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Peter M Blumberg - One of the best experts on this subject based on the ideXlab platform.
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Neristatin 1 Provides Critical Insight into Bryostatin 1 Structure− Function Relationships
2016Co-Authors: Peter M BlumbergAbstract:ABSTRACT: Bryostatin 1, a complex macrocyclic lactone isolated from Bugula neritina, has been the subject of multiple clinical trials for cancer. Although it functions as an activator of protein kinase C (PKC) in vitro, Bryostatin 1 paradoxically antagonizes most responses to the prototypical PKC activator, the phorbol esters. The bottom half of the Bryostatin 1 structure has been shown to be sufficient to confer binding to PKC. In contrast, we have previously shown that the top half of the Bryostatin 1 structure is necessary for its unique biological behavior to antagonize phorbol ester responses. Neristatin 1 comprises a top half similar to that of Bryostatin 1 together with a distinct bottom half that confers PKC binding. We report here that neristatin 1 is Bryostatin 1-like, not phorbol ester-like, in its biological activity on U937 promyelocytic leukemia cells. We conclude that the top half of the Bryostatin 1 structure is largely sufficient for Bryostatin 1-like activity, provided the molecule also possesses an appropriate PKC binding domain
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Biological activity of the Bryostatin analog Merle 23 on mouse epidermal cells and mouse skin.
Molecular Carcinogenesis, 2016Co-Authors: Jessica S. Kelsey, Christophe Cataisson, David O. Baumann, Mark E. Petersen, Jin Qiu Chen, Michelle A Herrmann, Kevin M. Mcgowan, Gary E Keck, Stuart H Yuspa, Peter M BlumbergAbstract:: Bryostatin 1, a complex macrocyclic lactone, is the subject of multiple clinical trials for cancer chemotherapy. Although Bryostatin 1 biochemically functions like the classic mouse skin tumor promoter phorbol 12-myristate 13-acetate (PMA) to bind to and activate protein kinase C, paradoxically, it fails to induce many of the typical phorbol ester responses, including tumor promotion. Intense synthetic efforts are currently underway to develop simplified Bryostatin analogs that preserve the critical functional features of Bryostatin 1, including its lack of tumor promoting activity. The degree to which Bryostatin analogs maintain the unique pattern of biological behavior of Bryostatin 1 depends on the specific cellular system and the specific response. Merle 23 is a significantly simplified Bryostatin analog that retains Bryostatin like activity only to a limited extent. Here, we show that in mouse epidermal cells the activity of Merle 23 was either similar to Bryostatin 1 or intermediate between Bryostatin 1 and PMA, depending on the specific parameter examined. We then examined the hyperplastic and tumor promoting activity of Merle 23 on mouse skin. Merle 23 showed substantially reduced hyperplasia and was not tumor promoting at a dose comparable to that for PMA. These results suggest that there may be substantial flexibility in the design of Bryostatin analogs that retain its lack of tumor promoting activity. © 2016 Wiley Periodicals, Inc.
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Neristatin 1 provides critical insight into Bryostatin 1 structure-function relationships.
Journal of Natural Products, 2015Co-Authors: Noemi Kedei, Matthew B. Kraft, Noeleen Melody, Gary E Keck, Cherry L Herald, George R Pettit, Peter M BlumbergAbstract:Bryostatin 1, a complex macrocyclic lactone isolated from Bugula neritina, has been the subject of multiple clinical trials for cancer. Although it functions as an activator of protein kinase C (PKC) in vitro, Bryostatin 1 paradoxically antagonizes most responses to the prototypical PKC activator, the phorbol esters. The bottom half of the Bryostatin 1 structure has been shown to be sufficient to confer binding to PKC. In contrast, we have previously shown that the top half of the Bryostatin 1 structure is necessary for its unique biological behavior to antagonize phorbol ester responses. Neristatin 1 comprises a top half similar to that of Bryostatin 1 together with a distinct bottom half that confers PKC binding. We report here that neristatin 1 is Bryostatin 1-like, not phorbol ester-like, in its biological activity on U937 promyelocytic leukemia cells. We conclude that the top half of the Bryostatin 1 structure is largely sufficient for Bryostatin 1-like activity, provided the molecule also possesses ...
