ERK5 kinase activity is dispensable for cellular immune response and proliferation | PNAS Edited by Stephen J. Benkovic, The Pennsylvania State University, University Park, PA, and approved August 23, 2016 (received for review June 6, 2016) SignificanceWhole protein deletion and pharmacological inhibition are frequently used to functionally annotate enzymes. Each has limitations: whole protein deletion removes both enzymatic and nonenzymatic functions, and small molecule inhibitors can have unrecognized off-target activities. When both approaches agree, it’s nearly incontrovertible support for protein function. Here we describe a counterexample. ERK5 knockdown and inhibition supported a role for this kinase in a number of biological processes. We show that previously reported ERK5 compounds inhibit bromodomain-containing proteins (BRDs) sufficiently to account for their phenotypic effects. We describe highly specific inhibitors of ERK5 that do not inhibit BRDs. With these, we show that cellular inflammation and proliferation are not dependent on ERK5 catalytic activity, thus making ERK5 unique among the MAP kinases.AbstractUnlike other members of the MAPK family, ERK5 contains a large C-terminal domain with transcriptional activation capability in addition to an N-terminal canonical kinase domain. Genetic deletion of ERK5 is embryonic lethal, and tissue-restricted deletions have profound effects on erythroid development, cardiac function, and neurogenesis. In addition, depletion of ERK5 is antiinflammatory and antitumorigenic. Small molecule inhibition of ERK5 has been shown to have promising activity in cell and animal models of inflammation and oncology. Here we report the synthesis and biological characterization of potent, selective ERK5 inhibitors. In contrast to both genetic depletion/deletion of ERK5 and inhibition with previously reported compounds, inhibition of the kinase with the most selective of the new inhibitors had no antiinflammatory or antiproliferative activity. The source of efficacy in previously reported ERK5 inhibitors is shown to be off-target activity on bromodomains, conserved protein modules involved in recognition of acetyl-lysine residues during transcriptional processes. It is likely that phenotypes reported from genetic deletion or depletion of ERK5 arise from removal of a noncatalytic function of ERK5. The newly reported inhibitors should be useful in determining which of the many reported phenotypes are due to kinase activity and delineate which can be pharmacologically targeted.ERK5bromodomaininflammationproliferationkinaseExtracellular signal-regulated kinase 5 (ERK5, BMK1) is a member of the mitogen-activated protein kinase (MAPK) family, which includes ERK1/2, JNK1/2/3, and p38α/β/δ/γ (1). However, unlike the other MAPK members, ERK5 contains a unique 400-amino-acid C-terminal domain in addition to the kinase domain. Through the MAPK signaling cascade, mitogen-activated protein kinase kinase 5 (MEK5) activates ERK5 by phosphorylating the TEY motif in the N-terminal activation loop (2). This event unlocks the N- and C-terminal halves, allowing ERK5 to auto-phosphorylate multiple sites in its C-terminal region, which can then regulate nuclear shuttling and gene transcription (3, 4). Noncanonical pathways (including cyclin-dependent kinases during mitosis and ERK1/2 during growth factor stimulation) also exist for phosphorylation of sites in the ERK5 tail (5⇓–7). Although ERK5 has been demonstrated to directly phosphorylate transcription factors (8⇓–10), the noncatalytic C-terminal tail of ERK5 can also interact with transcription factors and influence gene expression (4, 11, 12).ERK5 can be activated in response to a range of mitogenic stimuli [e.g., growth factors, G protein-coupled receptor (GPCR) agonists, cytokines] and cellular stresses (e.g., hypoxia, shear stress) (13). Like most kinases including MAPK members, ERK5 function is assumed to be driven by its kinase activity. ERK5 deletion is embryonic lethal in mice and a variety of tissue- or development-stage restricted KOs have shown clear phenotypes, suggesting that the catalytic function and/or an aspect of the nonkinase domain(s) have key roles in development and mature organ function (14⇓⇓⇓–18). The availability of the first ERK5 inhibitor XMD8-92 enabled the study of phenotypes resulting from direct kinase inhibition (19, 20). Effects of this inhibitor on cell proliferation were shown to be comparable to overexpression of a dominant negative ERK5 mutant (20, 21) or to siRNA-mediated ERK5 knockdown (21, 22), implicating a key role of the kinase function. Multiple