Project Title: Bioinformatics analysis on Bryostatin
By: Lim Yun Ping

Introduction

The marine ecosystem has increasingly been the focus of interest for new discoveries in the field that are expected to be of significant therapeutic impact in cancer patients. Researchers have mined the wealth of natural compounds found in marine organisms and discovered many with potential for use in pharmaceuticals.

Examples include

• A compound derived from mangrove tunicates as a potential potent anti-tumor treatment; those from corals have potent anti-cancer activity.
• A bacteria living inside the brown bryozoans colonies called Bugula neritina - may be the source of a new family of drugs, bryostatins, being developed to fight a variety of cancers. Bryostatin is now in clinical trials for use in humans. Bugula neritina colonies are also shown to be effective against leukaemia
• The compound manoalide from a Pacific sponge, for example, has spawned more than 300 chemical analogs, with a significant number of these going on to clinical trials as anti-inflammatory agents.

Bryostatins are a group of macrocyclic lactones first discovered in the late 1960s in a species of bryozoan, Bugula neritina. It is believed to be produced by symbiont bacteria to protect the bryozoan larva from predation, they have cytotoxic properties and are under investigation as anti-cancer agents and as a memory enhancement agent. Bryostatin has been shown to be a potent activator of protein kinase C.

In vitro trials have shown bryostatins to act synergistically with other anti-cancer drugs and to modulate protein kinase C (PKC) activity, with a potent antileukemic effect and action against lung, prostate and non-Hodgkin's lymphoma tumor cells. Human clinical trials have been less promising, but suggest bryostatins to have a potentially useful synergistic action with other chemotherapeutic agents.

Bryostatin has appeared very promising enhancing memory in animal models. Bryostatin was able to increase the duration of memory retention of the marine slug Hermissenda Crassicornis by over 50%, and was able to dramatically increase the rate of learning in rats. It has been rumored that it is now being investigated in human testing, possibly for treatment of Alzheimer's disease. Bryostatin is thought to potentiate memory by activating PKC.

The low concentration in bryozoans (to extract one gram of bryostatin, roughly one tonne of the raw bryozoans is needed) makes extraction unviable for large scale production. Due to the structural complexity, synthesis has proved difficult, with only a few total syntheses reported so far. However, structurally simpler synthetic analogs have been prepared which exhibit similar biological profile and in some cases greater potency, which may provide a practical supply for clinical use.

More information was obtained using
1. Entrez as the starting point,

From Pubmed:

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. Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6624-9.

Dendritic cells (DC) play a major role in priming naive T cells and modulating the immune response. We have previously reported that bryostatin-1, a potent immune modulator with antitumor activity, activates monocytes and lymphocytes to produce cytokines. Studies have shown that tumor-bearing hosts have a Th1/Th2 cytokine pattern that is associated with decreased production of IFN-gamma. We investigated the expression of IFN-gamma in bryostatin-1-treated human DC. Bryostatin-1 induced both IFN-gamma and T-bet mRNA expression in a dose- and time-dependent manner. As little as 1 ng/ml bryostatin-1 induced IFN-gamma and T-bet transcripts within 3 h and protein at 12 h. Treatment of DC with cycloheximide revealed that bryostatin-1-induced T-bet expression requires de novo protein synthesis, but bryostatin-1-induced IFN-gamma expression is independent of protein synthesis. Furthermore, dexamethasone inhibits bryostatin-1-induced IFN-gamma mRNA expression but increases bryostatin-1-induced T-bet mRNA expression. Experiments with ERK-1/2 inhibitors demonstrated that bryostatin-1 induction of IFN- gamma and T-bet was ERK-dependent and IL-12-independent. Similar results were obtained from both normal donors and cancer patients. In summary, our results suggest that bryostatin-1-induced IFN-gamma expression is T-bet independent. They also suggest for the first time that IFN- gamma and T-bet can be induced in human DC through an ERK-dependent pathway.

Bryostatin-1-induced IFN- gamma may play a crucial role in the initiation of the immune response, before specific recognition by T cells that could be beneficial in the treatment of cancer.
ref: J Immunol 2006 Sep 15;177(6):3554-63.

Pathway: http://www.biocarta.com/pathfiles/h_erk5Pathway.asp

Bryostatin 1, like phorbol 12-myristate 13-acetate (PMA), is a potent activator of protein kinase C (PKC). Modulation of PKC represents a novel approach to cancer therapy. Protein kinase C (PKC) plays an important role in cell proliferation, differentiation, and apoptosis. Protein kinase C (PKC) is a family of serine-threonine protein kinases that are involved in signal transduction pathways that regulate growth factor response, proliferation, and apoptosis. Its central role in these processes, which are closely involved in tumor initiation, progression, and response to antitumor agents, makes it an attractive therapeutic target in cancer.

Bryostatin-1 is a powerful protein kinase C (PKC) agonist, activating PKC isozymes at nanomolar concentrations. Pharmacological studies of bryostatin-1 have mainly been focused on its action in preventing tumor growth. Emerging evidence suggests, however, that bryostatin-1 exhibits additional important pharmacological activities. In preclinical studies bryostatin-1 has been shown at appropriate doses to have cognitive restorative and antidepressant effects. The underlying pharmacological mechanisms may involve an activation of PKC isozymes, induction of synthesis of proteins required for long-term memory, restoration of stress-evoked inhibition of PKC activity, and reduction of neurotoxic amyloid accumulation and tau protein hyperphosphorylation. The therapeutic potential of bryostatin-1 as a CNS drug should be further explored. CNS Drug Rev. 2006 Spring;12(1):1-8. Review.

Bryostatin-1 is a macrocyclic lactone derived from a marine invertebrate that binds to the regulatory domain of protein kinase C. Short-term exposure to bryostatin-1 promotes activation of PKC, whereas prolonged exposure promotes significant downregulation of PKC. In numerous hematological and solid tumor cell lines, bryostatin-1 inhibits proliferation, induces differentiation, and promotes apoptosis. Furthermore, preclinical studies indicate that bryostatin-1 potently enhances the effect of chemotherapy. In many cases, this effect is sequence specific. Bryostatin-1 is currently in phase I and phase II clinical trials. The major toxicities are myalgias, nausea, and vomiting. Although there is minimal single-agent activity, combinations with standard chemotherapy are providing very encouraging results and indicate a new direction in cancer therapy. Cancer Invest 2003;21(6):924-36.

Bryostatin induces protein kinase C modulation, Mcl-1 up-regulation and phosphorylation of Bcl-2 resulting in cellular differentiation and resistance to drug-induced apoptosis in B-cell chronic lymphocytic leukemia cells. Leuk Lymphoma 2004 May;45(5):997-1008.

STAT transcription factors as important mediators of Bryostatin 1-induced differentiation of CLL cells and could possibly lead to improved therapeutic approaches for the treatment of chronic lymphocytic leukemia (CLL). Blood 2003 Oct 15;102(8):3016-24. Epub 2003 Jul 10.

Further bioinformatics analysis was carried out to find out more about the Bryostatin gene and protein.