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abstract 1642 Bryostatin 1 and the simplified analog merle 23 have similar and opposing properties on mouse epidermal cells and mouse skin
Cancer Research, 2014Co-Authors: Jessica S. Kelsey, Noemi Kedei, Christophe Cataisson, Mark E. Petersen, Gary E Keck, Stuart H Yuspa, Peter M BlumbergAbstract:Protein kinase C (PKC) is differentially regulated in a range of cancers and has become an attractive therapeutic target. A number of natural compounds exist that have been found to regulate PKCs, and the biological responses of each compound can vary. The PKC activator PMA is an established tumor promoter, while others, such as the Bryostatins, are non-tumor promoting and have inhibitory effects on the PKC pathway. Because of its effects on the PKC pathway, Bryostatin 1 is currently in clinical trials as an anti-cancer agent. Limited availability and the difficulty involved in synthesizing Bryostatin 1 make its use as an anti-cancer agent less attractive. Merle 23, a simplified synthetic analog of Bryostatin 1, could be an alternative to Bryostatin 1 if it acted similarly. Initial investigations of Merle 23 have revealed biological responses both similar to Bryostatin 1 and similar to PMA, depending on the system. In U937 leukemia cells Merle 23 acts similar to PMA, and in the LNCaP prostate cancer cell line it behaves more like Bryostatin 1. Its behavior in mouse skin is of particular interest, since this is the system in which the tumor promoting activity of PMA and the anti-tumor promoting activity of Bryostatin 1 have been characterized. The activity of Merle 23 was compared to PMA and Bryostatin 1 in mouse epidermal cells and mouse skin. In mouse primary keratinocytes PMA produces a prolonged morphological response, while the morphological response to Bryostatin 1 is transient. The response to Merle 23 is intermediate, but more similar to Bryostatin 1. When applied simultaneously, Merle 23, like Bryostatin 1, is able to protect the cells from the morphological response induced by PMA. In the PKC pathway, all three compounds promote similar levels of initial PKCδ activation and ERK1/2 phosphorylation. By 24 hrs, however, the responses differ. At high concentrations, Bryostatin 1 protects PKCδ from down regulation whereas Merle 23 does not. Bryostatin 1 is also much more potent for down regulation of PKCα, whereas Merle 23 and PMA are similar. The inflammatory response was measured by monitoring the transcript levels of various genes involved in inflammation. PMA induced the largest increase in transcript levels, while Merle 23 was more similar to Bryostatin 1. Short-term in vivo analysis reveals no epidermal thickening in response to low doses of Merle 23 whereas the corresponding dose of PMA induced significant hyperplasia. The in vivo analysis of Merle 23 is ongoing. The results presented suggest that Merle 23 behaves more similarly to Bryostatin 1 in the mouse epidermal cells at the level of biological response, raising the possibility that it may likewise be non-promoting. The results emphasize that the structure activity relations responsible for the distinct behavior between PMA and Bryostatin 1 are distinct for different cellular systems and different biological endpoints. Citation Format: Jessica Kelsey, Noemi Kedei, Christophe Cataisson, Mark Petersen, Stuart Yuspa, Gary Keck, Peter Blumberg. Bryostatin 1 and the simplified analog Merle 23 have similar and opposing properties on mouse epidermal cells and mouse skin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1642. doi:10.1158/1538-7445.AM2014-1642
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Abstract 2454: Comparison of the activity of the Bryostatin derivative Merle 23 with that of Bryostatin 1 and phorbol ester in mouse epidermal cells.
Cancer Research, 2013Co-Authors: Jessica S. Kelsey, Noemi Kedei, Christophe Cataisson, Mark E. Petersen, Gary E Keck, Stuart H Yuspa, Peter M BlumbergAbstract:Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Tumor promoting phorbol esters, such as PMA, are potent activators of the PKC pathway. The Bryostatins, marine natural products, activate PKCs similarly to phorbol esters but paradoxically inhibit most phorbol ester responses, including skin tumor promotion. Because of this inhibitory activity on the PKC pathway, Bryostatin 1 has been the subject of extensive clinical trials. Serious impediments to clinical development, however, have been the scarcity of natural supplies coupled with great structural complexity, making synthesis problematic. Recent synthetic advances are now yielding simplified structural derivatives. Here, we compare the behavior of the Bryostatin derivative Merle 23 with that of Bryostatin 1 and phorbol 12-myristate 13-acetate (PMA) in mouse epidermal cells. While having similar binding activity on PKC as does Bryostatin 1, Merle 23 behaves like PMA rather than like Bryostatin 1 in U937 human leukemia cells but conversely behaves more like Bryostatin 1 in the human prostate cancer cell line LNCaP. As a first step in assessing whether Merle 23 possesses or lacks skin tumor promoting activity, its activity in mouse primary keratinocyte cultures was assessed. PMA induces a prominent, prolonged morphological response in keratinocytes. Bryostatin 1 induces a similar initial response, which is however very transient. The morphological response to Merle 23 is more prolonged than for Bryostatin 1 but much shorter than for PMA. For initial activation of PKCδ or induction of ERK1/2 phosphorylation, all three ligands show similar activity. By 24 hrs, however, Bryostatin 1 is much more potent for down regulation of PKCα, whereas Merle 23 and PMA are similar. For PKCδ, Bryostatin 1 at higher concentrations protects PKCδ from down regulation whereas Merle 23 does not. Transcriptional responses of various inflammation genes were monitored by qPCR. PMA causes the greatest increase in transcript levels for all genes investigated. In many cases Bryostatin 1 induces a smaller response and the response to Merle 23 generally resembles that of Bryostatin 1. While these studies are ongoing, they suggest that Merle 23 largely behaves like Bryostatin 1 in the mouse epidermal cells. Our findings emphasize the important role of cellular context in determining the pattern of behavior of Merle 23. An important implication is that different cellular environments reveal different structure activity relations for Bryostatin 1-like behavior. Finally, our studies raise the possibility that Merle 23, which is significantly simplified relative to Bryostatin 1 in its upper two rings, may be non-promoting, at least in the mouse skin tumor promotion system. Citation Format: Jessica Kelsey, Noemi Kedei, Christophe Cataisson, Mark Petersen, Stuart Yuspa, Gary Keck, Peter Blumberg. Comparison of the activity of the Bryostatin derivative Merle 23 with that of Bryostatin 1 and phorbol ester in mouse epidermal cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2454. doi:10.1158/1538-7445.AM2013-2454
George R Pettit - One of the best experts on this subject based on the ideXlab platform.
