Topical, immunomodulatory epoxy-tiglianes induce biofilm disruption and healing in acute and chronic skin wounds.

The management of antibiotic-resistant, bacterial biofilm infections in chronic skin wounds is an increasing clinical challenge. Despite advances in diagnosis, many patients do not derive benefit from current anti-infective/antibiotic therapies. Here, we report a novel class of naturally occurring and semisynthetic epoxy-tiglianes, derived from the Queensland blushwood tree (Fontainea picrosperma), and demonstrate their antimicrobial activity (modifying bacterial growth and inducing biofilm disruption), with structure/activity relationships established against important human pathogens. In vitro, the lead candidate EBC-1013 stimulated protein kinase C (PKC)-dependent neutrophil reactive oxygen species (ROS) induction and NETosis and increased expression of wound healing-associated cytokines, chemokines, and antimicrobial peptides in keratinocytes and fibroblasts. In vivo, topical EBC-1013 induced rapid resolution of infection with increased matrix remodeling in acute thermal injuries in calves. In chronically infected diabetic mouse wounds, treatment induced cytokine/chemokine production, inflammatory cell recruitment, and complete healing (in six of seven wounds) with ordered keratinocyte differentiation. These results highlight a nonantibiotic approach involving contrasting, orthogonal mechanisms of action combining targeted biofilm disruption and innate immune induction in the treatment of chronic wounds.

EBC-232 and 323: A Structural Conundrum Necessitating Unification of Five In Silico Prediction and Elucidation Methods.

Structurally unique halimanes EBC-232 and EBC-323, isolated from the Australian rainforest plant Croton insularis, proved considerably difficult to elucidate. The two diastereomers, which consist an unusual oxo-6,7-spiro ring system fused to a dihydrofuran, were solved by unification and consultation of five in silico NMR elucidation and prediction methods [i.e., ACDLabs, olefin strain energy (OSE), DP4, DU8+ and TD DFT CD]. Structure elucidation challenges of this nature are prime test case examples for empowering future AI learning in structure elucidation.

BRN2 expression increases anoikis resistance in melanoma.

Melanoma tumors are highly heterogeneous, comprising of many cell populations that vary in their potential for growth and invasion. Differential transcription factor expression contributes to these phenotypic traits. BRN2, a member of the POU domain family of transcription factors is thought to play important roles in melanoma invasion and metastasis. However, the function of BRN2 during the metastatic process of melanoma remains largely unknown. We therefore investigated the effect of BRN2 expression in melanoma cells with no or low constitutive expression using a doxycycline-inducible system. Induction of BRN2 expression led to reduced proliferation and partial resistance to an inhibitor of mutated BRAF. Whole-genome profiling analysis revealed novel targets and signaling pathway changes related to prevention of cell death induced by detachment from the extracellular matrix, known as anoikis resistance. Further investigation confirmed increased survival of BRN2-expressing cell lines in non-adherent conditions. Functionally, expression of BRN2 promoted induction of c-MET levels as well as increased phosphorylation of STAT3. Treatment with crizotinib, a c-MET inhibitor, decreased cellular viability of BRN2-expressing cells under non-adherent conditions to death by anoikis. Alternative inhibitors of c-MET showed similar results. These results highlight the importance of a largely overlooked transcription factor in the progression and metastasis of melanoma, and may suggest a strategy to target BRN2-expressing cells resistant to therapy and cell death by anoikis.

New Casbanes and the First trans-Cyclopropane seco-Casbane from the Australian Rainforest Plant Croton insularis.

Investigation of the Australian rainforest plant Croton insularis revealed seven new cis-, two new trans-cyclopropane casbanes, and the first trans-cyclopropane seco-casbane. The relative configuration of the cyclopropane moiety for all compounds (EBC-182, 217, 218, 220, 343, 357, 358, 361, 365, 373; EBC=EcoBiotics Compound) was assigned using 13 C NMR data. Comparison of the experimental electronic circular dichroism (ECD) spectra with the theoretical curves, calculated by TD-DFT at the B3LYP/6-31+G**//B3LYP/6-31+G** level, in conjunction with NOE data afforded the absolute configuration. EBC-180, 181 and 220 displayed potent activity against cervical carcinoma (HeLa cells).

