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. 2019 Feb 21;26(2):278-288.e6.
doi: 10.1016/j.chembiol.2018.10.026. Epub 2018 Dec 20.

Allosteric Inhibition of Ubiquitin-like Modifications by a Class of Inhibitor of SUMO-Activating Enzyme

Yi-Jia Li  1 , Li Du  1 , Jianghai Wang  1 , Ramir Vega  1 , Terry D Lee  2 , Yunan Miao  2 , Grace Aldana-Masangkay  1 , Eric R Samuels  1 , Baozong Li  1 , S Xiaohu Ouyang  3 , Sharon A Colayco  4 , Ekaterina V Bobkova  4 , Daniela B Divlianska  4 , Eduard Sergienko  4 , Thomas D Y Chung  4 , Marwan Fakih  5 , Yuan Chen  6
Affiliations

Allosteric Inhibition of Ubiquitin-like Modifications by a Class of Inhibitor of SUMO-Activating Enzyme

Yi-Jia Li et al. Cell Chem Biol. .

Abstract

Ubiquitin-like (Ubl) post-translational modifications are potential targets for therapeutics. However, the only known mechanism for inhibiting a Ubl-activating enzyme is through targeting its ATP-binding site. Here we identify an allosteric inhibitory site in the small ubiquitin-like modifier (SUMO)-activating enzyme (E1). This site was unexpected because both it and analogous sites are deeply buried in all previously solved structures of E1s of ubiquitin-like modifiers (Ubl). The inhibitor not only suppresses SUMO E1 activity, but also enhances its degradation in vivo, presumably due to a conformational change induced by the compound. In addition, the lead compound increased the expression of miR-34b and reduced c-Myc levels in lymphoma and colorectal cancer cell lines and a colorectal cancer xenograft mouse model. Identification of this first-in-class inhibitor of SUMO E1 is a major advance in modulating Ubl modifications for therapeutic aims.

Keywords: E1; KRas; SUMO; activating enzyme; allosteric inhibitor; c-Myc; cancer; covalent inhibitor; therapeutics; ubiquitin-like modification.

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Conflict of interest statement

