Summary /

ML364 reversibly inhibits USP2 in a biochemical assay. A high throughput screen resulted in the identification of a sulfamidobenzamide chemical series that inhibited USP2 biochemical activity. Further optimization through medicinal chemistry led to the development of the active compound ML364 and a structurally related inactive counterpart compound 2 (Fig. 1A). Using internally quenched fluorescent di-ubiquitin substrates (Di-Ub IQF; Fig. 1B), ML364 was determined to have an IC50 of 1.1 uM for the Lys-48-linked substrate and 1.7 uM for the Lys-63-linked substrate, whereas compound 2 was inactive. The compounds did not have an effect on the detection system alone. The interaction of ML364 with USP2 was reversible as determined by measuring the recovery of enzyme activity following a large dilution of the USP2-ML364 complex (Fig. 1G). ML364 was also tested in other protease assays to assess selectivity (Table 1 and Fig. 1C); it was inactive against caspase 6, caspase 7, MMP1, MMP9, and USP15, but it did inhibit USP8 with an IC50 of 0.95 uM, in agreement with the similarity between the active sites of these two isozymes.

FIGURE 1. ML364 binds to USP2 and inhibits its activity, although a related chemical analog 2 does not. A, chemical structures of ML364 and compound 2. B, plot of inhibition of USP2 biochemical activity of ML364 and compound 2, assessed using Lys-48- and Lys-63-linked IQF Di-Ub substrates. Colors indicate the compound/substrate combinations, as follows: blue, ML364/Lys-48-4; green, ML364/Lys-63-3; orange, compound 2/Lys-48-4; and red, compound 2/Lys-63-3. C, inhibition of activity of caspase 6 (orange), caspase 7 (purple), MMP1 (red), MMP9 (yellow), USP8 (green), and USP15 (blue) by ML364. D–F, demonstration of ML364 binding to USP2 through microscale thermophoresis. Microscale thermophoresis curves (normalized fluorescence versus time (s)) and resultant concentration responses were plotted as log (concentration) in M versus normalized thermophoresis without temperature jump ((average hot)/(average cold)1,000, where hot is the average value between the pink vertical lines and cold is the average between the blue vertical lines) for ML364 (D) and compound 2 (E). LED was 20%, and laser power was 17%. F, microscale thermophoresis curves (normalized fluorescence versus time (s)) and resultant IC50 curves plotted as log (concentration, M) versus normalized thermophoresis without temperature jump ((average hot)/(average cold).1,000, where hot is the average value between the pink vertical lines and cold is the average between the blue vertical lines) for titration of USP2 alone. LED20% and MST power 60%. G, ML364 and compound 2 bind reversibly to USP2. The fluorescence over time is plotted for ML364 (blue), compound 2 (red), enzyme alone (green), and no enzyme (purple).

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In a panel of 102 kinases that included regulators of the cell cycle there was no binding observed to any of the enzymes tested using 10 uM ML364 (Table 2 and Fig. 2).

FIGURE 2. Kinomescan visualization for ML364 (10 uM) tested for binding to 102 kinases.

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ADME Characterization of ML364—Preliminary ADME properties were determined, and although ML364 had low solubility in PBS 7.4 buffer (<2 uM), its solubility was more than 10-fold higher in the assay buffer (28.9 uM; 20mM Tris, pH 8, 2 mM beta-mercaptoethanol, 0.05% CHAPS), where it was stable for more than 48 h (Table 3). The logD at pH 7.4 (a measure of lipophilicity) was 2.31, and the PAMPA permeability was 82.2 x 10-6 cm/s, indicating that the compound has properties that should allow for cell membrane permeation. Although the stability in rat microsomes was modest (t1⁄2 = 15 min), the stability in mouse (61% remaining at 30 min) and human (84% remaining at 30 min) microsomes and in mouse plasma (100% remaining at 30 min) was quite good (Table 4).

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Effect of USP2 Inhibition by ML364 in Cells Investigated by Western Blotting—To determine the effect of USP2 inhibition on cyclin D1 stability, HCT116 colorectal cancer cells were treated with ML364, and cyclin D1 protein levels were monitored by both Western blotting (Fig. 3, A–C) and in-cell ELISA. Whereas cyclin D1 protein level remained unchanged in cells treated with the inactive compound 2, it decreased in both a time- and dose-dependent manner following treatment with ML364. Quantification of the Western blotting results revealed an IC50 value of 0.97 uM, which is consistent with the results from the biochemical assays discussed above (Fig. 1 and Table 1). The similar potencies for ML364 in the cell-based and biochemical assays are in concert with the expected cell permeability of the inhibitor and support the notion that ML364 engages USP2 in the cell. ML364-mediated cyclin D1 destabilization was not due to a reduction in USP2 protein levels (Fig. 3A) and was proteasome dependent, as the addition of the proteasome inhibitor MG132 restored cyclin D1 protein levels to those observed in the DMSO control (Fig. 3C). To test whether ML364 promoted cyclin D1 degradation in a cell line that relies on this protein for transformation, the above experiments were repeated using the mantle cell lymphoma cell line, Mino. Again, ML364, but not compound 2, reduced cyclin D1 protein levels in a time-, dose-, and proteasome-dependent manner (Fig. 3, D–F).

