Bryostatin-1 attenuates intestinal ischemia/reperfusion-induced intestinal barrier dysfunction, inflammation, and oxidative stress via activation of Nrf2/HO-1 signaling.

Bryostatin-1 (Bryo-1) exerts antioxidative stress effects in multiple diseases, and we confirmed that it improves intestinal barrier dysfunction in experimental colitis. Nevertheless, there are few reports on its action on intestinal ischemia/reperfusion (I/R). In this study, we mainly explored the effect of Bryo-1 on intestinal I/R injury and determined the mechanism. C57BL/6J mice underwent temporary superior mesenteric artery (SMA) obturation to induce I/R, on the contrary, Caco-2 cells suffered to oxygen and glucose deprivation/reperfusion (OGD/R) to establish the in vitro model. RAW264.7 cells were stimulated with LPS to induce macrophage inflammation. The drug gradient experiment was used to demonstrate in vivo and in vitro models. Bryo-1 ameliorated the intestinal I/R-induced injury of multiple organs and epithelial cells. It also alleviated intestinal I/R-induced barrier disruption of intestines according to the histology, intestinal permeability, intestinal bacterial translocation rates, and tight junction protein expression results. Bryo-1 significantly inhibited oxidative stress damages and inflammation, which may contribute to the restoration of intestinal barrier function. Further, Bryo-1 significantly activated Nrf2/HO-1 signaling in vivo. However, the deletion of Nrf2 in Caco-2 and RAW264.7 cells attenuated the protective functions of Bryo-1 and significantly abolished the anti-inflammatory effect of Bryo-1 on LPS-induced macrophage inflammation. Bryo-1 protects intestines against I/R-induced injury. It is associated with intestinal barrier protection, as well as inhibition of inflammation and oxidative stress partly through Nrf2/HO-1 signaling.

Biological evaluation of indolactams for in vitro bryostatin 1-like activity.

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.

Bryostatin-1 Decreases HIV-1 Infection and Viral Production in Human Primary Macrophages.

While combination antiretroviral therapy maintains undetectable viremia in people living with HIV (PLWH), a lifelong treatment is necessary to prevent viremic rebound after therapy cessation. This rebound seemed mainly caused by long-lived HIV-1 latently infected cells reverting to a viral productive status. Reversing latency and elimination of these cells by the so-called shock-and-kill strategy is one of the main investigated leads to achieve an HIV-1 cure. Small molecules referred to as latency reversal agents (LRAs) proved to efficiently reactivate latent CD4+ T cells. However, the LRA impact on de novo infection or HIV-1 production in productively infected macrophages remains elusive. Nontoxic doses of bryostatin-1, JQ1, and romidepsin were investigated in human monocyte-derived macrophages (MDMs). Treatment with bryostatin-1 or romidepsin resulted in a downregulation of CD4 and CCR5 receptors, respectively, accompanied by a reduction of R5 tropic virus infection. HIV-1 replication was mainly regulated by receptor modulation for bryostatin-1, while romidepsin effects rely on upregulation of SAMHD1 activity. LRA stimulation of chronically infected cells did not enhance HIV-1 production or gene expression. Surprisingly, bryostatin-1 caused a major decrease in viral production. This effect was not viral strain specific but appears to occur only in myeloid cells. Bryostatin-1 treatment of infected MDMs led to decreased amounts of capsid and matrix mature proteins with little to no modulation of precursors. Our observations revealed that bryostatin-1-treated myeloid and CD4+ T cells respond differently upon HIV-1 infection. Therefore, additional studies are warranted to more fully assess the efficiency of HIV-1 eradicating strategies. IMPORTANCE HIV-1 persists in a cellular latent form despite therapy that quickly propagates infection upon treatment interruption. Reversing latency would contribute to eradicate these cells, closing the gap to a cure. Macrophages are an acknowledged HIV-1 reservoir during therapy and are suspected to harbor latency establishment in vivo. However, the impact of latency reversal agents (LRAs) on HIV-1 infection and viral production in human macrophages is poorly known but nonetheless crucial to probe the safety of this strategy. In this in vitro study, we discovered encouraging antireplicative features of distinct LRAs in human macrophages. We also described a new viral production inhibition mechanism by protein kinase C agonists that is specific to myeloid cells. This study provides new insights into HIV-1 propagation restriction potentials by LRAs in human macrophages and underline the importance of assessing latency reversal strategy on all HIV-1-targeted cells.

