Abstract
The unfolded protein response (UPR) homeostatically matches endoplasmic reticulum (ER) protein-folding capacity to cellular secretory needs. However, under high or chronic ER stress, the UPR triggers apoptosis. This cell fate dichotomy is promoted by differential activation of the ER transmembrane kinase/endoribonuclease (RNase) IRE1α. We previously found that the RNase of IRE1α can be either fully activated or inactivated by ATP-competitive kinase inhibitors. Here we developed kinase inhibitors, partial antagonists of IRE1α RNase (PAIRs), that partially antagonize the IRE1α RNase at full occupancy. Biochemical and structural studies show that PAIRs promote partial RNase antagonism by intermediately displacing the helix αC in the IRE1α kinase domain. In insulin-producing β-cells, PAIRs permit adaptive splicing of Xbp1 mRNA while quelling destructive ER mRNA endonucleolytic decay and apoptosis. By preserving Xbp1 mRNA splicing, PAIRs allow B cells to differentiate into immunoglobulin-producing plasma cells. Thus, an intermediate RNase-inhibitory ‘sweet spot’, achieved by PAIR-bound IRE1α, captures a desirable conformation for drugging this master UPR sensor/effector.
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Data availability
Kinome selectivity data are provided in Supplementary Dataset 1. Coordinates and structure factors have been deposited in the PDB under accession code 7BMK. Cell line RNA-seq data have been deposited to the Gene Expression Omnibus (GEO)48 and are accessible through GEO Series accession number GSE164496 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE164496). The KEGG database used in this study was acquired from http://www.genome.jp/kegg/. Source data are provided with this paper.
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Acknowledgements
This work was supported by the Leona M. and Harry B. Helmsley Charitable Trust (G-3231 to F.R.P.), the Juvenile Diabetes Research Foundation (JDRF 2-SRA-2016-234-S-N to F.R.P.) and the NIH (R01DK116064 to D.J.M. and F.R.P.; R01DK100623 to B.J.B, D.J.M. and F.R.P.; U01DK123609 to D.J.M. and F.R.P.; R01HL145037 to F.R.P.; K08HL157654 to V.C.A.; R01AR069520 to J.Z. and R01AI148487 to J.Z.).
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Authors and Affiliations
Contributions
H.C.F. executed all enzymatic and biochemical analyses. H.C.F. and B.J.B. designed compounds. H.C.F. and V.N.V. synthesized and characterized compounds. R.G., J-H.K. and A.O. performed cellular analysis of XBP1 splicing, RIDD and apoptosis. V.C.A. conducted RNA-seq analyses. H.C.F. and Z.E.P. performed target engagement studies. H.C.F. and B.G.K.P. performed chemoproteomic profiling. Plasma cell differentiation studies were designed by J.Z. and performed by J.L.M. J.Z., F.R.P. and D.J.M. supervised experiments. H.C.F., F.R.P. and D.J.M. wrote the manuscript with input from all authors. F.R.P. and D.J.M. conceived of and led this study.
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Competing interests
B.J.B., F.R.P. and D.J.M. are founders, equity holders and consultants for OptiKIRA, LLC (Cleveland, OH), a biotech company founded on the treatment of ER-stress-induced retinal degeneration; no company funding or chemical matter supported the work in this manuscript. J.Z. serves on the SAB for Walking Fish Therapeutics.
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Peer review information Nature Chemical Biology thanks Nathanael Gray, Boaz Tirosh and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 KIRAs monomerize IRE1α*.
(a) Chemical structure of compound 2. (b) Inhibition of IRE1α*’s kinase activity by compound 2 (Ki = 480 nM). Data points shown are the mean of n = 3 independent experiments ± SEM. (c) Kdimer curve of the IRE1α*-compound 2 complex (teal). Kdimer curves of apo IRE1α* (dark gray) and the IRE1α*-KIRA8 complex (light blue) from Fig. 1e are shown for comparison. Data points shown are the mean of n = 3 independent experiments ± SEM.
