Structure‐Based Design of Fluorogenic Substrates Selective for Human Proteasome Subunits

Abstract Proteasomes are established therapeutic targets for hematological cancers and promising targets for autoimmune diseases. In the past, we have designed and synthesized mechanism‐based proteasome inhibitors that are selective for the individual catalytic activities of human constitutive proteasomes and immunoproteasomes: β1c, β1i, β2c, β2i, β5c and β5i. We show here that by taking the oligopeptide recognition element and substituting the electrophile for a fluorogenic leaving group, fluorogenic substrates are obtained that report on the proteasome catalytic activity also targeted by the parent inhibitor. Though not generally applicable (β5c and β2i substrates showing low activity), effective fluorogenic substrates reporting on the individual activity of β1c, β1i, β2c and β5i subunits in Raji (human B cell) lysates and purified 20S proteasome were identified in this manner. Our work thus adds to the expanding proteasome research toolbox through the identification of new and/or more effective subunit‐selective fluorogenic substrates.


Lysate preparation
Lysates of cells were prepared by treating cell pellets with 4 volumes of lysis buffer containing 50 mM TRIS-HCl pH 7.5, 2 mM DTT, 5 mM MgCl2, 10% glycerol, 2 mM ATP, and 0.05% digitonin for 15-60 min, followed by centrifugation at 20000g to squeeze out the soluble proteome from the cells. Protein concentration was determined using Qubit® protein assay kit (Thermofisher).

Fluorogenic substrate activity assay
In a polypropylene 96-well plate, assay mixture is prepared by mixing 100 μL assay buffer (lacking purified proteasome or lysate) with 2 μL substrate (10 mM stock). Addition of 100 μL assay buffer (pre-mixed with lysate or purified 20S proteasome) yielded the desired final 100 μM substrate concentration. All activity measurements were directly recorded on a pre-heated TECAN plate reader at 37 o C in quadruplo using λex = 365 nm and λem = 450 nm for ACC substrates (as determined by Table S3) and λex = 345 nm and λem = 445 nm for AMC substrates in 1 min intervals for a 2 h period. Michaelis-Menten calculations were performed using a non-linear fit in Prism v.6.0. Pre-incubation of the lysate assay buffer with the desired proteasome inhibitor for 1 h at 37 o C delivered the proteasome subunit inactivated lysate for substrate analysis.

Competitive ABPP by enzyme precipitation and SDS-PAGE
After the fluorogenic substrate activity assay, 10 µL 10x ABP-cocktail (Cy5-NC-001, BODIPY(FL)-LU-112, BODIPY(TMR)-NC-005-VS) was added to each well and incubated for 60 min at 37 o C to label the residual active proteasome subunits. The wells were transferred to individual Eppendorf tubes, proteins were precipitated by CHCl3/MeOH according to Wessel andFlüggel (doi: 10.1016/0003-2697(84)90782-6). The pellet was washed twice with PBS and dissolved in 20 µL 0.01% SDS in PBS followed by 5 min boiling with 10 µL gel-loading buffer. Electrophoresis was performed on 12.5% SDS-PAGE gel for 15 min at 80 V and then 2 h at 130 V. On each gel, 2.5 µl of page ruler was used. Multiplex fluorescent detection of residual ABPs was performed on a ChemiDoc™ MP System with Cy5, Cy3 and Cy2 channels.

Synthetic procedures
All reagents were of commercial grade and used as received unless stated otherwise. Solvents used in synthesis were dried and stored over 4Å molecular sieves, except MeOH and ACN which were stored over 3Å molecular sieves. Triethylamine (Et3N) and di-isopropylethylamine (DiPEA) were stored over KOH pellets.
High-resolution mass spectrometry (HRMS) was performed on a Thermo Scientific Q Exactive HF Orbitrap mass spectrometer equipped with an electrospray ion source in positive-ion mode (source voltage 3.5 kV, sheath gas flow 10, capillary temperature 275°C) with resolution R = 240.000 at m/z 400 (mass range of 150-6000) correlated to an external calibration, or on a Waters Synapt G2-Si (TOF) equipped with an electrospray ion source in positive mode (source voltage 3.5 kV) and LeuEnk (m/z = 556.2771) as internal lock mass. 1 H and 13 C NMR spectra were recorded on a Bruker AV-400 NMR, a Bruker DMX-400 NMR instrument (400 and 101 MHz respectively), a Bruker AV-500 NMR instrument (500 and 126 MHz respectively), and Bruker AV-600 NMR instrument (600 and 151 MHz respectively). Chemical shifts (δ) are given in ppm relative to tetramethylsilane as internal standard or the residual signal of the deuterated solvent. Coupling constants (J) are given in Hz. All given 13 C-APT spectra are proton decoupled. Assignment of NMR spectra was based on 1 H-COSY and 1 H-13 C-HSQC.
HPLC purification was performed on a Gilson HPLC system coupled to a Magerey-Nagel Nucleodur C18 Gravity 5 μm 250 ×10 mm column, or on an Agilent 1200 HPLC/6130 MS system coupled to a Magerey-Nagel Nucleodur C18 Gravity 5μm 250×10 mm column or on a Waters autopurifier HPCL/MS system coupled to a Phenomenex Gemini 5μm 150×21.2 mm column.

