TI-3D Research Highlights : Walter Fast : Pseudomonas DDAH
Tissue, cell and small-molecule libraries for diagnosing and treating melanoma
In collaboration with Suhendan Ekmekcioglu
Our goals for this project have been to 1) to develop small-molecule inhibitors of DDAH, along with a paired diagnostic reagent; and 2) to develop a melanoma cell line microarray and a plate-based panel of melanoma cell lines to evaluate the potency, efficacy and biochemical pathways involved in killing melanoma by these DDAH/NOS inhibitors.
At UT-Austin, we have developed high-throughput screens for two different DDAH isoforms (Z’ factors > 0.8). 6500 compounds have been screened as inhibitors of one DDAH isoform, with a second isoform underway. 50 compounds inhibit ≥ 50%. Secondary assays and hit analyses are now prioritizing future work. This work has already provided adequate preliminary data to justify an NIH screening grant, although patent protection for some compounds is under consideration.
Along with collaborators at UT- MDACC, we have established a melanoma cell line microarray (MCL-MA) containing 60 lines, including radial and vertical growth phases of primary melanoma lines, metastatic melanoma cells and normal control cells, such as human melanocytes, fibroblasts and keratinocytes. Initial stainings to show tissue (in this case, individual cell line pellets) recovery and to test staining techniques, such as antigen retrieval process, were successful. We have established a database to dissect characteristics of these lines, such as mutational status, some of which play a role in NO signaling. We are currently standardizing conditions for a 96-well format to determine DDAH inhibitor efficacy for growth inhibition and cell killing, and to correlate these to the molecular aspects of the biologically diverse cell lines.