FIGURE 3. ML364 reduces cyclin D1 protein levels in a time-, dose-, and proteasome-dependent manner in HCT116 cells and Mino cells. A, HCT116 cells were treated with 10 uM ML364 for the indicated amount of time (top panel) or with the indicated concentration of ML364 (bottom panel) for 4 h. B, HCT116 cells were treated with 10M compound 2 for the indicated amount of time (top panel) or with the indicated concentration of compound 2 (bottom panel) for 4h. C, HCT116 cells were treated with 10uM ML364 or 10M 2 for 4 h in the absence or presence of 10uM MG132.D, Mino cells were treated with 10uM ML364 for the indicated amount of time (top panel) or with the indicated concentration of ML364 (bottom panel) for 4 h. E, Mino cells were treated with 10 uM compound 2 for the indicated amount of time (top panel) or with the indicated concentration of compound 2 (bottom panel) for 4 h. F, Mino cells were treated with 10 uM ML364 or 10 uM compound 2 for 4 h in the absence or presence of 10 uM MG132. Cyclin D1 and USP2 protein levels were assessed by Western blotting using a GAPDH control. Bands were quantitated by ImageJ.

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Assessment of cell viability in HCT116 and Mino cells using cellular ATP content as the readout also showed that ML364 decreased cell viability (Fig. 6A). In addition to HCT116 and Mino cells, LnCAP cells (prostate cancer cells) and MCF7 cells (breast cancer cells) also showed a decrease in cell viability upon exposure to ML364 in a dose-dependent manner (Fig. 6, B and C). The decrease in cell viability in these cell lines correlated with cyclin D1 degradation (Fig. 6D). We next determined whether additional targets of USP2, namely MDM2 and FAS, are affected by ML364. Interestingly, FAS but not MDM2 protein levels were moderately reduced after ML364 exposure in LnCAP prostate cancer cells (Fig. 6, E and F).

FIGURE 6. ML364 exposure decreases cell viability and promotes cyclin D1 degradation in cancer cell lines. A, effect of ML364 on cell viability of Mino (squares) and HCT116 (circles) cells as measured by ATP content using CellTiter GloTM. B and C, effect of ML364 on cell viability of LnCAP (circles) and MCF7 (squares) cells as measured by ATP content using CellTiter GloTM. The cells were exposed to ML364 for 24 h (B) or 48 h (C). D, ML364 promotes degradation of cyclin D1 in LnCAP cells and MCF7 cells. Cells were treated with ML364 for 24 h, and cyclin D1 protein levels were assessed by Western blotting using a tubulin control. E and F, ML364 exposure results in a moderate decrease in FAS levels. LnCAP cells were treated with ML364 for 24 h, and FAS, MDM2, and cyclin D1 protein levels were assessed by Western blotting using a vinculin control.

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Effect of USP2 Inhibition on Homologous Recombination (HR)-mediated DNA Repair—Recent studies suggest that cyclin D1 regulates DNA damage response. Cyclin D1 interacts with RAD51, a critical component of HR-mediated DNA repair. Reduction of cyclin D1 levels in cancer cells results in impaired HR-mediated DNA repair and increased DNA damage. Wetherefore tested whether inactivation of USP2 by ML364 alters DNA repair. We used a cell-based DR-GFP reporter assay to measure the activity of HR-mediated DNA repair. Interestingly, ML364 caused a decrease in HR-mediated DNA repair in DR-GFP U2OS cells (Fig. 7A). We next analyzed RAD51 foci, a surrogate marker for efficient HR-mediated DNA repair, after inhibition of USP2 by ML364. Consistent with the results from the DR-GFP reporter assay, ML364 exposure in HeLa cells caused a decrease in IR-induced RAD51 foci (Fig. 7, B and C). Together, these data indicate that inhibition of USP2 by ML364 causes a decrease in HR suggesting the role of USP2 in DNA repair.

FIGURE 7. Inhibition of USP2 activity by ML364 causes a decrease in HR-mediated DNA repair. A, U2OS-DRGFP cells were transfected with a plasmid encoding I-SceI endonuclease and cultured for 24 h followed by exposure to ML364 at the indicated concentration for 24 h. Cells were then subjected to flow cytometric analysis. The relative GFP-positive cells normalized by solvent vehicle-treated group are shown. B and C, HeLa cells were treated with ML364 (5uM) for 24 h before exposing them with IR (10 gray). Eight hours after IR, cells were analyzed for RAD51 foci by immunofluorescence. Representative images (B) and quantification (C) of RAD51 foci are shown. At least 100 cells were scored for RAD51 foci for each replicate, and three replicates were scored.