Phenylhydrazone-based endoplasmic reticulum proteostasis regulator compounds with enhanced biological activity
- Department of Chemistry, The Scripps Research Institute, United States
- The Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, United States
- Department of Molecular and Cellular Biology, The Scripps Research Institute, United States
Figures
Figure 1 with 1 supplement
AA263 covalently modifies protein disulfide isomerase (PDI) family members.
(A) Mechanism of AA263 metabolic activation and covalent protein modification. (B) Activation of the ERSE.FLuc ATF6 reporter in HEK293T cells treated for 18 hr with AA263 (10 µM) or thapsigargin (Tg, 500 nM) in the presence or absence of β-mercaptoethanol (BME; 55 or 110 μM). Error bars show SEM for N > 6 replicates. *p < 0.05, **p < 0.01 for unpaired t-test. (C) Activation of the ERSE.FLuc ATF6 reporter in HEK293T cells treated for 18 hr with AA263 (10 µM) or Tg (500 nM) in the presence or absence of resveratrol (2.5 µM). Error bars show SEM for N > 6 replicates. *p < 0.05 for unpaired t-test. E. (D) Structure of AA263yne. (E) Activation of the ERSE.FLuc ATF6 reporter in HEK293T cells treated for 18 hr with the indicated dose of AA263 or AA263yne. Error bars show SEM for n = 3 replicates. The EC50 is shown. (F) Activation of the ERSE.FLuc ATF6 reporter in HEK293T cells treated with AA263yne (10 µM) in the presence or absence of BME (55 μM) or resveratrol (2.5 μM). *p < 0.05, **p < 0.01 for unpaired t-test. (G) Representative SDS–PAGE gel of Cy5-conjugated proteins from HEK293T cells treated for 4 hr with vehicle (0.1% DMSO), AA263yne (5 µM), or the combination of AA263yne (5 µM) and AA263 (20 μM). Coomassie-stained gel is shown below. (H) Venn diagram of identified targets of AA132yne and AA263yne. Hits defined as proteins with a significant fold change greater than 3 (p < 0.01) that were identified in two independent biological experiments. (I) TMT reporter ion enrichment ratio of select PDIs from comparative chemoproteomic experiment in HEK293T cells treated with the indicated compound relative to DMSO (n = 8 biological replicates). ***p < 0.005 for a two-way ANOVA. See also Figure 1—source data 1.
-
Figure 1—source data 1
Excel spreadsheet showing raw data used to generate panels Figure 1B, C, E, F, and I.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig1-data1-v1.zip
-
Figure 1—source data 2
Uncropped gel shown in Figure 1G.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig1-data2-v1.zip
-
Figure 1—source data 3
Uncropped gel of Figure 1G with the same annotations shown in Figure 1G.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig1-data3-v1.zip
Figure 1—figure supplement 1
AA263 covalently modifies protein disulfide isomerase family members.
(A) Activation of the ERSE.FLuc ATF6 reporter in HEK293T cells treated for 18 hr with vehicle (0.1% DMSO), Tg (500 nM), AA263 (10 µM), or AA263-1 (10 μM). Structures of AA263 and AA263-1 are shown to the right. Error bars show SEM for n > 3 replicates. (B) Activation of the ERSE.FLuc ATF6 reporter in HEK293T cells treated for 18 hr with vehicle (0.1% DMSO), Tg (500 nM), AA263 (10 µM), or AA263yne (10 μM), and/or Ceapin-A7 (CP7; 10 µM). Error bars show SEM for n = 3 replicates. ***p < 0.005 for two-way ANOVA. (C) Representative SDS–PAGE gel of Cy5-conjugated proteins from HEK293T cells treated for 4 hr with vehicle (0.1% DMSO), AA263yne (10 µM), or the combination of AA263yne (10 µM) and either resveratrol (10 μM) or BME (55 μM). (D) Representative SDS–PAGE gel of Cy5-conjugated proteins from lysates prepared from HEK293T treated for 1 hr with vehicle, AA147yne, or AA263yne (5 μM). See also Figure 1—figure supplement 1—source data 1.
