Recent work identified an ATP mimetic that activates mammalian IRE1α's RNase activity in vitro (Wang et al., 2012; Sanches et al., 2014). These results, along with the co-crystal structure of S. cerevisiae Ire1 with the aminopyrazole-based inhibitor APY29 (PDB ID: 3FBV) (Korennykh et al., 2009), provided a starting point to develop more selective and more potent IRE1 activators. We reasoned that (1) the cyclopropyl substituent on the pyrazole ring, which binds to the gatekeeper pocket in the S. cerevisiae Ire1 structure, would be a key determinant of human IRE1α binding, (2) interactions of the hinge-binding element of the APY29 scaffold would be essential to stabilizing IRE1α's kinase domain in a conformation leading to RNase activation, and (3) the pyrimidine ring, which occupies the adenine pocket in the S. cerevisiae structure, would provide an appropriate space filling moiety that further enhances affinity to the ATP binding pocket (Figure 1A). We therefore kept these three elements constant in further optimizations and explored varying substituents attached to the pyrimidine ring for their ability to improve properties of the compounds.

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To this end, we modified the molecule by substituting urea-linked and variably m- and p-substituted phenyl groups for the benzimidazole of APY29. Diphenyl urea linkers have previously been used successfully in the design of kinase inhibitors (Dar et al., 2008; Dar and Shokat, 2011; Korennykh et al., 2012). In exploring the addition of this chemotype on the APY29 scaffold, we generated a panel of 10 variants (Figure 1B). Each member of the panel was tested in an RNase activity assay using recombinant IRE1α-KR43 and a labeled short hairpin RNA substrate derived from human XBP1 mRNA. IRE1α-KR43 comprises the soluble cytoplasmic portion of IRE1α, composed of the kinase and RNase domains plus 43 amino acids of the linker that bridges the kinase to its transmembrane domain.

As expected from the chemical design strategy, we produced a series of IRE1α RNase activators. We observed that compounds bearing m-trifluoromethyl, p-methyl, p-ethyl, m-metoxy, and p-methoxy groups on the terminal phenyl ring (cmp1, cmp2, cmp3, cmp4, and cmp5) activated IRE1α-KR43 activity 10–100-fold under assay conditions, as compared to fivefold by the parent compound APY29 (Figure 1B). These results were surprising, because the presence of an m-trifluoromethyl group was previously shown to be important to retain activity of related kinase inhibitors but, as shown here, is not essential for activation of IRE1α-KR43. We were also surprised that the activity was improved further by the presence of a p-substituted thioether group and, similarly albeit weaker, by the presence of a p-iodo group (cmp6 and cmp7, 900-fold RNase activation under assay conditions). We conclude that IRE1α prefers a substituent with polarizable character at the para-position of the terminal phenyl ring, perhaps indicating that the ring occupies a hydrophobic region in IRE1α's active site. Increasing bulkiness at the para-position resulted in decreased activities (cmp8, cmp9, and cmp10), suggesting that the size of this pocket must be limited.

We decided to further characterize compound cmp6, based on its robust activation properties. Henceforth, we refer to cmp6 as IPA (for IRE1/PERK Activator) for reasons to be discussed below. To validate that binding of IPA to IRE1α's kinase domain is critical for IRE1α RNase activation, we generated a control compound, IPAx. In IPAx, the pyrazole ring bears an additional N1-methyl group that is predicted to sterically interfere with binding to the gatekeeper pocket in IRE1α (Figure 1A). Indeed, when assayed for activation of IRE1α RNase activity, IPAx was inactive showing an indistinguishable effect from the DMSO control (Figure 1B).

ATP mimetics, such as APY29, induce IRE1α-KR43 oligomerization, which in turn activates the RNase activity (Korennykh et al., 2011; Wang et al., 2012). To assess whether IPA acts through a corresponding mechanism, we measured RNase activity as a function of enzyme concentration in the presence of saturating compound concentrations (20 μM; Figure 1C and Figure 1—figure supplement 1). When no activator was added, apo-IRE1α–KR43 RNase activity, measured as k_obs under single-turnover conditions, increased sharply with increasing enzyme concentration, reaching ½ k_{max, obs} at 8 μM (Figure 1C, purple circles). The measured Hill coefficient of the activation was n = 3.3, indicating that, consistent with previous work (Li et al., 2010), enzymatic activation parallels oligomerization. By contrast, in the presence of cmp1 or IPA, IRE1α–KR43 RNase activity reached ½ k_{max, obs} at 3- and 20-fold lower enzyme concentrations, respectively (½ k_{max, obs} = 3 μM and 0.43 μM; and n = 3.8 and 4.2, Figure 1C, red and blue circles), indicating that cmp1 and IPA binding significantly predispose IRE1α–KR43 to oligomerization and activation. By contrast, addition of IPAx did not enhance IRE1α activation, showing activation kinetics that were in all respects indistinguishable from the no-compound control (Figure 1C, pink circles), consistent with its inability to bind to IRE1α-KR43 (Figure 1C and Figure 1—figure supplement 1).

Kinome-wide screening of cmp1 and IPA demonstrated a dramatic improvement of selectivity for both molecules when compared to APY24 (Figure 1D and Figure 1—figure supplement 1), a close analog of APY29, containing the same core-scaffold. In this assay, compounds were screened at a fixed concentration of 1 μM against a panel of 266 kinases, and the number of those inhibited by ≥ 80% was scored (Figure 1D). We also included in the analysis several bench-mark inhibitors, such as the promiscuous natural product staurosporine (STS) and several clinically approved kinase inhibitors. We note that both cmp1 and IPA show better selectivity profiles when compared to the clinically approved compounds Sunitinib and Dasatinib.

To examine the effects of IPA in living cells, we monitored the UPR in HEK293T cells using RT-PCR to measure splicing of XBP1 mRNA. We found that IPA-induced XBP1 mRNA splicing in a time- and dose-dependent manner (Figure 2A). At a concentration of 2 μM IPA, splicing was induced within 2 hr (lane 5). As expected, IPAx did not induce XBP1 mRNA splicing (Figure 2B, lane 3). We used tunicamycin (Tm), which induces ER stress by blocking N-linked glycosylation, as a positive control to induce the UPR (Figure 2A, lanes 7, and Figure 2B, lane 4).

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To further rule out off-target effects through which IPA might induce ER stress indirectly, we use a recently discovered IRE1α inhibitor, AD60, that binds to the ATP binding pocket of IRE1α's kinase domain, locking it into its inactive DFG/αC-out conformation and inhibiting its RNase activity (Dar et al., 2008; Korennykh et al., 2012). Using an XBP1 mRNA-splicing reporter that produces an XBP1-luciferase fusion protein only after IRE1-mediated splicing of its mRNA, we determined an IC₅₀ of 0.75 μM for AD60 in HEK293T cells (Figure 2C and Figure 2—figure supplement 1). As expected, AD60 inhibited Tm-induced XBP1 mRNA splicing (Figure 2C, lane 3). Likewise, AD60 fully ablated IPA-induced XBP1 mRNA splicing (Figure 2C, lanes 9–12), even at the highest IPA concentration tested. Taken together, these results confirm that IPA binds to the ATP-pocket of IRE1α to induce XBP1 mRNA splicing in cells.