reports have shown promising and corroborating effects of ERK5 knockdown and pharmacological inhibition in controlling inflammation and tumor growth (20, 22⇓⇓⇓–26).Given the proposed therapeutic uses for ERK5 inhibitors, we set out to develop improved compounds with high selectivity and potency. With these ERK5 compounds, we show that first-generation ERK5 inhibitors actually derived the bulk of their biological activity from off-target activity on bromodomains (BRDs). Surprisingly, selective inhibition of the kinase activity alone had no effect on cellular immune response or proliferation, in contrast to whole ERK5 protein knockdown. The newly developed ERK selective inhibitors should prove useful in determining which phenotypes derive from catalytic activity and which derive from other functions of this multidomain kinase.Results and DiscussionInhibitors of ERK5.We synthesized derivatives of the benzopyrimidodiazepinone XMD8-92, a first-generation ERK5 kinase inhibitor (Fig. 1). Among these compounds were ATP-competitive inhibitors that potently inhibit ERK5 with IC50 values ranging from 8 to 190 nM using the chemoproteomics platform KiNativ, which profiles global kinase inhibition in complex lysates (27, 28). At 1-μM screening concentration, there was no significant inhibition of any off-target kinases among the greater than 100 kinases profiled in a single cellular lysate, indicating excellent specificity (Fig. S1).Download figureOpen in new tabDownload powerpointFig. 1. Compound structures and potencies against ERK5 in cell lysate and in live cells treated with compound.The ERK5 inhibitors were also evaluated in an additional KiNativ experimental format wherein compound is incubated with live cells before washing, lysis, and analysis. Such experiments provide an indication of the cellular permeability of the compound and of intracellular target (and off-target) engagement. As seen in Fig. 1, intracellular ERK5 IC50 values were very similar to lysate ERK5 IC50 values, demonstrating that the compounds effectively reached their intracellular target. The most selective ERK5 inhibitor AX15836 was also profiled using live cell KiNativ across several cell types, including peripheral blood mononuclear cells (PBMCs), endothelial cells, and oncogenic cell lines, and verified to maintain its intracellular potency (4–9 nM) across all cells tested.The compounds were next tested for their ability to inhibit epidermal growth factor (EGF)-mediated auto-phosphorylation of endogenous ERK5 in HeLa cells (4). HeLa cells are commonly used to study ERK5 regulation in part due to the ability to clearly observe ERK5 activation resulting from triggers such as growth factors and mitosis (5, 29, 30). In this assay, activated ERK5 migrates more slowly than unactivated ERK5 on SDS/PAGE by virtue of the protein being phosphorylated. As seen in Fig. 2, when treated at 2 μM, approximately fivefold over the weakest intracellular ERK5 IC50, all of the ERK5 inhibitors substantially blocked the formation of phosphorylated ERK5 on EGF stimulation. Because ERK5 is an intracellular target, this provided additional evidence, together with the KiNativ results, that the compounds were able to effectively engage their intracellular target.Download figureOpen in new tabDownload powerpointFig. 2. HeLa cell ERK5 auto-phosphorylation assay. Stimulation of HeLa cells with EGF induced a slower migrating, auto-phosphorylated ERK5 band (upper arrow) separated by SDS/PAGE and detected by Western blot. This p-ERK5 was prevented by pharmacological ERK5 inhibition. Compounds were screened at 2 μM. Figure is a composite of lanes from nonadjacent samples on the same blot.Inhibitor Characterization in Cellular Inflammation Models.ERK5 has been recently studied as a target for mediating inflammation (23, 24, 26). We determined the activity of the ERK5 compounds in cellular assays of inflammatory response. To function in the recruitment of neutrophils and monocytes, the endothelial cell adhesion molecule E-selectin is rapidly synthesized in response to inflammatory stimulation (31). Primary human umbilical vein endothelial cells (HUVECs) were incubated with compounds before stimulation with the Toll-like receptor (TLR1/2) agonist Pam3CSK4. Up-regulation of cell-surface E-selectin was quantified by flow cytometry (Table 1). Similar to that reported by Wilhelmsen and colleagues (23), we found the ERK5 inhibitor XMD8-92 to inhibit up to 38% of the E-selectin expression. Likewise, two other ERK5 inhibitors, AX15839 and AX15910, were effective in reducing E-selectin. Surprisingly, two of the more potent