

Guitarrins A-E (1-5), the first natural 5-azaindoles, and aluminumguitarrin A (1a), the first aluminum-containing compound from marine invertebrates, were isolated from the sponge Guitarra fimbriata. The structures of these compounds were established using detailed analysis of 1D and 2D NMR data, mass spectra, and X-ray analysis of 1 and 1a. Compound 3 was proved to be a natural inhibitor of alkaline phosphatase.

New Trisulfated Steroids from the Vietnamese Marine Sponge Halichondria vansoesti and Their PSA Expression and Glucose Uptake Inhibitory Activities.

Seven new unusual polysulfated steroids-topsentiasterol sulfate G (1), topsentiasterol sulfate I (2), topsentiasterol sulfate H (3), bromotopsentiasterol sulfate D (4), dichlorotopsentiasterol sulfate D (8), bromochlorotopsentiasterol sulfate D (9), and 4ß-hydroxyhalistanol sulfate C (10), as well as three previously described-topsentiasterol sulfate D (7), chlorotopsentiasterol sulfate D (5) and iodotopsentiasterol sulfate D (6) have been isolated from the marine sponge Halichondria vansoesti. Structures of these compounds were determined by detailed analysis of 1D- and 2D-NMR and HRESIMS data, as well as chemical transformations. The effects of the compounds on human prostate cancer cells PC-3 and 22Rv1 were investigated.

Absolute Configuration of the Cytotoxic Marine Alkaloid Monanchocidin A.

The absolute configuration of the cytotoxic guanidine alkaloid monanchocidin A with 11 stereogenic centers from the marine sponge Monanchora pulchra was determined as 5 R, 8 S, 10 S, 13 R, 14 S, 15 R, 19 R, 23 R, 37 S, 42 S, 43 R after extensive reductive degradation and conversion of the resulting alcohols to MTPA derivatives.

Melonoside B and Melonosins A and B, Lipids Containing Multifunctionalized ω-Hydroxy Fatty Acid Amides from the Far Eastern Marine Sponge Melonanchora kobjakovae.

Melonoside B (1) and melonosins B (2) and A (3), new lipids based on polyoxygenated fatty acid amides, and known melonoside A (4) were isolated from two different collections of the marine sponge Melonanchora kobjakovae. The structures of these compounds, including their absolute configurations, were established using detailed analysis of 1D and 2D NMR, ECD, and mass spectra as well as chemical transformations. Melonosins 2 and 3 inhibit AP-1- and NF-kB-dependent transcriptional activities in JB6 Cl41 cells at noncytotoxic concentrations, demonstrating potential cancer preventive activity.

Proteomic-based investigations on the mode of action of the marine anticancer compound rhizochalinin.

Rhizochalinin (Rhiz) is a novel marine natural sphingolipid-like compound, which shows promising in vitro and in vivo activity in human castration-resistant prostate cancer. In the present study, a global proteome screening approach was applied to investigate molecular targets and biological processes affected by Rhiz in castration-resistant prostate cancer. Bioinformatical analysis of the data predicted an antimigratory effect of Rhiz on cancer cells. Validation of proteins involved in the cancer-associated processes, including cell migration and invasion, revealed downregulation of specific isoforms of stathmin and LASP1, as well as upregulation of Grp75, keratin 81, and precursor IL-1ß by Rhiz. Functional analyses confirmed an antimigratory effect of Rhiz in PC-3 cells. Additionally, predicted ERK1/2 activation was confirmed by Western blotting analysis, and revealed prosurvival effects in Rhiz-treated prostate cancer cells indicating a potential mechanism of resistance. A combination of Rhiz with MEK/ERK inhibitors PD98059 (non-ATP competitive MEK1 inhibitor) and FR180204 (ATP-competitive ERK1/2 inhibitor) resulted in synergistic effects. This work provides further insights into the molecular mechanisms underlying Rhiz bioactivity. Furthermore, our research is exemplary for the ability of proteomics to predict drug targets and mode of action of natural anticancer agents.

Anti-migratory activity of marine alkaloid monanchocidin A - proteomics-based discovery and confirmation.

Monanchocidin A (MonA) is a novel marine alkaloid with promising anti-cancer properties. We recently demonstrated its high efficacy in human urogenital cancers including germ cell tumors. Here, we applied a global proteome screening approach to investigate molecular targets and biological processes affected by MonA in the human cisplatin-resistant germ cell cancer cell line NCCIT-R. Bioinformatical analysis of the proteomics data predicted an effect of MonA on cancer cell migration. Thus, proteins known to be involved in cancer cell migration and invasion were chosen for further validation. The protein alterations identified by proteomics resulted from both, regulation of the total protein expression and post-transcriptional modifications. Among others, regulation of an isoform of vimentin, up-regulation of multiple apolipoprotein E isoforms, and inhibition of hypusination of eukaryotic translation initiation factor 5A-1 were found upon treatment with MonA. Further functional analyses were performed and revealed decreased cell migration and colony formation of cancer cells treated with MonA at non-cytotoxic and non-antiproliferative concentrations. This work provides further insights into the molecular mechanisms behind MonA bioactivity. Furthermore, our research is exemplary for the ability of proteomics to predict drug targets and mode of action of natural anti-cancer agents.