We next used a T-REx293 cell line, containing a genome-integrated doxycycline-inducible IRE1α-GFP fusion gene, to monitor IRE1α oligomerization in vivo (Li et al., 2010). In the presence of doxycycline, IRE1α-GFP was expressed and localized to the ER (Figure 2D). As previously described, Tm rapidly induced the relocalization of IRE1α-GFP into discrete foci, indicative of IRE1α oligomerization and activation (Li et al., 2010). Furthermore, the foci dissolved by 8 hr of treatment as cells attenuated IRE1α signaling even in the presence of unmitigated ER stress (Figure 2D, upper panels) (Li et al., 2010). As expected, IPA caused relocalization of IRE1α-GFP into foci, consistent with sustained IRE1 activation (Figure 2D, lower panels). While Tm-induced foci became larger and fewer over time as previously observed, IPA-induced foci remained small and numerous. Interestingly, IPA-induced foci remained stable even at late time points, consistent with the results obtained for XBP1 mRNA splicing (Figure 2A). These results suggest that IPA-binding to IRE1α stabilizes IRE1α's oligomeric state, interfering with its attenuation.

We next tested the effect of IPA on cell viability. As shown in Figure 2E, IPA killed cells with an LD₅₀ of 0.82 μM. This strong toxicity was surprising considering that previous studies have shown that IRE1α has a cytoprotective role and that IRE1α knock-out cells are viable. The result therefore is best explained by a possible off-target effect connected to ER stress, likely to be mediated through another kinase in the cell. Moreover, as the interplay between the different UPR branches controls the switch over from cytoprotection to apoptosis (Lin et al., 2007), we next probed for possible effect of IPA on PERK and ATF6 signaling.

To assess the effects of IPA on the activity of the two other UPR branches, we first examined activation of ATF6. To this end, we monitored mRNA levels by RT-PCR for two ATF6-driven transcriptional target genes, HERP1 and DERL3, in HEK293T cells. As expected, treatment with Tm induced the expression of these two genes (Figure 3A,B, lanes 6–9). By contrast, IPA caused no detectable induction (Figure 3A,B, lanes 2–5), indicating that IPA does not induce general ER stress.

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To evaluate effects of IPA on the PERK branch, we measured the phosphorylation state of PERK and eIF2α by Western blotting. Upon activation, PERK becomes hyperphosphorylated and displays a characteristic mobility shift in SDS-PAGE gels, as observed in cells treated with Tm (Figure 3C, lane 7). Unexpectedly, we observed that IPA also induced a shift in the mobility of PERK (Figure 3C, lanes 2–4). This shift, however, was only observed at low-to-intermediate concentrations (0.1, 0.5, and 1 µM), whereas at higher concentrations (2 and 4 μM, Figure 3C, lanes 5 and 6), PERK phosphorylation was severely diminished or not detectable. We also monitored PERK activation by phosphorylation of eIF2α, detected with a phospho-eIF2α-specific antibody (Figure 3C). These results paralleled those obtained for PERK phosphorylation. By contrast, equivalent concentrations of IPAx did not stimulate PERK or eIF2α phosphorylation (Figure 3D, lane 3).

PERK activation by IPA could be due to direct binding of IPA to PERK, or it could be caused by indirect activation of PERK by IRE1. If the latter were true, IPA should not trigger PERK activation in the absence of IRE1. To test this notion, we treated mouse embryonic fibroblasts (MEFs) derived from Ire1α^−/− and Perk^−/− knock-out mice with IPA and monitored XBP1 mRNA splicing, and PERK and eIF2α phosphorylation. As in HEK293T cells, treatment of wild-type MEFs with IPA induced both the PERK and IRE1 branches of the UPR. As expected, we observed no IPA-induced XBP1 mRNA splicing in Ire1α^−/− MEFs (Figure 3E, lane 4) and only a trace amount of IPA-induced eIF2α phosphorylation in Perk^−/− MEFs (Figure 3F, lane 6). By contrast, we observed no IPA-induced XBP1 mRNA splicing, but pronounced PERK phosphorylation (indicated by its mobility shift) and eIF2α phosphorylation in the Ire1α^−/− knock-out MEFs. These results indicate that IPA activates the IRE1 and PERK branches of the UPR independently.

To test whether IPA binds PERK at its ATP binding site, we purified the cytosolic domain of PERK as a GST-PERK fusion protein. This preparation purified as a stable GST-PERK dimer, which in the presence of radiolabelled [γ-³²P]ATP efficiently phosphorylated eIF2α in vitro. We next tested the effects of IPA and IPAx using this kinase assay. IPA inhibited the PERK kinase reaction with an IC₅₀ = 2.8 μM (Figure 4A, solid red circles and Figure 4—figure supplement 1). These data suggest that IPA binds to PERK directly, where it would act as a competitive inhibitor for ATP and thus block its phosphorylation activity.

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The observation of IPA-induced PERK inhibition (shown in Figure 3C and Figure 4A) poses the conundrum of why we observed IPA-induced activation in the low micromolar IPA range, producing an unusual, bell-shaped dose response (Figure 3C). This behavior could be explained by a model posing that at lower IPA concentrations, IPA occupies the ATP-binding sites of a subset of PERK molecules, triggering kinase activation of neighboring, unoccupied PERK molecules, as previously shown for other kinases (Hall-Jackson et al., 1999; Hatzivassiliou et al., 2010; Poulikakos et al., 2010). At higher IPA concentrations, IPA would saturate all PERK molecules, thereby inhibiting the pathway. We treated HEK293T with both Tm and IPA, expecting that Tm would not be able to activate PERK signaling in the presence of sufficiently high IPA concentration. Indeed, at 4 μM IPA, we observed that Tm addition did not activate PERK (Figure 4B, lane 6). Moreover, at 1 µM IPA, which induces PERK activation (Figure 3C and Figure 3D), we found that Tm did not enhance the PERK mobility shift or eIF2α phosphorylation further than what was achieved by IPA alone (Figure 4B, lane 5). IPA inhibition of PERK was reversible, since washing IPA out allowed for activation by Tm (Figure 4B, lane 9).

