Small-Molecule Targeting of E3 Ligase Adaptor SPOP in Kidney Cancer Zhong-Qiang Guo, Tong Zheng, Baoen Chen, Cheng Luo, Sisheng Ouyang, Shouzhe Gong, Jiafei Li, Liu-Liang Mao, Fulin Lian, Yong Yang, Yue Huang, Li Li, Jing Lu, Bidong Zhang, Luming Zhou, Hong Ding, Zhiwei Gao, Liqun Zhou, Guoqiang Li, Ran Zhou, Ke Chen, Jingqiu Liu, Yi Wen, Likun Gong, Yuwen Ke, Shang-Dong Yang, Xiao-Bo Qiu, Naixia Zhang, Jin Ren, Dafang Zhong, Cai-Guang Yang, Jiang Liu, Hualiang Jiang  Cancer Cell  Volume 30, Issue 3, Pages 474-484 (September 2016) DOI: 10.1016/j.ccell.2016.08.003 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cancer Cell 2016 30, 474-484DOI: (10.1016/j.ccell.2016.08.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Development of SPOP Inhibitors (A) Work flow of the structure-based virtual screening of inhibitors of SPOP-substrate interactions. (B) Structure of inhibitors as identified through virtual screening and subsequent synthetic optimizations. Compound 6a shows moderate inhibitory activity, 6b is the most active inhibitor, and 6c is the negative control. (C) Inhibitors competitively inhibit puc_SBC1 peptide binding to SPOP23−337 measured by FP assay. (D) In a pull-down assay, 6b disrupts protein binding between SPOP and PTEN. In the negative control experiment, protein interaction is intact in the presence of compound 6c. (E) Proliferation experiment on A498 cells in the presence of inhibitors. IC50 (μM) values of inhibitors were calculated from three independent experiments. Error bars represent mean ± SD. See also Figure S1. Cancer Cell 2016 30, 474-484DOI: (10.1016/j.ccell.2016.08.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 SPOP Inhibitor Binds to SPOP (A) 6b binds to SPOP23−337 protein as shown by surface plasmon resonance measurements. Graphs of equilibrium response unit responses versus compound concentrations are plotted. The estimated KD is 19.1 μM. (B) NMR measurement of direct binding between 6b and SPOP. T1ρ NMR spectra for 6b (red), 6b in the presence of SPOP at 2.5 μM (blue), 5 μM (cyan), and 10 μM (green). The STD spectrum for 6b is recorded in the presence of 5 μM SPOP. (C) Representative western blots for the stabilization of the SPOP protein. CETSA was assayed in HEK293 cell lysate and in intact cells in the presence of 20 and 5 μM 6b, respectively. See also Figure S2. Cancer Cell 2016 30, 474-484DOI: (10.1016/j.ccell.2016.08.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 SPOP Inhibitors Suppress the Proliferation of ccRCC Cell Lines and the Growth of Primary ccRCC Cancer Cells (A) SPOP localizes in the nucleus of HK-2 cells. (B) The relative abundance of SPOP protein minimally varies in six ccRCC cells. Much less SPOP was detectable in HK-2 cells. GAPDH is used as a loading control. (C) Cell proliferations of six ccRCC cell lines and one non-tumor HK-2 cell line in the presence of 6a-c. (D) Inhibition of the proliferation of the primary ccRCC cells isolated from seven patients. The proliferation of A498 cells is considered a positive control, and proliferation of HK-2 cells is considered a negative control. The cell proliferations assays were performed in triplicate, and error bars represent mean ± SD. See also Figure S3. Cancer Cell 2016 30, 474-484DOI: (10.1016/j.ccell.2016.08.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 SPOP Inhibitors Inhibit the Ubiquitination and Degradation of Tumor Repressors In Vivo (A) 6a disrupts SPOP-PTEN interaction in HEK293 cells. (B) SPOP-PTEN and SPOP-DUSP7 protein interactions are inhibited in the presence of 6b in a concentration-dependent manner. These interactions are intact in the presence of 6c. (C) 6a inhibits PTEN ubiquitination in HEK293 cells. (D) 6b inhibits the ubiquitination of PTEN and DUSP7 in a dose-dependent manner. 6c is inactive and does not inhibit the ubiquitination of PTEN or DUSP7. (E) 6b inhibits the degradation of PTEN and DUSP7 in a concentration-dependent manner in A498 cells. The downstream p-AKT and p-ERK1/2 abundances are observed to decrease. (F) 6a increases the accumulation of PTEN in both a time- and concentration-dependent manner in A498 cells. (G) 6a increases the accumulation of PTEN, while it decrease the levels of p-AKT in Caki-2 cells. See also Figure S4. Cancer Cell 2016 30, 474-484DOI: (10.1016/j.ccell.2016.08.003) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 6b Dose-Dependently Impairs the Growth of Xenografted A498 Cells in Nude Mice (A) Volume measurements of A498 xenograft tumors treated with vehicle or compound 6b at the indicated dosages for 21 days. 6b was administered by intraperitoneal injection once per day at 60 or 80 mg/kg. Changes in mean tumor volume are given relative to untreated tumor volumes. Error bars represent mean ± SD, with n = 6 for vehicle control, and 60 and 80 mg/kg groups. p Values were obtained by unpaired t test. (B) Volume distribution at day 21 of A498 xenograft tumors treated with vehicle or 6b. (C) Quantification of PTEN and DUSP7 accumulation and repression of p-AKT and p-ERK at day 21 of A498 xenograft tumors treated with vehicle or 6b. (D) Schematic overview of the mechanism that the small-molecule inhibitor uses to block SPOP-substrate protein interactions in ccRCC cancer. 6b disrupts the SPOP-mediated protein interactions, inhibits the ubiquitination and degradation of tumor repressors PTEN and DUSP7, and decreases the downstream phosphorylation of AKT and ERK; ultimately, this process leads to the death of the ccRCC cells. See also Figure S5 and Tables S1–S4. Cancer Cell 2016 30, 474-484DOI: (10.1016/j.ccell.2016.08.003) Copyright © 2016 Elsevier Inc. Terms and Conditions