USP25(IC50= 0.7 μM);USP28(IC50= 0.6 μM)

The in vitro profiles of AZ1, AZ2, and AZ4 show them to be active against the USP28 enzyme and to bind to USP28 by iso-thermal calorimetry (ITC) and microscale thermophoresis (MST). They were also shown to be selective over USP2a. A fourth analogue (AZ3) was shown to be significantly less potent at inhibiting USP28 but retained selectivity over USP2a. The data shown in the table were generated from one experimental replicate, unless stated otherwise[1].

The USP25/28 inhibitor AZ1 (AZ1; 40 mg/kg; gavage; daily; for 7 days) prevents diarrhea and weight loss brought on by dextran sulfate sodium (DSS) as well as impaired colon shortening[1].
Colon tumor numbers are significantly decreased by USP25/28 inhibitor AZ1 (20 mg/kg/day; gavage; 6 times a week in the 1, 3, and 6 weeks) treatment. Tumors exhibit elevated SOCS3 levels, decreased pSTAT3 levels, and Wnt-related gene expression. AZ1 gavage is not effective in treating spontaneous colitis in Il10-/-mice or DSS-induced colitis in Usp25-/-mice[1].
The USP25/28 inhibitor AZ1 (20 mg/kg/day; gavage; every 3 days from 13–20 weeks) prolongs the survival of AOM/Vil-Cre;Trp53fl/fl (VP) mice and significantly inhibits colon tumorigenesis. Treatment with AZ1 has little effect on tumorigenesis in the background deficient in USP25[1].

For these binding studies, two independent biophysical techniques were utilized, the first of which was isothermal titration calorimetry (ITC). This label free methodology directly measures the heat of binding for the interaction that occurs when a ligand binds to a target protein and can be used to confirm ligand binding and calculate the equilibrium dissociation constant (Kd) and stoichiometry of the interaction. These parameters are additionally useful in characterizing the binding interaction and demonstrating the functional integrity of the enzyme. Under the conditions of our experiments, Kd values of 0.2, 0.9, and 2.7 μM were derived for AZ1, AZ2, and AZ4, respectively (Figure 1a–c; Table 1). These values are consistent with the biochemical activity data described previously. In addition, corresponding stoichiometry values of 0.6, 0.7, and 0.8 were derived for AZ1, AZ2, and AZ4, respectively (Figure 1a–c) in line with a nonspurious and specific mode of binding, hence supporting our initial observations from the “ratio test” experiments. For comparison, two compounds determined by the “ratio test” as potentially acting via alternative mechanisms were also tested and produced noisy and difficult to interpret data (data not shown). The second approach taken to confirm binding of the compounds to USP28 utilized the NanoTemper Microscale Thermophoresis (MST) methodology, which produced comparable data. In this instance, Kd values of 3.7 and 10.3 μM were determined for AZ1 and AZ2, respectively (Supporting Information Figure S2a,b and Table 1). In agreement with the ITC data, AZ3 failed to produce a determinable result. The binding constants for the compounds were determined using the protein’s intrinsic fluorescence. We believe this resulted in a less sensitive detection system than the fluorescently labeled version of the approach. This may in part account for the apparent discrepancy in Kd estimates between MST and ITC. In contrast, AZ4 generated a Kd using the labeled protein, which was in much closer agreement with the ITC result (3.6 vs 2.7 μM; Table 1). Despite Kd values being higher than the IC50 values obtained in the in vitro enzyme assay and the Kd values from our ITC experiments, these data further confirm target-ligand engagement. Taken together, these data derived from two independent methodologies demonstrate that AZ1, AZ2, and AZ4 interact with and bind to USP28 in a nonspurious and specific manner[1].

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Binding Assays—Isothermal Titration Calorimetry [3]
The USP28 protein and test compounds were dialyzed in 40 mM HEPES (pH 7.5) and 150 mM NaCl, in order to minimize heat effects due to buffer mismatch or ionization. ITC experiments were carried out with 20 μM USP28 protein, contained in the cell of a Microcal iTC200 instrument, titrated with 200 μM test compound, contained in the instrument injection syringe. The interaction of USP28 with the test compound was quantified using a Microcal ITC 200. The titration data were recorded at 25 °C in 40 mM HEPES at pH 7.5, 150 mM NaCl, and 2% DMSO. Aliquots of 200 μM ligand stocks were added to 20 μM USP28 in multiple 2 μL intervals. Data were analyzed using nonlinear least-squares regression using Microcal Origin software.