We next demonstrated that it is possible to keep the PERK pathway off in the presence of IPA. To this end, we added the selective PERK inhibitor GSK2606414 (Axten et al., 2012) (henceforth abbreviated as ‘GSK’) to HEK293T cells treated with IPA, at concentrations that exert the greatest effect on PERK pathway activation. As expected, GSK abolished IPA-induced PERK and eIF2α phosphorylation (Figure 5A; lanes 5 and 6 and Figure 5—figure supplement 4).

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The phosphorylation of eIF2α resulting from PERK activation inhibits translation initiation. Indeed, when we incubated HEK293T cells with IPA in the low concentration range where PERK becomes activated, incorporation of [³⁵S]-methionine into newly synthesized proteins was impaired (Figure 5B, lanes 3 and 5), consistent with translation attenuation. Interestingly, treatment with GSK-restored translation to baseline levels (Figure 5B, lanes 4 and 6 and Figure 5—figure supplement 1). This pharmacological manipulation therefore allowed us to experimentally uncouple IPA-induced IRE1α and PERK activation. In particular, we were interested in testing whether the unexpected toxicity of IPA (Figure 2E) could, at last in part, be explained by PERK activation, which is known to induce apoptosis (Lin et al., 2007). To explore this notion, we tested cell viability at increasing IPA concentrations in the presence GSK. GSK inhibits PERK in cells with an EC₅₀ of 200 nM (Axten et al., 2012; Moreno et al., 2013) and shows no cell toxicity at 1 μM (Figure 5C and Figure 5—figure supplement 2). Indeed, GSK added at 1 μM protected cells from IPA-induced cell death, shifting the LD₅₀ from 0.8 μM to 6.2 μM (Figure 5C). As a further control, we tested whether GSK would also rescue cells treated with STS, a rather pleiotropic kinase inhibitor. GSK had no effect under these conditions (Figure 5C and Figure 5—figure supplement 3).

To address directly whether PERK activation resulted from IPA binding to PERK itself, we recombinantly expressed the cytosolic portion of human PERK and eIF2α, the only known PERK substrate, and reconstituted the phosphorylation reaction in vitro. To this end, the cytosolic portion of PERK was dephosphorylated and then proteolytically severed and chromatographically separated from its glutathione S-transferase tag, known to promote-constitutive multimerization. The resulting PERK cytosolic fragment was responsive to activation, by contrast to the constitutively active, phosphorylated PERK-GST fusion protein assayed in Figure 4—figure supplement 1.

As shown in Figure 6A, we observed a large increase in the rate of PERK phosphorylation of eIF2α, which peaked at 0.5 μM IPA (3.2-fold increase over DMSO control) and then declined at higher IPA concentrations, whereas IPAx had no effect (Figure 6A, Figure 6—figure supplements 1, 2). The in vitro bell-shaped activity profile mimicked closely the effect observed in intact cells (Figure 3C). Furthermore using chemical crosslinking, we observed that IPA—but not its inactive analog IPAx—significantly increased PERK oligomerization, apparent as an enhanced formation of cross-linked species migrating at the size expected for PERK dimers, trimers, and tetramers (Figure 6B). The formation of higher-ordered PERK oligomers during ER stress was previously reported (Bertolotti et al., 2000; Marciniak et al., 2006).

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HEK293T, T-REx293 and wild type, Ire1^−/− and Perk^−/− cells MEF cells were maintained at 37°C, 5% CO₂ in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 10 units/ml penicillin and 10 μg/ml streptomycin (Life Technologies, San Francisco, CA). Tm was obtained from Sigma (Milwaukee, WI). Transient and stable transfections were performed using the Lipofectamine 2000 (Invitrogen, Carlsbad CA) and FuGene6 reagent (Roche). Stable cell lines expressing IRE1-3F6HGFP T-REx293 were described previously (Li et al., 2010).

T-REx293 cells were split 2 days before imaging onto glass-bottom micro-well dishes (MatTek) at 5 × 10⁴ cells/dish. Doxycyclin-containing medium (10 nM doxycycline) was added for 24 hr, withdrawn before imaging and replaced with imaging media (Hank's Balanced Salt Solution [Gibco], 2% FBS, and 5 mM HEPES pH 7.0). Images were acquired on a spinning-disk confocal microscope as described (Li et al., 2010).

HEK293T cells (500,000 cells/ml) were plated in 12-well flat bottom culture cluster dishes (Costar, Fisher Scientific) 24 hr before the experiment. IPA (1 μM), or a combination of IPA with GSK2606414 (TRC inc. Toronto Canada), were added to the cells culture for no longer than 2 hr. 15 min prior to lysing cells, 4 μCi of Express [³⁵S] protein-labeling mix (Perkin–Elmer) was added. Media were removed, and cells were immediately lysed in a buffer containing 25 mM Tris-HCl pH 8, 8 mM MgCl₂, 1 mM dithiothreitol (DTT), 1% Triton X-100, 15% glycerol, 10 mM leupeptin, 153 μM aprotenin, and 1 mM phenylmethanesulfonyl fluoride. Cells lysates were kept on ice for 30 min before high-speed centrifugation followed by denaturation at 95°C. The samples were resolved on an SDS-polyacrylamide gel, which were then stained with Coomassie blue to ascertain protein loading. Dry gels were exposed to a blank phosphorscreen and scanned using a Typhoon variable mode imager (GE Healthcare). The resulting autoradiograms were quantified using ImageQuant software (Molecular Dynamics).

HEK293T cells were grown in DMEM (Sigma) complete media containing 10% FBS, 10 units/ml penicillin (Invitrogen), and 10 μg/ml streptomycin (Invitrogen). Cells were plated at a density of 30,000 cells per well in 96-well black plates with clear flat bottoms (Corning, Sigma-Aldrich) 24 hr before the experiment. A 12-point semi-logarithmic dilution series of compounds was made starting at 30 μM not to exceed 0.1% DMSO after final dilution into the growth media. Viability assays were conducted over the course of 24 hr. Cell viability was determined using CellTiter-Blue (Promega) following the manufacturer's instructions. A Spectramax5 Microplate reader equipped with SoftMax Pro 5 software (Molecular Devices) was used to read out viability, and data were plotted using SigmaPlot software package (Systat Software). Experiments were done in triplicate, and mean values were computed for each data point.