MITF and BRN2 contribute to metastatic growth after dissemination of melanoma.

Melanoma tumors are highly heterogeneous, comprising of different cell types that vary in their potential for growth and invasion. Heterogeneous expression of the Microphthalmia-associated Transcription Factor (MITF) and the POU domain transcription factor BRN2 (POU3F2) has been found in malignant melanoma. Changing expression of these transcription factors as the disease progresses has been linked to the metastatic mechanism of phenotype switching. We therefore investigated the effects of MITF and BRN2 expression in melanoma growth and metastasis. Depletion of MITF resulted in a cell population that had a slowed cell cycle progression, was less invasive in vitro and had hindered tumor and metastasis forming ability in mouse xenograft studies. BRN2 depletion left a cell population with intact proliferation and invasion in vitro; however metastatic growth was significantly reduced in the mouse xenograft model. These results suggest that the proliferative population within melanoma tumors express MITF, and both MITF and BRN2 are important for metastatic growth in vivo. This finding highlights the importance of BRN2 and MITF expression in development of melanoma metastasis.

The First Casbane Hydroperoxides EBC-304 and EBC-320 from the Australian Rainforest.

Investigation of the Australian rainforest plant Croton insularis led to isolation of the first casbane hydroperoxide diterpenes EBC-304 and EBC-320. Extensive DFT and electronic circular dichroism (ECD) calculations in combination with 2D NMR spectroscopy determined the absolute configurations. EBC-304 and EBC-320 both display significant cytotoxicity.

Expression profiling of cutaneous squamous cell carcinoma with perineural invasion implicates the p53 pathway in the process.

Squamous cell carcinoma (SCC) is the second most common cancer worldwide and accounts for approximately 30% of all keratinocyte cancers. The vast majority of cutaneous SCCs of the head and neck (cSCCHN) are readily curable with surgery and/or radiotherapy unless high-risk features are present. Perineural invasion (PNI) is recognized as one of these high-risk features. The molecular changes during clinical PNI in cSCCHN have not been previously investigated. In this study, we assessed the global gene expression differences between cSCCHN with or without incidental or clinical PNI. The results of the analysis showed signatures of gene expression representative of activation of p53 in tumors with PNI compared to tumors without, amongst other alterations. Immunohistochemical staining of p53 showed cSCCHN with clinical PNI to be more likely to exhibit a diffuse over-expression pattern, with no tumors showing normal p53 staining. DNA sequencing of cSCCHN samples with clinical PNI showed no difference in mutation number or position with samples without PNI, however a significant difference was observed in regulators of p53 degradation, stability and activity. Our results therefore suggest that cSCCHN with clinical PNI may be more likely to contain alterations in the p53 pathway, compared to cSCCHN without PNI.

IL-1 Contributes to the Anti-Cancer Efficacy of Ingenol Mebutate.

Ingenol mebutate is approved for the topical treatment of actinic keratoses and may ultimately also find utility in treating skin cancers. Here we show that relapse rates of subcutaneous B16 melanoma tumours treated topically with ingenol mebutate were not significantly different in C57BL/6 and Rag1-/- mice, suggesting B and T cells do not play a major role in the anti-cancer efficacy of ingenol mebutate. Relapse rates were, however, significantly increased in MyD88-/- mice and in C57BL/6 mice treated with the anti-IL-1 agent, anakinra. Ingenol mebutate treatment induces a pronounced infiltration of neutrophils, which have been shown to have anti-cancer activity in mice. Herein we provide evidence that IL-1 promotes neutrophil recruitment to the tumour, decreases apoptosis of infiltrating neutrophils and increases neutrophil tumour killing activity. These studies suggest IL-1, via its action on neutrophils, promotes the anti-cancer efficacy of ingenol mebutate, with ingenol mebutate treatment causing both IL-1ß induction and IL-1α released from keratinocytes.