-
Figure 1—figure supplement 1—source data 1
Excel spreadsheet showing raw data used to generate Figure 1—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig1-figsupp1-data1-v1.zip
-
Figure 1—figure supplement 1—source data 2
Uncropped gels shown in Figure 1—figure supplement 1C and D.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig1-figsupp1-data2-v1.zip
-
Figure 1—figure supplement 1—source data 3
Uncropped gels shown in Figure 1—figure supplement 1C and D with the same annotations shown in Figure 1—figure supplement 1C and D.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig1-figsupp1-data3-v1.zip
Figure 2 with 1 supplement
Identification of AA263 analogs that show enhanced ATF6 activation.
(A) Structures of AA263 analogs. (B) Activation of the ERSE.Fluc ATF6 reporter in HEK293T cells reporter treated for 18 hr with vehicle, Tg (0.5 μM), or the indicated analog (10 µM). Error bars show SEM for n = 3–6 biological replicates. ***p < 0.005 from one-way ANOVA. (C) Expression, measured by RNAseq, of gene sets comprising target genes regulated downstream of the ATF6 (left), IRE1/XBP1s (middle), or PERK/ISR (right) arms of the unfolded protein response (UPR) in HEK293T cells treated for 6 hr with 10 µM AA263, AA263yne, or AA263-5. Full RNAseq data and genesets used in this analysis are shown in Supplementary file 1. *p < 0.05, ***p < 0.005 for one-way ANOVA. See also Figure 2—source data 1.
-
Figure 2—source data 1
Excel spreadsheet showing raw data used to generate Figure 2B.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig2-data1-v1.zip
Figure 2—figure supplement 1
Identification of AA263 analogs that show enhanced ATF6 activation.
(A) Expression, measured by qPCR, of the ATF6 target gene BiP, the IRE1/XBP1s target gene DNAJB9, and the PERK/ISR target gene CHOP in HEK293T cells treated for 6 hr with the indicated compound (10 µM). Error bars show SEM for n = 3 biological replicates. *p < 0.05, ***p < 0.001 for one-way ANOVA relative to vehicle-treated cells. (B) Activation of the ERSE-FLuc ATF6 reporter in HEK293T cells treated for 18 hr with the indicated concentration of AA263, AA263yne, or AA263-5. Error bars show SEM for n = 3 replicates. The data for AA263 and AA263yne is the same as that shown in Figure 1E and are included for comparison. (C) Expression, measured by RNAseq, of gene sets comprising target genes regulated downstream of the heat shock response (HSR, left) or the oxidative stress response (OSR, right) in HEK293 cells treated for 6 hr with 10 µM AA263, AA263yne, or AA263-5. Full RNAseq data and genesets used in this analysis are shown in Supplementary file 1. See also Figure 2—figure supplement 1—source data 1.
-
Figure 2—figure supplement 1—source data 1
Excel spreadsheet showing raw data used to generate Figure 2—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig2-figsupp1-data1-v1.zip
Figure 3 with 1 supplement
Diversification of the AA263 B-ring affords improved AA263 analogs.
(A) Structures of AA263 analogs. (B) Heat map showing activation of the ERSE-FLuc ATF6 reporter in HEK293T cells treated for 18 hr with the indicated dose of compound. (C) Activation of the ERSE.Fluc ATF6 reporter in HEK293T cells treated for 18 hr with the indicated dose of compound. Error bars show SEM for n = 6 replicates. (D) Expression, measured by qPCR, of the ATF6 target gene BiP in HEK293T cells treated with indicated AA263 analog (10 µM) for 6 hr. Error bars show SEM for n = 3 independent biological replicates. *p < 0.05, ***p < 0.001 for one-way ANOVA. See also Figure 3—source data 1.
-
Figure 3—source data 1
Excel spreadsheet showing raw data used to generate Figure 3B–D.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig3-data1-v1.zip
Figure 3—figure supplement 1
Diversification of the AA263 B-ring affords improved AA263 analogs.