Guanidine Alkaloids from the Marine Sponge Monanchora pulchra Show Cytotoxic Properties and Prevent EGF-Induced Neoplastic Transformation in Vitro.

Guanidine alkaloids from sponges Monanchora spp. represent diverse bioactive compounds, however, the mechanisms underlying bioactivity are very poorly understood. Here, we report results of studies on cytotoxic action, the ability to inhibit EGF-induced neoplastic transformation, and the effects on MAPK/AP-1 signaling of eight rare guanidine alkaloids, recently isolated from the marine sponge Monanchora pulchra, namely: monanchocidin A (1), monanchocidin B (2), monanchomycalin C (3), ptilomycalin A (4), monanchomycalin B (5), normonanchocidin D (6), urupocidin A (7), and pulchranin A (8). All of the compounds induced cell cycle arrest (apart from 8) and programmed death of cancer cells. Ptilomycalin A-like compounds 1-6 activated JNK1/2 and ERK1/2, following AP-1 activation and caused p53-independent programmed cell death. Compound 7 induced p53-independent cell death without activation of AP-1 or caspase-3/7, and the observed JNK1/2 activation did not contribute to the cytotoxic effect of the compound. Alkaloid 8 induced JNK1/2 (but not ERK1/2) activation leading to p53-independent cell death and strong suppression of AP-1 activity. Alkaloids 1-4, 7, and 8 were able to inhibit the EGF-induced neoplastic transformation of JB6 P⁺ Cl41 cells. Our results suggest that investigated guanidine marine alkaloids hold potential to eliminate human cancer cells and prevent cancer cell formation and spreading.

Pyridine Nucleosides Neopetrosides A and B from a Marine Neopetrosia sp. Sponge. Synthesis of Neopetroside A and Its ß-Riboside Analogue.

Neopetrosides A (1) and B (2), new naturally occurring ribosides of nicotinic acid with extremely rare α-N-glycoside linkages and residues of p-hydroxybenzoic and pyrrole-2-carboxylic acids attached to C-5', were isolated from a marine Neopetrosia sp. sponge. Structures 1 and 2 were determined by NMR and MS methods and confirmed by the synthesis of 1 and its ß-riboside analogue (3). Neopetroside A (1) upregulates mitochondrial functions in cardiomyocytes.

6-bromohypaphorine from marine nudibranch mollusk Hermissenda crassicornis is an agonist of human α7 nicotinic acetylcholine receptor.

6-Bromohypaphorine (6-BHP) has been isolated from the marine sponges Pachymatisma johnstoni, Aplysina sp., and the tunicate Aplidium conicum, but data on its biological activity were not available. For the nudibranch mollusk Hermissenda crassicornis no endogenous compounds were known, and here we describe the isolation of 6-BHP from this mollusk and its effects on different nicotinic acetylcholine receptors (nAChR). Two-electrode voltage-clamp experiments on the chimeric α7 nAChR (built of chicken α7 ligand-binding and glycine receptor transmembrane domains) or on rat α4ß2 nAChR expressed in Xenopus oocytes revealed no action of 6-BHP. However, in radioligand analysis, 6-BHP competed with radioiodinated α-bungarotoxin for binding to human α7 nAChR expressed in GH4C1 cells (IC50 23 ± 1 µM), but showed no competition on muscle-type nAChR from Torpedo californica. In Ca2+-imaging experiments on the human α7 nAChR expressed in the Neuro2a cells, 6-BHP in the presence of PNU120596 behaved as an agonist (EC50 ~80 µM). To the best of our knowledge, 6-BHP is the first low-molecular weight compound from marine source which is an agonist of the nAChR subtype. This may have physiological importance because H. crassicornis, with its simple and tractable nervous system, is a convenient model system for studying the learning and memory processes.

Molecular Cloning, Heterologous Expression, Purification, and Evaluation of Protein-Ligand Interactions of CYP51 of Candida krusei Azole-Resistant Fungal Strain.

Due to the increasing prevalence of fungal diseases caused by fungi of the genus Candida and the development of pathogen resistance to available drugs, the need to find new effective antifungal agents has increased. Azole antifungals, which are inhibitors of sterol-14α-demethylase or CYP51, have been widely used in the treatment of fungal infections over the past two decades. Of special interest is the study of C. krusei CYP51, since this fungus exhibit resistance not only to azoles, but also to other antifungal drugs and there is no available information about the ligand-binding properties of CYP51 of this pathogen. We expressed recombinant C. krusei CYP51 in E. coli cells and obtained a highly purified protein. Application of the method of spectrophotometric titration allowed us to study the interaction of C. krusei CYP51 with various ligands. In the present work, the interaction of C. krusei CYP51 with azole inhibitors, and natural and synthesized steroid derivatives was evaluated. The obtained data indicate that the resistance of C. krusei to azoles is not due to the structural features of CYP51 of this microorganism, but rather to another mechanism. Promising ligands that demonstrated sufficiently strong binding in the micromolar range to C. krusei CYP51 were identified, including compounds 99 (Kd = 1.02 ± 0.14 µM) and Ch-4 (Kd = 6.95 ± 0.80 µM). The revealed structural features of the interaction of ligands with the active site of C. krusei CYP51 can be taken into account in the further development of new selective modulators of the activity of this enzyme.