Development of a High Throughput Essay for Inhibitor of ACSVL3, a Therapeutic Target in Malignant Glioma

Gliomas account for more than half of brain tumors, and nearly two-thirds of gliomas are highly aggressive glioblastomas. Despite recent therapeutic advances, glioblastoma carries a very poor prognosis. Thus, new treatment strategies and novel therapeutic targets are urgently needed. We recently found that ACSVL3, an enzyme of fatty acid metabolism that is not expressed in normal glia, is found at extremely high levels in human gliomas and glioblastoma cell lines. Depletion of ACSVL3 in glioblastoma cells by RNA interference decreased their malignant behavior both in culture (decreased growth rate; decreased colony formation in soft agar) and in both subcutaneous and intracranial xenografts (decreased tumor initiation; decreased tumor growth).

Importantly, cancer stem-like cells (neurospheres) prepared from three glioma cell lines had even higher ACSVL3 expression levels than the tumor cell lines; differentiation of stem-like cells in culture with retinoic acid reduced ACSVL3 expression to low to undetectable levels. Taken together, our data suggests that ACSVL3, one of 26 human fatty acyl-CoA synthetases, is a viable therapeutic target. However, there are no known small molecule inhibitors of ACSVL3.

In an effort to work toward developing high-throughput screening of chemical libraries to identify candidate ACSVL3 inhibitors, we propose to pursue the following goals:

1. To produce purified, stable, enzymatically active recombinant ACSVL3 for use in high-throughput screening assays
2. To develop and validate a colorimetric assay for ACSVL3 enzyme activity in 96-well format that is suitable for identification of small molecule inhibitors.

To achieve goal 1, we will first express ACSVL3 with various N-terminal (hydrophobic, membrane-association domain) deletions in E. coli and assess enzyme activity, solubility, and stability. Expression in the yeast Pichia pastoris and in mammalian cells are alternatives. The effects of various enzyme stabilizing agents will be assessed. Currently, enzyme activity is assessed using a radiochemical assay that is labor-intensive and impractical for high-throughput screening. Thus, our second goal will be to adapt a colorimetric assay that uses a dye, malachite green (MG), to quantitate enzymes whose products include either inorganic phosphate or pyrophosphate. The assay will be scaled to 96-well format and validated with respect to linearity with time and enzyme concentration, optimization of substrate concentration, and lack of interference by DMSO or serum albumin.

If successful, this proposal will allow us to proceed immediately to screening of chemical libraries for inhibitory compounds using high-throughput liquid handling robotic platforms.