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USP Selectivity Assessment [3]
The selectivity of compounds across the DUB family was analyzed through testing in the DUBProfiler panel at Ubiquigent (www.ubiquigent.com). This involved inclusion of test compounds at a concentration of 10 μM in a range of Ub-Rho110 in vitro enzyme assays. Enzyme assays were generated and run for the following DUB-family members: 14 USPs (USPs 1, 2, 4, 5, 7, 11, 15, 19, 20, 25, 28, 36, 45, and CYLD), four UCHs (UCHL1, UCHL3, UCHL5, and BAP1), three OTUs (OTUB2, OTUD6B), and one JAMM (AMSH-LP). Data generated are displayed as a percentage inhibition of total enzyme activity for each enzyme. Dose response testing against USP28 and USP25 was performed in the same way, testing compounds in the range of 100–0.03 μM. Selectivity of AZ1 against cysteine proteases including caspases 1/2/4/5/8 and cathepsins H/L/S was tested at a fixed screening concentration of 30 μM and data reported as % of enzyme activity relative to DMSO control.

HCT116 cells were pretreated with cycloheximide (100 μg/mL) and the proteasome inhibitor MG132 (20 μM) and subsequently exposed to AZ1 (60 μM) or vehicle control (DMSO). Cells were lysed at the indicated time points (from 0 to 180 min) and samples analyzed by Western blotting probing for c-Myc. The half-life values of c-Myc were determined by densitometry analysis based on these blots. β-Actin was included as a loading control. These data are representative data from at least three independent experiments[1].

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Cell Assays [3]
In order to assess cellular activity of the compounds, HCT116 cells were treated with USP28 inhibitor compounds for a period of 3 h. Following this incubation, cells were harvested, lysed, and subjected to Western Blot analysis. Samples were probed for c-Myc protein levels and also probed for β-actin levels, as a loading control. In order to determine the half-life of c-Myc in cells, HCT116 cells were treated for 3 h with USP28 inhibitors in the presence of cycloheximide (100 μg/mL) to block nascent protein synthesis. Thus any alteration in c-Myc levels would be indicative of changes in degradation as opposed to protein synthesis. Following compound treatment, cells were harvested and lysed prior to Western blot analysis

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Reagents for Cellular Characterization [3]
The USP28 inhibitors (AZ1, AZ2, AZ3, and AZ4) were prepared as 100 mM DMSO stocks for cell culture experiments. Cycloheximide was used at a final concentration of 100 μg/mL. MG132 and propidium iodide (PI) were used at a final concentration of 20 μM and 10 μg/mL respectively. RNaseA was used at a final concentration of 250 μg/mL. CellTiter-Glo (cell viability assay) was purchased from Promega. Ubiquitin-Vinyl Sulfone (Ub-VS) was used at a final concentration of 32 μg/mL.

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Target Engagement Assay [3]
HCT116 cells were treated with USP28 inhibitors for 2 h. Following incubation, cells were harvested in TE lysis buffer containing 50 mM Tris (pH7.4), 150 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, 0.5% NP40, 10% glycerol, and 2 mM DTT and clarified cell lysates (40 μg) incubated with ubiquitin-vinyl sulfone in assay buffer containing 50 mM Tris (pH7.6), 5 mM MgCl2, 250 mM sucrose, 0.5 mM EDTA, and 2 mM DTT for 1 h on ice. The reaction was terminated by the addition of LDS sample buffer and heated to 70 °C. Samples were then analyzed by Western blotting.

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Proliferation Assays [3]
Cells were typically seeded in 96 well plate format (typically 4000–6000 cells/well) and treated after 24 h with increasing concentration of compound from 0 to 100 μM in 1/2 log unit increments. Cell viability was assessed after 72 h by CellTiter-Glo as recommended by the manufacturer’s instructions. Analysis and EC50 values were derived using GraphPadPrism.

Animal Model: Male Usp25+/+ and Usp25-/- mice aged 12 weeks[1]
Dosage: 40 mg/kg
Administration: Gavage; daily; for 7 days
Result: In comparison to control mice, the colons of Usp25-/-mice showed increased expression of proinflammatory cytokines and antibacterial peptides, and they were shielded from the weight loss and diarrhea caused by dextran sulfate sodium (DSS). They also showed impaired colon shortening.

[1]. ACS Chem Biol. 2017 Dec 15;12(12):3113-3125.

[2]. Nature Cancer volume 1, pages811–825(2020).

[3]. ACS Chem Biol. 2017 Dec 15;12(12):3113-3125. doi: 10.1021/acschembio.7b00334.