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Neristatin 1 provides critical insight into Bryostatin 1 structure-function relationships.
Journal of Natural Products, 2015Co-Authors: Noemi Kedei, Matthew B. Kraft, Noeleen Melody, Gary E Keck, Cherry L Herald, George R Pettit, Peter M BlumbergAbstract:Bryostatin 1, a complex macrocyclic lactone isolated from Bugula neritina, has been the subject of multiple clinical trials for cancer. Although it functions as an activator of protein kinase C (PKC) in vitro, Bryostatin 1 paradoxically antagonizes most responses to the prototypical PKC activator, the phorbol esters. The bottom half of the Bryostatin 1 structure has been shown to be sufficient to confer binding to PKC. In contrast, we have previously shown that the top half of the Bryostatin 1 structure is necessary for its unique biological behavior to antagonize phorbol ester responses. Neristatin 1 comprises a top half similar to that of Bryostatin 1 together with a distinct bottom half that confers PKC binding. We report here that neristatin 1 is Bryostatin 1-like, not phorbol ester-like, in its biological activity on U937 promyelocytic leukemia cells. We conclude that the top half of the Bryostatin 1 structure is largely sufficient for Bryostatin 1-like activity, provided the molecule also possesses ...
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chapter 12 biosynthetic products for anticancer drug design and treatment the Bryostatins
Anticancer Drug Development, 2002Co-Authors: George R Pettit, Cherry L Herald, Fiona HoganAbstract:The chapter focuses on the use of Bryostatins for anticancer drug design and treatment. The field of anticancer drug discovery based on natural products is vast and covers many areas, including isolation, chemical identification, and synthesis, preclinical evaluation, both in vitro and in vivo, and clinical trials. The U.S. National Cancer Institute (NCI) research programs directed at discovery of new and clinically useful animal, plant, and microorganism anticancer constituents were implemented in September 1957, and quickly demonstrated that 2–4% of plant specimens produce a great variety of anticancer agents. Because of this vitally important NCI endeavor, new antineoplastic and/or cytotoxic biosynthetic products are now being discovered worldwide at an increasing rate, and the potential for discovery of new animal, plant, and microorganism biosynthetic products for treatment of human cancer is truly extraordinary, and offers great promise of many curative approaches to the cancer problem. The experiments conducted by Dr. Blumberg in the NCI showed that the potent anticancer activity of Bryostatin and Bryostatins was primarily based on modulation of protein kinase C (PKC). Compared to various PKC interactive substances, Bryostatin has proved to be a very potent antitumor-promoting agent, and a very promising anticancer drug. Bryostatin was also found to be an immune stimulant. Bryostatin was also found to stimulate the normal production of interleukin-2 (IL-2) and interferon, and it has provided curative levels of activity against a variety of murine experimental cancer systems.
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the design computer modeling solution structure and biological evaluation of synthetic analogs of Bryostatin 1
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Paul A Wender, Jef Debrabander, Patrick G Harran, Juan Miguel Jimenez, Michael F T Koehler, Blaise Lippa, Cheolmin Park, Carsten Siedenbiedel, George R PettitAbstract:The Bryostatins are a unique family of emerging cancer chemotherapeutic candidates isolated from marine bryozoa. Although the biochemical basis for their therapeutic activity is not known, these macrolactones exhibit high affinities for protein kinase C (PKC) isozymes, compete for the phorbol ester binding site on PKC, and stimulate kinase activity in vitro and in vivo. Unlike the phorbol esters, they are not first-stage tumor promoters. The design, computer modeling, NMR solution structure, PKC binding, and functional assays of a unique class of synthetic Bryostatin analogs are described. These analogs (7b, 7c, and 8) retain the putative recognition domain of the Bryostatins but are simplified through deletions and modifications in the C4-C14 spacer domain. Computer modeling of an analog prototype (7a) indicates that it exists preferentially in two distinct conformational classes, one in close agreement with the crystal structure of Bryostatin 1. The solution structure of synthetic analog 7c was determined by NMR spectroscopy and found to be very similar to the previously reported structures of Bryostatins 1 and 10. Analogs 7b, 7c, and 8 bound strongly to PKC isozymes with Ki = 297, 3.4, and 8.3 nM, respectively. Control 7d, like the corresponding Bryostatin derivative, exhibited weak PKC affinity, as did the derivative, 9, lacking the spacer domain. Like Bryostatin, acetal 7c exhibited significant levels of in vitro growth inhibitory activity (1.8–170 ng/ml) against several human cancer cell lines, providing an important step toward the development of simplified, synthetically accessible analogs of the Bryostatins.
