ABSTRACT
Small molecule activators of protein kinase C (PKC) have traditionally been classified as either tumor promoters or suppressors. Although bryostatin 1 has well established anti-cancer activity, most natural products that target the PKC regulator domain exhibit tumor promotion properties. In this study, we examine a focused library of indolactam analogues in cell-based assays to establish the structural features of the scaffold that enhance bryostatin 1-like activity. These systematic biological assessments identified specific indole substitution patterns that impart diminished tumor promotion behavior in vitro for indolactam analogues, while still maintaining nanomolar potency for PKC.
Subject(s)
Lactams , Neoplasms , Protein Kinase C , Humans , Bryostatins/pharmacology , Bryostatins/chemistry , Bryostatins/metabolism , Lactones , Protein Kinase C/metabolism , Tetradecanoylphorbol Acetate , Lactams/chemistry , Lactams/pharmacologyABSTRACT
Neuronal damage in iron-sensitive brain regions occurs as a result of iron dyshomeostasis. Increased iron levels and iron-related pathogenic triggers are associated with neurodegenerative diseases, including Alzheimer's disease (AD). Ferritin is a key player involved in iron homeostasis. Major pathological hallmarks of AD are amyloid plaques, neurofibrillary tangles (NFTs) and synaptic loss that lead to cognitive dysfunction and memory loss. Natural compounds persist in being the most excellent molecules in the area of drug discovery because of their different range of therapeutic applications. Bryostatins are naturally occurring macrocyclic lactones that can be implicated in AD therapeutics. Among them, Bryostatin 1 regulates protein kinase C, a crucial player in AD pathophysiology, thus highlighting the importance of bryostatin 1 in AD management. Thus, this study explores the binding mechanism of Bryotstain 1 with ferritin. In this work, the molecular docking calculations revealed that bryostatin 1 has an appreciable binding potential towards ferritin by forming stable hydrogen bonds (H-bonds). Molecular dynamics simulation studies deciphered the binding mechanism and conformational dynamics of ferrritin-bryostatin 1 system. The analyses of root mean square deviation, root mean square fluctuations, Rg, solvent accessible surface area, H-bonds and principal component analysis revealed the stability of the ferritin-bryostatin 1 docked complex throughout the trajectory of 100 ns. Moreover, the free energy landscape analysis advocated that the ferritin-bryostatin 1 complex stabilized to the global minimum. Altogether, the present work delineated the binding of bryostatin 1 with ferritin that can be implicated in the management of AD.Communicated by Ramaswamy H. Sarma.
Subject(s)
Alzheimer Disease , Humans , Alzheimer Disease/metabolism , Bryostatins/pharmacology , Bryostatins/chemistry , Bryostatins/metabolism , Ferritins/therapeutic use , Molecular Docking Simulation , Iron/metabolismABSTRACT
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,ß-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.
Subject(s)
Bryostatins/chemistry , Bryostatins/chemical synthesis , Halogenation , Bryostatins/metabolism , Chemistry Techniques, Synthetic , Models, Molecular , Molecular Conformation , Protein Kinase C/metabolismABSTRACT
Bugula neritina is a common invasive cosmopolitan bryozoan that harbors (like many sessile marine invertebrates) a symbiotic bacterial (SB) community. Among the SB of B. neritina, "Candidatus Endobugula sertula" continues to receive the greatest attention, because it is the source of bryostatins. The bryostatins are potent bioactive polyketides, which have been investigated for their therapeutic potential to treat various cancers, Alzheimer's disease, and AIDS. In this study, we compare the metagenomics sequences for the 16S ribosomal RNA gene of the SB communities from different geographic and life cycle samples of Chinese B. neritina. Using a variety of approaches for estimating alpha/beta diversity and taxonomic abundance, we find that the SB communities vary geographically with invertebrate and fish mariculture and with latitude and environmental temperature. During the B. neritina life cycle, we find that the diversity and taxonomic abundances of the SB communities change with the onset of host metamorphosis, filter feeding, colony formation, reproduction, and increased bryostatin production. "Ca. Endobugula sertula" is confirmed as the symbiont of the Chinese "Ca. Endobugula"/B. neritina symbiosis. Our study extends our knowledge about B. neritina symbiosis from the New to the Old World and offers new insights into the environmental and life cycle factors that can influence its SB communities, "Ca. Endobugula," and bryostatins more globally.
