Divergent Proteome Reactivity Influences Arm-Selective Activation of the Unfolded Protein Response by Pharmacological Endoplasmic Reticulum Proteostasis Regulators

Pharmacological activation of the activating transcription factor 6 (ATF6) arm of the unfolded protein response (UPR) has proven useful for ameliorating proteostasis deficiencies in cellular and mouse models of numerous etiologically diverse diseases. Previous high-throughput screening efforts identified the small molecule AA147 as a potent and selective ATF6 activating compound that operates through a mechanism involving metabolic activation of its 2-amino-p-cresol substructure affording a quinone methide, which then covalently modifies a subset of endoplasmic reticulum (ER) protein disulfide isomerases (PDIs). Another compound identified in this screen, AA132, also contains a 2-amino-p-cresol moiety; however, this compound showed less transcriptional selectivity, instead globally activating all three arms of the UPR. Here, we show that AA132 activates global UPR signaling through a mechanism analogous to that of AA147, involving metabolic activation and covalent modification of proteins including multiple PDIs. Chemoproteomic-enabled analyses show that AA132 covalently modifies PDIs to a greater extent than AA147. However, the extent of PDI labeling by AA147 approaches a plateau more rapidly than PDI labeling by AA132. These observations together suggest that AA132 can access a larger pool of proteins for covalent modification, possibly because its activated form is less susceptible to quenching than activated AA147. In other words, the lower reactivity of activated AA132 allows it to persist longer and modify more PDIs in the cellular environment. Collectively, these results suggest that AA132 globally activates the UPR through increased engagement of ER PDIs. Consistent with this, reducing the cellular concentration of AA132 decreases PDI modifications and enables selective ATF6 activation. Our results highlight the relationship between metabolically activatable-electrophile stability, ER proteome reactivity, and the transcriptional response observed with the enaminone chemotype of ER proteostasis regulators, enabling continued development of next-generation ATF6 activating compounds.

Full GO analysis is included in Table S4.

