Previous work performed by the Drug Discovery Group, together with QBiotics Ltd. (an Australian based biotech company situated in North Queensland), has led to the identification of a novel protein kinase C (PKC)-activating agent, EBC-46, from a rain forest tree. Intratumoral injection of EBC-46 gives long term ablation, approaching cure, of cutaneous tumors in dogs, cats and horses. We have also shown at QIMR Berghofer that intratumoral injection of EBC-46 ablates subcutaneous tumor xenografts of human cell lines in nude mice, with few recurrences. Commercial development of the compound is underway, with a Phase I trial in humans. Localised hemorrhagic necrosis appears to be the overt mechanism of efficacy in vivo but the molecular subtleties behind this and the potential to exploit them for enhancing efficacy are still being explored. A number of analogues of EBC-46 have been isolated from the same plant, and many more have been generated by semisynthesis. These include candidates in development for indications other than cancer.
Although EBC-46 is a bone fidePKC activating agent we have a selection of in vitrodata which suggests that the anti-tumor efficacy of this drug may also involve PKC-independent pathways. To this extent, we are interested in identifying further molecular targets of EBC-46. The following aims are a guide to the work that could be done in this area, although this may be subject to change.
- Identification of potential EBC-46 targets.
- Development of a suitable protocol to isolate EBC-46 interacting proteins from cells in culture. Various EBC-46 analogues, modified for use in click chemistry/Halotag based assays, will be used to pull down EBC-46 interacting proteins for subsequent mass spectroscopy (MS) based identification.
- Identification of interacting proteins using MS based peptide mass fingerprinting. Subsequent pulldowns will be processed via SDS-PAGE/on column digests and identification performed using MS/MS based peptide sequencing and database searches.
- Confirmation of MS derived hits. Drug conjugated resin will be used in pull down experiments with cell lysates and potential targets identified via MS will be confirmed via immunoblotting.
- Determine physiological significance of identified targets using siRNA/CRISPR knockdown/knockout methodology.
- 1 or 2 targets identified in Aim 1 will be ablated using either siRNA/CRISPR based technology, to understand their role in EBC-46 mediated anti-tumor efficacy.
Supervisor: Dr Jason Cullen: +61 7 3845 3741, Jason.Cullen@qimrberghofer.edu.au