Subject(s)
Bryozoa/microbiology , Gammaproteobacteria/classification , Gammaproteobacteria/isolation & purification , Gammaproteobacteria/metabolism , Metagenomics , Symbiosis , Animals , Biodiversity , Bryostatins/metabolism , Bryozoa/growth & development , China , DNA, Bacterial/isolation & purification , Ecology , Gammaproteobacteria/genetics , Geography , Larva/microbiology , Life Cycle Stages , RNA, Ribosomal, 16S/geneticsABSTRACT
ß-Branching is an expansion upon canonical polyketide synthase extension that allows for the installation of diverse chemical moieties in several natural products. Several of these moieties are unique among natural products, including the two vinyl methylesters found in the core structure of bryostatins. This family of molecules is derived from an obligate bacterial symbiont of a sessile marine bryozoan, Bugula neritina. Within this family, bryostatin 1 has been investigated as an anticancer, neuroprotective, and immunomodulatory compound. We have turned to the biosynthetic gene cluster within the bacterial symbiont to investigate the biosynthesis of bryostatins. Recent sequencing efforts resulted in the annotation of two missing genes: bryT and bryU. Using novel chemoenzymatic techniques, we have validated these as the missing enoyl-CoA hydratase and donor acyl carrier protein, essential components of the ß-branching cassette of the bryostatin pathway. Together, this cassette installs the vinyl methylester moieties essential to the activity of bryostatins.
Subject(s)
Biochemistry/methods , Bryostatins/metabolism , Enzymes/metabolism , Acyl Carrier Protein/genetics , Acyl Carrier Protein/metabolism , Animals , Bryostatins/biosynthesis , Bryozoa/genetics , Bryozoa/metabolism , Enoyl-CoA Hydratase/genetics , Enoyl-CoA Hydratase/metabolism , Enzymes/genetics , Magnetic Resonance Spectroscopy , Metabolic Networks and Pathways , Methylation , Multigene Family , Polyketides/metabolismABSTRACT
Marine bryozoans play an important role for the discovery of novel bioactive compounds among marine organisms. In this review, we summarize 164 new secondary metabolites including macrocyclic lactones, sterols, alkaloids, sphingolipids and so forth from 24 marine bryozoans in the last two decades. The structural features, bioactivity, structure-activity relationship, mechanism and strategies to address the resupply of these scarce secondary metabolites are discussed. The structural and bioactive diversity of the secondary metabolites from marine bryozoans indicated the possibility of using these compounds, especially bryostatin 1 (1), bryostatin analog (BA1), alkaloids (50, 53, 127-128 and 134-139), sphingolipids sulfates (148 and 149) and sulfur-containing aromatic compound (160), as the starting points for new drug discovery.
Subject(s)
Alkaloids/pharmacology , Biological Products/pharmacology , Bryostatins/pharmacology , Bryozoa/metabolism , Drug Discovery , Sphingolipids/pharmacology , Sterols/pharmacology , Alkaloids/chemistry , Alkaloids/metabolism , Animals , Biological Products/chemistry , Biological Products/metabolism , Bryostatins/chemistry , Bryostatins/metabolism , Bryozoa/chemistry , Drug Discovery/methods , Humans , Hydrocarbons, Aromatic/chemistry , Hydrocarbons, Aromatic/metabolism , Hydrocarbons, Aromatic/pharmacology , Secondary Metabolism , Sphingolipids/chemistry , Sphingolipids/metabolism , Sterols/chemistry , Sterols/metabolismABSTRACT
Oxidative stress and amyloid-ß (Aß) oligomers have been implicated in Alzheimer's disease (AD). The growth and maintenance of neuronal networks are influenced by brain derived neurotrophic factor (BDNF) expression, which is promoted by protein kinase C epsilon (PKCÉ). We investigated the reciprocal interaction among oxidative stress, Aß, and PKCÉ levels and subsequent PKCÉ-dependent MnSOD and BDNF expression in hippocampal pyramidal neurons. Reduced levels of PKCÉ, MnSOD, and BDNF and an increased level of Aß were also found in hippocampal neurons from autopsy-confirmed AD patients. In cultured human primary hippocampal neurons, spherical aggregation of Aß (amylospheroids) decreased PKCÉ and MnSOD. Treatment with t-butyl hydroperoxide (TBHP) increased superoxide, the oxidative DNA/RNA damage marker, 8-OHG, and Aß levels, but reduced PKCÉ, MnSOD, BDNF, and cultured neuron density. These changes were reversed with the PKCÉ activators, bryostatin and DCPLA-ME. PKCÉ knockdown suppressed PKCÉ, MnSOD, and BDNF but increased Aß. In cultured neurons, the increase in reactive oxygen species (ROS) associated with reduced PKCÉ during neurodegeneration was inhibited by the SOD mimetic MnTMPyP and the ROS scavenger NAc, indicating that strong oxidative stress suppresses PKCÉ level. Reduction of PKCÉ and MnSOD was prevented with the PKCÉ activator bryostatin in 5-6-month-old Tg2576 AD transgenic mice. In conclusion, oxidative stress and Aß decrease PKCÉ expression. Reciprocally, a depression of PKCÉ reduces BDNF and MnSOD, resulting in oxidative stress. These changes can be prevented with the PKCÉ-specific activators.