Cells were lysed and total RNA was collected (Total RNA Kit 1, EZ-RNA, EZNA [USA]). PolyA⁺ mRNA was reverse transcribed with M-MLV (Invitrogen), and the resulting cDNA was used as a template for PCR amplification across the fragment of XBP1 cDNA containing the intron. Primers used to amplify human XBP1: 5′-TTACGAGAGAAAACTCATGGC-3′ and 5′-GGGTCCAAGTTGTCCAGAATGC-3′. To amplify murine XBP1: 5′-GAACCAGGAGTTAAGAACACG-3′ and 5′-AGGCAACAGTGTCGAGTCC-3′. PCR conditions were as follows: 95°C for 5 min, 95°C for 1 min, 58°C for 30 s, 72°C for 30 s, and 72°C for 5 min, with 20 cycles of amplification. PCR products were resolved on a 2.5% agarose tris-acetate EDTA (TAE) gel. As previously reported, a hybrid amplicon species consisting of unspliced Xbp-1 annealed to spliced Xbp-1 can also be produced through this PCR reaction and was visible as a slower migrating band above the unspliced amplicon. The same cDNA was used as template for PCR of DERLIN-3 and HERPUD1, both ATF6 targets, and GAPDH, which was used as loading control. Primers used included the following: DERLIN-3, 5′-AGTTCCACTCTTTGATGGAGGGCA-3′ and 5′-AGCCAGCTGTGAGGAATATGGGAA-3′; HERPUD1-1, 5′-ACAAGGTGGCCCTATTGTGGAAGA-3′ and 5′-AGTCCATTCCTGTCAAAGCCTCCA-3′; GAPDH, 5′-CCATGTTCGTCATGGGTGTGA-3′ and 5′-CATGGACTGTGGTCATGAGT-3′. PCR conditions were: 95°C × 5 min, 95°C × 1 min, 56°C × 30 s, 72°C for 30 s for 25 cycles, 72°C for 5 min at 4°C. PCR products were resolved on a 2% agarose TAE gel.

Human IRE1α-KR43 with a hexa-histidine (6xHis) tag in its N-terminus was expressed and purified in SF21 cells as described (Li et al., 2010). Cells were lysed in buffer containing 20 mM Tris-HCl, pH 7.5, 600 mM NaCl, 2 mM MgCl₂, 3 mM imidazole, 10% glycerol, 1% Triton X-100, 3 mM β-mercaptoethanol, COMPlete protease inhibitors (Roche), and PhosSTOP phosphatase inhibitor cocktail (Roche) and passed through an AVESTIN emulsiflex-C3 3× times for complete lysis. The lysate was cleared using centrifugation at 100,000×g. Clear lysate was allowed to incubate on Ni-NTA agarose (Qiagen) beads for 2 hr before being washed 3× times with buffer containing 20 mM Tris-HCl, pH7.5, 600 mM NaCl, 2 mM MgCl₂, 30 mM imidazole, 10% glycerol, and 3 mM β-mercaptoethanol. Protein was eluted from the column by raising the imidazole concentration to 250 mM. The eluate was then passed through a HiTrap desalting column (GE Healthcare). Lastly, the 6xHis was cleaved as described (Li et al., 2010) and the resulting protein was then loaded onto a HisTrap HP, 5 × 5 ml column to remove the uncleaved protein. The cleaved protein was then loaded on a Mono-S 5/50 GL column (GE Healthcare), and the eluate was then concentrated to 5 mg/ml and loaded on a Superdex 200 HR 10/300 (GE Healthare) column in buffer containing 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5% Glycerol and 5 mM tris(2-carboxyethyl)phosphine (TCEP). The pure protein solution was aliquoted and stored at −80°C.

To express the cytosolic kinase domain of PERK, the murine PERK kinase domain (580–1077aa) was cloned into a pGEX4T1 vector to create a fusion protein containing N-terminal GST. The plasmid was transformed into Escherichia coli strain BL21DE3 RIPL (Agilent Technologies). Cells were grown in LB medium containing ampicillin and chloramphenicol until OD600 = 0.6. Expression was induced with 0.2 mM IPTG at 18°C for 16 hr. Cells were harvested by centrifugation, resuspended in buffer A (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 3 mM β-mercaptoethanol, 2.5% Glycerol) and lysed by sonication. After centrifugation, the supernatant was applied to a GST-Sepharose column and washed with buffer A and buffer A containing 500 mM KCl and 1 mM ATP. Sample was eluted with buffer A containing 20 mM glutathione. GST-PERK kinase domain was then concentrated and further purified on a Superdex 200 10/300 gel filtration column equilibrated in buffer B (50 mM Tris pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 3 mM DTT, 1% Glycerol). Fractions containing GST-PERK kinase domain were concentrated and flash-frozen in liquid nitrogen and stored at −80°C.

Full-length cytosolic human PERK was codon-optimized for E. coli expression by Genewiz Inc. A construct was then cloned into a Pgex-6p-2 vector for expression using two rounds of In-Fusion cloning (Clontech) (535–1093 Δ660–868). The cytosolic portion of PERK, lacking the unstructured loop region (amino acids 535–1093 Δ660–868) was then co-expressed with a tagless lambda phosphatase to produce a fully dephosphorylated PERK protein in BL21 star (DE3) (Life Technologies) (Dar et al., 2008). Cells were grown to an OD₆₀₀ of 0.5 before induction with 0.1 mM IPTG at 15°C for 25 hr. Cells were harvested and lysed using AVESTIN Emulsiflex-C3 in a buffer containing 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10% glycerol, 5 mM TCEP (buffer A), and EDTA-free COMPlete protease inhibitor cocktail (Roche). Lysate was cleared by centrifugation at 100,000×g before batch binding to a GST-Sepharose resin. The resin was washed 5× times with buffer A, and on-column tag cleavage was preformed using PreScission protease (GE Healthcare). The protein was loaded onto a HiTrap Q HP column to remove remaining protease. The PERK (535–1093 Δ660–868) protein was then concentrated and fractionated on a Superdex 200 GL (GE Healthcare) to remove uncleaved GST-PERK protein.

Cells were lysed in 50 mM Tris-HCl, 2% SDS, 10% glycerol, 0.01% bromophenol blue, pH 6.8 supplemented with phosphatase inhibitors (Sigma) and protease inhibitors (Roche). 30 μg to 50 μg of total protein was loaded on each lane of 8% or 10% SDS-PAGE gels. The separated proteins were then transferred onto nitrocellulose membranes for immunoblotting. The following antibodies and dilutions were used: anti-β-actin 1:10,000 (A5441, Sigma), anti-total PERK at 1:1000 (C33E10, Cell Signaling Technology), anti-phospho eIF2α at 1:1000 (3597S, Cell Signaling Technology), anti-total eIF2α at 1:1000 (L57A5, Cell Signaling Technology). After overnight incubation with primary antibody, membranes were washed in PBS with 0.05% Tween and incubated in HRP-coupled secondary antibody anti-rabbit (611–1302, Rockland) or anti-mouse (610–1302, Rockland) diluted at 1:5000 in wash buffer. Immunoreactive bands were detected by chemiluminescence.