The ubiquitin proteasome system is widely postulated to be a new and important field of drug discovery for the future, with the ubiquitin specific proteases (USPs) representing one of the more attractive target classes within the area. Many USPs have been linked to critical axes for therapeutic intervention, and the finding that USP28 is required for c-Myc stability suggests that USP28 inhibition may represent a novel approach to targeting this so far undruggable oncogene. Here, we describe the discovery of the first reported inhibitors of USP28, which we demonstrate are able to bind to and inhibit USP28, and while displaying a dual activity against the closest homologue USP25, these inhibitors show a high degree of selectivity over other deubiquitinases (DUBs). The utility of these compounds as valuable probes to investigate and further explore cellular DUB biology is highlighted by the demonstration of target engagement against both USP25 and USP28 in cells. Furthermore, we demonstrate that these inhibitors are able to elicit modulation of both the total levels and the half-life of the c-Myc oncoprotein in cells and also induce apoptosis and loss of cell viability in a range of cancer cell lines. We however observed a narrow therapeutic index compared to a panel of tissue-matched normal cell lines. Thus, it is hoped that these probes and data presented herein will further advance our understanding of the biology and tractability of DUBs as potential future therapeutic targets.[1]

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Bacterial infection or abnormal colonization in the gastrointestinal system is associated with subsets of inflammatory bowel disease and colorectal cancer. Here we demonstrated essential roles of ubiquitin-specific protease 25 (USP25) in experimental colitis, bacterial infections and colon cancer. Knockout or pharmacologic inhibition of USP25 potentiated immune responses after induction of experimental colitis or bacterial infections that promoted clearance of infected bacteria and resolution of inflammation and attenuated Wnt and SOCS3–pSTAT3 signaling, which inhibited colonic tumorigenesis. USP25 levels were positively or negatively correlated with Fusobacterium nucleatum colonization and β-catenin levels or SOCS3 levels in human colorectal tumor biopsies, respectively, and predicted poor prognosis of patients with cancers in the gastrointestinal system. Our findings suggest USP25 as a promoter and druggable target for gastrointestinal infections and cancers.[2]

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The ubiquitin proteasome system is widely postulated to be a new and important field of drug discovery for the future, with the ubiquitin specific proteases (USPs) representing one of the more attractive target classes within the area. Many USPs have been linked to critical axes for therapeutic intervention, and the finding that USP28 is required for c-Myc stability suggests that USP28 inhibition may represent a novel approach to targeting this so far undruggable oncogene. Here, we describe the discovery of the first reported inhibitors of USP28, which we demonstrate are able to bind to and inhibit USP28, and while displaying a dual activity against the closest homologue USP25, these inhibitors show a high degree of selectivity over other deubiquitinases (DUBs). The utility of these compounds as valuable probes to investigate and further explore cellular DUB biology is highlighted by the demonstration of target engagement against both USP25 and USP28 in cells. Furthermore, we demonstrate that these inhibitors are able to elicit modulation of both the total levels and the half-life of the c-Myc oncoprotein in cells and also induce apoptosis and loss of cell viability in a range of cancer cell lines. We however observed a narrow therapeutic index compared to a panel of tissue-matched normal cell lines. Thus, it is hoped that these probes and data presented herein will further advance our understanding of the biology and tractability of DUBs as potential future therapeutic targets.[3]

C17H16BRF4NO2

422.2121

421.0301

C, 48.36; H, 3.82; Br, 18.92; F, 18.00; N, 3.32; O, 7.58

2165322-94-9

2165322-94-9

135397656

White to off-white solid powder

3.7

2

7

7

25

399

0

BrC1C([H])=C([H])C(=C(C=1[H])C([H])([H])N([H])C([H])([H])C([H])([H])O[H])OC([H])([H])C1C([H])=C([H])C(=C(C(F)(F)F)C=1[H])F

ITHSFXDGKQYOED-UHFFFAOYSA-N

InChI=1S/C17H16BrF4NO2/c18-13-2-4-16(12(8-13)9-23-5-6-24)25-10-11-1-3-15(19)14(7-11)17(20,21)22/h1-4,7-8,23-24H,5-6,9-10H2

2-(5-Bromo-2-(4-fluoro-3-(trifluoromethyl)benzyloxy)benzylamino)ethanol

AZ1; AZ-1; USP25/28 inhibitor AZ1; 2165322-94-9; USP25 and 28 inhibitor AZ-1; Ethanol, 2-[[[5-bromo-2-[[4-fluoro-3-(trifluoromethyl)phenyl]methoxy]phenyl]methyl]amino]-; CHEMBL4442615; 2-[[5-bromo-2-[[4-fluoro-3-(trifluoromethyl)phenyl]methoxy]phenyl]methylamino]ethanol; 2-((5-bromo-2-((4-fluoro-3-(trifluoromethyl)benzyl)oxy)benzyl)amino)ethanol; C17H16BrF4NO2; AZ 1; AZ1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 84~250 mg/mL ( 198.95~592.12 mM )
Ethanol : ~84 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.93 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.08 mg/mL (4.93 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.93 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)