Declaration of Interests

S.X.O. is an employee and shareholder of SUMO Biosciences, Inc. Y.C. is a founder of SUMO Biosciences, Inc. and a member of its advisory board. Other authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Identification of a selective SUMOylation inhibitor by HTS
(A) Summary of the assays that the HTS hit was most potent for human proteins based on IC50 values among 780 assays tested as shown by data deposited in PubChem. (B and C) In vitro SUMOylation assay of SUMO conjugation to RanGAP1 detected by AlphaScreen (B) and ubiquitylation assay detected by time-resolved fluorescence resonance energy transfer (C, see also PubChem AID 2658). One representative curve from four experiments is shown for SUMOylation (B). The results from five experiments are shown for ubiquitylation (C). The structure of COH000 is shown to the right in (B).
Figure 2.
Figure 2.. COH000 is an allosteric covalent inhibitor of the SUMO E1
(A) ATP:PPi exchange assay to show that COH000 inhibits the SUMO adenylation step of SUMO E1 catalysis. The assay was conducted with various concentrations of the compound at increasing concentrations of ATP (upper panel) or SUMO-1 (lower panel). (B) Fragment ion assignments for the MS/MS spectrum of the 2+ charge state of peptide COH000 conjugated peptide (residues 14–35 of SAE2, AVAGGRVLVVGAGGIGCELLKN, m/z 849.47). The potential conjugation sites of COH000 with Cys30 are shown to the right. (C) SUMO E1 catalyzed Ubc9•SUMO thioester formation is inhibited by COH000 in a dose-dependent manner for WT enzyme but not C30S mutant. 0.25 μM E1 WT or C30S was incubated with 2 μM Ubc9, 5 μM SUMO-1, 100 μM ATP, 5 mM MgCl2, 50 mM NaCl, 0.05% Tween-20, 20 mM HEPES pH 7.6 for 15 minutes at 37oC. The figure shows a Coomassie stained SDS-PAGE gel. (D) Analysis of KI and kinact of COH000 inhibition of SUMO E1. Natural logarithm of the rate of Ubc9·SUMO-1 thioester is plotted against the pre-incubation time of SUMO E1 with the indicated concentrations of COH000 (left panel). After incubation, homogeneous time-resolved fluorescence readings were taken. The negative slope in the graph from the upper panel was used to determine the inactivation rate kobs (right panel) that was used for non-linear regression analysis that obtained KI and kinact. Data are presented as means ± SD, in three independent experiments.
Figure 3.
Figure 3.. COH000 enhances SAE2 degradation in cells
(A) The COH000 covalent attachment site Cys30 is shown in spheres colored according to the atom types (green, carbon; yellow, sulfur), and the bound ATP or ATP analog (red) and SUMO (magenta) are shown according to published crystal structures in the adenylation active (upper panel, pdb id: 1Y8R) and thioester formation active (lower panel, pdb id: 3KYD) conformations that differ on both the domain orientations and active site structures. (B) Differential scanning fluorimetry of SUMO E1 with and without fully inhibited by COH000. The derivative of the raw data is shown. (C) COH000 increased degradation of SAE2 in cells as indicated by pulse-chase experiment. Upper panel, the gel image. Lower panel, the gel band intensity normalized by total input protein detected by Coomassie staining relative to time zero. (D) MG132 (+) or vehicle (−) was added to cells for 2 hours prior to COH000 treatment at indicated concentrations. After 2 hours of COH000 treatment, the cells were harvested and directly lysed in SDS-sample buffer. SAE2 protein was then analyzed by Western blot. Bracket indicates modified SAE2, likely including ubiquitylation. GAPDH, shown on the same membrane, was used for loading control.
Figure 4.
Figure 4.. COH000 inhibits SUMOylation in cells
(A) COH000 inhibits SUMOylation in HCT-116 cells in a dose-dependent manner upon 18 hours treatment. (B) COH000 selectively inhibits the SUMO E1 in cells. Inhibition of SUMO, Nedd8, and ubiquitin activating enzymes in cells was assessed by Western blot analysis of Ubc9•SUMO, ubiquitin•Ubc5, and Nedd8•Ubc12 thioester formation in HCT-116 cell lysates. (C) Quantification of thermo-stable SAE2 Western blots from cellular thermal-shift assay (CETSA) to determine COH000 target engagement in cells as previously described (Jafari et al., 2014). Blot intensity was measured by Li-Cor Odyssey software and normalized to intensity of the 42 °C samples for each concentration. Data are presented as means ± STDEV, n = 3, in independent experiments. (D) Parental, SAE2 -overexpressed or –knockdown HCT116 cells were treated with vehicle (DMSO) or 10 μM COH000 for 20 h. Percentile of apoptotic HCT116 cells were shown in the bar graph. SAE2 overexpression reduced the percentage of apoptotic cells. In contrast, knockdown of SAE2 increased the percentage of apoptotic cells. The data shown are the means of three independent experiments ±STDEV. (E and F) Correlation of in vitro biochemical activity with cell proliferation. Biochemical assays of SUMO-Ubc9 thioester formation after 10 min reactions with the indicated compounds at 10 and 20 μM as described for Figure 2C (F). Compound A and B are close analogs of COH000 as shown by the structures. “-” and “+” indicate reactions without and with ATP, respectively, as negative and positive controls. COH000, 54 and 55 were added to cell culture media at the indicated concentrations and MTS assays were performed 72 hours later (E). Data shown are the means of three independent experiments ±STDEV.
Figure 5.
Figure 5.. COH000 increases miR-34b expression and reduces c-Myc mRNA and protein levels
(A) Lymphoma (Raji) and colorectal cancer (HCT116) cells were treated COH000 at the indicated concentrations. miR-34b and c-Myc mRNA expression levels were measured by RT-qPCR after 2 days of inhibitor treatments. c-Myc protein levels were detected by Western blots. (B) HCT116 xenografted Es1e/SCID mice were treated with s.c. peritumoral injection of vehicle or 10 mg/kg COH000. Tumor growth was monitored (left panel). Statistics of apoptosis were detected by TUNEL staining of in tumor tissue from vehicle and COH000-treated mice (right panel). miR-34b levels in tumor cells of the mouse model were also measured. Two-tail Student’s t-test was used to calculate p value. *, p < 0.05, ** p< 0.01. (C) Representative IHC staining images for SAE2, c-Myc, and TUNEL of xenograft tumor tissues in Es1e/SCID mice from COH000 treatments. Red arrows indicate TUNEL positive staining cells and scale bar presents 50μm.
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