Validating Eaton's Hypothesis: Cubane as a Benzene Bioisostere.

Pharmaceutical and agrochemical discovery programs are under considerable pressure to meet increasing global demand and thus require constant innovation. Classical hydrocarbon scaffolds have long assisted in bringing new molecules to the market place, but an obvious omission is that of the Platonic solid cubane. Eaton, however, suggested that this molecule has the potential to act as a benzene bioisostere. Herein, we report the validation of Eaton's hypothesis with cubane derivatives of five molecules that are used clinically or as agrochemicals. Two cubane analogues showed increased bioactivity compared to their benzene counterparts whereas two further analogues displayed equal bioactivity, and the fifth one demonstrated only partial efficacy. Ramifications from this study are best realized by reflecting on the number of bioactive molecules that contain a benzene ring. Substitution with the cubane scaffold where possible could revitalize these systems, and thus expedite much needed lead candidate identification.

Ectopic expression of protein kinase C-ß sensitizes head and neck squamous cell carcinoma to diterpene esters.

BACKGROUND: The objective of this study was to examine the effect of specific Protein kinase C (PKC) isoform re-expression in solid malignancies, particularly head and neck squamous cell carcinoma cell lines, and the impact this may have on treatment with known activators of PKC. MATERIALS AND METHODS: The constitutive expression of PKC isoforms were determined in six head and neck squamous cell carcinoma (SCC) cell lines. Cytotoxicity of the prototypic phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and the novel diterpene ester PEP005 was established. Viral transduction to re-express PKCß isoforms in two of these cell lines was performed, and its effect on the sensitivity to the compounds was quantified. RESULTS: Tongue and hypopharyngeal SCC cell lines were resistant to both TPA and PEP005, with the concentration required to inhibit growth by 50% (IC50) being >1,000 ng/ml. CAL-27 (tongue SCC) and FaDu (hypopharyngeal SCC) cell lines re-expressing PKCßI and -ßII isoforms demonstrated IC50 of 1-5 ng/ml with TPA or PEP005. CONCLUSION: Re-expression of PKCß in head and neck SCC cell lines leads to cells one thousand-times more sensitive to the cytotoxic effects of phorbol or diterpene esters in culture. This highlights the importance of the isoform in tumor progression and presents the potential benefit of these compounds in malignancies expressing the protein, and in combination therapy.

Intra-lesional injection of the novel PKC activator EBC-46 rapidly ablates tumors in mouse models.

Intra-lesional chemotherapy for treatment of cutaneous malignancies has been used for many decades, allowing higher local drug concentrations and less toxicity than systemic agents. Here we describe a novel diterpene ester, EBC-46, and provide preclinical data supporting its use as an intra-lesional treatment. A single injection of EBC-46 caused rapid inflammation and influx of blood, followed by eschar formation and rapid tumor ablation in a range of syngeneic and xenograft models. EBC-46 induced oxidative burst from purified human polymorphonuclear cells, which was prevented by the Protein Kinase C inhibitor bisindolylmaleimide-1. EBC-46 activated a more specific subset of PKC isoforms (PKC-ßI, -ßII, -α and -γ) compared to the structurally related phorbol 12-myristate 13-acetate (PMA). Although EBC-46 showed threefold less potency for inhibiting cell growth than PMA in vitro, it was more effective for cure of tumors in vivo. No viable tumor cells were evident four hours after injection by ex vivo culture. Pharmacokinetic profiles from treated mice indicated that EBC-46 was retained preferentially within the tumor, and resulted in significantly greater local responses (erythema, oedema) following intra-lesional injection compared with injection into normal skin. The efficacy of EBC-46 was reduced by co-injection with bisindolylmaleimide-1. Loss of vascular integrity following treatment was demonstrated by an increased permeability of endothelial cell monolayers in vitro and by CD31 immunostaining of treated tumors in vivo. Our results demonstrate that a single intra-lesional injection of EBC-46 causes PKC-dependent hemorrhagic necrosis, rapid tumor cell death and ultimate cure of solid tumors in pre-clinical models of cancer.