(A) Expression, measured by qPCR, of the IRE1/XBP1s target gene DNAJB9 and the PERK/ISR target gene CHOP in HEK293T cells treated for 6 hr with the indicated AA263 analog (10 µM). Error bars show SEM for n = 3 independent biological replicates. (B) Representative SDS–PAGE gel of Cy5-conjugated proteins from HEK293T cells treated for 4 hr with vehicle, AA263yne (10 μM), or co-treatment of AA263yne (10 μM) with the indicated analog (40 μM). See also Figure 3—figure supplement 1—source data 1.
-
Figure 3—figure supplement 1—source data 1
Excel spreadsheet showing raw data used to generate Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig3-figsupp1-data1-v1.zip
-
Figure 3—figure supplement 1—source data 2
Uncropped gel shown in Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig3-figsupp1-data2-v1.zip
-
Figure 3—figure supplement 1—source data 3
Uncropped gel shown in Figure 3—figure supplement 1B with the same annotations shown in Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig3-figsupp1-data3-v1.zip
Figure 4 with 1 supplement
AA263 analogs improve secretory proteostasis for the disease-associated AAT-Z variant.
Intracellular AAT-Z polymer levels (A), extracellular AAT-Z polymer levels in conditioned media (B), and elastase inhibition activity of AAT-Z in conditioned media (C) from Huh7.5Z cells treated for 24 hr with AA263 (10 µM), AA263yne (10 µM), or AA263-20 (10 µM). Error bars show SEM for n > 5 replicates. Data are shown normalized to vehicle-treated cells. *p < 0.05, **p < 0.01, ***p < 0.005 for one-way ANOVA compared to vehicle-treated cells. See also Figure 4—source data 1.
-
Figure 4—source data 1
Excel spreadsheet showing raw data used to generate Figure 4A–C.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig4-data1-v1.zip
Figure 4—figure supplement 1
AA263 analogs improve secretory proteostasis for the disease-associated AAT-Z variant.
Expression, measured by qPCR, of the ATF6 target gene BiP in Huh7.5Z cells treated for 6 hr with 10 µM of AA263, AA263yne, or AA263-20. Error bars show SEM for n = 3 replicates. *p < 0.05, **p < 0.01, ***p < 0.005 for one-way ANOVA compared to vehicle-treated cells are shown. See also Figure 4—figure supplement 1—source data 1.
-
Figure 4—figure supplement 1—source data 1
Excel spreadsheet showing raw data used to generate Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig4-figsupp1-data1-v1.zip
Figure 5 with 1 supplement
Enhanced AA263 analogs promote the trafficking and plasma membrane activity of destabilized, disease-associated GABAA receptors.
(A) Representative immunoblot (above) and quantification (below) of surface biotinylated γ2 in HEK293T cells stably expressing α1β2γ2(R177G) GABAA receptors treated for 24 hr with 10 µM AA263yne or AA263-5. Na+/K+ ATPase serves as a loading control. (B) Representative immunoblot showing surface γ2 expression in HEK293T cells transiently transfected with α1β2γ2(R177G) receptors and treated with indicated AA263 analogs 10 µM, 24 hr. Na+/K+ ATPase serves as a loading control. (C) Representative evoked inhibitory postsynaptic current (eIPSC) traces for α1β2γ2(WT) GABAA receptors and α1β2γ2(R177G) GABAA receptors treated for 24 hr with DMSO, AA263yne (10 µM), or AA263-20 (10 µM). 10 mM GABA (saturating condition) was applied to the recorded cells to evoke currents. Histograms showing changes in eIPSC peak amplitude (D) and peak current density (E) for the indicated groups. Band intensities were quantified using ImageJ software, normalized to the DMSO control condition. Each data point is reported as mean ± SEM. One-way ANOVA followed by post hoc Dunnett’s test was used for statistical analysis for A and B. Kruskal–Wallis test followed by post hoc Dunn’s test was used for statistical analysis for D and E. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure 5—source data 1 and Figure 5—source data 2.