IRE1 endoribonuclease activity was detected employing two in vitro assays. k_obs and Hill coefficients were determined from the cleavage kinetics of [³²P]-labeled RNA substrates as previously described (Korennykh et al., 2009). The assay was started by adding 1 μl of [³²P]-labeled RNA to 9 μl of premixture containing 20 mM HEPES pH 7.4, 70 mM NaCl, 2 mM MgCl₂, 4 mM DTT, 5% glycerol, 1 µl of 10 μM compound in DMSO. Reactions were performed at 30°C. Reactions were conducted under single turnover conditions. They contained ≤1 pM [³²P]-labeled RNA and increasing concentrations of purified IRE1α-KR43. Reactions were quenched at time intervals with 6 μl stop solution (10 M urea, 0.1% SDS, 0.1 mM EDTA, 0.05% xylene cyanol, and 0.05% bromophenol blue). Samples were analyzed in 10–20% Urea-PAGE gels. Gels were scanned using a Typhoon variable mode imager (GE Healthcare) and quantified using the ImageQuant and GelQuant software packages (Molecular Devices). The data were plotted and fit to exponential curves using SigmaPlot software package (Systat Software) to determine observed rate constants as previously described (Korennykh et al., 2009). EC₅₀ values were determined using a FRET-based cleavage assay. A FRET probe (FRET_IDT_17 56- FAM/rCrArCrCrUrCrUrGrCrArGrCrArGrGrUrG/IABlk_FQ) was purchased from IDT (RNase free HPLC purification) and dissolved in RNase-free H₂O. Excitation: 485 (480–490) and emission: 517 (512–520). The reactions were started by addition of RNA FRET probe (to a final concentration of 100 nM) to 9.4 μl of premixture containing 20 mM HEPES, pH 7.4, 70 mM NaCl, 2 mM MgCl₂, 4 mM DTT, 5% glycerol, and compound in DMSO not to exceed 1%. Reactions were performed at 30°C and contained 150 nM enzyme under single turnover conditions. Reactions were quenched at time intervals with equal volumes of formamide. Samples were analyzed using a SpectraMax M5 plate reader equipped with SoftMax Pro 5 and data acquisition software (Molecular Devices). The data were plotted using SigmaPlot software package (Systat Software). Experiments were repeated 3–4 times and mean values were computed.

A plasmid encoding a modified ER stress reporter construct consisting of a C-terminally FLAG-tagged firefly luciferase coding sequence fused to an N-terminal hemagglutinin-tagged tandem repeat of a partial sequence of XBP1 of human origin lacking its DNA-binding domain and containing the IRE1-cognate intron (pCAX-HA-2xXBP1ΔDBD(anATG)-LUC-F, kind gift of Takao Iwawaki [Hosoda et al., 2010]), was used as a template to generate a retroviral expression construct. The coding sequence of the reporter was amplified by PCR using primers with engineered BamHI and EcoRI sites and was subsequently cloned into the cognate sites of the retroviral expression vector pBABE.puro (Addgene) to generate construct DAA-A171. DAA-A171 was used to generate recombinant retroviral particles using standard methods and the resulting retroviral supernatant was used to transduce HEK293T cells, which were then subsequently selected with puromycin to create a stable reporter cell line.

IPA and cmp1 were assayed by Invitrogen to derive percent inhibition of kinase activity. All compounds were screened at 1 μM and raw values are shown in supplementary tables. Detailed procedures for kinase reactions, ATP concentrations used and Z′-LYTE or Adapta assay formats are described in SelectScreen Customer Protocol (http://www.invitrogen.com/kinaseprofiling). Kinase-inhibition data for 1 μM inhibitor of each of the clinical and tool compounds STS, Sunitinib, Dasatinib, Imatinib, SB202190, Erlotinib and Gefitinib were provided by Invitrogen based on stock results and not an independent comparison conducted or commissioned by the authors. Raw data of percent inhibition of all 266 kinases can be located in Supplementary file 1. Screening analysis for APY24 was previously reported (Statsuk et al., 2008).

A solution of PERK cytosolic domain (535–1093 Δ660–868) (2 μM) was incubated with IPA or IPAx for 15 min before the addition of 250 μM of the amino group-reactive cross-linking agent bis[sulfosuccinimidyl] suberate (BS2g-d0, Thermo Scientific). After 30-min incubation at room temperature Tris-HCl pH 7.5 was added to a final concentration of 55 mM. Samples were then boiled in Laemmli sample buffer and loaded into an Any kD Mini-protean TGX gel (Bio Rad). Cross-linked protein was visualized using Colloidal Coomassie G-250 stain.

GST-PERK kinase domain (580–1077 aa) was pre-incubated with compounds for 30 min. The kinase reaction was initiated by addition of ATP including 0.2 mCi γ-[³²P]-ATP. The final concentrations of the reactants were 50 nM GST-PERK, 50 μM eIF2α, 100 μM ATP and varying concentrations of compound. At 30 min, 2 μl of each reaction was spotted onto a P81 phospho-cellulose membrane. The membrane was washed in 1% phosphoric acid and the sheets were washed five times in buffer, dried, and transferred radioactivity was measured using a Typhoon variable mode imager (GE Healthcare) and quantified using the ImageQuant (Molecular Devices). Titration data were fit to a sigmoidal dose response to derive IC₅₀ values using the SigmaPlot software package (Systat software). Dose responses were based on a 12-point inhibitor titration, using a semi-logarithmic dilution series starting from 30 μM. Experiments were completed 2–4 times and mean values were computed.

PERK activation was measured using PERK (535–1093 Δ660–868) in a buffer containing 20 mM Tris-HCl, 150 mM NaCl, 4 mM MgCl_2, 5 mM TCEP, 1% glycerol, pH 8.0. PERK concentration was 1 μM, and activity was measured at 30°C. PERK was pre-incubated with 50 μM of cold ATP in the presence or absence of IPA or IPAx. The reaction was then initiated by adding S. cerevisiae eIF2α (1–180) and 0.2 mCi γ-[³²P]-ATP. The reaction was monitored over a time course of 30 min, during which 1/6 of the reaction volume was removed at 0.5, 1, 5, 10, 20, and 30 min and stopped in Laemmli buffer supplemented with 50 mM EDTA. Reactions were then boiled and loaded onto a 12.5% Criterion precast gel (Bio-rad). The gel was then dried before imaging using a Typhoon variable mode imager (GE Healthcare). Gels were quantified using ImageQuant software and data were processed using SigmaPlot software package. Rates were determined using non-linear regression. Experiments were performed 3 times, and mean errors were determined.

The fraction PERK bound by IPA was calculated using the standard Langmuir isotherm equation. Using the calculated IC₅₀ described in Figure 4A.