Prodrugs of PKC modulators show enhanced HIV latency reversal and an expanded therapeutic window.

AIDS is a pandemic disease caused by HIV that affects 37 million people worldwide. Current antiretroviral therapy slows disease progression but does not eliminate latently infected cells, which resupply active virus, thus necessitating lifelong treatment with associated compliance, cost, and chemoexposure issues. Latency-reversing agents (LRAs) activate these cells, allowing for their potential clearance, thus presenting a strategy to eradicate the infection. Protein kinase C (PKC) modulators-including prostratin, ingenol esters, bryostatin, and their analogs-are potent LRAs in various stages of development for several clinical indications. While LRAs are promising, a major challenge associated with their clinical use is sustaining therapeutically meaningful levels of the active agent while minimizing side effects. Here we describe a strategy to address this problem based on LRA prodrugs, designed for controllable release of the active LRA after a single injection. As intended, these prodrugs exhibit comparable or superior in vitro activity relative to the parent compounds. Selected compounds induced higher in vivo expression of CD69, an activation biomarker, and, by releasing free agent over time, significantly improved tolerability when compared to the parent LRAs. More generally, selected prodrugs of PKC modulators avoid the bolus toxicities of the parent drug and exhibit greater efficacy and expanded tolerability, thereby addressing a longstanding objective for many clinical applications.

Insertion Depth Modulates Protein Kinase C-δ-C1b Domain Interactions with Membrane Cholesterol as Revealed by MD Simulations.

Protein kinase C delta (PKC-δ) is an important signaling molecule in human cells that has both proapoptotic as well as antiapoptotic functions. These conflicting activities can be modulated by two classes of ligands, phorbol esters and bryostatins. Phorbol esters are known tumor promoters, while bryostatins have anti-cancer properties. This is despite both ligands binding to the C1b domain of PKC-δ (δC1b) with a similar affinity. The molecular mechanism behind this discrepancy in cellular effects remains unknown. Here, we have used molecular dynamics simulations to investigate the structure and intermolecular interactions of these ligands bound to δC1b with heterogeneous membranes. We observed clear interactions between the δC1b-phorbol complex and membrane cholesterol, primarily through the backbone amide of L250 and through the K256 side-chain amine. In contrast, the δC1b-bryostatin complex did not exhibit interactions with cholesterol. Topological maps of the membrane insertion depth of the δC1b-ligand complexes suggest that insertion depth can modulate δC1b interactions with cholesterol. The lack of cholesterol interactions suggests that bryostatin-bound δC1b may not readily translocate to cholesterol-rich domains within the plasma membrane, which could significantly alter the substrate specificity of PKC-δ compared to δC1b-phorbol complexes.

The ε-Isozyme of Protein Kinase C (PKCε) Is Impaired in ALS Motor Cortex and Its Pulse Activation by Bryostatin-1 Produces Long Term Survival in Degenerating SOD1-G93A Motor Neuron-like Cells.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and ultimately fatal neurodegenerative disease, characterized by a progressive depletion of upper and lower motor neurons (MNs) in the brain and spinal cord. The aberrant regulation of several PKC-mediated signal transduction pathways in ALS has been characterized so far, describing either impaired expression or altered activity of single PKC isozymes (α, ß, ζ and δ). Here, we detailed the distribution and cellular localization of the ε-isozyme of protein kinase C (PKCε) in human postmortem motor cortex specimens and reported a significant decrease in both PKCε mRNA (PRKCE) and protein immunoreactivity in a subset of sporadic ALS patients. We furthermore investigated the steady-state levels of both pan and phosphorylated PKCε in doxycycline-activated NSC-34 cell lines carrying the human wild-type (WT) or mutant G93A SOD1 and the biological long-term effect of its transient agonism by Bryostatin-1. The G93A-SOD1 cells showed a significant reduction of the phosphoPKCε/panPKCε ratio compared to the WT. Moreover, a brief pulse activation of PKCε by Bryostatin-1 produced long-term survival in activated G93A-SOD1 degenerating cells in two different cell death paradigms (serum starvation and chemokines-induced toxicity). Altogether, the data support the implication of PKCε in ALS pathophysiology and suggests its pharmacological modulation as a potential neuroprotective strategy, at least in a subgroup of sporadic ALS patients.

Protein kinase Cα activation switches YAP1 from TEAD-mediated signaling to p73-mediated signaling.