Subject(s)
Alzheimer Disease/pathology , Brain-Derived Neurotrophic Factor/metabolism , Down-Regulation/physiology , Hippocampus/pathology , Neurons/metabolism , Protein Kinase C-epsilon/deficiency , Adjuvants, Immunologic/pharmacology , Aged , Aged, 80 and over , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/toxicity , Animals , Bryostatins/metabolism , Bryostatins/pharmacology , Cells, Cultured , Female , Fetus/anatomy & histology , Hippocampus/cytology , Hippocampus/metabolism , Humans , Male , Metalloporphyrins/pharmacology , Mice , Middle Aged , Morpholinos/pharmacology , Protein Kinase C-epsilon/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolism , Superoxide Dismutase/metabolism , Transfection , tert-Butylhydroperoxide/pharmacologyABSTRACT
The synthesis and biological evaluation of chromane-containing bryostatin analogues WN-2-WN-7 and the previously reported salicylate-based analogue WN-8 are described. Analogues WN-2-WN-7 are prepared through convergent assembly of the chromane-containing fragment B-I with the "binding domain" fragment A-I or its C26-des-methyl congener, fragment A-II. The synthesis of fragment B-I features enantioselective double C-H allylation of 1,3-propanediol to form the C2-symmetric diol 3 and Heck cyclization of bromo-diene 5 to form the chromane core. The synthesis of salicylate WN-8 is accomplished through the union of fragments A-III and B-II. The highest binding affinities for PKCα are observed for the C26-des-methyl analogues WN-3 (Ki = 63.9 nM) and WN-7 (Ki = 63.1 nM). All analogues, WN-2-WN-8, inhibited growth of Toledo cells, with the most potent analogue being WN-7. This response, however, does not distinguish between phorbol ester-like and bryostatin-like behavior. In contrast, while many of the analogues contain a conserved C-ring in the binding domain and other features common to analogues with bryostatin-like properties, all analogues evaluated in the U937 proliferation and cell attachment assays displayed phorbol ester-like and/or toxic behavior, including WN-8, for which "bryostatin-like PKC modulatory activities" previously was suggested solely on the basis of PKC binding. These results underscore the importance of considering downstream biological effects, as tumor suppression cannot be inferred from potent PKC binding.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Bryostatins/chemistry , Bryostatins/pharmacology , Chromans/chemistry , Hydrogen/chemistry , Antineoplastic Agents/metabolism , Bryostatins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Proto-Oncogene Proteins c-akt/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
As an initial step in designing a simplified bryostatin hybrid molecule, three bryostatin analogues bearing a diacylglycerol lactone-based C-ring, which possessed the requisite pharmacophores for binding to protein kinase C (PKC) together with a modified bryostatin-like A- and B-ring region, were synthesized and evaluated. Merle 46 and Merle 47 exhibited binding affinity to PKC alpha with Ki values of 7000 ± 990 and 4940 ± 470 nM, respectively. Reinstallation of the trans-olefin and gem-dimethyl group present in bryostatin 1 in Merle 48 resulted in improved binding affinity, 363 ± 42 nM. While Merle 46 and 47 were only marginally active biologically, Merle 48 showed sufficient activity on the U937 cells to confirm that it was PMA-like for growth and attachment, as predicted by the substitution pattern of its A- and B-rings.