Extended Data Fig. 2 KIRA8 completely suppresses XBP1 splicing in ER-stressed INS-1 cells.
(a) Quantification of spliced XBP1 in untreated parent INS-1 cells and parent INS-1 cells treated with brefeldin A (BFA) or BFA and KIRA8. Values shown are the mean of n = 3 biologically independent samples ± SEM. (b) Representative example of an EtBr-stained agarose gel of XBP1 cDNA amplicons from INS-1 cells subjected to the conditions described in (a). (c) Quantification of spliced XBP1 in untreated parent INS-1 cells and parent INS-1 cells treated with thapsigargin (Tg) or Tg and KIRA8. Values shown are the mean of n = 3 biologically independent samples ± SEM. (d) Representative example of an EtBr-stained agarose gel of XBP1 cDNA amplicons from INS-1 cells subjected to the conditions described in (c).
Extended Data Fig. 3 Structurally-related PAIRs and KIRAs.
(a) Chemical structure of (left) and inhibition of IRE1α*’s kinase activity (right, Ki = 140 nM) by compound 6. (b) Chemical structure of (left) and inhibition of IRE1α*’s kinase activity (right, Ki = 98 nM) by compound 7. (c) Chemical structure of (left) and inhibition of IRE1α*’s kinase activity (right, Ki = 220 nM) by compound 8 (PAIR1). (d) Chemical structure of (left) and inhibition of IRE1α*’s kinase activity (right, Ki = 160 nM) by compound 9 (KIRA9). Inhibition of IRE1α*’s RNase activity by (e) compounds 4 and 5, and (f) PAIR1 and KIRA9. All data points shown are the mean of n = 3 independent experiments ± SEM.
Extended Data Fig. 4 Partial antagonism of RNase activity is directly related to R3-group size.
Three-way correlation of kinase inhibition (a proxy for ATP-binding site occupancy), residual RNase activity, and R3-group size measured as Connolly molecular surface area (Å2) from matched inhibitor sets: (a) 4 and 5, (b) 10 (a previously described KIRA), 6, 7, and (c) KIRA9 and PAIR1. Kinase inhibition and residual RNase activity are shown as the mean of n = 3 independent experiments ± SEM. Note: values are also shown in Fig. 2f.
Extended Data Fig. 5 PAIR1 stabilizes an intermediately displaced helix-αC.
(a) Superimposition of the helix-αCs of active IRE1α* (gray, PDB: 5HGI) with the PAIR1-IRE1α* complex (orange), and the KIRA8-IRE1α* complex (blue, PDB: 6URC). Inset displays the displacement of Glu612 within the helix-αC relative to active IRE1α* (gray, PDB: 5HGI). (b) 2Fo-Fc electron density maps of Glu612 from active IRE1α* (gray, PDB: 5HGI), PAIR1-IRE1α* (orange), and AMG-16-IRE1α* (teal, PDB: 4U6R), contoured to 1.0σ shown as gray isomesh, revealing strong electron density for Glu612, making it a suitable residue to measure approximate helix-αC movement. (c) Displacement of β-carbons from helix-αC residues (606-617) from co-crystal structures of IRE1α* with PAIR1 (orange), AMG-16 (teal, PDB: 4U6R), and KIRA8 (blue, PDB: 6URC) relative to active IRE1α* (gray, PDB: 5HGI).
Extended Data Fig. 6 The PAIR1-bound IRE1α* dimer interface is partially disrupted through Asp 620 displacement.
