Abstract 1489: Targeting the mitochondrial enzyme proline dehydrogenase with a mechanism-based irreversible inhibitor induces selective mitochondrial stress and enhances breast cancer cell death under hypoxia
Authors: Scott Gary K. , Frazier Katya , Yau Christina , Becker Beatrice , Ortega Mauricio , Benz Christopher C. 
Published: July 2017

Proline dehydrogenase (PRODH) is a p53-inducible inner mitochondrial membrane flavoprotein functionally linked to electron transport for proline catabolism and intracellular ATP production, particularly under nutrient stress conditions. We have previously shown that in breast cancer cells, PRODH and glutaminase (GLS1) transcript levels are inversely correlated, supplying anaplerotic glutamate to triple-negative (GLS1) and luminal (PRODH) breast cancer subtypes by different means; and that siRNA knockdown or competitive inhibition of PRODH induces synthetic lethal interactions with both GLS1 inhibition and p53 upregulation in various malignant (ZR-75-1, DU4475, MCF7) but not normal (MCF10A) breast epithelial models. In the present study we have synthesized and structurally modeled a novel mechanism-based irreversible (suicide) inhibitor of PRODH, N-propargylglycine (PPG), that shows more than 2-fold greater capacity to inhibit PRODH activity in isolated mitochondrial assays when compared to competitive PRODH inhibitors (L-tetrahydrofuroic acid, THFA; or 5-oxo-2-tetrahydrofurancarboxylic acid, 5-oxo). Modeling human PRODH predicts a post-reactive PPG structure with PPG covalently linked to the enzyme pocket’s FAD moiety, producing pocket distortion that does not occur with competitive PRODH inhibitors. Reflecting PPG’s irreversible binding to PRODH, mitochondria isolated from PPG treated ZR-75-1 cells are unable to catabolize proline despite being able to efficiently catabolize malate; in contrast, isolated mitochondria from 5-oxo treated cells remain efficient at catabolizing both proline and malate. Unexpectedly, we observed that PPG but not the competitive inhibitors induces selective degradation of mitochondrial PRODH protein levels within 24 h of cell culture treatment followed by loss of other mitochondrial proteins like complex-I NDUFS1 but not by concomitant loss of cytosolic FAD-containing proteins like MTHRF. MitoTracker assays confirm the selective cellular loss of mitochondrial mass in ZR-75-1 cells within 24 h of PPG treatment. To confirm that suicide inhibition of PRODH can also induce synthetically lethal metabolic interactions, breast cancer cells (MCF7) were cultured under normoxic (20% O2) or hypoxic (1% O2) conditions and then treated with PPG and a p53 upregulator known to induce PRODH expression (MI-63). Cell viability demonstrated that during hypoxia PPG with p53 upregulation synergistically reduces cell survival at 48 h, confirming the synthetic lethality of this treatment combination. Altogether, these findings support the preclinical development of suicide PRODH inhibitors as potential cancer therapeutics capable of inducing mitochondrial stress, exploiting synthetically lethal metabolic conditions, and selectively enhancing cancer cell death.

Citation Format: Gary K. Scott, Katya Frazier, Christina Yau, Beatrice Becker, Mauricio Ortega, Christopher C. Benz. Targeting the mitochondrial enzyme proline dehydrogenase with a mechanism-based irreversible inhibitor induces selective mitochondrial stress and enhances breast cancer cell death under hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1489. doi:10.1158/1538-7445.AM2017-1489