Subject(s)
Bryostatins/chemical synthesis , Bryostatins/pharmacology , Diglycerides/chemistry , Lactones/chemistry , Bryostatins/metabolism , Carbon-13 Magnetic Resonance Spectroscopy , Protein Kinase C/metabolism , Proton Magnetic Resonance Spectroscopy , Spectrometry, Mass, Electrospray Ionization , Substrate SpecificityABSTRACT
Bryostatin-1 is a marine natural product that has demonstrated medicinal activity in pre-clinical and clinical trials for the treatment of cancer, Alzheimer's disease, effects of stroke, and HIV. In this study, iron-bryostatin-1 was obtained using a pharmaceutical aquaculture technique developed by our lab that cultivates marine bacteria for marine natural product extraction. Analytical measurements (1)H and (13)C NMR, mass spectrometry, and flame atomic absorption were utilized to confirm the presence of an iron-bryostatin-1 complex. The iron-bryostatin-1 complex produced was then tested against the National Cancer Institute's 60 cell line panel. Adding iron to bryostatin-1 lowered the anti-cancer efficacy of the compound.
Subject(s)
Antineoplastic Agents/pharmacology , Bryostatins/chemistry , Bryostatins/pharmacology , Iron/chemistry , Antineoplastic Agents/chemistry , Bryostatins/isolation & purification , Bryostatins/metabolism , Cell Line, Tumor , Humans , Magnetic Resonance Spectroscopy , Microbiological TechniquesABSTRACT
Protein kinase C (PKC) isozymes are important regulatory enzymes that have been implicated in many diseases, including cancer, Alzheimer's disease, and in the eradication of HIV/AIDS. Given their potential clinical ramifications, PKC modulators, e.g. phorbol esters and bryostatin, are also of great interest in the drug development. However, structural details on the binding between PKC and its modulators, especially bryostatin - the highly potent and non-tumor promoting activator for PKCs, are still lacking. Here, we report the first comparative molecular dynamics study aimed at gaining structural insight into the mechanisms by which the PKC delta cys2 activator domain is used in its binding to phorbol ester and bryostatin-1. As anticipated in the phorbol ester binding, hydrogen bonds are formed through the backbone atoms of Thr242, Leu251, and Gly253 of PKC. However, the opposition of H-bond formation between Thr242 and Gly253 may cause the phorbol ester complex to become less stable when compared with the bryostatin binding. For the PKC delta-bryostatin complex, hydrogen bonds are formed between the Gly253 backbone carbonyl and the C30 carbomethoxy substituent of the ligand. Additionally, the indole Nε1 of the highly homologous Trp252 also forms an H-bond to the C20 ester group on bryostatin. Backbone fluctuations also suggest that this latter H-bond formation may abrogate the transient interaction between Trp252 and His269, thus dampening the fluctuations observed on the nearby Zn(2+)-coordinating residues. This new dynamic fluctuation dampening model can potentially benefit future design of new PKC modulators.
Subject(s)
Bryostatins/chemistry , Models, Molecular , Molecular Conformation , Phorbol Esters/chemistry , Protein Kinase C/chemistry , Binding Sites , Bryostatins/metabolism , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Phorbol Esters/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Kinase C/metabolismABSTRACT
Although an effective combination of antiretroviral therapy (cART) controls HIV-1 viraemia in infected patients, viral latency established soon after infection hinders HIV-1 eradication. It has been shown that bryostatin-1 (BRY) inhibits HIV-infection in vitro and reactivates the latent virus through the protein kinase C-NF-κB pathway. We determined the in vitro potential effect of BRY in combination with currently used antiretroviral drugs. BRY alone or in combination with maraviroc (MVC)/Atripla (ATP) was tested for its capacity to reactivate latent virus and inhibit new infections. JLTRG-R5 cells and two latent HIV-1-infected cell lines, J89GFP and THP89GFP, were used as latency models. To quantify HIV infection, the reporter cell line TZM-bl was used. We found that BRY reactivates HIV-1 even in combination with MVC or ATP. Antiretroviral combinations with BRY do not interfere with BRY activity (i.e., the reactivation of latently infected cells) or with the antiviral activity of antiretroviral drugs. In addition, BRY-mediated down-modulation of surface CD4 and CXCR4 was not affected when it was used in combination with other antiretrovirals, and no hyperactivation or high-proliferation effects were observed in primary T cells. Moreover, the BRY treatment was able to reactivate HIV-1 in CD4+ T cells from HIV-1-infected patients under cART. Thus, we propose the use of BRY to purge the viral reservoir and recommend its combination with current antiretroviral treatments.