-
Figure 5—source data 1
Excel spreadsheet showing raw data used to generate Figure 5A, B, D, E.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig5-data1-v1.zip
-
Figure 5—source data 2
Uncropped gels shown in Figure 5A and B with the same annotations shown in Figure 5A and B.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig5-data2-v1.zip
-
Figure 5—source data 3
Uncropped gels shown in Figure 5A and B.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig5-data3-v1.zip
Figure 5—figure supplement 1
Enhanced AA263 analogs promote the trafficking and plasma membrane activity of destabilized, disease-associated GABAA receptors.
(A) Representative immunoblot (above) of HEK293T cells stably expressing α1β2γ2(R177G) GABAA receptors treated for 24 hr with the indicated AA263 analog (10 µM). Quantification of band intensities (below) for cells treated with AA263yne or AA263-5 is shown. (B) Immunoblot and quantification of γ2 and BiP in lysates prepared on HEK293T cells stably expressing α1β2γ2(R177G) GABAA receptors treated for 24 hr with AA263yne (10 µM) and/or Ceapin-A7 (CP7; 10 µM). (C) Immunoblot showing the intensity of γ2 over time following cycloheximide (CHX)-chase application (0–4 hr, 100 μg/ml) in HEK293T cells transiently transfected with α1β2γ2(WT) receptors (top) and HEK293T cells stably expressing α1β2γ2(R177G) receptor variant treated with vehicle (middle) or AA263yne (bottom). (D) Representative immunoblot showing total γ2 expression in HEK293T cells stably expressing α1β2γ2(R177G) receptor variant treated with indicated AA263yne analogs (10 µM, 24 hr). One-way ANOVA followed by post hoc Dunnett’s test was used for statistical analysis. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001. See also Figure 5—figure supplement 1—source data 1 and Figure 5—figure supplement 1—source data 2.
-
Figure 5—figure supplement 1—source data 1
Excel spreadsheet showing raw data used to generate Figure 5—figure supplement 1A, B, and D.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig5-figsupp1-data1-v1.zip
-
Figure 5—figure supplement 1—source data 2
Uncropped gels shown in Figure 5—figure supplement 1A–D with the same annotations shown in Figure 5—figure supplement 1A–D.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig5-figsupp1-data2-v1.zip
-
Figure 5—figure supplement 1—source data 3
Uncropped gels shown in Figure 5—figure supplement 1A–D.
- https://cdn.elifesciences.org/articles/107000/elife-107000-fig5-figsupp1-data3-v1.zip
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (human) | HEK293-Trex overexpressing ERSE.Fluc | Plate et al., 2016 | ||
| Cell line (human) | HEK293-Trex overexpressing XBP1.RLuc | Plate et al., 2016 | ||
| Cell line (human) | HEK293T overexpressing ATF4.FLuc | Yang et al., 2023 | ||
| Cell line (human) | HEK293T | ATCC | ||
| Cell line (human) | HEK293T cells stably expressing α1β2γ2(R177G) GABAA receptors | This manuscript | See Materials and methods | |
| Cell line (human) | Huh7.5 cells stably expressing AAT-Z | Lu et al., 2022 | ||
| Antibody | Rabbit anti-GABAAR-γ2 polyclonal antibody (#AB5559) | Millipore | RRID:AB_11211236 | 1:1000 |
| Antibody | Rabbit monoclonal anti-Na+/K+-ATPase (#ab76020) | Abcam | RRID:AB_1310695 | 1:10,000 |
| Antibody | Mouse anti-human AAT monoclonal antibody 2C1 (Cat #HM2289) | Hycult Biotech | 1:1000 | |
| Antibody | Mouse anti-human AAT monomer-specific monoclonal antibody 16F8 | Balch Lab (Scripps Research) | 1:1000 | |
| Antibody | Rhodamine anti-actin primary antibody (#12004163) | Bio-Rad | RRID:AB_2861334 | 1:8000 |
