73-Deoxychondropsin A: A specific inhibitor of resorption and osteosarcoma?

Previous work by the Long/Grigoriadis labs have identified a novel role for 73-deoxychondropsin A (73-DOC) in bone biology. 73-DOC has been isolated from the Great Barrier Reef sponge, I. ramosa, and purified by Paul Long in collaboration with the Australian Institute of Marine Sciences (AIMS). 73-DOC exhibits potent inhibitory activity to mammalian V-ATPases. V-ATPases have a role in fundamental cellular physiology processes through regulation of intra/extracellular pH. Additionally, for bone tissue, V-ATPases are critically important in bone-resorbing osteoclasts which require V-ATPases for normal function. Preliminary and yet unpublished results from the Long/Grigoriadis labs have established a unique role for 73-DOC in targeting osteoclasts, although due to the complexity of the V-ATPase protein complex, the precise targets of 73-DOC and its mechanism of action are not yet known. This project will investigate the specific effects of 73-DOC on osteoclastic resorption and extend this to a bone cancer model in which osteoclasts are implicated in tumour progression.
The hypothesis of this project is that 73-DOC specifically inhibits osteoclast resorption, without affecting bone remodeling by targeting V-ATPase subunit isoforms shared between osteoclasts and tumors such as osteosarcoma and thus should effectively inhibit resorption and tumor pathogenesis. Overall, the effects of 73-DOC administration on osteoclast resorption and osteosarcoma will be characterized comprehensively using a combined phenotypic, biochemical, and proteomic approach.

Specific aims:

Characterize the effect of 73-DOC administration on the phenotype and functioning of osteoclast and osteosarcoma cells.

Determine whether 73-DOC inhibits V-ATPase activity by targeting specific subunit isoforms and determine the molecular mechanics of 73-DOC mediated V-ATPase inhibition.

Assess whether 73-DOC mediated inhibition of specific V-ATPases isoforms is a viable approach to therapeutically managing osteolytic diseases and osteosarcoma.
The plan of work will follow a logical progression from purification of new 73-DOC compound, in vitro assays on osteoclast differentiation from haematopoietic stem cells, functional resorption assays and quantification by surface profilometry, osteosarcoma cell growth and activity, cytoskeletal analysis/confocal imaging, mass spec/proteomics analysis, and in vivo tumour modelling. The project therefore will provide the student with a variety of diverse and complementary skills, including cell biology and mammalian cell culture, molecular analyses (qPCR, RNAi strategies), protein biochemistry/mass spec, confocal microscopy, and in vivo models of tumour development, including histological and immunolocalisation analyses. These research methods will ensure that the specified aims are met within the time-frame of the PhD.