Abstract 1795: Small molecule SBI-601 inhibits Siah1/2 ubiquitin ligases, attenuates HIF1α expression and growth of melanoma and prostate cancer cells

The ubiquitin ligases Siah1/2 have been implicated in the control of key cellular pathways, including hypoxia (through the control of prolyl hydroxylase 1/3 stability), mitochondrial fission (through the control of AKAP121 stability) and Ras/Raf signaling (through its control of Sprouty 2 stability). Expression or activity of Siah1/2 is upregulated in number of cancer types and correspondingly, inhibition of Siah1/2 effectively attenuates growth of prostate, melanoma, lung and pancreatic tumors. To identify possibly Siah1/2 inhibitors, we have performed a HTS screen using affinity selection-mass spectrometry (AS-MS). Using a select set of 32,000 drug-like molecules in mass-encoded mixtures of 400 compounds per assay well, we identified 36 distinct clusters of structurally related scaffolds that demonstrate high-binding affinity to full-length bacterially produced purified and active Siah2. Of those, 9 out of 21 closely related analogs were evaluated in functional assays. One compound, SBI-0640601, showed potent and reproducible inhibition of Siah2, as measured by the reduction in HIF1α levels and activity, measured by HRE-Luciferase assays in both melanoma and prostate cancer cultures. SBI-0640601 is a natural product known as betulonic acid (BA) and is one of a family of triterpenoid natural medicinal products derived from birch trees (genera Betula). Independent binding studies (differential scanning calorimetry) confirmed that SBI-0640601 binds to full-length bacterially produced Siah2 and to a RING domain-deleted version of Siah1. In vitro, SBI-0640601 attenuated the binding of PHD3 to Siah2, in keeping with the observation that ubiquitination of most substrates requires binding to the Siah2 substrate-binding domain. SBI-0640601 inhibited growth of melanoma and prostate cancer cells in 2D and 3D cultures. Further, SBI-0640601 effectively attenuated neuroendocrine differentiation phenotype in adenocarcinoma cells, consistent with the effect seen upon inhibition of Siah1/2 expression. RPPA analysis identified that SBI-0640601 effectively attenuates AMPK and mTOR signaling pathways, in addition to affecting MAPK signaling. Overall, SBI-0640601 appears to inhibit key processes previously shown to be regulated by Siah1/2, holding promise for further assessment in preclinical setting. Note: This abstract was not presented at the meeting. Citation Format: Yongmei Feng, Tal Varsano, Hampton Sessions, Michael Davies, Greg Roth, Ze'ev Ronai. Small molecule SBI-6