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Bryostatin 1 Induces Biphasic Activation of Protein Kinase D in Intact Cells
Journal of Biological Chemistry, 1997Co-Authors: Sharon A. Matthews, George R Pettit, Enrique RozengurtAbstract:Abstract Bryostatin 1 and phorbol esters are both potent activators of protein kinase C (PKC), although their specific biological effects can differ in many systems. Here, we report that Bryostatin 1 activates protein kinase D (PKD), a novel serine/threonine protein kinase, in intact Swiss 3T3 cells and secondary mouse embryo fibroblasts and in COS-7 cells transiently transfected with a PKD expression construct. The dose response of PKD activation induced by Bryostatin 1 follows a striking biphasic pattern with maximal activation achieved at a concentration of 10 nm. Higher concentrations of Bryostatin 1 (100 nm) reduced PKD activation induced by phorbol 12,13-dibutyrate to levels stimulated by Bryostatin 1 alone. Bryostatin 1-induced PKD activation was markedly attenuated by treatment of cells with the PKC inhibitors bisindolylmaleimide I and Ro 31-8220. However, these agents did not inhibit PKD activity when added directly to in vitro kinase assays, suggesting that Bryostatin 1 stimulates PKD activation through a PKC-dependent pathway in intact cells. Our results raise the possibility that activated PKD in intact cells could mediate some of the multiple biphasic biological responses induced by Bryostatin 1.
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the Bryostatins inhibit growth of b16 f10 melanoma cells in vitro through a protein kinase c independent mechanism dissociation of activities using 26 epi Bryostatin 1
Cancer Research, 1996Co-Authors: Zoltan Szallasi, Linh Du, Rachel Levine, Phi Nga Nguyen, Nancy E. Lewin, Michael D. Williams, George R Pettit, Peter M BlumbergAbstract:Bryostatin 1 is a potential cancer chemotherapeutic agent in Phase II clinical trials, with positive responses observed for malignant melanoma, among other tumors. The Bryostatins are known to be potent ligands for protein kinase C (PKC), functioning as partial antagonists. In the present study, we explore the mechanism by which the Bryostatins inhibit growth of B16/F10 mouse melanoma cells in vitro . Three experimental approaches suggest that the growth inhibition is independent of PKC. First, we characterized in detail the translocation and down-regulation of the PKC isozymes α, δ, and e in response to phorbol ester and Bryostatin 1 in these cells. Although the dose-response curves obtained for the translocation-activation of PKC isozymes showed good correlation with the growthenhancing activity of phorbol 12-myristate 13-acetate, for no PKC isozyme was there a good correlation with the growth-inhibitory activity of Bryostatin 1. Second, inhibition of PKC enzymatic activity by the specific PKC inhibitor bisindolyl-maleimide I did not block the inhibition of thymidine incorporation induced by Bryostatin 1. Finally, 26-epi-Bryostatin 1, a stereoisomer of the naturally occurring Bryostatin 1 designed to have markedly reduced affinity for PKC, inhibited the growth of the B16/F10 melanoma cell lines with potency similar to that of Bryostatin 1. We confirmed here that 26-epi-Bryostatin 1 showed 60-fold reduced affinity for PKC and 30-60-fold reduced potency to translocate and downregulate PKC isozymes compared with Bryostatin 1. We presume that the principal toxicity of Bryostatin 1 reflects its interaction with PKC, and we would thus predict that epi-Bryostatin 1 would be less toxic. Indeed, we found at least 10-fold reduced toxicity of 26-epi-Bryostatin 1 in C57BL/6 mice compared with Bryostatin 1. We conclude that the growth inhibition of the Bryostatins, at least in this system, does not result from interaction with PKC. As exemplified by 26-epi-Bryostatin 1, this insight permits the design of analogues with comparable growth inhibition to Bryostatin 1 but with reduced toxicity.
Margo G. Haygood - One of the best experts on this subject based on the ideXlab platform.
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Bryostatins biological context and biotechnological prospects
Current Opinion in Biotechnology, 2010Co-Authors: Amaro E Trindadesilva, Grace E Limfong, Koty H. Sharp, Margo G. HaygoodAbstract:Bryostatins are a family of protein kinase C modulators that have potential applications in biomedicine. Found in miniscule quantities in a small marine invertebrate, lack of supply has hampered their development. In recent years, Bryostatins have been shown to have potent bioactivity in the central nervous system, an uncultivated marine bacterial symbiont has been shown to be the likely natural source of the Bryostatins, the Bryostatin biosynthetic genes have been identified and characterized, and Bryostatin analogues with promising biological activity have been developed and tested. Challenges in the development of Bryostatins for biomedical and biotechnological application include the cultivation of the bacterial symbiont and heterologous expression of Bryostatin biosynthesis genes. Continued exploration of the biology as well as the symbiotic origin of the Bryostatins presents promising opportunities for discovery of additional Bryostatins, and new functions for Bryostatins.