| Recombinant DNA reagent | pCMV6 plasmids containing human GABAA receptor α1 | Origene | Uniprot No. P14867-1 | |
| Recombinant DNA reagent | pCMV6 plasmids containing human GABAA receptor β2 (isoform 2) | Origene | Uniprot No. P47870-1 | |
| Recombinant DNA reagent | pCMV6 plasmids containing human GABAA receptor γ2 (isoform 2) subunits | Origene | Uniprot No. P18507-2 | |
| Recombinant DNA reagent | pCMV6 encoding human GABAA receptor γ2 subunit missense mutation R177G | Constructed using QuikChange II site-directed mutagenesis Kit (Agilent Genomics) | ||
| Peptide, recombinant protein | Human neutrophil elastase (Cat # IHUELASD100UG) | Innovation Research | ||
| Commercial assay or kit | QuikChange II site-directed mutagenesis Kit (#200523) | Agilent Genomics | ||
| Commercial assay or kit | Firefly luciferase assay reagent-1 | Targeting Systems | ||
| Commercial assay or kit | Renilla luciferase assay reagent-1 | Targeting Systems | ||
| Commercial assay or kit | QuickRNA Miniprep Kit (R1055) | Zymo | ||
| Commercial assay or kit | High-Capacity cDNA Reverse Transcription Kit | Applied Biosystems | ||
| Commercial assay or kit | PowerSYBR Green PCR Master Mix | Applied Biosystems | ||
| Commercial assay or kit | Micro BCA protein assay (#23235) | Thermo Fisher | ||
| Commercial assay or kit | Tandem Mass Tag (TMT) 10plex (Cat #90110) | Thermo Scientific | ||
| Chemical compound, drug | Cycloheximide (Cat #01810) | Sigma-Aldrich | ||
| Chemical compound, drug | γ-Aminobutyric acid (GABA) (#A2129) | Sigma-Aldrich | ||
| Chemical compound, drug | MG-132 (#A2585) | ApexBio | ||
| Chemical compound, drug | Thapsigargin | Sigma-Aldrich | ||
| Chemical compound, drug | AA263 and AA263 analogs | Synthesized in this manuscript | See Supplementary file 3 | |
| Chemical compound, drug | Protease inhibitor cocktail (#4693159001) | Roche, Indianapolis, IN | ||
| Chemical compound, drug | Cy5-azide | Click Chemistry Tools, Scottsdale, AZ | ||
| Chemical compound, drug | 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 | ||
| Chemical compound, drug | Elastase substrate (Z-Ala4)2Rh110 (Cat No. 11675) | Cayman Chemical | ||
| Chemical compound, drug | Sulfo-NHS SS-Biotin (#A8005) | ApexBIO |
Additional files
-
Supplementary file 1
RNAseq analysis of HEK293 cells treated with AA263 (10 µM), AA263yne (10 µM), or AA263-5 (10 µM).
There are four sheets in this Excel workbook. DESEQ outputs for RNAseq in HEK293 cells treated with 10 µM AA263, AA263yne, or AA263-5 for 6 hr and a sheet showing the geneset profiling of different stress-responsive signaling pathways from these RNAseq data.
- https://cdn.elifesciences.org/articles/107000/elife-107000-supp1-v1.xlsx
-
Supplementary file 2
GO analysis of RNAseq from HEK293 cells treated with AA263 (10 µM), AA263yne (10 µM), or AA263-5 (10 µM).
There are three sheets in this Excel workbook showing the GO analysis results of RNAseq data from HEK293 cells treated for 6 hr with 10 µM AA263, AA263yne, or AA263-5. GO analysis was performed on genes induced greater than 1.5-fold with an adjusted p value less than 0.05.
- https://cdn.elifesciences.org/articles/107000/elife-107000-supp2-v1.xlsx
-
Supplementary file 3
Document describing the synthesis and characterization of AA263 analogs discussed in this article.
- https://cdn.elifesciences.org/articles/107000/elife-107000-supp3-v1.pdf
-
MDAR checklist
- https://cdn.elifesciences.org/articles/107000/elife-107000-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Phenylhydrazone-based endoplasmic reticulum proteostasis regulator compounds with enhanced biological activity
eLife 14:RP107000.
https://doi.org/10.7554/eLife.107000.3
