Biochemistry and Chemical Biology

Phenylhydrazone-based Endoplasmic Reticulum Proteostasis Regulator Compounds with Enhanced Biological Activity

Gabriel M Kline
Lisa Boinon
Adrian Guerrero
Sergei Kutseikin
Gabrielle Cruz
Marnie P Williams
Ryan J Paxman
William E Balch
Jeffery W Kelly author has email address
Tingwei Mu author has email address
R Luke Wiseman author has email address

Department of Chemistry, The Scripps Research Institute, La Jolla, United States
The Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, United States
Department of Molecular and Cellular Biology, The Scripps Research Institute, La Jolla, United States

https://doi.org/10.7554/eLife.107000.1

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Figures and data

AA263 covalently modifies protein disulfide isomerase 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 μM 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 AA263^yne E. Activation of the ERSE.FLuc ATF6 reporter in HEK293T cells treated for 18 h with the indicated dose of AA263 or AA263^yne. Error bars show SEM for n=6 replicates. The EC₅₀ is shown. F. Activation of the ERSE.FLuc ATF6 reporter in HEK293T cells treated with AA263^yne (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 h with vehicle (0.1% DMSO), AA263^yne (5 µM), or the combination of AA263^yne (5 µM) and AA263 (20 μM). Coomassie stained gel is shown below. H. Venn diagram of identified targets of AA132^yne and AA263^yne. 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.

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 h with vehicle, Tg (0.5 μM), or the indicated analog (10 µM). Error bars show SEM for n=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 UPR in HEK293T cells treated for 6 h with 10 µM AA263, AA263^yne, or AA263-5. Full RNAseq data and genesets used in this analysis are shown in Table S1. *p<0.05, ***p<0.005 for one-way ANOVA.

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 h with the indicated dose of compound. C. Activation of the ERSE.Fluc ATF6 reporter in HEK293T cells treated for 18 h 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 h. Error bars show SEM for n=3 independent biological replicates. *p<0.05, ***p<0.001 for one-way ANOVA.

AA263 analogs improve secretory proteostasis for the disease-associated AAT-Z variant.
A-C. 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 h with AA263 (10 µM), AA263^yne (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.

Enhanced AA263 analogs promote the trafficking and plasma membrane activity of destabilized, disease-associated GABA_A receptors.
A. Representative immunoblot (above) and quantification (below) of surface biotinylated γ2 in HEK293T cells stably expressing α1β2γ2(R177G) GABA_A receptors treated for 24h with 10 µM AA263^yne 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, 24hrs. Na⁺/K⁺ ATPase serves as a loading control. C. Representative eIPSC traces for α1β2γ2(WT) GABA_A receptors and, α1β2γ2(R177G) GABA_A receptors treated for 24 h with DMSO, AA263^yne (10 µM), or AA263-20 (10 µM). 10 mM GABA (saturating condition) was applied to the recorded cells to evoke currents. D,E. 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.

(Supplement to Figure 1). AA263 covalently modifies protein disulfide isomerase family members.
A. Activation of the ERSE.FLuc ATF6 reporter in HEK293T cells treated for 18 h with vehicle (0.1% DMSO), Tg (500 nM), AA263(10 µM), or AA263-1 (10 μM). Structures of AA263 and AA263-1 shown to the right. B. Representative SDS-PAGE gel of Cy5-conjugated proteins from HEK293T cells treated for 4 h with vehicle (0.1% DMSO), AA263^yne (10 µM), or the combination of AA263^yne (10 µM) and either resveratrol (10 μM) or BME (55 μM). C. Representative SDS-PAGE gel of Cy5-conjugated proteins from lysates prepared from HEK293T treated for 1h with vehicle, AA147^yne, or AA263^yne (5 μM).

(Supplement to Figure 2). 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 h 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 h with the indicated concentration of AA263, AA263^yne, or AA263-5. Error bars show SEM for n= 3 replicates. 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 h with 10 µM AA263, AA263^yne, or AA263-5. Full RNAseq data and genesets used in this analysis are shown in Table S1

(Supplement to Figure 3). 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 h 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 h with vehicle, AA263^yne (5 μM) or cotreatment of AA263^yne (5μM) with the indicated analog (20 μM).

(Supplement to Figure 4). 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 h with 10 µM of AA263, AA263^yne, or AA263-20*p<0.05, **p<0.01, ***p<0.005 for one-way ANOVA compared to vehicle-treated cells are shown.

(Supplement to Figure 5). Enhanced AA263 analogs promote the trafficking and plasma membrane activity of destabilized, disease-associated GABA_A receptors.
A. Representative immunoblot (above) of HEK293T cells stably expressing α1β2γ2(R177G) GABA_A receptors treated for 24h with the indicated AA263 analog (10 µM). Quantification of band intensities (below) for cells treated with AA263^yne or AA263-5 is shown. B. Immunoblot showing the intensity of γ2 over time following cycloheximide (CHX)-chase application (0 to 4 hours, 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 AA263^yne (bottom). C. Representative immunoblot showing total γ2 expression in HEK293T cells stably expressing α1β2γ2(R177G) receptor variant treated with indicated AA263^yne analogs (10 µM, 24hrs). One-way ANOVA followed by post-hoc Dunnett’s test was used for statistical analysis. *p≤0.05; **p≤0.01; ****p≤0.0001.

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