Abstract A127: The identification and development of selective natural product inhibitors of hypoxia inducible factor-2α for the treatment of renal cell carcinoma
Authors: Lee Molly M. , Rabe Daniel C. , McKee Tawnya C. , Woldemichael Girma M. , Vasselli James R. , McMahon James B. , Linehan W. Marston , Bottaro Donald P. 
Published: August 2018

Abstract
Clear cell renal cell carcinoma (ccRCC) is the most prevalent form of kidney cancer and is frequently associated with loss of von Hippel Lindau (VHL) gene function, resulting in the aberrant accumulation of the hypoxia inducible factor-alpha subunit (HIF-α), which contributes to tumor growth, angiogenesis, and metastasis. Recent studies suggest that this normoxic stabilization of HIF-1α is not sufficient to reproduce tumorigenesis, and HIF-1α is thought to act as a tumor suppressor in ccRCC. Conversely, HIF-2α acts as an oncogene, turning on transcription of a large number of HIF-regulated genes, and upregulation of HIF-2α is often associated with poor prognosis. To this end, two HIF-2α selective inhibitors are currently in clinical trials for RCC. To isolate additional HIF-2α selective compounds for use as research tools and drug development leads, a cell-based high-throughput screen (HTS) of the NCI Natural Products Repository was performed. Stably transformed clones of the human ccRCC-derived cell line 786-0 expressing a luciferase reporter construct downstream of the human vascular endothelial growth factor (VEGF) gene promoter were used to identify natural product samples with inhibitory activity and minimal cellular toxicity. HTS leads were chromatographically separated into component structures yielding ~40 pure compounds with micromolar or submicromolar IC50 values, &gt;80% inhibition, and &lt;10% cell toxicity. These hits were then screened for their ability to inhibit VEGF-A protein secretion by VHL- and HIF1A-negative 786-0 cells, and several compounds showed &gt;90% inhibition of VEGF secretion at micromolar doses under normoxic conditions. Patterns of mRNA expression of HIF-1α, HIF-2α, and several HIF target genes in cell lines engineered to express either HIF alone or both were studied to help identify HIF-2α-selective inhibitors. Several lead compounds reduced VEGF transcription and secretion, and downregulated HIF-2α
target genes. To further characterize the HTS leads, ICM-Pro protein/small-molecule docking software was used to dock the ligands of interest to high-resolution HIF-2α and HIF-1α 3D structures. The HIF-2α selective inhibitor currently undergoing clinical investigation, PT2385, has been found to bind directly to a hydrophobic pocket of the HIF-2α:ARNT dimerization. Using the PT2385 binding mode as a benchmark, docking studies of the HTS leads were performed and the results have been linked to empirical data in an effort to explain selectivity for HIF-2α over HIF-1α, reveal key favorable interactions between the protein and ligand, define mode of action, and aid in future drug development studies.
Citation Format: Molly M. Lee, Daniel C. Rabe, Tawnya C. McKee, Girma M. Woldemichael, James R. Vasselli, James B. McMahon, W. Marston Linehan, Donald P. Bottaro. The identification and development of selective natural product inhibitors of hypoxia inducible factor-2α for the treatment of renal cell carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A127.