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. 2012 Apr 10;109(15):E869-78.
doi: 10.1073/pnas.1115623109. Epub 2012 Feb 6.

The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule

Benedict C S Cross  1 , Peter J Bond, Pawel G Sadowski, Babal Kant Jha, Jaroslav Zak, Jonathan M Goodman, Robert H Silverman, Thomas A Neubert, Ian R Baxendale, David Ron, Heather P Harding
Affiliations

The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule

Benedict C S Cross et al. Proc Natl Acad Sci U S A. .

Abstract

IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4μ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Coumarin-based inhibition of IRE1. (A) Structure of CB5305630 [7-hydroxy-4-methyl-8-((pyridine-2-ylimino)methyl)-2H-chromenone], the most potent IRE1 inhibitor identified in a screen of 238,287 compounds. Note the imine bond between the umbelliferone and the pyridine. (B) A plot of IRE1’s RNase initial velocity (Vi) as a function of RNA substrate concentration in the presence of the indicated concentration of CB5305630 (mean ± SD, n = 2). Km for the RNA substrate is approximately 37 nM and is unaffected by the inhibitor, whereas the Vmax is inhibited in a concentration-dependent manner with a Ki of approximately 60 nM. (C) A plot of the concentration-dependent inhibition of IRE1’s RNase activity by CB5305630 (mean ± SD, n = 4). (D) Autoradiograph of 32P-labeled RNA substrate following cleavage by IRE1 in the absence or presence of the indicated concentration of CB5305630.
Fig. 2.
Fig. 2.
Stable Schiff base formation between 4μ8C and IRE1. (A) Predicted reaction schemes for Schiff base formation of 4μ8C to form a 4μ8C-lysyl imine (in reaction 1) that can be reduced to the stable lysyl amine by borohydride (NaBH4, reaction 2). Reductive inactivation of 4μ8C to the 8-hydroxymethyl is depicted in reaction 3. (B) Plot of the relationship between the incubation time of IRE1 with 4μ8C and the apparent IC50 in the in vitro RNase assay (mean ± SEM, n = 2). (C) In vitro RNase activity of IRE1 following incubation with 4μ8C and 4μ8C that had been previously reduced with borohydride (NaBH4; mean ± SEM, n = 2). (D) Absorbance trace of material eluting from a size exclusion chromatography column loaded with IRE1 or IRE1 and 200 μM 4μ8C. Note the emergence of a novel absorbance peak at 350 nm that comigrates with IRE1 in the 4μ8C-treated sample. (E) Fluorescent micrograph (excitation 340 nm, emission 450 nm) of an SDS-PAGE loaded with IRE1, following exposure 120 μM 4μ8C for the indicated times with or without subsequent reduction by borohydride. The sample in lane 6 was reacted with 4μ8C that had been previously reductively inactivated by borohydride.
Fig. 3.
Fig. 3.
Two critical lysines in IRE1 are selectively targeted by 4μ8C. (A) Absorption traces of peptides eluting from a C18 reverse-phase HPLC. The column was loaded with a tryptic digest of baculovirus expressed IRE1 that had or had not been exposed to 120 μm 4μ8C followed by reduction with borohydride. Note the appearance of two peaks at 320 nm (labeled “A” and “B”) in the sample exposed to 4μ8C. (B) MALDI-TOF spectra of the material recovered in peaks “A” and “B” from A above. Peaks, corresponding in mass to predicted singly charged 4μ8C-modified peptides NH2-Asn-Lys-Lys-COOH (labeled NKK + 4μ8C in upper trace) and NH2-Asp-Val-Ala-Val-Lys-Arg-COOH (labeled DVAVKR + 4μ8C in lower trace) are singled out for attention. The unmodifed DVAVKR peptide is also detectable, likely resulting from in-source loss of the modification. (C) Absorption traces at 320 nm of peptides derived as in A from samples of immunopurified FLAG-tagged IRE1WT that had not been modified (−4μ8C, panel i) or IRE1WT, IREK907A, and IRE1K599A that had been modified with 120 μm 4μ8C (panels ii–iv). Note the absence of peak “A” in the IRE1K907A sample and the absence of peak “B” in the IRE1K599A sample. (D) Position of K599 and K907 in human IRE1α (from PDB ID code 3P23 A chain). (E) (Upper) Autoradiograph of SDS-PAGE of IRE1 following incubation with γ32P-ATP in the presence of decreasing concentrations of 4μ8C in twofold dilutions from 32 μM. (Lower) Coomassie Brilliant Blue (CBB) staining of the same gel. (F) Absorption traces (320 nm) of peptides derived from wildtype baculovirus expressed IRE1 or immunopurified Flag-IRE1 purified in the absence of either 4μ8C (ii) or EDTA (iii). (G) As in C and F. Where indicated, the nucleotide-binding pocket ligands ADP (ii) and staurosporine (iii) were preincubated prior to addition of 4μ8C.
Fig. 4.
Fig. 4.
Stable binding of 4μ8C to IRE1 lysine 907. (A) Absorption traces of peptides eluting from a C18 reverse-phase HPLC (as in Fig. 3). Where indicated, the recently described IRE1 inhibitors MK0186893 (31) and STF083010 (32) were allowed to compete with 4μ8C for IRE1 binding. (B) Snapshots of the IRE1 RNase domain at the indicated time points from a molecular dynamics simulation of the apo state and with 4μ8C attached to K907 via an imine. Note the relative flexibility of the apo state and stable conformation in the presence of 4μ8C. Residues highlighted are Y892, F889, N906, K907, and H910. Five additional repeat simulations with different starting velocities were carried out for both protomers in PDB ID code 3P23, and yielded comparable results. (C) Predicted solvent accessibility (SAS) and hydrogen bonding to water of lysine residues in IRE1 (based on PDB ID code 3P23) in the apo state and in presence of the indicated bound ligands derived from the MD simulations.