Croton insularis introduces the seco-casbane class with EBC-329 and the first casbane endoperoxide EBC-324.

Investigation of Croton insularis afforded the first in class seco-casbane diterpene, EBC-329. A highly oxidised casbane, EBC-324, was also isolated. The structural motif within EBC-324, which consists of an epoxidised hemi-acetal endoperoxide, is new to the casbane family.

Unprecedented 1,14-seco-crotofolanes from Croton insularis: oxidative cleavage of crotofolin C by a putative homo-Baeyer-Villiger rearrangement.

EBC-162 isolated from Croton insularis, obtained from the northern rainforest of Australia, was structurally affirmed as crotofolin C (4). Novel oxidative degradation products, EBC-233 and EBC-300, which are the first crotofolane endoperoxides, were also isolated. Both endoperoxides were found to be stable intermediates, which are proposed to undergo an unprecedented homo-Baeyer-Villiger biosynthetic rearrangement to give a new class of 1,14-seco-crotofolane diterpenes. Prolonged storage of all isolates assisted in authenticating their natural product status. Anticancer activities of reported compounds are presented.

EBC-219: a new diterpene skeleton, crotinsulidane, from the Australian rainforest containing a bridgehead double bond.

EBC-219 (4), isolated from Croton insularis (Baill), was established by spectroscopic and DFT methods as the first member of a new diterpene skeletal class, uniquely defined by the presence of a bicyclo[10.2.1] bridgehead olefin. The proposed biogenetic pathway to 4 from the co-isolated natural products EBC-131 (1), EBC-180 (2) and EBC-181 (3) is highly likely. EBC-180 (2) and EBC-181 (3) showed moderate to strong cytotoxic activity against various cancer cell lines.

Isolation and confirmation of the proposed cleistanthol biogentic link from Croton insularis.

The proposed cleistanthol biosynthetic intermediate en route to spruceanol, and other related family members, was isolated for the first time from Croton insularis, confirming the Jacobs-Reynolds hypothesis. Anticancer evaluation of the new isolates and their aerial oxidation products is also reported.

The induction of senescence-like growth arrest by protein kinase C-activating diterpene esters in solid tumor cells.

We previously identified the induction of senescence in melanoma cell lines sensitive to diterpene esters, indicating a therapeutic potential. Here we compared the cytostatic effects of two diterpene esters: the prototypic PKC-activating drug TPA (12-O-tetradecanoylphorbol-13-acetate), and the novel compound PEP008 (20-O-acetyl-ingenol-3-angelate) in cell lines derived from melanoma, breast cancer and colon cancer. The diterpene esters induced permanent growth arrest with characteristics of senescence in a subset of cell lines in all three solid tumor models at 100-1000 ng/ml. Use of the PKC inhibitor bisindolylmaleimide-l demonstrated that activation of PKC was required for growth arrest. Full genome expression profiling identified pivotal genes involved in DNA synthesis and cell cycle control down-regulated by treatment in all three sensitive tumor models. At the protein level, prolonged down-regulation of E2F-1 and proliferating cell nuclear antigen (PCNA), sustained expression of p21(WAF1/CIP1) and dephosphorylation of retinoblastoma (Rb) occurred in the sensitive cells. Additionally, the type II tumor suppressor HRASLS3, which has a role in mitogen-activated protein kinase (MAPK) pathway suppression, was constitutively elevated in cell lines resistant to the senescence effects compared to their sensitive counterparts. Together, these results demonstrate that both TPA and the novel PKC-activating drug PEP008 induce growth arrest with characteristics of senescence in solid tumor cell lines derived from a variety of tissue types, and by a similar mechanism. PKC-activating diterpene esters may therefore have therapeutic potential in a subset of breast cancer, colon cancer and melanoma tumors.