Subject(s)
Anti-Retroviral Agents/metabolism , Bryostatins/metabolism , Drug Interactions , HIV-1/drug effects , HIV-1/physiology , Virus Latency/drug effects , Adult , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/virology , Cells, Cultured , Cyclohexanes/metabolism , Efavirenz, Emtricitabine, Tenofovir Disoproxil Fumarate Drug Combination/metabolism , Humans , Male , Maraviroc , Middle Aged , Triazoles/metabolismABSTRACT
Protein kinase C (PKC) modulators are currently of great importance in preclinical and clinical studies directed at cancer, immunotherapy, HIV eradication, and Alzheimer's disease. However, the bound conformation of PKC modulators in a membrane environment is not known. Rotational echo double resonance (REDOR) NMR spectroscopy could uniquely address this challenge. However, REDOR NMR requires strategically labeled, high affinity ligands to determine interlabel distances from which the conformation of the bound ligand in the PKC-ligand complex could be identified. Here we report the first computer-guided design and syntheses of three bryostatin analogues strategically labeled for REDOR NMR analysis. Extensive computer analyses of energetically accessible analogue conformations suggested preferred labeling sites for the identification of the PKC-bound conformers. Significantly, three labeled analogues were synthesized, and, as required for REDOR analysis, all proved highly potent with PKC affinities (â¼1 nM) on par with bryostatin. These potent and strategically labeled bryostatin analogues are new structural leads and provide the necessary starting point for projected efforts to determine the PKC-bound conformation of such analogues in a membrane environment, as needed to design new PKC modulators and understand PKC-ligand-membrane structure and dynamics.
Subject(s)
Bryostatins/chemical synthesis , Bryostatins/metabolism , Drug Design , Protein Kinase C/metabolism , Rotation , Bryostatins/chemistry , Bryostatins/pharmacology , Chemistry Techniques, Synthetic , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Protein Kinase C/chemistry , Protein Structure, TertiaryABSTRACT
Protein kinase C (PKC), a validated therapeutic target for cancer chemotherapy, provides a paradigm for assessing structure-activity relations, where ligand binding has multiple consequences for a target. For PKC, ligand binding controls not only PKC activation and multiple phosphorylations but also subcellular localization, affecting subsequent signaling. Using a capillary isoelectric focusing immunoassay system, we could visualize a high resolution isoelectric focusing signature of PKCδ upon stimulation by ligands of the phorbol ester and bryostatin classes. Derivatives that possessed different physicochemical characteristics and induced different patterns of biological response generated different signatures. Consistent with different patterns of PKCδ localization as one factor linked to these different signatures, we found different signatures for activated PKCδ from the nuclear and non-nuclear fractions. We conclude that the capillary isoelectric focusing immunoassay system may provide a window into the integrated consequences of ligand binding and thus afford a powerful platform for compound development.
Subject(s)
Bryostatins/metabolism , Isoelectric Focusing , Phorbol Esters/metabolism , Protein Kinase C-delta/metabolism , Cell Line, Tumor , Humans , Immunoassay/methods , Ligands , Phosphorylation , Protein Binding , Structure-Activity RelationshipABSTRACT
Mutualism, a beneficial relationship between two species, often requires intimate interaction between the host and symbiont to establish and maintain the partnership. The colonial marine bryozoan Bugula neritina harbors an as yet uncultured endosymbiont, "Candidatus Endobugula sertula," throughout its life stages. The bacterial symbiont is the putative source of bioactive complex polyketide metabolites, the bryostatins, which chemically defend B. neritina larvae from predation. Despite the presence of "Ca. Endobugula sertula" in all life stages of the host, deterrent bryostatins appear to be concentrated in reproductive portions of the host colony, suggesting an interaction between the two partners to coordinate production and distribution of the metabolites within the colony. In this study, we identified host genes that were differentially expressed in control colonies and in colonies cured of the symbiont. Genes that code for products similar to glycosyl hydrolase family 9 and family 20 proteins, actin, and a Rho-GDP dissociation inhibitor were significantly downregulated (more than twice) in antibiotic-cured non-reproductive zooids compared to control symbiotic ones. Differential expression of these genes leads us to hypothesize that the host B. neritina may regulate the distribution of the symbiont within the colony via mechanisms of biofilm degradation and actin rearrangement, and consequently, influences bryostatin localization to bestow symbiont-associated protection to larvae developing in the reproductive zooids.