Fig. 5.
Fig. 5.
Selective targeting of the IRE1 RNase by 4μ8C in vivo. (A) Fluorescent stained agarose gel of an RT-PCR assay of the Xbp1 mRNA from MEF cells treated simultaneously with increasing concentrations of 4μ8C and either the ER stress causing agent tunicamycin (Tm 2.5 μg/mL) or a vehicle control for 6 h. The migration of the spliced (Xbp1S) and unspliced (Xbp1U) forms are indicated. (B) Immunoblot of endogenous IRE1α immunopurified from MEF cells treated simultaneously with thapsigargin (Tg 0.5 μM) and 4μ8C (32 μM) or a vehicle control for the indicated times analyzed by SDS-PAGE or PhosTag™-PAGE and probed either with phosphospecific antsera to IRE1S724 or total IRE1 as indicated. The lower panel is an RT-PCR of endogenous (mouse) Xbp1 mRNA from the same cells. (C) Quantitative PCR (qPCR) analysis of mRNAs expressed in MEF cells following 6-h treatment with vehicle control (dark gray bar); tunicamycin (2.5 μg/mL) alone (light gray) or with cotreatment of twofold increasing concentrations of 4μ8C (black bars), from 0.125 to 64 μM (mean ± SEM, n = 2). (D) Plots of tunicamycin-induced Xbp1 splicing and Erdj4 expression in cells cotreated with the indicated concentration of 4μ8C for 6 h. Note the similarity in the IC50 for both in vivo read outs of 4μ8C activity (mean ± SEM, n = 2 for Erdj4).
Fig. 6.
Fig. 6.
Inhibition of RIDD by 4μ8C. (A) Autoradiograph of in vitro transcribed 32P-labeled mouse Xbp1 or Insulin2 (Ins2) mRNA separated by electrophoresis after incubation with the indicated concentration of IRE1 in the absence or presence of 4μ8C (10 μM). (B) RNase L activity measured with a FRET assay in the presence of 2–5A cofactor and in the presence or absence of the indicated concentrations of 4μ8C or Sunitinib (45). (C) Expression levels determined by qPCR of known IRE1 RIDD target genes following 6 h of treatment with tunicamycin (2.5 μg/mL) in the presence or absence of coapplied 32 μM 4μ8C (mean ± SEM, n = 2). (D) Plot of mRNA level of the RIDD target gene Scara3 in tunicamycin-treated cells exposed simultaneously to the indicated concentrations of 4μ8C (mean ± SEM, n = 2) for 6 h. (E) Immunoblot of puromycinylated proteins recovered from the membrane fraction of semipermeabilized wild-type and Perk-/- mutant MEFs that had been exposed to thapsigargin (500 nM) or tunicamycin (2.5 μg/mL) in the absence and presence of 32 μM 4μ8C for the indicated times. The intensity of the signal, integrated over the surface of each lane, is plotted under the image of the immunoblot.
Fig. 7.
Fig. 7.
The expansion of secretory capacity is selectively inhibited by 4μ8C. (A) Viable cell mass of MEF following 24-h exposure to escalating (2-fold) concentrations of 4μ8C (from 1 to 128 μM) or thapsigargin (from 16 to 1,024 nM; mean ± SEM, n = 3). (B) Viable cell mass of MEF of the indicated genotypes following 24-h exposure to escalating (2-fold) concentrations of 4μ8C (from 1 to 128 μM; mean ± SEM, n = 3). (C) Viable cell mass of three different clones of wild-type MEFs following 24-h exposure to escalating (2-fold) concentrations of tunicamycin (from 16 to 2,048 ng/mL), and in the absence or presence of 4μ8C (32 μM; mean ± SEM, n = 3). (D) Time-dependent growth of multiple myeloma MM.1R cells exposed to the indicated concentrations of 4μ8C in the absence or presence of bortezomib (Bz, 6 nM) as assessed by WST1 assay (mean ± SEM, n = 3). (E) Representative electron micrographs of AR42J cells treated for 48 h with dexamethansone (100 nM, an inducer of differentiation to the secretory state) in the absence or presence of 4μ8C (32 μM) (F) Quantitation showing the ratio of ER cross-section area to cytosol cross-section area (AU) in the samples treated as in E (mean ± SEM, n = 5; * indicates p < 0.05 by T test). (G) Amylase secretion detected in the media of AR42J cells cultured for 48 h in the absence or presence of dexamethasome (DEX, 100 nM) with or without 4μ8C (32 μM; mean ± SEM, n = 3). (H) Amylase secretion as in G, with dexamethasome (DEX, 100 nM) and a range of concentrations of 4μ8C (mean ± SEM, n = 3).
Fig. P1.
Fig. P1.
Targeted inhibition of IRE1. 8-formyl-7-hydroxy-4-methylcoumarin (abbreviated 4μ8C, see inset to graph), identified by high-throughput screening, was found to inhibit the endonuclease activity of mammalian IRE1 with high selectivity in both an in vitro FRET-derepression assay (see graph) and cultured cells. The compound binds to a critical lysine in the endonuclease active site of IRE1 by formation of an unusually stable Schiff base. IRE1 K907-4μ8C modification constrains the flexibility of the endonuclease site by formation of stacking interactions with F889 and interjects between essential catalytic residues, inactivating the enzyme. Structure shows human IRE1 protomer (Protein Data Bank ID code 3P23, Left) and the detail (Lower Right, residues 870 to 939) shows computationally docked 4μ8C (green) at K907, with residues F889, Y892, N906, K907, and H910 highlighted.
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References

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