Figure 8

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2,4-dichloropyrimidine 2 (13.30 g, 89.31 mmol) and 3-amino-5-cyclopropylpyrazole 1 (11.00 g, 89.31 mmol) were dissolved in tetrahydrofuran (TFA) (100 ml) followed by the addition of deionized water (100 ml). The solution was then treated with potassium acetate (261.9 g, 2.5 mol). The resulting mixture was kept at 55°C for 48 hr. The mixture was filtered to remove excess potassium acetate. The organic layer was separated and dried using magnesium sulfate and concentrated before loading on an AnaLogix silica column. Intermediate 3 was separated as previously reported (Hosoda et al., 2010). ¹H NMR (400 MHz, DMSO) δ 12.14 (s, 1H), 10.23 (s, 1H), 1.84 (m, 1H), 0.88 (m, 2H), 0.64 (m, 2H). ¹³C NMR (100 MHz, DMSO) δ 161.4, 160.7, 160.0, 153.9, 148.6, 147.8, 146.8, 8.4, 8.2. MS calculated for C₁₀H₁₀N₅ 235.06, found 236.15 (M⁺).

Figure 9

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Pyrimidine monochloride 3 (4 g, 16.9 mmol) (Oakwood chemical) and p-phenylene-diamine 4 (1.9 g, 16.9 mmol) (Sigma) were dissolved in butanol (BuOH) (50 ml) (Sigma) followed by the addition of concentrated HCl (0.1 ml) (Fisher Scientific). The resulting mixture was kept at 100°C overnight. Purple precipitate was collected by filtration, washed with 30 ml of cold BuOH and dried under vacuum yielding intermediate 5, which was used in the next step without further purification. MS calculated for C₁₆H₁₇N₇ 307.15, found 308.5.

Figure 10

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Intermediate 5 (153 mg, 0.5 mmol) was dissolved in 10 ml of dry dimethylformamide (DMF) (Sigma). The flask was placed in an ice bath before addition of 3-(trifluoromethyl)phenyl isocyanate (93.5 mg; 0.069 ml; 0.5 mmol) 6 (Sigma) drop wise using a Hamilton syringe under argon. The resulting mixture was allowed to warm to room temperature overnight. Crude compound was isolated by the addition of cold H₂O (30 ml) and isolated by filtration. Solid was washed with 100% CH₃CN and then suspended in 1:1 CH₃CN and distilled water (dH₂O) for reverse-phase purification using HPLC. A gradient from 2–80% CH₃CN:H₂O (0.1% TFA) was used to further during purification on a C18 column (Agilent Technologies) followed by lyophilization to yielded cmp1. ¹H NMR (400 MHz, DMSO-d₆): δ 12.30 (s, 1H), 10.00 (s, 2H), 8.70 (s, 3H), 7.43 (m, J = 2.3 Hz, 6H), 6.62 (d, J = 2.1 Hz, 5H), 5.29 (s, 1H) 2.51 (m, J = 10.2 Hz, 4H), 1.79 (t, J = 9.6 Hz, 1H), 0.87 (s, 4H) ¹³C NMR (151 MHz, DMSO-d₆) δ 155.7, 152.8, 141.8, 138.4, 135.5, 133.6, 130.2, 129.3, 128.9, 118.8, 112.6, 97.6, 8.9; ESI-MS m/z [M + H] found 495.49 calculated 494.49.

Figure 11

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Intermediate 5 (153 mg, 0.5 mmol) was dissolved in 10 ml of dry DMF. Flask was placed in an Ice bath before addition of p-Toyl isocyanate (0.066 mg; 0.063 ml; 0.5 mmol) 7 (Sigma) drop wise using a Hamilton syringe under argon. Resulting mixture was allowed to warm to room temperature overnight. Crude compound was isolated by addition of cold H₂O (30 ml) and isolated by filtration. Solid was washed with 100% CH₃CN and then suspended in 1:1 CH₃CN and dH₂O for reverse-phase purification using HPLC. A gradient from 2–80% CH₃CN:H₂O (0.1% TFA) was used to further during purification on a C18 column (Agilent Technologies) followed by lyophilization to yield cmp2. ¹H NMR (400 MHz, DMSO-d₆): δ 12.21 (s, 1H), 9.13 (s, 1H), 8.58 (q, J = 1.3 Hz, 9H), 8.35 (d, J = 21.6 Hz, 3H), 6.54 (s, 3H) 2.51 (m, J = 10.2 Hz, 4H), 1.89 (s, 1H) 1.29 (t, J= 1.1 Hz, 3H), 0.87 (s, 4H); ¹³C NMR (151 MHz DMSO-d₆) δ 155.4, 153.8, 146.8, 140.3, 139.7, 136.7, 131.2, 127.8, 117.3, 115.4, 63.5, 33.5, 32.1 8.1; ESI-MS m/z [M + H] found 440.28 calculated 440.21.

Figure 12

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Intermediate 5 (153 mg, 0.5 mmol) was dissolved in 10 ml of dry DMF. The flask was placed in an ice bath before addition of 4-Ethylphenyl isocyante (0.073 mg; 0.072 ml; 0.5 mmol) 8 (Sigma) drop wise using a Hamilton syringe under argon. The resulting mixture was allowed to warm to room temperature overnight. Crude compound was isolated by addition of cold H₂O (30 ml) and isolated by filtration. Solid was washed with 100% CH₃CN and then suspended in 1:1 CH₃CN and dH₂O for reverse-phase purification using HPLC. A gradient from 2–80% CH₃CN:H₂O (0.1% TFA) was used to further during purification on a C18 column (Agilent Technologies) followed by lyophilization to yield product cmp3. ¹H NMR (400 MHz, DMSO-d₆): δ 12.30 (s, 1H), 10.00 (s, 2H), 7.50 (d, J = 2.1 Hz, 3H), 7.33 (s, 6H), 7.08 (t, J = 7.2 Hz, 3H), 1.79 (s, 2H), 1.13 (m, J = 13.4 Hz, 5H), 0.87 (s, 4H); ¹³C NMR (151 MHz DMSO-d₆) δ 162.4, 153.0, 141.16, 130.4, 130.0, 129.9, 123.8, 122.21, 118.4, 114.5, 113.9, 35.6, 8.4; ESI-MS m/z [M + H] found 455.5 calculated 454.31.