Yes-associated protein 1 (YAP1) interacts with TEAD transcription factor in the nucleus and upregulates TEAD-target genes. YAP1 is phosphorylated by large tumor suppressor (LATS) kinases, the core kinases of the Hippo pathway, at 5 serine residues and is sequestered and degraded in the cytoplasm. In human cancers with the dysfunction of the Hippo pathway, YAP1 becomes hyperactive and confers malignant properties to cancer cells. We have observed that cold shock induces protein kinase C (PKC)-mediated phosphorylation of YAP1. PKC phosphorylates YAP1 at 3 serine residues among LATS-mediate phosphorylation sites. Importantly, PKC activation recruits YAP1 to the cytoplasm even in LATS-depleted cancer cells and reduces the cooperation with TEAD. PKC activation induces promyelocytic leukemia protein-mediated SUMOylation of YAP1. SUMOylated YAP1 remains in the nucleus, binds to p73, and promotes p73-target gene transcription. Bryostatin, a natural anti-neoplastic reagent that activates PKC, induces YAP1/p73-mediated apoptosis in cancer cells. Bryostatin reverses malignant transformation caused by the depletion of LATS kinases. Therefore, bryostatin and other reagents that activate PKC are expected to control cancers with the dysfunction of the Hippo pathway.

Development of a Novel In Vitro Primary Human Monocyte-Derived Macrophage Model To Study Reactivation of HIV-1 Transcription.

Latent HIV reservoirs persist in people living with HIV despite effective antiretroviral therapy and contribute to rebound viremia upon treatment interruption. Macrophages are an important reservoir cell type, but analysis of agents that modulate latency in macrophages is limited by lack of appropriate in vitro models. We therefore generated an experimental system to investigate this by purifying nonproductively infected human monocyte-derived macrophages (MDM) following in vitro infection with an M-tropic enhanced green fluorescent protein reporter HIV clone and quantified activation of HIV transcription using live-cell fluorescence microscopy. The proportion of HIV-infected MDM was quantified by qPCR detection of HIV DNA, and GFP expression was validated as a marker of productive HIV infection by colabeling of HIV Gag protein. HIV transcription spontaneously reactivated in latently infected MDM at a rate of 0.22% ± 0.04% cells per day (mean ± the standard error of the mean, n = 10 independent donors), producing infectious virions able to infect heterologous T cells in coculture experiments, and both T cells and TZM-bl cells in a cell-free infection system using MDM culture supernatants. Polarization to an M1 phenotype with gamma interferon plus tumor necrosis factor resulted in a 2.3-fold decrease in initial HIV infection of MDM (P < 0.001, n = 8) and a 1.4-fold decrease in spontaneous reactivation (P = 0.025, n = 6), whereas M2 polarization using interleukin-4 prior to infection led to a 1.6-fold decrease in HIV infectivity (P = 0.028, n = 8) but a 2.0-fold increase in the rate of HIV reactivation in latently infected MDM (P = 0.023, n = 6). The latency reversing agents bryostatin and vorinostat, but not panobinostat, significantly induced HIV reactivation in latently infected MDM (P = 0.031 and P = 0.038, respectively, n = 6). IMPORTANCE Agents which modulate latent HIV reservoirs in infected cells are of considerable interest to HIV cure strategies. The present study characterizes a robust, reproducible model enabling quantification of HIV reactivation in primary HIV-infected human MDM which is relatively insensitive to the monocyte donor source and hence suitable for evaluating latency modifiers in MDM. The rate of initial viral infection was greater than the rate of HIV reactivation, suggesting that different mechanisms regulate these processes. HIV reactivation was sensitive to macrophage polarization, suggesting that cellular and tissue environments influence HIV reactivation in different macrophage populations. Importantly, latently infected MDM showed different susceptibilities to certain latency-reversing agents known to be effective in T cells, indicating that dedicated strategies may be required to target latently infected macrophage populations in vivo.

Selective BCL-XL Antagonists Eliminate Infected Cells from a Primary-Cell Model of HIV Latency but Not from Ex Vivo Reservoirs.