Subject(s)
Bryozoa/genetics , Gammaproteobacteria/physiology , Symbiosis/genetics , Actins/genetics , Animals , Base Sequence , Bryostatins/genetics , Bryostatins/metabolism , Bryozoa/metabolism , Gammaproteobacteria/drug effects , Gammaproteobacteria/genetics , Gene Expression , Molecular Sequence Data , Polymerase Chain ReactionABSTRACT
The total synthesis of bryostatin 9 was accomplished using a uniquely step-economical and convergent Prins-driven macrocyclization strategy. At 25 linear and 42 total steps, this is currently the most concise and convergent synthesis of a potent bryostatin.
Subject(s)
Bryostatins/chemical synthesis , Bryostatins/chemistry , Bryostatins/metabolism , Cyclization , Protein Kinase C/metabolismABSTRACT
Amino acid- and inosine-induced germination of Bacillus cereus ATCC 14579 spores was reversibly inhibited in the presence of 3 mM undissociated sorbic acid. Exposure to high hydrostatic pressure, Ca-dipicolinic acid (DPA), and bryostatin, an activator of PrkC kinase, negated this inhibition, pointing to specific blockage of signal transduction in germinant receptor-mediated germination.
Subject(s)
Bacillus cereus/growth & development , Bacillus cereus/metabolism , Sorbic Acid/metabolism , Spores, Bacterial/growth & development , Spores, Bacterial/metabolism , Amino Acids/metabolism , Bryostatins/metabolism , Hydrostatic Pressure , Inosine/metabolism , Metabolic Networks and Pathways , Picolinic Acids/metabolism , Signal TransductionABSTRACT
Bryostatin-1 (Bryo-1), a natural macrocyclic lactone, is clinically used as an anti-cancer agent. In this study, we demonstrate for the first time that Bryo-1 acts as a Toll-like receptor 4 (TLR4) ligand. Interestingly, activation of bone marrow-derived dendritic cells (in vitro with Bryo-1) led to a TLR4-dependent biphasic activation of nuclear factor-κB (NF-κB) and the unique induction of cytokines (IL-5, IL-6, and IL-10) and chemokines, including RANTES (regulated on activation normal T cell expressed and secreted) and macrophage inflammatory protein 1α (MIP1-α). In addition, EMSA demonstrated that Bryo-1-mediated induction of RANTES was regulated by NF-κB and the interferon regulatory factors (IRF)-1, IRF-3, and IRF-7 to the RANTES independently of myeloid differentiation primary response gene-88 (MyD88). Bryo-1 was able to induce the transcriptional activation of IRF-3 through the TLR4/MD2-dependent pathway. In vivo administration of Bryo-1 triggered a TLR-4-dependent T helper cell 2 (Th2) cytokine response and expanded a subset of myeloid dendritic cells that expressed a CD11c(high)CD8α(-) CD11b(+)CD4(+) phenotype. This study demonstrates that Bryo-1 can act as a TLR4 ligand and activate innate immunity. Moreover, the ability of Bryo-1 to trigger RANTES and MIP1-α suggests that Bryo-1 could potentially be used to prevent HIV-1 infection. Finally, induction of a Th2 response by Bryo-1 may help treat inflammatory diseases mediated by Th1 cells. Together, our studies have a major impact on the clinical use of Bryo-1 as an anti-cancer and immunopotentiating agent.
Subject(s)
Bryostatins/metabolism , Bryostatins/pharmacology , Chemokines/biosynthesis , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Toll-Like Receptor 4/metabolism , Animals , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Biological Products/metabolism , Biological Products/pharmacology , Bone Marrow Cells/cytology , Chemokines/genetics , Chemokines/metabolism , Dendritic Cells/immunology , Female , HEK293 Cells , Humans , Immunity, Innate/drug effects , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/metabolism , Ligands , Mice , Myeloid Differentiation Factor 88/metabolism , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Phenotype , Protein Binding , Transcriptional Activation/drug effects , Up-Regulation/drug effectsABSTRACT
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.
Subject(s)
Bryostatins/metabolism , Animals , Bacteria/growth & development , Bacteria/metabolism , Invertebrates/microbiology , Symbiosis/physiologyABSTRACT
Superdormant spores of Bacillus cereus and Bacillus subtilis germinated just as well as dormant spores with pressures of 150 or 500 MPa and with or without heat activation. Superdormant B. subtilis spores also germinated as well as dormant spores with peptidoglycan fragments or bryostatin, a Ser/Thr protein kinase activator.