(a) Superimposition of the active IRE1α* (gray, PDB: 5HGI) and PAIR1-IRE1α* complex (orange) dimers. (b,c) Zoom-in views of the dimer interface contacts that are similar between active IRE1α* (gray, PDB: 5HGI) and the PAIR1-IRE1α* complex (orange) dimers. (d) 2Fo-Fc electron density maps of Asp620, contoured to 1.0σ shown as gray isomesh, reveal equal distribution of Asp620 between ‘in’ and ‘out’ conformations. (e) Superimposition of dimer interface residue Asp620 from active IRE1α* (gray, PDB: 5HGI) and the PAIR1-IRE1α* complex (orange) shows that Asp620-IN is in a similar conformation as active IRE1α* and forms a salt bridge with the side-chains of Arg594 and Arg627 of the adjacent IRE1α* protomer. In the other conformation, Asp620-OUT, the side-chain of Asp620 is displaced 11.6 Å and can no longer form a salt bridge with Arg594 and Arg627. This inter-dimer salt bridge between Asp620 and Arg594/Arg627 is essential for RNase active dimer formation and although Asp620 is found equally in conformations productive (Asp620-IN) and unproductive (Asp620-OUT) for RNase active dimer formation, the cumulative effect is partial disruption of the RNase active dimer interface. (f) Superimposition of kinase catalytic residues: K599, Glu612, and the DFG-motif (Asp712, Gly713, Phe714) from the PAIR1-IRE1α* complex (orange) and the AMG-16-IRE1α* complex (teal, PDB:4U6R) reveal that residues outside of the helix-αC (K599 and the DFG-motif) are in similar conformations, suggesting that differences in the pharmacology between PAIRs and KIRAs stems mainly from helix-αC movement.
Extended Data Fig. 7 PAIR1 and KIRA9 equipotently block ER stress-induced IRE1α autophosphorylation.
Immunoblots for total IRE1α (bottom) from parent INS-1 cells treated with various concentrations of PAIR1 or KIRA9 followed by the addition of 200 ng/μL Brefeldin-A (BFA) for 2 hours. Samples were subjected to phostag SDS-PAGE and % non-phosphorylated IRE1α (top) was determined from the ratio of dephosphorylated IRE1α relative to total IRE1α. Values shown are the mean of n = 3 biologically independent samples ± SEM.
Extended Data Fig. 8 Characterization of the highly potent PAIR, PAIR2.
(a) Chemical structure of PAIR2. (b) Inhibition of IRE1α*’s kinase activity by PAIR2 (Ki = 8.8 nM, values shown as the mean of n = 3 independent experiments ± SEM) and KIRA8 (Ki = 12 nM, values shown as the mean of n = 2 independent experiments). (c) Immunoblots for total IRE1α (bottom) from the INS-1 cells described in Fig. 4b treated with various concentrations of PAIR2 or KIRA8 followed by the addition of 5 ng/ml doxycycline (Dox) for 6 hours. Samples were subjected to phostag SDS-PAGE and % phosphorylated IRE1α (top) was determined from the ratio of phosphorylated IRE1α relative to total IRE1α. Values shown are the mean of n = 3 independent experiments ± SEM. (d) Percent spliced XBP1 (quantified from PstI-digested XBP1 cDNA amplicons) from parent INS-1 cells treated with DMSO, PAIR2, or KIRA8 (0.5, 1, 3, 10 μM) followed by the addition of 2 mM 1,4-dithiothreitol (DTT) (top). Data shown are the mean of n = 3 biologically independent samples ± SEM. A representative EtBr-stained agarose gel of XBP1 cDNA amplicons is shown below. (e) Percent spliced XBP1 (quantified from PstI-digested XBP1 cDNA amplicons) from parent INS-1 cells treated first with DMSO (lanes 1-4) or 10 μM PAIR2 (lanes 5-7) for 30 minutes followed by treatment with either DMSO (lanes 1, 2, and 5) or 0.5 or 1 μM KIRA8 (lanes 3, 4, 6, and 7) for 30 minutes followed by the addition of 2 mM 1,4-dithiothreitol (DTT) (top). Data shown are the mean of n = 3 biologically independent samples ± SEM. A representative EtBr-stained agarose gel of XBP1 cDNA amplicons is shown below. (f) Kinome selectivity of PAIR2 as determined by kinobead profiling. Kinases that were identified in the profiling experiment are shown with gray circles. Circle size and color have been scaled to the log2 ratio (difference in LFQ intensity) between DMSO and treatment with 10 and 2.5 μM of PAIR2 (mean of three and four replicates, respectively). Kinases reported as being drug-sensitive (Log2 Difference > 2) were also required to show significance from a two-sample T-test with FDR of 0.05. (g) Heat map displaying kinome selectivity from (f). P values calculated in (d), (e) are versus DTT treated cells and were determined using a two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
Extended Data Fig. 9 Flow cytometric analysis of lipopolysaccharide (LPS)-treated mouse splenocytes.