HIV persists, despite immune responses and antiretroviral therapy, in viral reservoirs that seed rebound viremia if therapy is interrupted. Previously, we showed that the BCL-2 protein contributes to HIV persistence by conferring a survival advantage to reservoir-harboring cells. Here, we demonstrate that many of the BCL-2 family members are overexpressed in HIV-infected CD4+ T cells, indicating increased tension between proapoptotic and prosurvival family members-and suggesting that inhibition of prosurvival members may disproportionately affect the survival of HIV-infected cells. Based on these results, we chose to study BCL-XL due to its consistent overexpression and the availability of selective antagonists. Infection of primary CD4+ T cells with HIV resulted in increased BCL-XL protein expression, and treatment with two selective BCL-XL antagonists, A-1155463 and A-1551852, led to selective death of productively infected CD4+ T cells. In a primary cell model of latency, both BCL-XL antagonists drove reductions in HIV DNA and in infectious cell frequencies both alone and in combination with the latency reversing agent bryostatin-1, with little off-target cytotoxicity. However, these antagonists, with or without bryostatin-1 or in combination with the highly potent latency reversing agent combination phorbol myristate acetate (PMA) + ionomycin, failed to reduce total HIV DNA and infectious reservoirs in ex vivo CD4+ T cells from antiretroviral therapy (ART)-suppressed donors. Our results add to growing evidence that bona fide reservoir-harboring cells are resistant to multiple "kick and kill" modalities-relative to latency models. We also interpret our results as encouraging further exploration of BCL-XL antagonists for cure, where combination approaches, including with immune effectors, may unlock the ability to eliminate ex vivo reservoirs. IMPORTANCE Although antiretroviral therapy (ART) has transformed HIV infection into a manageable chronic condition, there is no safe or scalable cure. HIV persists in "reservoirs" of infected cells that reinitiate disease progression if ART is interrupted. Whereas most efforts to eliminate this reservoir have focused on exposing these cells to immune-mediated clearance by reversing viral latency, recent work shows that these cells also resist being killed. Here, we identify a "prosurvival" factor, BCL-XL, that is overexpressed in HIV-infected cells, and demonstrate selective toxicity to these cells by BCL-XL antagonists. These antagonists also reduced reservoirs in a primary-cell latency model but were insufficient to reduce "natural" reservoirs in ex vivo CD4+ T cells-adding to growing evidence that the latter are resilient in a way that is not reflected in models. We nonetheless suggest that the selective toxicity of BCL-XL antagonists to HIV-infected cells supports their prioritization for testing in combinations aimed at reducing ex vivo reservoirs.

Trimethylene Methane Dianion Equivalent for the Asymmetric Consecutive Allylation of Aldehydes: Applications to Prins-Driven Macrocyclizations for the Synthesis of Bryostatin 1 and Analogues.

We report a one-step (one-flask) generation and reaction of a bifunctional allylating reagent, a trimethylene methane dianion equivalent, that provides a route for the asymmetric 2-(trimethylsilylmethyl) allylation of aldehydes. The product of the first aldehyde allylation process is then set to engage in a second separate aldehyde allylation, providing an improved Prins macrocyclization strategy both for the scalable synthesis of bryostatin 1 and for the total synthesis of a new potent bryostatin analogue.

Spatiotemporal distribution of PKCα, Cdc42, and Rac1 before directed cell migration.

Cdc42 is a key factor in directed cell migration and accumulates at the leading edge of migrating cells. However, what kind of proteins control Cdc42 and when is unclear. After mechanical wounding, protein kinase C α (PKCα), a conventional PKC isozyme, begins to accumulate at the edges of cells adjacent to the wounded cells (WCs). In this study, we hypothesized that PKCα may be implicated in directed cell migration at an early stage before Cdc42 controls the migration. We focused on the spatiotemporal distribution of PKCα, Cdc42, and Rac1 before cell migration. After wounding, at the edges of cells adjacent to the WCs, PKCα accumulation, Cdc42 accumulation, Rac1 accumulation, and filopodia formation occurred in that order. The PKCα inhibitor suppressed Cdc42 accumulation at the cell edges. These results suggest that inhibition of PKCα activity inhibits cell migration. In addition, it is not Cdc42 but PKCα that may decide the direction of cell migration.

Rebound increases in chemokines by CXCR2 antagonist in breast cancer can be prevented by PKCδ and PKCε activators.