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localization of candidatus endobugula sertula and the Bryostatins throughout the life cycle of the bryozoan bugula neritina
The ISME Journal, 2007Co-Authors: Seana K Davidson, Koty H. Sharp, Margo G. HaygoodAbstract:‘Candidatus Endobugula sertula,’ the uncultivated γ-proteobacterial symbiont of the marine bryozoan Bugula neritina, synthesizes Bryostatins, complex polyketides that render B. neritina larvae unpalatable to predators. Although the symbiosis is well described, little is known about the locations of ‘E. sertula’ or the Bryostatins throughout larval settlement, metamorphosis and early development. In this study, we simultaneously localized ‘E. sertula’ and the Bryostatins in multiple stages of the B. neritina life cycle, using a novel Bryostatin detection method based on its known ability to bind mammalian protein kinase C. Our results suggest that the Bryostatins are deposited onto the exterior of B. neritina larvae during embryonic development, persist on the larval surface throughout metamorphosis and are shed prior to cuticle formation. During metamorphosis, ‘E. sertula’ remains adhered to the larval pallial epithelium and is incorporated into the preancestrula cystid tissue layer, which ultimately develops into a bud and gives rise to the next zooid in the colony. Colocalization of Bryostatin signal with aggregates of ‘E. sertula’ in buds of ancestrulae suggested new synthesis of Bryostatins in ancestrulae. In adult B. neritina colonies, symbiont microcolonies were observed in the funicular cords of rhizoids, which likely result in asexual transmission of ‘E. sertula’ to regenerated colonies. Furthermore, Bryostatin signal was detected on the surface of the rhizoids of adult B. neritina colonies. Through simultaneous localization of the Bryostatins and the ‘E. sertula,’ we determined how ‘E. sertula’ is transmitted, and identified shifts in Bryostatin localization throughout the life cycle of the host B. neritina.
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Localization of ‘Candidatus Endobugula sertula’ and the Bryostatins throughout the life cycle of the bryozoan Bugula neritina
The ISME Journal, 2007Co-Authors: Koty H. Sharp, Seana K Davidson, Margo G. HaygoodAbstract:‘ Candidatus Endobugula sertula,’ the uncultivated γ-proteobacterial symbiont of the marine bryozoan Bugula neritina , synthesizes Bryostatins, complex polyketides that render B. neritina larvae unpalatable to predators. Although the symbiosis is well described, little is known about the locations of ‘ E. sertula ’ or the Bryostatins throughout larval settlement, metamorphosis and early development. In this study, we simultaneously localized ‘ E. sertula ’ and the Bryostatins in multiple stages of the B. neritina life cycle, using a novel Bryostatin detection method based on its known ability to bind mammalian protein kinase C. Our results suggest that the Bryostatins are deposited onto the exterior of B. neritina larvae during embryonic development, persist on the larval surface throughout metamorphosis and are shed prior to cuticle formation. During metamorphosis, ‘ E. sertula ’ remains adhered to the larval pallial epithelium and is incorporated into the preancestrula cystid tissue layer, which ultimately develops into a bud and gives rise to the next zooid in the colony. Colocalization of Bryostatin signal with aggregates of ‘ E. sertula ’ in buds of ancestrulae suggested new synthesis of Bryostatins in ancestrulae. In adult B. neritina colonies, symbiont microcolonies were observed in the funicular cords of rhizoids, which likely result in asexual transmission of ‘ E. sertula ’ to regenerated colonies. Furthermore, Bryostatin signal was detected on the surface of the rhizoids of adult B. neritina colonies. Through simultaneous localization of the Bryostatins and the ‘ E. sertula ,’ we determined how ‘ E. sertula ’ is transmitted, and identified shifts in Bryostatin localization throughout the life cycle of the host B. neritina .
