National Committee on Marine Sciences (NCMS)
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- Genomic and targeted approaches unveil the cell membrane as a major target of the antifungal cytotoxin amantelide AElsadek, Lobna A.; Matthews, James H.; Nishimura, Shinichi; Nakatani, Takahiro; Ito, Airi; Gu, Tongjun; Luo, Danmeng; Salvador-Reyes, Lilibeth A.; Paul, Valerie J.; Kakeya, Hideaki; Luesch, Hendrik (Wiley, 2021-03-23)Amantelide A, a polyhydroxylated macrolide isolated from a marine cyanobacterium, displays broad-spectrum activity against mammalian cells, bacterial pathogens, and marine fungi. We conducted comprehensive mechanistic studies to identify the molecular targets and pathways affected by amantelide A. Our investigations relied on chemical structure similarities with compounds of known mechanisms, yeast knockout mutants, yeast chemogenomic profiling, and direct biochemical and biophysical methods. We established that amantelide A exerts its antifungal action by binding to ergosterol-containing membranes followed by pore formation and cell death, a mechanism partially shared with polyene antifungals. Binding assays demonstrated that amantelide A also binds to membranes containing epicholesterol or mammalian cholesterol, thus suggesting that the cytotoxicity to mammalian cells might be due to its affinity to cholesterol-containing membranes. However, membrane interactions were not completely dependent on sterols. Yeast chemogenomic profiling suggested additional direct or indirect effects on actin. Accordingly, we performed actin polymerization assays, which suggested that amantelide A also promotes actin polymerization in cell-free systems. However, the C-33 acetoxy derivative amantelide B showed a similar effect on actin dynamics in vitro but no significant activity against yeast. Overall, these studies suggest that the membrane effects are the most functionally relevant for amantelide A mechanism of action.This research was supported by the National Institutes of Health (grant R01CA172310 to H.L.), the Debbie and Sylvia DeSantis Chair Professorship (H.L), and a Grant-in Aid for Scientific Research (no. 17H06401 to S.N. and H.K.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. L.E. was supported by the NIH/NIGMS T32GM136583 “Chemistry-Biology Interface Training Program at the University of Florida”. We thank Dr. Yanping Zhang (University of Florida) from the UF ICBR NextGen DNA Sequencing core facility for carrying out the nextgeneration sequencing, Dr. Maya Schuldiner (Weizmann Institute of Science, Israel) for providing the triple yeast deletion library, Dr. Kaoru Takegawa (Kyushu University, Japan) for providing the ergosterol mutants, Dr. Charles Boone laboratory (University of Toronto, Canada) for providing the wild-type S. cerevisiae Y7092 and Dr. Kalina Atanasova (CNPD3, University of Florida) for assistance with the imaging.
- Anti-inflammatory activity of monosubstituted xestoquinone analogues from the marine sponge Neopetrosia compactaSusana, Shalice R.; Salvador-Reyes, Lilibeth A. (MDPI, 2022-03-22)Chronic inflammation is recognized as a contributor to multiple chronic diseases, such as cancer, cardiovascular, and autoimmune disorders. Here, a natural products-initiated discovery of anti-inflammatory agents from marine sponges was undertaken. From the screening of 231 crude extracts, a total of 30 extracts showed anti-inflammatory activity with no direct cytotoxic effects at 50 μg/mL on RAW 264.7 (ATCC®TIB-71™) murine macrophage cells stimulated with 1 μg/mL lipopolysaccharide (LPS). Bioactivity-guided purification of the anti-inflammatory extract from the sponge Neopetrosia compacta led to the isolation of xestoquinone (1), adociaquinone B (2), adociaquinone A (3), 14-hydroxymethylxestoquinone (4), 15-hydroxymethylxestoquinone (5), and an inseparable 2:1 mixture of 14-methoxyxestoquinone and 15-methoxyxestoquinone (6). Compounds 1–6 caused a concentration-dependent reduction of nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells, with 4–6 having low micromolar IC50 and acceptable selectivity index. Gene expression analysis using qRT-PCR showed that 1, 5, and 6 downregulated Il1b and Nos2 expression by 2.1- to 14.8-fold relative to the solvent control at 10 μM. Xestoquinone (1) and monosubstituted analogues (4–6), but not the disubstituted adociaquinones (2 and 3), caused Nrf2 activation in a luciferase reporter MCF7 stable cells. Compounds 5 and 6 caused a modest increase in Nqo1 gene expression at 10 μM. The anti-inflammatory activity of xestoquinone (1) and monosubstituted analogues (4–6) may, in part, be mediated by Nrf2 activation, leading to attenuation of inflammatory mediators such as IL-1β and NOS2.Samples were collected under gratuitous permit numbers GP-0084-15 and GP-0123-17, issued by the Department of Agriculture of the Philippines. We thank the municipalities of Bolinao, Pangasinan, and Puerto Galera, Oriental Mindoro for permission for sample collection. We acknowledge assistance from Z. L. Malto and DDHP chemical ecology group in obtaining the mass spectrometric data and sample collection, respectively.