Activation of CXCR2 by chemokines such as CXCL1 and CXCL2 increases aggressiveness of breast cancer, inducing chemoresistance, hence CXCR2 antagonists are in clinical trials. We previously reported that inhibition of CXCR2 increases MIP-2 (CXCL2), which may inhibit anti-tumoral effects of CXCR2 antagonists. This seems to be due to inhibition of protein kinase C (PKC) by CXCR2 antagonist since specific inhibitor of PKC also enhances MIP-2 secretion. We here examined whether CXCR2 inhibitor also increases KC (CXCL1) secretion, ligand for CXCR2 involved in metastasis and PKC activators can prevent increases in chemokine secretion. We used SB 225002, which is a specific CXCR2 antagonist. The effects of PKC activators that have documented anti-tumoral effects and activates multiple isozymes of PKC such as Ingenol-3-angelate (I3A) and bryostatin-1 were examined here. In addition, FR236924, PKCε selective and 7α-acetoxy-6ß-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz), PKCδ selective activators were also tested. The effects of activators were determined using brain metastatic (4TBM) and heart metastatic (4THM) subset of 4T1 breast carcinoma cells because these aggressive carcinoma cells with cancer stem cell features secrete high levels of KC and MIP-2. Inhibition of CXCR-2 activity increased KC (CXCL1) secretion. PKC activators prevented SB225002-induced increases in KC and MIP-2 secretion. Different activators/modulators induce differential changes in basal and SB225002-induced chemokine secretion as well as cell proliferation and the activators that act on PKCδ and/or PKCε such as bryostatin 1, FR236924 and Roy-Bz are the most effective. These activators alone also decrease cell proliferation or chemokine secretion or both. Given the role of KC and MIP-2 in drug resistance including chemotherapeutics, activators of PKCε and PKCδ may prevent emerging of resistance to CXCR2 inhibitors as well as other chemotherapeutics.

Bryostatin-1 alleviates experimental multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disorder targeting the central nervous system (CNS). The relapsing-remitting phase of MS is largely driven by peripheral activation of autoreactive T-helper (Th) 1 and Th17 lymphocytes. In contrast, compartmentalized inflammation within the CNS, including diffuse activation of innate myeloid cells, characterizes the progressive phase of MS, the most debilitating phase that currently lacks satisfactory treatments. Recently, bryostatin-1 (bryo-1), a naturally occurring, CNS-permeable compound with a favorable safety profile in humans, has been shown to act on antigen-presenting cells to promote differentiation of lymphocytes into Th2 cells, an action that might benefit Th1-driven inflammatory conditions such as MS. In the present study, we show that bryo-1 provides marked benefit in mice with experimental autoimmune encephalomyelitis (EAE), an experimental MS animal model. Preventive treatment with bryo-1 abolishes the onset of neurologic deficits in EAE. More strikingly, bryo-1 reverses neurologic deficits after EAE onset, even when treatment is initiated at a late stage of disease when peak adaptive immunity has subsided. Treatment with bryo-1 in vitro promotes an anti-inflammatory phenotype in antigen-presenting dendritic cells, macrophages, and to a lesser extent, lymphocytes. These findings suggest the potential for bryo-1 as a therapeutic agent in MS, particularly given its established clinical safety. Furthermore, the benefit of bryo-1, even in late treatment of EAE, combined with its targeting of innate myeloid cells suggests therapeutic potential in progressive forms of MS.

HIV-1 infection and latency-reversing agents bryostatin-1 and JQ1 disrupt amyloid beta homeostasis in human astrocytes.

Since the introduction of the combined antiretroviral therapy, HIV-1 infection has become a manageable chronic disease in which patients display a life expectancy almost identical to the general population. Nevertheless, various age-related pathologies such as neurocognitive disorders have emerged as serious complications. A "shock and kill" strategy using latency-reversing agents (LRA) to reactivate HIV-1 has been proposed to eliminate the viral reservoir in such chronically infected patients. However, the impact of LRA on the central nervous system remains elusive. Given that an increased amyloid beta (Aß) deposition is a feature of HIV-1-infected brains, we investigated the consequences of HIV-1 infection and treatment with two LRA (bryostatin-1 and JQ1) on the capacity of human astrocytes to engulf and clear Aß. We show here that HIV-1-infected astrocytes accumulate a very high amount of Aß compared to uninfected cells, but the engulfed peptide in degraded very slowly. The LRA bryostatin-1 induces a reduction in Aß endocytosis, whereas JQ1 treatment results in a very slow degradation of the ingested material associated with a reduced expression of the endopeptidase neprilysin. An exposure to JQ1 also induces a sustained release of Aß-loaded microvesicles. Thus, both HIV-1 infection and treatment with some LRA could contribute to the reported Aß accumulation in the brain of HIV-1-infected persons.