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evidence for the biosynthesis of Bryostatins by the bacterial symbiont candidatus endobugula sertula of the bryozoan bugula neritina
Applied and Environmental Microbiology, 2001Co-Authors: Seana K Davidson, Scott W Allen, Christine Anderson, Margo G. HaygoodAbstract:The marine bryozoan, Bugula neritina, is the source of the Bryostatins, a family of macrocyclic lactones with anticancer activity. Bryostatins have long been suspected to be bacterial products. B. neritina harbors the uncultivated gamma proteobacterial symbiont “Candidatus Endobugula sertula.” In this work several lines of evidence are presented that show that the symbiont is the most likely source of Bryostatins. Bryostatins are complex polyketides similar to bacterial secondary metabolites synthesized by modular type I polyketide synthases (PKS-I). PKS-I gene fragments were cloned from DNA extracted from the B. neritina-“E. sertula” association, and then primers specific to one of these clones, KSa, were shown to amplify the KSa gene specifically and universally from total B. neritina DNA. In addition, a KSa RNA probe was shown to bind specifically to the symbiotic bacteria located in the pallial sinus of the larvae of B. neritina and not to B. neritina cells or to other bacteria. Finally, B. neritina colonies grown in the laboratory were treated with antibiotics to reduce the numbers of bacterial symbionts. Decreased symbiont levels resulted in the reduction of the KSa signal as well as the Bryostatin content. These data provide evidence that the symbiont E. sertula has the genetic potential to make Bryostatins and is necessary in full complement for the host bryozoan to produce normal levels of Bryostatins. This study demonstrates that it may be possible to clone Bryostatin genes from B. neritina directly and use these to produce Bryostatins in heterologous host bacteria.
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identification of sibling species of the bryozoan bugula neritina that produce different anticancer Bryostatins and harbor distinct strains of the bacterial symbiont candidatus endobugula sertula
The Biological Bulletin, 1999Co-Authors: Seana K Davidson, Margo G. HaygoodAbstract:Although the cosmopolitan marine bryozoan Buga chemotype M lacks Bryostatins with the octa-2,4- dienoate substituent. B. neritina contains a symbiotic y-pro- teobacterium "Cundidutus Endobugula sertula," and it has been proposed that Bryostatins may be synthesized by bac- terial symbionts. In this study, B. neritina populations along the California coast were sampled for genetic variation and Bryostatin content. Colonies that differ in chemotype also differ genetically by 8% in the mitochondrial cytochrome c oxidase subunit 1 (CO I) gene; this difference is sufficient to suggest that the chemotypes represent different species. Each species contains a distinct strain of "E. sertula" that differs at four nucleotide sites in the small subunit ribo- somal RNA (SSU rRNA) gene. These results indicate that the chemotypes have a genetic basis rather than an environ- mental cause. Gene sequences from an Atlantic sample matched sequences from the California chemotype M col- onies, suggesting that this type may be cosmopolitan due to transport on boat hulls.
Eric J Thomas - One of the best experts on this subject based on the ideXlab platform.
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towards 20 20 difluorinated Bryostatin synthesis and biological evaluation of c17 c27 fragments
Organic and Biomolecular Chemistry, 2019Co-Authors: Paul R Mears, Steven Hoekman, Claire E Rye, Fiona P Bailey, Dominic P Byrne, Patrick A Eyers, Eric J ThomasAbstract:Bryostatins with modified C17-C27 fragments have not been widely studied. The synthesis of 20,20-difluorinated analogues was therefore investigated. Such substitution would inhibit dehydration involving the C19-hydroxyl group and stabilise the ring-closed hemiacetal tautomers. Following preliminary studies, allyldifluorination was used to prepare difluorinated alkenols. Oxidation followed by stereoselective Wittig reactions of the resulting α,α-difluorinated ketones gave (E)-α,β-unsaturated esters that were taken through to complete syntheses of 2-hydroxytetrahydropyrans corresponding to C17-C27 fragments of 20,20-difluorinated Bryostatin. These compounds showed modest binding to protein kinase Cα isozyme. Attempts were also undertaken to synthesise macrocyclic 20,20-difluorinated analogues. During preliminary studies, allyldifluorination was carried out using a 2-alkyl-3-bromo-1,1-difluoropropene.
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synthesis of c16 c27 fragments of Bryostatins modified by 20 20 difluorination
Tetrahedron Letters, 2015Co-Authors: Paul R Mears, Eric J ThomasAbstract:2-Hydroxytetrahydropyrans corresponding to the C16–C27 fragment of Bryostatins which have been difluorinated at C20 (Bryostatin numbering) have been synthesised. The fluorine substituents were introduced by difluoroallylation. An (E)-selective Wittig reaction using a stabilised ylide provided the required methoxycarbonylmethylene substituent with excellent stereoselectivity.
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Synthesis of C16–C27-fragments of Bryostatins modified by 20,20-difluorination
Tetrahedron Letters, 2015Co-Authors: Paul R Mears, Eric J ThomasAbstract:2-Hydroxytetrahydropyrans corresponding to the C16–C27 fragment of Bryostatins which have been difluorinated at C20 (Bryostatin numbering) have been synthesised. The fluorine substituents were introduced by difluoroallylation. An (E)-selective Wittig reaction using a stabilised ylide provided the required methoxycarbonylmethylene substituent with excellent stereoselectivity.
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Synthesis of C16–C27-fragments of Bryostatins modified by 20,20-difluorination
Tetrahedron Letters, 2015Co-Authors: Paul R Mears, Eric J ThomasAbstract:2-Hydroxytetrahydropyrans corresponding to the C16–C27 fragment of Bryostatins which have been difluorinated at C20 (Bryostatin numbering) have been synthesised. The fluorine substituents were introduced by difluoroallylation. An (E)-selective Wittig reaction using a stabilised ylide provided the required methoxycarbonylmethylene substituent with excellent stereoselectivity.