Mouse splenocytes were loaded with vital dye and subsequently cultured +/− 1 μg/mL LPS for three days in the presence or absence of KIRA8, KIRA9, PAIR1, or PAIR2 at noted concentrations. Following culture, cells were stained to detect surface marker expression and subsequently permeabilized to detect XBP1s intracellular expression. Samples were mixed with a fixed number of counting beads and collected by flow cytometry. Representative plots are displayed. (a) Splenocytes and beads were first gated on the basis of forward scatter (FSC) and side scatter (SSC) for the purpose of absolute quantification of cell number. (b) Live B-cells were then identified on the basis of CD19 expression and exclusion of live/dead dye. (c) Plasmablasts were identified as CD19+ cells with downregulation of B220 and upregulation of CD138 expression. (d) Plots depict vital dye dilution as a marker of cell division and XBP1s + gate. Corresponding quantification of these cell populations is shown in Fig. 6 and Extended Data Fig. 10.
Extended Data Fig. 10 Effect of PAIRs and KIRAs on B-cell division.
Quantification of CD19 positive cells following treatment of mouse splenocytes with 1 μg/mL LPS for three days in the presence or absence of various concentrations of (a) PAIR1 (orange) or KIRA9 (teal) or (b) PAIR2 (dark orange) or KIRA8 (dark blue) as gated in Extended Data Fig. 9. (c, d) Division index of CD19 + cells treated as in (a, b) was calculated via Flowjo on the basis of vital dye dilution (as shown in Extended Data Fig. 9). P values were calculated using one-tailed Student’s t-test. *P < 0.033, **P < 0.002, ***P < 0.001. Data for 20 μM KIRA8 could not be generated due to cellular toxicity. All data shown are the mean of n = 3 biologically independent samples ± SEM.
Supplementary information
Supplementary Information
Supplemental Figs. 1–18, Table 1 and Note (with Datasets and Chemistry Methods).
Supplementary Data
Complete proteomic dataset for kinome selectivity profiling using HEK293T and HCT116 cells treated with inhibitors 2, 3, 6, 7, PAIR1, KIRA9 or PAIR2. Relevant for Fig. 4a, Supplemental Fig. 9 and Extended Data Fig. 8.
Source data
Source Data Fig. 4
Unprocessed westerns and agarose gels for Fig. 4b,c,i.
Source Data Fig. 5
Unprocessed westerns for Fig. 5b.
Source Data Extended Data Fig. 2
Unprocessed agarose gels for Extended Data Fig. 2.
Source Data Extended Data Fig. 7
Unprocessed westerns for Extended Data Fig. 7.
Source Data Extended Data Fig. 8
Unprocessed westerns and agarose gels for Extended Data Fig. 8c,d,e.
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Feldman, H.C., Ghosh, R., Auyeung, V.C. et al. ATP-competitive partial antagonists of the IRE1α RNase segregate outputs of the UPR. Nat Chem Biol 17, 1148–1156 (2021). https://doi.org/10.1038/s41589-021-00852-0
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DOI: https://doi.org/10.1038/s41589-021-00852-0
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