Unlocking the Drug Potential of the Bryostatin Family: Recent Advances in Product Synthesis and Biomedical Applications.

Bryostatins are a class of naturally occurring macrocyclic lactones with a unique fast developing portfolio of clinical applications, including treatment of AIDS, Alzheimer's disease, and cancer. This comprehensive account summarizes the recent progress (2014-present) in the development of bryostatins, including their total synthesis and biomedical applications. An emphasis is placed on the discussion of bryostatin 1, the most-studied analogue to date. This review highlights the synthetic and biological challenges of bryostatins and provides an outlook on their future development.

Function-Oriented Synthesis: Design, Synthesis, and Evaluation of Highly Simplified Bryostatin Analogues.

Using a function-oriented synthesis strategy, we designed, synthesized, and evaluated the simplest bryostatin 1 analogues reported to date, in which bryostatin's A- and B-rings are replaced by a glutarate linker. These analogues, one without and one with a C26-methyl group, exhibit remarkably different protein kinase C (PKC) isoform affinities. The former exhibited bryostatin-like binding to several PKC isoforms with Ki's < 5 nM, while the latter exhibited PKC affinities that were up to ∼180-fold less potent. The analogue with bryostatin-like PKC affinities also exhibited bryostatin-like PKC translocation kinetics in vitro, indicating rapid cell permeation and engagement of its PKC target. This study exemplifies the power of function-oriented synthesis in reducing structural complexity by activity-informed design, thus enhancing synthetic accessibility, while still maintaining function (biological activity), collectively providing new leads for addressing the growing list of therapeutic indications exhibited by PKC modulators.

Munc13 Is a Molecular Target of Bryostatin 1.

Bryostatin 1 is a natural macrolide shown to improve neuronal connections and enhance memory in mice. Its mechanism of action is largely attributed to the modulation of novel and conventional protein kinase Cs (PKCs) by binding to their regulatory C1 domains. Munc13-1 is a C1 domain-containing protein that shares common endogenous and exogenous activators with novel and conventional PKC subtypes. Given the essential role of Munc13-1 in the priming of synaptic vesicles and neuronal transmission overall, we explored the potential interaction between bryostatin 1 and Munc13-1. Our results indicate that in vitro bryostatin 1 binds to both the isolated C1 domain of Munc13-1 ( Ki = 8.07 ± 0.90 nM) and the full-length Munc13-1 protein ( Ki = 0.45 ± 0.04 nM). Furthermore, confocal microscopy and immunoblot analysis demonstrated that in intact HT22 cells bryostatin 1 mimics the actions of phorbol esters, a previously established class of Munc13-1 activators, and induces plasma membrane translocation of Munc13-1, a hallmark of its activation. Consistently, bryostatin 1 had no effect on the Munc13-1H567K construct that is insensitive to phorbol esters. Effects of bryostatin 1 on the other Munc13 family members, ubMunc13-2 and bMunc13-2, resembled those of Munc13-1 for translocation. Lastly, we observed an increased level of expression of Munc13-1 following a 24 h incubation with bryostatin 1 in both HT22 and primary mouse hippocampal cells. This study characterizes Munc13-1 as a molecular target of bryostatin 1. Considering the crucial role of Munc13-1 in neuronal function, these findings provide strong support for the potential role of Munc13s in the actions of bryostatin 1.

Bryostatin-1 ameliorated experimental colitis in Il-10-/- Mice by protecting the intestinal barrier and limiting immune dysfunction.

Bryostatin-1 (Bry-1) has been proven to be effective and safe in clinical trials of a variety of immune-related diseases. However, little is known about its effect on Crohn's disease (CD). We aimed to investigate the impact of Bry-1 on CD-like colitis and determine the mechanism underlying this effect. In the present study, 15-week-old male Il-10-/- mice with spontaneous colitis were divided into positive control and Bry-1-treated (Bry-1, 30 µg/kg every other day, injected intraperitoneally for 4 weeks) groups. Age-matched, male wild-type (WT) mice were used as a negative control. The effects of Bry-1 on colitis, intestinal barrier function and T cell responses as well as the potential regulatory mechanisms were evaluated. We found that the systemic delivery of Bry-1 significantly ameliorated colitis in Il-10-/- mice, as demonstrated by decreases in the disease activity index (DAI), inflammatory score and proinflammatory mediator levels. The protective effects of Bry-1 on CD-like colitis included the maintenance of intestinal barrier integrity and the helper T cell (Th)/regulatory T cell (Treg) balance. These effects of Bry-1 may act in part through nuclear factor erythroid 2-related factor 2 (Nrf2) signalling activation and STAT3/4 signalling inhibition. The protective effect of Bry-1 on CD-like colitis suggests Bry-1 has therapeutic potential in human CD, particularly given the established clinical safety of Bry-1.