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a preliminary evaluation of a metathesis approach to Bryostatins
Tetrahedron Letters, 2006Co-Authors: Matthew Ball, Thomas J. Gregson, Raphael Dumeunier, Benjamin Bradshaw, Hiroki Omori, Somhairle Maccormick, Eric J ThomasAbstract:Abstract Preliminary investigations into the synthesis of Bryostatins using ring-closing metathesis to form the C(16)–C(17) double bond led to a synthesis of the Bryostatin analogue 51 ; precursors 26 and 52 , which possess the geminal dimethyl group at C-18, did not undergo the required ring-closing metathesis.
Seana K Davidson - One of the best experts on this subject based on the ideXlab platform.
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localization of candidatus endobugula sertula and the Bryostatins throughout the life cycle of the bryozoan bugula neritina
The ISME Journal, 2007Co-Authors: Seana K Davidson, Koty H. Sharp, Margo G. HaygoodAbstract:‘Candidatus Endobugula sertula,’ the uncultivated γ-proteobacterial symbiont of the marine bryozoan Bugula neritina, synthesizes Bryostatins, complex polyketides that render B. neritina larvae unpalatable to predators. Although the symbiosis is well described, little is known about the locations of ‘E. sertula’ or the Bryostatins throughout larval settlement, metamorphosis and early development. In this study, we simultaneously localized ‘E. sertula’ and the Bryostatins in multiple stages of the B. neritina life cycle, using a novel Bryostatin detection method based on its known ability to bind mammalian protein kinase C. Our results suggest that the Bryostatins are deposited onto the exterior of B. neritina larvae during embryonic development, persist on the larval surface throughout metamorphosis and are shed prior to cuticle formation. During metamorphosis, ‘E. sertula’ remains adhered to the larval pallial epithelium and is incorporated into the preancestrula cystid tissue layer, which ultimately develops into a bud and gives rise to the next zooid in the colony. Colocalization of Bryostatin signal with aggregates of ‘E. sertula’ in buds of ancestrulae suggested new synthesis of Bryostatins in ancestrulae. In adult B. neritina colonies, symbiont microcolonies were observed in the funicular cords of rhizoids, which likely result in asexual transmission of ‘E. sertula’ to regenerated colonies. Furthermore, Bryostatin signal was detected on the surface of the rhizoids of adult B. neritina colonies. Through simultaneous localization of the Bryostatins and the ‘E. sertula,’ we determined how ‘E. sertula’ is transmitted, and identified shifts in Bryostatin localization throughout the life cycle of the host B. neritina.
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Localization of ‘Candidatus Endobugula sertula’ and the Bryostatins throughout the life cycle of the bryozoan Bugula neritina
The ISME Journal, 2007Co-Authors: Koty H. Sharp, Seana K Davidson, Margo G. HaygoodAbstract:‘ Candidatus Endobugula sertula,’ the uncultivated γ-proteobacterial symbiont of the marine bryozoan Bugula neritina , synthesizes Bryostatins, complex polyketides that render B. neritina larvae unpalatable to predators. Although the symbiosis is well described, little is known about the locations of ‘ E. sertula ’ or the Bryostatins throughout larval settlement, metamorphosis and early development. In this study, we simultaneously localized ‘ E. sertula ’ and the Bryostatins in multiple stages of the B. neritina life cycle, using a novel Bryostatin detection method based on its known ability to bind mammalian protein kinase C. Our results suggest that the Bryostatins are deposited onto the exterior of B. neritina larvae during embryonic development, persist on the larval surface throughout metamorphosis and are shed prior to cuticle formation. During metamorphosis, ‘ E. sertula ’ remains adhered to the larval pallial epithelium and is incorporated into the preancestrula cystid tissue layer, which ultimately develops into a bud and gives rise to the next zooid in the colony. Colocalization of Bryostatin signal with aggregates of ‘ E. sertula ’ in buds of ancestrulae suggested new synthesis of Bryostatins in ancestrulae. In adult B. neritina colonies, symbiont microcolonies were observed in the funicular cords of rhizoids, which likely result in asexual transmission of ‘ E. sertula ’ to regenerated colonies. Furthermore, Bryostatin signal was detected on the surface of the rhizoids of adult B. neritina colonies. Through simultaneous localization of the Bryostatins and the ‘ E. sertula ,’ we determined how ‘ E. sertula ’ is transmitted, and identified shifts in Bryostatin localization throughout the life cycle of the host B. neritina .
