External ID: UCHL1 FAST DUB HTS Confirmation

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of UCHL1: Confirmation Screen
1. Compounds in 8-point, half-log dose response were transferred from source plates to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076) starting at an initial concentration of 25 uM (columns 45-46: DMSO control)
2. Reagent, 2.5 uL of UCHL1 in assay buffer (final concentration of 1 nM) to columns 1-46
3. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
4. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 7 nM) to all wells
5. Centrifuge plates (10s, 500 rpm)
6. Time, Incubate at room temperature for 60 minutes
7. Detection, Fluorescence, PheraStar, FITC Module

Comment: Each compound was tested in triplicate in the dose-response confirmation assay. Normalization of raw data to positive and negative control wells, systematic pattern models, and curve fitting were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018). For measuring compound potency, a four-parameter sigmoid Hill curve model was fitted to the data (Gubler et al., 2018) and the absolute IC50 was used for potency measurements, that is the concentration of compound where the data showed 50% inhibition. If no data points achieved 50% inhibition, then the absolute IC50 was reported as >25 uM (the highest tested concentration). Maximal efficacy of each compound was reported as a percent inhibition based on normalization to plate controls.

Primary screen hits were confirmed as active if they demonstrated greater than or equal to 30% inhibition of UCHL1 (UCHL1_EFFICACY >/= 30). Active compounds were given a score based on potency.

External ID: USP8 FAST DUB HTS Primary

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of USP8: Primary Screen
1.#Compound, 20 nL of screening compounds (final concentration of 20 uM) to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076) (columns 45-46: DMSO control)
2.#Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
3.#Reagent, 2.5 uL of USP8 in assay buffer (final concentration of 1 nM) to columns 1-46
4.#Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 50 nM) to all wells
5.#Time, Incubate at room temperature for 30 minutes
6.#Detection, Fluorescence, PheraStar, FITC Module

Comment: Normalization of raw data to positive and negative control wells were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018)

Compounds demonstrating greater than or equal to 30% inhibition of USP8 (USP8_EFFICACY >/= 30) were called as active, compounds demonstrating activity below this threshold were called as inactive.

External ID: USP17 FAST DUB HTS Primary

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of USP17: Primary Screen
1. Compound, 50 nL of screening compounds (final concentration of 50 uM) to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076) (columns 45-46: DMSO control)
2. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
3. Reagent, 2.5 uL of USP17 in assay buffer (final concentration of 1 nM) to columns 1-46
4. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 40 nM) to all wells
5. Time, Incubate at room temperature for 3 hours
6. Reagent, 2.5 uL of stop buffer to all wells
7. Centrifuge plates (5s, 350 rpm)
8. Detection, Fluorescence, PheraStar, FITC Module

Comment: Normalization of raw data to positive and negative control wells were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018)

Compounds demonstrating greater than or equal to 30% inhibition of USP17 (USP17_EFFICACY >/= 30) were called as active, compounds demonstrating activity below this threshold were called as inactive.

External ID: USP7 FAST DUB HTS Primary

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of USP7: Primary Screen
1. Reagent, 2.5 uL of USP7 in assay buffer (final concentration of 4 nM) to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076)
2. Compound, 20 nL of screening compounds (final concentration of 20 uM) to columns 1-46 (columns 45-46: DMSO control)
3. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
4. Time, Incubate at room temperature for 60 minutes
5. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 40 nM) to all wells
6. Time, Incubate at room temperature for 10 minutes
7. Detection, Fluorescence, PheraStar, FITC Module

Comment: Normalization of raw data to positive and negative control wells were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018)

Compounds demonstrating greater than or equal to 30% inhibition of USP7 (USP7_EFFICACY >/= 30) were called as active, compounds demonstrating activity below this threshold were called as inactive.

External ID: USP28 FAST DUB HTS Primary

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of USP28: Primary Screen
1. Compound, 25 nL of screening compounds (final concentration of 25 uM) to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076) (columns 45-46: DMSO control)
2. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
3. Reagent, 2.5 uL of USP28 in assay buffer (final concentration of 0.5 nM) to columns 1-46
4. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 12 nM) to all wells
5. Time, Incubate at room temperature for 30 minutes
6. Reagent, 2.5 uL of stop buffer to all wells
7. Centrifuge plates (5s, 350 rpm)
8. Detection, Fluorescence, PheraStar, FITC Module

Comment: Normalization of raw data to positive and negative control wells were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018)

Compounds demonstrating greater than or equal to 30% inhibition of USP28 (USP28_EFFICACY >/= 30) were called as active, compounds demonstrating activity below this threshold were called as inactive.