Cellular Thermal Shift Assay
ALMC2 cells grown to concentration of 2 million cells/mL and 15 mL cell suspension incubated in T75 flask with indicated compound (10 µM, 2h, 37° C).After treatment, cells were pelleted by centrifugation (3 min, 300 g) and washed with PBS before resuspension in PBS at 30 million cells/mL.100 µL cell suspension was added to 0.2 mL PCR tube before heat treatment at indicated temperature for 3 min and room temperature incubation for 3 min.Samples snap frozen at -80 C were lysed by sequential freeze-thaw cycles and centrifuged (15,000 g, 10 min) to pellet insoluble material.Soluble fractions were boiled for 5 min in Laemmli buffer with 100 mM DTT before loading onto SDS-PAGE gels.Proteins were transferred from gel slabs to PVDF membranes and blotted using rabbit anti-PDIA1 antibody (1:1000) Protein Tech) and visualized on the Odyssey Infrared Imaging System (Li-Cor Biosciences).
Cells pellets were resuspended in radioimmunoprecipitation assay (RIPA) buffer before sonication with a probe tip sonicator to lyse the cells (15 sec, 3 sec on/2 off, 30% amplitude).For each sample, 1 g lysate (500 µL) were reacted with click reagents to give final concentrations as follows: 100 µM of diazo biotin-azide (Click Chemistry Tools, Scottsdale, AZ), 800 µM copper (II) sulfate, 1.6 mM BTTAA ligand This stepwise process was repeated with an additional 100 µL 0.1 % TFA solution for a final volume of 300 µL.The pooled samples were fractionated using the Pierce high pH Reversed-Phase Fractionation Kit (Thermo Fisher Scientific 84868) according to manufacturer's instructions.The peptide fractions were eluted from the spin column with solutions of 0.1% triethylamine containing an increasing concentration of MeCN (5 -95% MeCN; 8 fractions).Samples were dried via vacuum centrifugation, reconstituted in 50 µL 0.1% formic acid, and stored at -80 °C until ready for mass spectrometry analysis.
For each sample, 100 μg of lysate were reacted with click reagents to give final concentrations as follows: 100 µM of diazo biotin-azide (Click Chemistry Tools, Scottsdale, AZ), 800 µM copper (II) sulfate, 1.6 mM BTTAA ligand (2-(4-((bis((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)acetic acid) (Albert Einstein College), and 5 mM sodium ascorbate.The reaction was placed on a shaker at 1000 rpm at 30 °C for 2 h.The proteins were then precipitated from the reaction mixture by adding an equal volume of 3:1 chloroform/methanol.The pellet was washed three times with 1:1 chloroform/methanol.The precipitate was suspended in 500 µL of 6 M urea with 25 mM ammonium bicarbonate and 140 µL of 10% SDS was added to this mixture to help solubilize the protein.50 µL of high-capacity streptavidin beads were washed with PBS and mixed with the protein solution in 6 mL of phosphate-buffered saline (PBS).This suspension was placed on a rotator or a shaker and agitated for 2 h.The beads were centrifuged and washed 5 times with PBS and 1% SDS.The protein was eluted from the beads by two washes of 50 mM sodium dithionite in 1% SDS for 1 h and then precipitated by chloroform/methanol precipitation as described above.
Air-dried pellets from the affinity precipitation were resuspended in 1% RapiGest SF (Waters) in 100 mM HEPES (pH 8.0).Proteins were reduced with 5 mM tris(2-carboxyethyl)phosphine hydrochloride (Thermo Fisher) for 30 min and alkylated with 10 mM iodoacetamide (Sigma Aldrich, St. Louis, MO) for 30 min at ambient temperature and protected from light.Proteins were digested for 18 h at 37 °C with 2 μg trypsin (Promega).After digestion, 20 μg of peptides from each sample were reacted for 1 h with the appropriate TMT-NHS isobaric reagent (ThermoFisher) in 40% (v/v) anhydrous acetonitrile and quenched with 0.4% NH4HCO3 for 1 h.Samples with different TMT labels were pooled and acidified with 5% formic acid.Acetonitrile was evaporated on a SpeedVac and debris was removed by centrifugation for 30 min at 18,000×g.MuDPIT (Multi-Dimensional Protein Identification Technology) microcolumns were prepared as described previously 79.LCMS/MS analysis was performed using a Q Exactive mass spectrometer equipped with an EASY nLC 1000 (Thermo Fisher).MuDPIT experiments were performed by 5 min sequential injections of 0, 20, 50, 80, 100% buffer C (500 mM ammonium acetate in buffer A) and a final step of 90% buffer C / 10% buffer B (20% water, 80% acetonitrile, 0.1% fomic acid, v/v/v) and each step followed by a gradient from buffer A (95% water, 5% acetonitrile, 0.1% formic acid) to buffer B. Electrospray ionization was performed directly from the analytical column by applying a voltage of 2.5 kV with an inlet capillary temperature of 275°C.Datadependent acquisition of MS/MS spectra was performed with the following settings: eluted peptides were scanned from 400 to 1800 m/z with a resolution of 30,000 and the mass spectrometer in a data dependent acquisition mode.The top ten peaks for each full scan were fragmented by HCD using a normalized collision energy of 30%, a 100 ms activation time, a resolution of 7500, and scanned from S13 100 to 1800 m/z.Dynamic exclusion parameters were 1 repeat count, 30 ms repeat duration, 500 exclusion list size, 120 s exclusion duration, and exclusion width between 0.51 and 1.51.Peptide identification and protein quantification was performed using the Integrated Proteomics Pipeline Suite (IP2, Integrated Proteomics Applications, Inc., San Diego, CA) as described previously.