Bryostatin-1 inhibits cell proliferation of hepatocarcinoma and induces cell cycle arrest by activation of GSK3ß.

Bryostatin-1, a macrolide lactone derived from marine organism Bugula neritina, has been shown to inhibit carcinogenesis in several prospective clinical trials. In the current study, the therapeutic potential of bryostatin-1 in inhibiting proliferation of hepatocarcinoma was evaluated by in vitro and in vivo studies. The mechanisms of action of bryostatin-1 were predicted by in silico assay and further validated by surface plasmon resonance and western blot assay. Our results show that bryostatin-1 (100, 200 nM) treatment can suppress cell proliferation and induce G1 cell cycle arrest in PLC/PRF/5 and SMCC7721 cell. We also found a significant inhibitory action of bryostatin-1 (100, 200 nM) on CyclinD1 activity in PLC/PRF/5 cells, and bryostatin-1 can promote ubiquitination-dependent protein degradation of CyclinD1 in PLC/PRF/5 cells. Western blot results confirmed that the active form phospho-GSK3ß Tyr216 expression was increased significantly after bryostatin-1 treatment. Activation of GSK3ß might be responsible for bryostatin-1 induced cyclinD1 degradation and cell cycle arrest. Taken together, bryostatin-1 may inhibit HCC cells proliferation by promoting cyclinD1 proteolysis and inducing cell cycle arrest.

Class 1-Selective Histone Deacetylase (HDAC) Inhibitors Enhance HIV Latency Reversal while Preserving the Activity of HDAC Isoforms Necessary for Maximal HIV Gene Expression.

Combinations of drugs that affect distinct mechanisms of HIV latency aim to induce robust latency reversal leading to cytopathicity and elimination of the persistent HIV reservoir. Thus far, attempts have focused on combinations of protein kinase C (PKC) agonists and pan-histone deacetylase inhibitors (HDIs) despite the knowledge that HIV gene expression is regulated by class 1 histone deacetylases. We hypothesized that class 1-selective HDIs would promote more robust HIV latency reversal in combination with a PKC agonist than pan-HDIs because they preserve the activity of proviral factors regulated by non-class 1 histone deacetylases. Here, we show that class 1-selective agents used alone or with the PKC agonist bryostatin-1 induced more HIV protein expression per infected cell. In addition, the combination of entinostat and bryostatin-1 induced viral outgrowth, whereas bryostatin-1 combinations with pan-HDIs did not. When class 1-selective HDIs were used in combination with pan-HDIs, the amount of viral protein expression and virus outgrowth resembled that of pan-HDIs alone, suggesting that pan-HDIs inhibit robust gene expression induced by class 1-selective HDIs. Consistent with this, pan-HDI-containing combinations reduced the activity of NF-κB and Hsp90, two cellular factors necessary for potent HIV protein expression, but did not significantly reduce overall cell viability. An assessment of viral clearance from in vitro cultures indicated that maximal protein expression induced by class 1-selective HDI treatment was crucial for reservoir clearance. These findings elucidate the limitations of current approaches and provide a path toward more effective strategies to eliminate the HIV reservoir.IMPORTANCE Despite effective antiretroviral therapy, HIV evades eradication in a latent form that is not affected by currently available drug regimens. Pharmacologic latency reversal that leads to death of cellular reservoirs has been proposed as a strategy for reservoir elimination. Because histone deacetylases (HDACs) promote HIV latency, HDAC inhibitors have been a focus of HIV cure research. However, many of these inhibitors broadly affect multiple classes of HDACs, including those that promote HIV gene expression (class 1 HDACs). Here, we demonstrate that targeted treatment with class 1-selective HDAC inhibitors induced more potent HIV latency reversal than broadly acting agents. Additionally, we provide evidence that broadly acting HDIs are limited by inhibitory effects on non-class 1 HDACs that support the activity of proviral factors. Thus, our work demonstrates that the use of targeted approaches to induce maximum latency reversal affords the greatest likelihood of reservoir elimination.