Identification of Inhibitors of Acsvl3, a Therapeutic Target in Glioma

Abstract

The objective of this study was to identify inhibitors of the enzyme Very Long Chain Acyl‐CoA Synthetase 3 (ACSVL3; SLC27A3). ACSVL3 is present in human glioma cells but not healthy glial cells, and knocking out ACSVL3 in the U87 human glioblastoma cell line has previously been shown to result in slower growth rate, fewer and smaller tumors formed in xenografts, and disrupted Akt signaling. Therefore, an inhibitor of its activity is desired for both understanding its mechanism of action in tumorigenesis and for potential chemotherapeutic usage. Several drugs and compounds that were previously shown to inhibit the homologous enzyme ACSVL1 (SLC27A2) were studied to see if they had similar inhibitory effects on ACSVL3. COS‐1 cells stably expressing ACSVL1 or ACSVL3 were harvested and cell pellets assayed for activation of [1‐14C]stearic acid (C18:0, a preferred ACSVL3 substrate) to its CoA derivative in the absence and presence of inhibitors. We also developed a fluorescence‐based assay using COS‐1 cells stably expressing a chimeric ACSVL1/3 protein containing the regulatory domain of ACSVL1 and the catalytic domain of ACSVL3. The regulatory domain of ACSVL1 conferred the ability to uptake the fluorescent fatty acid analog, C1‐BODIPY‐C12, in a process requiring functional ACSVL3 enzyme activity. Most ACSVL1 inhibitors tested also inhibited ACSVL3 using in vitro acyl‐CoA synthetase assays. These compounds inhibited BODIPY fatty acid uptake in COS‐1 cells expressing chimeric ACSVL1/3 in a dose‐dependent manner. One compound, Grassofermata/CB5, was a significantly more potent inhibitor of stearic acid activation by ACSVL3 than by ACSVL1 and was tested further. CB5 was the most potent inhibitor of stearic acid activation in U87 glioma cells. To assess effects on cell growth, CB5 was added to U87 cells in culture. At a concentration of 3 μM, CB5 drastically slowed cell growth rate but did not lead to increased cell mortality. The effects of CB5 on cell growth were reversible; when CB5 was removed from cells after several days of treatment, they reverted to a faster growth rate. A dose response curve of cell growth to CB5 concentration revealed a narrow therapeutic window. In conclusion, many known inhibitors of ACSVL1 are also potent inhibitors of ACSVL3, and the small molecule CB5 shows promise as a potential chemotherapeutic drug and tool to understand the mechanism(s) underlying the role of ACSVL3 in glioma.Support or Funding InformationKennedy Krieger institutional funds.