General Synthetic Procedures
All compounds and reagents were purchased from Sigma-Aldrich, Acros, Alfa Aesar, Combi-blocks, and EMD Millipore unless otherwise noted and were used without further purification.Thin layer chromatography with Merck silica plates (60-F254), using UV light as the visualizing agent, was used to monitor reaction progress.Flash column chromatography was carried out using a Teledyne Isco Combiflash Nextgen 300+ machine using Luknova SuperSep columns (SiO2,25 µm) with ethyl acetate and hexanes as eluents. 1 H NMR spectra were recorded on a Varian INOVA-400 400MHz spectrometer.Chemical shifts are reported in δ units (ppm) relative to residual solvent peak.Coupling constants (J) are reported in hertz (Hz).Characterization data are reported as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, br=broad, m=multiplet), coupling constants, number of protons, mass to charge ratio.The compound's identity was confirmed via high-resolution mass spectrometry.

Synthesis of AA132
To a flame dried flask added 1-Decyl-3-methylimidazolium chloride (3.52 g, 13.6 mmol, 3.2 eq) and aluminum chloride (1.02 g, 7.65 mmol, 1.8 eq) and stirred for 16 hours under Argon to make the chloroaluminate fluid.To this flask at 0 °C, added 4-fluorobenzoyl chloride (S1, 500 µL, 4.25 mmol, 1 eq) slowly and the mixture became syrupy.The mixture was warmed to 75 ° C and acetylene gas generated in a separate flask from calcium carbide was bubbled through for 2 hours to afford the ßchlorovinyl ketone.The crude was added to ice water and extracted with ether which was washed once with brine and concentrated to give a yellow oil (S2), which was used for the next step without further purification.

Synthesis of AA132 yne
To 4-Iodobenzoic acid (S3, 738 mg, 3 mmol, 1 eq) dissolved in 15 mL DCM added oxalyl chloride (321 µL, 3.75 mmol, 1.25 eq) dropwise at 0 °C before addition of several drops of DMF.Reaction allowed to warm to room temperature and stir for 3h before TLC indicated complete conversion to the acyl chloride.
Reaction stirred under argon at 40 °C overnight before washing with saturated NaHCO3, and brine before drying over MgSO4 and concentrating.The crude residue was purified by flash column chromatography (SiO2, 9:1 Hex/EtOAc) to give an orange solid (S4, 638 mg, 71 % yield).

Figure S5 .
Figure S5.AA132 yne Shows Slower Protein Labeling Kinetics as Compared to AA147 yne .A. Representative immunoblot of the soluble fraction of PDIA1 from heat-treated ALMC2 cells at the temperatures indicated (40-72°C) preincubated with listed compound (10 µM, 2h).B. Graph of percent soluble fraction (y-axis) versus temperature (x-axis) for data in Fig S5A.Fitted curves calculated using Boltzmann Sigmoidal Fit in Prism and plotted with 95% confidence intervals (dotted lines).Tm is temperature on calculated sigmoidal curve with 50% soluble PDIA1 fraction remaining.Error bars represent S.E.M (N = 3 biological replicates).C. Representative immunoblot of PDIA1 levels from HEK293T cells treated with AA147(10 µM), AA132 (10 µM), or AA132 (30 µM) for 6h.D. Quantification of Fig S5C.Error bars represent standard error of the mean.E. Representative SDS-PAGE gel of Cy5-conjugated proteins from ALMC2 cells treated at indicated time point with AA147 yne (10 µM).F. Representative SDS-PAGE gel of Cy5-conjugated proteins from ALMC2 cells treated at indicated time point with AA132 yne (10 µM).G. Representative gel of AA132 yne and AA147 yne labeled proteins in HEK293T cells cotreated with indicated concentrations of β-mercaptoethanol or resveratrol for 4h.H. Quantification of Fig S5G.Error bars represent SEM for n=3 replicates.Percent labeling calculated as lane intensity relative to cotreatment with vehicle (0.1% DPBS) *p < 0.05 for a two-way ANOVA.

