Abstract 3860: Discovery and characterization of novel anti-cancer small molecule inhibitors of Sec61

Abstract

Abstract Secreted and membrane proteins play key roles in malignant transformation and tumor growth, including autocrine growth factor expression, receptor oncogene signal transduction pathways, metastasis, and immune system evasion. The biogenesis of most secreted and transmembrane proteins involves cotranslational translocation of nascent polypeptides into the endoplasmic reticulum through the Sec61 translocon. Broad inhibition of protein secretion can be mediated by natural product-derived Sec61 inhibitors that also possess anti-tumor activity, suggesting that Sec61 represents an untapped therapeutic target for cancer treatment. Here we describe a process for discovering and evaluating novel Sec61 inhibitors with varying degrees of selectivity for therapeutically relevant targets in oncology. We used HEK293 reporter cell lines stably transfected to express secreted and transmembrane proteins of interest fused to luciferase to enable high-throughput screening of potential inhibitors of Sec61-dependent protein secretion. As expected, target selectivity was largely dependent on respective signal sequences. However, full-length protein constructs were found to be 5-10 fold less sensitive to inhibition compared to reporters containing only the signal sequence plus 10 amino acids, suggesting that additional interactions beyond the signal sequence are involved in promoting cotranslational translocation. To verify that novel compounds specifically targeted Sec61, we found that compounds were unable to inhibit the activity of luciferase localized to the cytosol and had reduced potency in reporter cells expressing a Sec61 mutant known to confer resistance to published inhibitors such as cotransin, mycolactone, and apratoxin. We assessed compound selectivity using a library of 37 transiently expressed Sec61-client proteins of therapeutic relevance, revealing 4 distinct chemical scaffolds differentiated by target selectivity profile. Consistent with data from published compounds, broad inhibitors of protein secretion had anti-tumor activity. Of particular interest were compounds able to block expression of multiple immune checkpoints including PD-1, PD-L1, CTLA-4, and CD96. Activity in primary cells, such as receptor expression on activated T-cells and cytokine release from stimulated PBMC, was assessed to verify specificity. Compounds with adequate in vitro metabolic stability were tested in mice for pharmacokinetics as well as inhibition of target protein secretion and global inhibition of Sec61 activity. Taken together, our data suggest that Sec61 is a novel therapeutic target which represents a unique opportunity to both selectively block expression of multiple proteins involved in tumor growth and globally modulate protein homeostasis, resulting in tumor cell death. Citation Format: Eric Lowe, Janet L. Anderl, Andrea R. Fan, Ying Fang, Jing Jiang, Henry W. Johnson, Christopher J. Kirk, Dustin McMinn, Tony Muchamuel, Meera Rao, Phillip P. Sharp, Jack Taunton, Jinhai Wang, Jennifer A. Whang. Discovery and characterization of novel anti-cancer small molecule inhibitors of Sec61 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3860.