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evidence for the biosynthesis of Bryostatins by the bacterial symbiont candidatus endobugula sertula of the bryozoan bugula neritina
Applied and Environmental Microbiology, 2001Co-Authors: Seana K Davidson, Scott W Allen, Christine Anderson, Margo G. HaygoodAbstract:The marine bryozoan, Bugula neritina, is the source of the Bryostatins, a family of macrocyclic lactones with anticancer activity. Bryostatins have long been suspected to be bacterial products. B. neritina harbors the uncultivated gamma proteobacterial symbiont “Candidatus Endobugula sertula.” In this work several lines of evidence are presented that show that the symbiont is the most likely source of Bryostatins. Bryostatins are complex polyketides similar to bacterial secondary metabolites synthesized by modular type I polyketide synthases (PKS-I). PKS-I gene fragments were cloned from DNA extracted from the B. neritina-“E. sertula” association, and then primers specific to one of these clones, KSa, were shown to amplify the KSa gene specifically and universally from total B. neritina DNA. In addition, a KSa RNA probe was shown to bind specifically to the symbiotic bacteria located in the pallial sinus of the larvae of B. neritina and not to B. neritina cells or to other bacteria. Finally, B. neritina colonies grown in the laboratory were treated with antibiotics to reduce the numbers of bacterial symbionts. Decreased symbiont levels resulted in the reduction of the KSa signal as well as the Bryostatin content. These data provide evidence that the symbiont E. sertula has the genetic potential to make Bryostatins and is necessary in full complement for the host bryozoan to produce normal levels of Bryostatins. This study demonstrates that it may be possible to clone Bryostatin genes from B. neritina directly and use these to produce Bryostatins in heterologous host bacteria.
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identification of sibling species of the bryozoan bugula neritina that produce different anticancer Bryostatins and harbor distinct strains of the bacterial symbiont candidatus endobugula sertula
The Biological Bulletin, 1999Co-Authors: Seana K Davidson, Margo G. HaygoodAbstract:Although the cosmopolitan marine bryozoan Buga chemotype M lacks Bryostatins with the octa-2,4- dienoate substituent. B. neritina contains a symbiotic y-pro- teobacterium "Cundidutus Endobugula sertula," and it has been proposed that Bryostatins may be synthesized by bac- terial symbionts. In this study, B. neritina populations along the California coast were sampled for genetic variation and Bryostatin content. Colonies that differ in chemotype also differ genetically by 8% in the mitochondrial cytochrome c oxidase subunit 1 (CO I) gene; this difference is sufficient to suggest that the chemotypes represent different species. Each species contains a distinct strain of "E. sertula" that differs at four nucleotide sites in the small subunit ribo- somal RNA (SSU rRNA) gene. These results indicate that the chemotypes have a genetic basis rather than an environ- mental cause. Gene sequences from an Atlantic sample matched sequences from the California chemotype M col- onies, suggesting that this type may be cosmopolitan due to transport on boat hulls.
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biology of the Bryostatins in the marine bryozoan bugula neritina symbiosis cryptic speciation and chemical defense
California Sea Grant College Program, 1999Co-Authors: Seana K DavidsonAbstract:Author(s): Davidson, Seana K. | Abstract: This dissertation investigates the identity and function of a bacterial symbiont described in the marine bryozoan Bugula neritina by R.M. Woollacott in 1981. B. neritina is the source of Bryostatins, unique cytotoxins suspected to have a bacterial source, and is considered a single species throughout its cosmopolitan temperate range. Bryostatins found from different collections of B. neritina vary, and only certain populations produce Bryostatins that possess an octa-2,4-dienoate substituent. In this dissertation the bacterial symbionts of the larvae are identified by small subunit ribosomal rRNA {SSU) gene sequences and named "Candidatus Endobugula sertula." The variable regions of these genes were used to design oligonucleotides specific to the symbiont. These specific oligonucleotides were used for in situ hybridization to the bacteria in the pallial sinus to confirm the origin of the sequence, and for specific amplification of symbiont SSU rRNA genes by PCR. Then the mitochondrial cytochrome c oxidase subunit I gene was used to identify two distinct species of B. neritina each harboring a different symbiont as determined by SSU rRNA sequence. Variation in the Bryostatin profiles is associated with this genetic difference. Only one B. neritina/"E. sertula" association can produce Bryostatins with an octa-2,4-dienoate substituent (Bryostatins 1-3, 12 and 15). In order to elucidate the possible involvement of the symbiont in production of Bryostatins, experiments were conducted to eliminate "E. sertula" from R. neritina to determine whether B. neritina can continue to grow normally without the symbiont, and/or continue to produce equivalent levels of Bryostatins. Symbiont levels were estimated using a symbiont-specific PCR assay, then Bryostatin activity levels were compared between control and treated B. neritina colonies. When symbiont levels were greatly reduced, Bryostatin activity declined by approximately 50%. Genetic evidence was discovered that indicates "E. sertuld" has the potential to synthesize complex polyketides like Bryostatin. Finally evidence was gathered to address the hypothesis that Bryostatins serve as defensive compounds for the bryozoan host. The distribution of Bryostatins in the colonies is suggestive of a defense, and it was found that predatory nudibranchs of B. neritina sequester Bryostatins and concentrate them in their egg ribbons. In summary, the symbiont's most likely function is to provide a chemical defense, Bryostatins, for the host.