Synthesis of ACC fluorogenic substrates
Procedure for Fmoc protection during solid phase synthesis The Fmoc-protected amino acids were prepared by treating the free amine with Fmoc-Cl in H2O/Organic solvent system. The choice of organic solvent varied based upon which free amine was used (THF or 1,4-dioxane). The free amine and NaHCO3 (1.2 eq) were added to H2O followed by the addition of the organic solvent. The H2O/organic solvent ratio used was 1/1 (v/v). The solution was cooled in an ice bath and stirred rapidly before addition of Fmoc-Cl (1.2 eq). Fmoc-Cl was dissolved in either THF or 1,4-dioxane and slowly added to the reaction mixture. After addition of the Fmoc-Cl the reaction was stirred at 0 o C for at least 1h. Then, the reaction mixture was allowed to warm to RT and stirred overnight. TLC was used to monitor the reaction until its completion. The reaction mixture was evaporated in vacuo and the remaining solid was dissolved in H2O. The solution was extracted with Et2O (4x) and the aqueous phase was acidified to pH 2 with 2 M HCl and extracted with EtOAc (2x). The combined organic fractions were washed once with brine, dried over MgSO4, filtered and evaporated in vacuo. The product was further purified using flash column chromatography.
Kaiser test procedure A set of 3 reagents was made as a stock for the Kaiser test. Solution A; 500 mg ninhydrin in 10 mL EtOH. Solution B; 20 g phenol in 10 mL EtOH. Solution 3; 200 μL 1 mM solution of KCN diluted to a total volume of 10 mL with pyridine. Three drops of each solution together with a small amount of resin was added to a LC-MS vial, mixed and stored at 80 o C for 5 min. The solution should be either blue (indication of free amines) or yellow (no free amines).

N-(Fluorenylmethoxycarbonyl)aminocoumarin-4-acetic acid (12).
Compound 23 (1.50 g, 6.87 mmol) and DCM (40 mL) were added to a 250 mL round-bottom flask equipped with a reflux condenser. Next, TMSCl (1.63 g, 15.1 mmol) and DIPEA (1.94 g, 15.1 mmol) were added while stirring and the reaction was heated to reflux for 3 h. After reflux the reaction mixture was cooled to 0 o C using an ice bath after which Fmoc-Cl (1.94 g, 7.53 mmol) was added in small portions. The reaction was left to stir at 0 o C for 1 h before it was allowed to warm to RT and stirred overnight. Addition of MeOH caused the formation of a white precipitate. The product was collected by filtration and washed with cold MeOH, Et2O and dried in a vacuum oven to yield compound 5 (2.71 g, 6.16 mmol, 90%

Procedure S-A: ACC-Rink Amide resin
Rink Amide resin (5.03 g, 1.21 mmol) was placed in two 20 mL syringes equipped with a filter and the resin was gently shaken in DMF for 0.5 h using a mechanical shaker. After filtration and washing with DMF (3x), a solution of 20% piperidine in DMF was added and the resin was shaken for 5 min. The resin was filtered, washed with DMF (3x) and new 20% piperidine in DMF was added. After 5 min the resin was filtered, washed with DMF (3x) and fresh 20% Piperidine in DMF was added and the resin was again shaken for 25 min followed by filtration and washing with DMF (6x). Compound 5 (1.32 g, 3.01 mmol), DICI (378 mg, 3.01 mmol), HOBt (465 mg, 3.01 mmol) were pre-activated in THF and added to the resin. The reaction mixture was stirred overnight. The resin was filtered and washed with DMF (6x) and then the reaction was repeated with compound 5 (1.32 g, 3.01 mmol), DICI (378 mg, 3.01 mmol), HOBt (465 mg, 3.01 mmol) in THF respectively to improve the yield of ACC coupling to the resin. After the second coupling the resin was washed with DMF (6x), MeOH (3x), DCM (3x) and Et2O (2x). Kaiser test was used to indicate completion of the coupling to the resin. For N-terminus acetylation, a capping solution of 5% (v/v %) Ac2O, 0.1 M DIPEA in DMF was used and the reaction was gently shaken for 0.5 h. A small amount of resin was subjected to cleavage using a mixture of TFA:TIPS:H2O (v/v/v % 95:2.5:2.5). The resin was cleaved in a LC-MS vial using 1 drop of cleaving solution and after the resin turned red, a mixture of H2O:ACN:tBuOH (v/v/v % 1/1/1) was added and mixed as a sample for LC-MS analysis.