Figure 13

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Intermediate 5 (153 mg, 0.5 mmol) was dissolved in 10 ml of dry DMF. The flask was placed in an ice bath before addition of 3-methoxyphenyl isocyanate (0.075 mg; 0.066 ml; 0.5 mmol) 9 (Sigma) drop wise using a Hamilton syringe under argon. The resulting mixture was allowed to warm to room temperature overnight. Crude compound was isolated by the addition of cold H₂O (30 ml) and isolated by filtration. Solid was washed with 100% CH₃CN and then suspended in 1:1 CH₃CN and dH₂O for reverse-phase purification using HPLC. A gradient from 2–80% CH₃CN:H₂O (0.1% TFA) was used to further during purification on a C18 column (Agilent Technologies) followed by lyophilization to yield cmp4. ¹H NMR (400 MHz, DMSO-d₆): δ 12.43 (s, 1H), 11.1 (s, 1H), 10.17 (s, 1H), 8.78 (d, J = 19 Hz, 2H), 8.68, 7.82 (s, 1H), 7.57 (q, j = 6.1 Hz, 2H), 7.41 (t, J = 8.9 Hz, 2H), 7.18 (q, J = 7.2 Hz, 3H), 6.84 (d, J = 5.8 Hz, 1H), 3.89 (s, 3H), 1.79 (s, 1H), 0.87 (s, 4H); ¹³C NMR (151 MHz DMSO-d₆) δ 160.1, 152.95, 141.4, 130.0, 119.9, 110.9, 107.6, 104.4, 55.3, 8.37; ESI-MS m/z [M + H] found 457.6 calculated 456.45.

Figure 14

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Intermediate 5 (153 mg, 0.5 mmol) was dissolved in 10 ml of dry DMF. The flask was placed in an ice bath before addition of 4-methoxyphenyl isocyanate (0.075 mg; 0.065; 0.5 mmol) 10 (Sigma) drop wise using a Hamilton syringe under argon. The resulting mixture was allowed to warm to room temperature overnight. Crude compound was isolated by the addition of cold H₂O (30 ml) and isolated by filtration. Solid was washed with 100% CH₃CN and then suspended in 1:1 CH₃CN and dH₂O for reverse-phase purification using HPLC. A gradient from 2–80% CH₃CN:H₂O (0.1% TFA) was used to further during purification on a C18 column (Agilent Technologies) followed by lyophilization to yield cmp5. ¹H NMR (400 MHz, DMSO-d₆): δ 12.40 (s, 1H), 11.18 (s, 1H), 10.20 (s, 1H), 9.73 (q, J = 8.6 Hz, 2H), 8.78 (d, J = 8.9 Hz, 3H), 7.62 (t, J = 2.5 Hz, 2H), 7.44 (t, j = 6.1 Hz, 3H), 6.98 (t, J = 7.2 Hz, 2H) 3.78 (m, J = 12.2 Hz, 3H), 1.79 (s, 1H), 0.87 (s, 4H), 0.54 (s, 2H); ¹³C NMR (151 MHz DMSO-d₆) δ 159.3, 153.1, 141.3, 125.3, 121.4, 110.4, 108.6, 104.3, 58.3, 8.4; ESI-MS m/z [M + H] found 457.5 calculated 456.51.

Figure 15

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Intermediate 5 (153 mg, 0.5 mmol) was dissolved in 10 ml of dry DMF. The flask was placed in an ice bath before addition of 4-(methylthio)phenyl isocyanate (0.082 mg; 0.07 ml; 0.5 mmol) 11 (Sigma) drop wise using a Hamilton syringe under argon. The resulting mixture was allowed to warm to room temperature overnight. Crude compound was isolated by addition of cold H₂O (30 ml) and isolated by filtration. Solid was washed with 100% CH₃CN and then suspended in 1:1 CH₃CN and dH₂O for reverse-phase purification using HPLC. A gradient from 2–80% CH₃CN:H₂O (0.1% TFA) was used to further during purification on a C18 column (Agilent Technologies) followed by lyophilization to yield IPA. ¹H NMR (400 MHz, DMSO-d₆): δ 12.10 (s, 1H), 9.95 (s, 2H), 8.61 (d, J = 3.7 Hz, 2H), 7.48 (d, J = 2.3 Hz, 8H), 7.22 (d, J = 21.3 Hz, 3H), 2.21 (m, J = 10.2 Hz, 3H), 1.79 (s, 1H), 0.87 (s, 4H); ¹³C NMR (151 MHz DMSO-d₆) δ 161.3, 155.3, 142.3, 140.6, 137.4, 135.8, 133.3, 131.2, 128.3, 119.5, 114.3, 16.5, 8.2; ESI-MS m/z [M + H] found 472.18 calculated 472.20.

Figure 16

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Intermediate 5 (153 mg, 0.5 mmol) was dissolved in 10 ml of dry DMF. The flask was placed in an ice bath before addition of 4-iodophenyl isocyanate (0.122 mg, 0.5 mmol) 12 (Sigma) drop wise using a Hamilton syringe under argon. The resulting mixture was allowed to warm to room temperature overnight. Crude compound was isolated by addition of cold H₂O (30 ml) and isolated by filtration. Solid was washed with 100% CH₃CN and then suspended in 1:1 CH₃CN and dH₂O for reverse-phase purification using HPLC. A gradient from 2–80% CH₃CN:H₂O (0.1% TFA) was used to further during purification on a C18 column (Agilent Technologies) followed by lyophilization to yield cmp7. ¹H NMR (400 MHz, DMSO-d₆): δ 12.21 (s, 1H), 9.10 (s, 1H), 8.81 (s, 1H), 8.79 (s, 1H), 8.52 (t, J = 8.5 Hz, 1H), 8.08 (S, 1H), 7.68 (m, J = 11.8 Hz, 4H), 7.38 (m, J = 10.2, 4H), 6.14 (d, J = 15.2 Hz, 2H), 1.79 (s, 1H), 0.87 (s, 4H); ¹³C NMR (151 MHz DMSO-d₆) δ 156.4, 152.9, 140.3, 137.7, 136.1, 133.5, 120.7, 120.2, 119.3, 116.9, 98.1, 84.7, 8.1; ESI-MS m/z [M + H] found 553.3 calculated 552.08.

Figure 17

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Intermediate 5 (153 mg, 0.5 mmol) was dissolved in 10 ml of dry DMF. The flask was placed in an ice bath before addition of 4-bromophenyl isocyanate (0.099 mg, 0.5 mmol) 13 (Sigma) drop wise using a Hamilton syringe under argon. The resulting mixture was allowed to warm to room temperature overnight. Crude compound was isolated by addition of cold H₂O (30 ml) and isolated by filtration. Solid was washed with 100% CH₃CN and then suspended in 1:1 CH₃CN and dH₂O for reverse-phase purification using HPLC. A gradient from 2–80% CH₃CN:H₂O (0.1% TFA) was used to further during purification on a C18 column (Agilent Technologies) followed by lyophilization to yield cmp8. ¹H NMR (400 MHz, DMSO-d₆): δ 12.21 (s, 1H), 9.51 (s, 1H), 8.81 (m, J = 21.8 HZ, 2H), 8.68 (d, J = 6.3 Hz, 1H), 7.82 (s, 1H), 7.48 (m, J = 22.5 Hz, 8H), 7.33 (d, J = 23.2 Hz, 2H), 7.08 (t, J = 7.2, 3H), 1.79 (s, 1H), 0.87 (s, 4H); ¹³C NMR (151 MHz DMSO-d₆) δ 155.3, 152.3, 140.9, 138.3, 137.6, 132.6, 122.4, 121.8, 120.8, 118.4, 95.4, 82.7, 8.2; ESI-MS m/z [M + H] found 505.5 calculated 504.10.