- Global mass spectrometric analysis reveals chemical diversity of secondary metabolites and 44-Methylgambierone production in Philippine Gambierdiscus strainsMalto, Zabrina Bernice L.; Benico, Garry A.; Batucan, Jeremiah D.; Dela Cruz, James; Romero, Marc Lawrence J.; Azanza, Rhodora V.; Salvador-Reyes, Lilibeth A. (Frontiers Media SA, 2022-02-04)Surveillance and characterization of emerging marine toxins and toxigenic dinoflagellates are warranted to evaluate their associated health risks. Here, we report the occurrence of the ciguatera poisoning-causative dinoflagellate Gambierdiscus balechii in the Philippines. Toxin production and chemical diversity of secondary metabolites in G. balechii GtoxSAM092414, G. balechii Gtox112513, and the recently reported Gambierdiscus carpenteri Gam1BOL080513 were assessed using targeted and untargeted UPLC-MS/MS analysis and radioligand receptor-binding assay (RBA). 44-methylgambierone was produced by all three strains, albeitwith different levels based on RBA and UPLC-HRMS/MS analysis. The fatty acid composition was similar in all strains, while subtle differences in monosaccharide content were observed, related to the collection site rather than the species. Molecular networking using the GNPS database identified 45 clusters belonging to at least ten compound classes, with terpene glycosides, carbohydrate conjugates, polyketides, and macrolides as major convergence points. Species-specific peptides and polyhydroxylated compounds were identified in G. balechii GtoxSAM092414 and G. carpenteri Gam1BOL080513, respectively. These provide a glimpse of the uncharacterized biosynthetic potential of benthic dinoflagellates and highlight the intricate and prolific machinery for secondary metabolites production in these organisms.We would like to thank H. Junio and the Secondary Metabolites Profiling Laboratory of the Institute of Chemistry, University of the Philippines Diliman and K. B. Davis for assistance in the conduct of this study.
- Modified oxylipins as inhibitors of biofilm formation in Staphylococcus epidermidisPeran, Jacquelyn E.; Salvador-Reyes, Lilibeth A. (Frontiers Media SA, 2024-05-23)New approaches to combating microbial drug resistance are being sought, with the discovery of biofilm inhibitors considered as alternative arsenal for treating infections. Natural products have been at the forefront of antimicrobial discovery and serve as inspiration for the design of new antibiotics. We probed the potency, selectivity, and mechanism of anti-biofilm activity of modified oxylipins inspired by the marine natural product turneroic acid. Structure-activity relationship (SAR) evaluation revealed the importance of the trans-epoxide moiety, regardless of the position, for inhibiting biofilm formation. trans-12,13-epoxyoctadecanoic acid (1) and trans-9,10 epoxyoctadecanoic acid (4) selectively target the early stage of biofilm formation, with no effect on planktonic cells. These compounds interrupt the formation of a protective polysaccharide barrier by significantly upregulating the ica operon’s transcriptional repressor. This was corroborated by docking experiment with SarA and scanning electron micrographs showing reduced biofilm aggregates and the absence of thread-like structures of extrapolymeric substances. In silico evaluation revealed that 1 and 4 can interfere with the AgrA-mediated communication language in Staphylococci, typical to the diffusible signal factor (DSF) capacity of lipophilic chains.
- Genomics and metabolomics-based assessment of the biosynthetic potential of the sponge-associated microorganism Streptomyces cacaoi strain R2A-843A from the PhilippinesMalto, Zabrina Bernice L.; Reyes, Joeriggo M.; Lo, Bernard Isaiah; Davis, Kevin Bossie S.; Concepcion, Gisela; Salvador-Reyes, Lilibeth A. (Philippine-American Academy of Science and Engineering, 2023-10-20)The biosynthetic machinery of the sponge-associated Streptomyces cacaoi strain R2A-843A was assessed using a combined genomics and metabolomics approach. Whole genome sequencing and molecular networking showed the high biosynthetic potential of this actinomycete. A significant proportion of the genome is dedicated to secondary metabolite production, with biosynthetic gene clusters for nonribosomal peptides, polyketides, and terpenes being the most represented. Seven cyclic pentapeptides, including a putative new analogue, and a glycosylated lanthipeptide were identified using HRMS and untargeted MS/MS analysis. To validate our genome and metabolome analysis, we undertook a mass spectrometry-guided purification and confirmed the production of the known peptides BE-18257A (1) and BE-18257B (2). The production of 1 and 2 and the growth of the microorganism were monitored for eight days. Compound 2 was produced at a higher concentration, starting at 48 h post-incubation. Both compounds were noncytotoxic against colorectal and breast cancer cell lines.The authors acknowledge funding support from the Department of Science and Technology - Philippine Council for Health Research and Development through the Discovery and Development of Health Products - Marine Component Program. Genome sequencing was made possible through the CHEDPCARI IHITM63 Project. We thank Ms. Shalice R. SusanaGuevarra for conducting the bioactivity assay. This work was done under the supervision of the Bureau of Fisheries and Aquatic Resources under Gratuitous Permit No. FBP-0035-10. This is MSI Contribution No. 501.