Procedure S-B: AA1-ACC-Rink Amide resin
To a syringe equipped with a filter containing ACC-Rink Amide resin (0.2 mmol), 20% piperidine/DMF was added (3x after 5, 15 and 25 min) with washing with DMF (3x) between each deprotection. After removal of the Fmoc-group, amino acid (0.8 mmol), HATU (304 mg, 0.8 mmol) and 2,4,6-trimethylpyridine (97 mg, 0.8 mmol) in DMF were activated for 2 min and added to the ACC-Rink amide resin. The reaction was gently shaken overnight, and the resin was washed with DMF (6x). At this point the resin was subjected to a Kaiser test using the described procedure. After the Kaiser test a capping solution (5% Ac2O, 0.1 M DIPEA in DMF) was added to the resin and the reaction was gently shaken for 30 min. After the reaction the resin was washed with DMF (3x), MeOH (3x), DCM (3x) and Et2O (2x). A small amount of resin was subjected to cleavage using a mixture of TFA:TIPS:H- 2O (v/v/v % 95:2.5:2.5). The resin was cleaved in a LC-MS vial using 1 drop of cleaving solution and after the resin turned red, a mixture of H2O:ACN:tBuOH (v/v/v % 1/1/1) was added and mixed as a sample for LC-MS analysis to confirm the AA coupling.

Procedure S-C: P2, P3, P4 -ACC-Rink Amide resin
To a syringe equipped with a filter containing AA1-ACC-Rink Amide resin (0.2 mmol), 20% piperidine/DMF was added (3x after 5, 15 and 25 min) with washing with DMF (3x) between each deprotection. After removal of the Fmoc-group, amino acid (0.8 mmol), HOBt (108 mg, 0.8 mmol) and DICI (101 mg, 0.8 mmol) in DMF were activated for 2 min and added to the ACC-Rink amide resin. The reaction was gently shaken overnight and the resin was washed with DMF (6x). At this point the resin was subjected to a Kaiser test using the described procedure. After the Kaiser test 20% piperidine in DMF was added (3x after 5, 15 and 25 min) to remove the Fmoc-group followed by addition of a capping solution (5% AC2O, 0.1 M DIPEA in DMF) and the reaction was gently shaken for 30 min. After the reaction the resin was washed with DMF (3x), MeOH (3x), DCM (3x) and Et2O (2x). A small amount of resin was subjected to cleavage using a mixture of TFA:TIPS:H2O (v/v/v % 95:2.5:2.5). The resin was cleaved in a LC-MS vial using 1 drop of cleaving solution and after the resin turned red, a mixture of H2O:ACN:tBuOH (v/v/v % 1/1/1) was added and mixed as a sample for LC-MS analysis to confirm the AA coupling. This procedure was repeated until all desired AAs were coupled.

Synthesis of AMC fluorogenic substrates
Scheme S1. General synthetic route towards peptide-AMC fluorogenic substrate. Reagents and conditions: A) POCl3, Pyr, Pg-AA-OH, THF; B) HCTU, DiPEA, Boc-AA-OH; C) TFA/DCM; D) NaN3, DMF, 50 o C Procedure A: AA-coupling to AMC AMC 15 (1.2 eq.) and free acid (1.0 eq.) were dissolved in dry THF and cooled to 0ºC. Pyridine (10 eq.) was added, followed by slow addition of POCl3 (3.7 eq.) over the course of 1 h. After stirring for an additional 1.5 h, the reaction was quenched by the addition of sat. aq. NaHCO3. Subsequently, THF was evaporated under vacuo and the mixture was redissolved in EtOAc, washed with 1 M HCl (2x), sat. aq. NaHCO3 (2x) and brine. The organic layer was dried over MgSO4, filtered and concentrated in vacuo and used without further purification.

Procedure B: AA-coupling
Free amine (1.0 eq.) and free acid (1.2 eq.) were dissolved in DCM, followed by addition of HCTU (1.2 eq.) and DiPEA (3.5 eq.). After stirring overnight, the reaction mixture was concentrated in vacuo and re-dissolved in EtOAc, washed with 1 M HCl (2x), sat. aq. NaHCO3 (2x) and brine (in case of morpholino acetic acid coupling, no 1 M HCl washing). The organic layer was dried over MgSO4, filtered and concentrated in vacuo. Purification by silica gel flash column chromatography yielded the title compound.

Procedure C: Boc-deprotection
The appropriate Boc-protected amino acid derivative was dissolved in TFA:DCM 1:1 and stirred for 20 min. Co-evaporation with toluene (3x) and CHCl3 (1x) afforded the TFAsalt, which was used without further purification unless stated otherwise.

Procedure D: Fmoc-deprotection
The appropriate Fmoc-protected amino acid derivative was dissolved in DMF (0.5 M) and N3 (1.2 eq.) was added. The reaction mixture was stirred overnight at 50ºC followed by evaporation of the solvent under vacuo. Purification by silica gel flash column chromatography yielded the title compound.