External ID: USP10 FAST DUB HTS Primary

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of USP10: Primary Screen
1. Compound, 20 nL of screening compounds (final concentration of 20 uM) to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076) (columns 45-46: DMSO control)
2. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
3. Reagent, 2.5 uL of USP10 in assay buffer (final concentration of 15 nM) to columns 1-46
4. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 15 nM) to all wells
5. Time, Incubate at room temperature for 60 minutes
6. Detection, Fluorescence, PheraStar, FITC Module

Comment: Normalization of raw data to positive and negative control wells were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018)

Compounds demonstrating greater than or equal to 30% inhibition of USP10 (USP10_EFFICACY >/= 30) were called as active, compounds demonstrating activity below this threshold were called as inactive.

External ID: UCHL1 FAST DUB HTS Primary

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of UCHL1: Primary Screen
1. Reagent, 2.5 uL of UCHL1 in assay buffer (final concentration of 1 nM) to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076)
2. Compound, 20 nL of screening compounds (final concentration of 20 uM) to columns 1-46 (columns 45-46: DMSO control)
3. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
4. Time, Incubate at room temperature for 60 minutes
5. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 7 nM) to all wells
6. Time, Incubate at room temperature for 10 minutes
7. Detection, Fluorescence, PheraStar, FITC Module

Comment: Normalization of raw data to positive and negative control wells were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018)

Compounds demonstrating greater than or equal to 30% inhibition of UCHL1 (UCHL1_EFFICACY >/= 30) were called as active, compounds demonstrating activity below this threshold were called as inactive.

External ID: OTUD3 FAST DUB HTS Primary

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of OTUD3: Primary Screen
1. Compound, 25 nL of screening compounds (final concentration of 25 uM) to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076) (columns 45-46: DMSO control)
2. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
3. Reagent, 2.5 uL of OTUD3 in assay buffer (final concentration of 12 nM) to columns 1-46
4. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 40 nM) to all wells
5. Time, Incubate at room temperature for 2 hours
6. Reagent, 2.5 uL of stop buffer to all wells
7. Centrifuge plates (5s, 350 rpm)
8. Detection, Fluorescence, PheraStar, FITC Module

Comment: Normalization of raw data to positive and negative control wells were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018)

Compounds demonstrating greater than or equal to 30% inhibition of OTUD3 (OTUD3_EFFICACY >/= 30) were called as active, compounds demonstrating activity below this threshold were called as inactive.

External ID: USP30 FAST DUB HTS Primary

Protocol: Deubiquitinases were purified from E. Coli. Ub-Rho substrate was purchased from Boston BioChem (Cat #: U-555). Assay buffer consisted of 50 mM Tris pH 8, 50 mM NaCl, 0.002% Tween20, 5 mM DTT in MilliQ water. Stop buffer consisted of assay buffer + 0.2% trifluoroacetic acid.

Protocol for Inhibitors of USP30: Primary Screen
1. Compound, 25 nL of screening compounds (final concentration of 25 uM) to columns 1-46 of a Greiner 1536 well Black, flat bottom, polystyrene microplate (Griener 782076) (columns 45-46: DMSO control)
2. Reagent, 2.5 uL of assay buffer to columns 47-48 on all plates (No enzyme control)
3. Reagent, 2.5 uL of USP30 in assay buffer (final concentration of 1 nM) to columns 1-46
4. Reagent, 2.5 uL of Ub-Rho in assay buffer (final concentration of 12 nM) to all wells
5. Time, Incubate at room temperature for 2 hours
6. Reagent, 2.5 uL of stop buffer to all wells
7. Centrifuge plates (5s, 350 rpm)
8. Detection, Fluorescence, PheraStar, FITC Module

Comment: Normalization of raw data to positive and negative control wells were applied to the data using Helios, a high-throughput screening data analysis program developed at Novartis.(Gubler et al., 2018)

Compounds demonstrating greater than or equal to 30% inhibition of USP30 (USP30_EFFICACY >/= 30) were called as active, compounds demonstrating activity below this threshold were called as inactive.