Figure 18

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Intermediate 5 (153 mg, 0.5 mmol) was dissolved in 10 ml of dry DMF. The flask was placed in an ice bath before addition of 4-isopropylphenyl isocyanate (0.080 mg; 0.08; 0.5 mmol) 14 (Sigma) drop wise using a Hamilton syringe under argon. The resulting mixture was allowed to warm to room temperature overnight. Crude compound was isolated by addition of cold H₂O (30 ml) and isolated by filtration. Solid was washed with 100% CH₃CN and then suspended in 1:1 CH₃CN and dH₂O for reverse-phase purification using HPLC. A gradient from 2–80% CH₃CN:H₂O (0.1% TFA) was used to further during purification on a C18 column (Agilent Technologies) followed by lyophilization to yield cmp9. ¹H NMR (400 MHz, DMSO-d₆): δ 12.11 (s, 1H), 8.51 (t, J = 13.6 Hz, 4H), 7.58 (m, J = 8.3 Hz, 2H), 7.22 (q, J = 12.6 Hz, 8H), 6.48 (d, J = 5.1 Hz, 1H), 1.13 (m, J = 13.4, 8H), 0.87 (m, J = 19.4 Hz, 4H); ¹³C NMR (151 MHz DMSO-d₆) δ 158.3, 151.6, 144.7, 139.3, 138.6, 136.9, 134.5, 124.8, 119.1, 118.5, 36.5, 32.5, 8.1; ESI-MS m/z [M + H] found 469.5 calculated 468.24.

Figure 19

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Intermediate 5 (200 mg, 0.5 mmol) was dissolved in 10 ml of dry DMF. The flask was placed in an ice bath before addition of 4-tert-butylphenyl isocyanate (0.087 mg; 0.089 ml; 0.5 mmol) 15 (Sigma) drop wise using a Hamilton syringe under argon. The resulting mixture was allowed to warm to room temperature overnight. Crude compound was isolated by addition of cold H₂O (30 ml) and isolated by filtration. Solid was washed with 100% CH₃CN and then suspended in 1:1 CH₃CN and dH₂O for reverse-phase purification using HPLC. A gradient from 2–80% CH₃CN:H₂O (0.1% TFA) was used to further during purification on a C18 column (Agilent Technologies) followed by lyophilization to yield cmp10. ¹H NMR (400 MHz, DMSO-d₆): δ 12.11 (s, 1H), 9.43 (s, 1H), 8.81 (s, 1H), 8.68 (m, J = 22.3 Hz, 2H), 7.92 (s, 1H), 7.43 (m, j = 28.1 Hz, 8H), 6.28 (t, J = 7.2 Hz, 2H) 1.79 (s, 1H), 1.13 (s, 9H), 0.87 (s, 4H); ¹³C NMR (151 MHz DMSO-d₆) δ 153.1, 144.3, 137.7, 135.8, 133.9, 125.8, 119.1, 118.3, 119.07, 118.3, 34.3, 31.7, 30.9, 8.1; ESI-MS m/z [M + H] found 483.5 calculated 482.25.

Figure 20

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Pyrimidine monochloride 3 (0.4 g, 1.67 mmol) was dissolved in dry round bottom flask containing 30 ml of dichloromethane (anhydrous) (Sigma). The flask was then chilled to 4°C in an ice bath under argon. Potassium carbonate (K₂CO₃) (0.92 g, 6.68 mmol) was added, and the reaction was stirred for 10 min. Iodomethane (0.425 g, 2.5 mmol) was then added drop-wise and kept at 4°C until determined complete by thin layer chromatography. The reaction was then filtered to remove excess K₂CO₃ and an equal volume of dH₂O was added to reaction mixture. The mixture was washed three times with brine solution and then dried using magnesium sulfate. The solution was then filtered and concentrated before being loaded on to an AnaLogix silica column. Monomethylated intermediate 16 was isolated using a solution of 5% methanol in chloroform over 30 min. ESI-MS m/z [M + H] found 250.3 calculated 249.09.

Figure 21

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Intermediate 16 (0.157 g, 0.633 mmol) and p-phenylene-diamine (0.068 g, 0.633 mmol) were dissolved in BuOH (3 ml) followed by the addition of concentrated HCl (0.01 ml) resulting mixture was kept at 100°C for 8 hr. Isolation and purification of 17 is as described for intermediate 3. ESI-MS m/z [M + H]⁺ found 322.5 calculated 321.19.

Figure 22

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Intermediate 17 (0.038 mg, 0.123 mmol) was dissolved in 5 ml of dry DMF. The flask was placed in a ice bath before addition of 4-(methylthio)phenyl isocyonate (0.023 mg, 0.123 mmol) (Sigma) drop wise using a Hamilton syringe under argon. The resulting mixture was allowed to warm to room temperature overnight. Crude compound was isolated by the addition of cold H₂O (30 ml) and isolated by filtration. Solid was washed with 100% CH₃CN and then suspended in 1:1 CH₃CN and dH₂O for reverse-phase purification using HPLC. A gradient from 2–80% CH₃CN:H₂O (0.1% TFA) was used to further during purification on a C18 column (Agilent Technologies) followed by lyophilization to yield IPAX. ¹H NMR (400 MHz, DMSO-d₆): δ 10.00 (s, 2H), 8.81 (s, 1H), 8.68 (d, J = 2.3 Hz, 1H), 7.82 (s, 1H), 7.50 (d, j = 2.1 Hz, 3H), 7.33 (s, 6H), 7.08 (t, J = 7.2, 3H), 6.08 (s, 2H), 1.79 (s, 1H), 0.87 (s, 4H); ¹³C NMR (151 MHz DMSO-d₆) δ 162.4, 153.0, 141.1, 131.3, 130.1, 129.3, 123.8, 122.1, 118.2, 115.2, 114.2, 8.4; ESI-MS m/z [M + H]⁺ found 487.5 calculated 486.21.

The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We thank the members of the Walter and Shokat labs for helpful advice and support. We also acknowledge Drs Erica L Cain, Jirka Peschek, Shelly Starck, and Chao Zhang for guidance in assay development and many invaluable insights. This work was funded by a Collaborative Innovation Award from the Howard Hughes Medical Institute to SB, KMS and PW and by PFB16/Fondecyt 1131137 from the Comisión Nacional de Investigación Científica y Tecnológica, Chile, to SB, JA and MAMS. KMS and PW are Investigators of the Howard Hughes Medical Institute.

© 2015, Mendez et al.

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