Figure S6 .
Figure S6.AA132 Selectively Activates ATF6 Transcriptional Signaling at Lower Doses. A. Fluorescence and coomassie-stained SDS-PAGE of lysates prepared from HEK293T cells treated with the indicated concentration of AA132 yne (4 h) and then conjugated to Cy-5.5-Azide.B-D.Top-10 GO terms for significantly induced genes (fold change >1.3, p<0.05) identified by RNAseq in HEK293T cells treated with 10 µM (A), 15 µM (B), or 30 µM (C) AA132 for 6 h.RNAseq data is included in TableS3Full GO analysis is included in TableS4.

( 2 -
(4-((bis((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)acetic acid) (Albert Einstein College), and 5 mM sodium ascorbate.The reaction was placed on a shaker at 1000 rpm at 30 °C for 90 The reaction was quenched with the sequential addition of cold methanol (4x volume), chloroform (1x volume), and DPBS (4x volume) to precipitate proteins.Proteins were pelleted by centrifugation (4,700 g, 10 min, 4 ˚C).The supernatant was discarded, and the pellets dried under air for 5 min.Protein pellets were resuspended in 6M urea in PBS (500 µL) with brief sonication.50 µL of high-capacity streptavidin beads were washed with PBS and mixed with the protein solution in 6 mL of phosphate-buffered saline (PBS).This suspension was placed on a rotator or a shaker and agitated for 2 h.The beads were centrifuged and washed 5 times with PBS and 1% SDS.The protein was eluted from the beads by two washes of 50 mM sodium dithionite in 1% SDS for 1 h and then precipitated by chloroform/methanol precipitation as described above.50 µL of freshly made 1:1 mixture 200 mM TCEP•HCl in DPBS and 600 mM K2CO3 in DPBS was added to each sample before incubation at 37 °C for 30 minutes while shaking.Alkylation of reduced thiols was achieved by addition of 70 µL freshly prepared 400 mM iodoacetamide in DPBS and incubation at room temperature while protected from light.The reaction was quenched by adding 130 µL of 10% SDS in DPBS and then diluted to approximately 0.2% SDS via DPBS (5.5 mL) and incubated with preequilibrated streptavidin agarose beads (3x1 mL PBS wash).The samples were rotated at room temperature for 1.5 hours, centrifuged at 2000 rpm for 2 minutes, and then washed sequentially with 5 mL 0.2% SDS in DPBS, 5 mL DPBS, and 5 mL 100 mM TEAB (Thermo Cat #90114) pH 8.5 to remove non-binding proteins.The beads were transferred to low-bind 1.5 mL Eppendorf tubes and the bound proteins digested overnight at 37 °C in 200 µL 100 mM TEAB containing 2 µg sequencing grade porcine trypsin, 1 mM CaCl2, and 0.01% ProteaseMax (Promega Cat #V2071).The beads were centrifuged at 2000 rpm for 5 minutes to separate the beads from the supernatant.200 µL supernatant was transferred to a new tube using a gel-loading tip, and the beads were washed with 100 µL TEAB buffer.The beads were centrifuged at 2000 rpm for 5 minutes and the supernatant combined with the previous.120 µL acetonitrile was added to each supernatant sample before addition of 80 µL (200 µg) of TMT 10 plex (Thermo Scientific, cat #90110) reconstituted in acetonitrile.The samples were incubated at room temperature for 1 hour and vortexed occasionally.7 µL of freshly prepared 5% hydroxylamine in water was added to each sample to quench the reaction, vortexed, and incubated for 15 minutes before quenching with addition of 5 µL MS-grade formic acid.The samples were then vacuum centrifuged to dryness.The samples were combined by redissolving the contents of one tube in 200 µL 0.1 % trifluoroacetic acid solution in water and sequential transfer to the respective multiplexed experiment until all samples were redissolved.
NMR Spectra for S51 H NMR for S5