ERK5 inhibitors (AX15836 and AX15892) were inactive in this assay, even at a concentration of compound at least 90-fold higher than the intracellular ERK5 IC50 (Fig. 1).View this table:View inlineView popupTable 1. Surface E-selectin expression on endothelial cells stimulated with TLR1/2 agonist Pam3CSK4Given these results, it seemed likely that an additional activity was responsible for the efficacy of AX15839, AX15910, and XMD8-92. We therefore performed a more extensive KiNativ experiment to expand the kinase coverage to more than 200 kinases and furthermore increased the compound screening concentration to 10 μM. However, we still did not find any significant shared inhibition of off-target kinases among these three inhibitors (Fig. S2). XMD8-92 was derived from the polo-like kinase (PLK1) inhibitor BI-2536 (19, 20). Recently, BI-2536 (as well as a number of other kinase inhibitors) has been shown to inhibit the interaction between BRD and acetyl-lysine binding (32⇓–34). BRDs are protein modules that bind to ε-N-acetylated lysine-containing sequences and modulate transcriptional processes. Members of the dual BRD-containing BET (bromo and extra terminal) proteins BRD2, BRD3, BRD4, and BRDT are targets of drugs currently pursued in oncology, neurological diseases, diabetes, atherosclerosis, and inflammation (35, 36). To determine whether XMD8-92 and other ERK5 kinase inhibitors can likewise inhibit BRDs, we screened the compounds against BRD4, the most well-studied and key member of the BET protein family.Table 2 shows the dissociation constants for compound binding to the first BRD of BRD4 [BRD4(1)]. Using the BROMOscan assay (DiscoveRx), two reference BRD inhibitors, JQ1 (37) and I-BET762 (38), exhibited potent BRD4 (1) Kd values. However, analysis of the ERK5 inhibitors using this method revealed a clear split. Compounds that were active in the E-selectin assay, AX15839, AX15910, and XMD8-92, potently interfered with the acetyl-lysine/ BRD4(1) interaction. These compounds thus represent dual inhibitors of ERK5 kinase and of BRD. In contrast, compounds that were potent on ERK5 but inactive in the E-selectin assay, AX15836 and AX15892, gave considerably higher BRD4 (1) dissociation constants, indicating loss of off-target BRD inhibition.View this table:View inlineView popupTable 2. Inhibitor characteristics and classificationKnowing that the dual ERK5/BRD inhibitors were efficacious in the E-selectin HUVEC assay, whereas the ERK5-selective inhibitors had no effect (Table 1), we returned to that assay to measure the activity of the two BRD-selective reference inhibitors. Using 1 μM of I-BET762 and JQ1, we observed E-selectin reductions of 27% and 29%, respectively, confirming the notion that TLR1/2-induced E-selectin expression in endothelial cells could be reduced by BRD inhibition but not by ERK5 inhibition. Neither BRD inhibitor was active against ∼100 kinases (including ERK5) when profiled at up to 10 μM using KiNativ (Fig. S3).We characterized the activities of several compounds from each classification type in additional cellular models of inflammation and found good consistency of response. For brevity, we show the results of three representative compounds: AX15836 as the ERK5-selective inhibitor, AX15839 as the dual ERK5/BRD inhibitor, and I-BET762 as the selective BRD inhibitor.To determine whether the compounds could suppress inflammatory cytokine response, endothelial cells were pretreated with compound and stimulated with Pam3CSK4. Culture supernatants were subjected to immunoassay for cytokines IL-6 and IL-8. As seen in Table 3, compounds with BRD inhibition (selective and dual) suppressed IL-6 and IL-8; however, the ERK5-specific inhibitor AX15836 was completely ineffective (EC50 » 10 µM), suggesting that it was the BRD inhibition component of the compounds that mediated cytokine reduction.View this table:View inlineView popupTable 3. EC50 values of compounds in reducing cytokines IL-6 and IL-8 produced by endothelial cells stimulated with TLR1/2 agonist Pam3CSK4To determine whether the lack of ERK5-specific effect was limited to a certain cell type and agonist, we repeated the experiment using a normal human bronchial epithelial cell line, BEAS-2B. ERK5 has been identified to be part of an IL-17–mediated signaling cascade that drives keratinocyte proliferation and tumorigenesis (39). IL-17A is also overexpressed in conditions of chronic inflammation such as asthma and is thought to mediate airway neutrophilia through the induction of additional cytokines from target cells (40⇓–42). After preincubation with compound, BEAS-2B cells were stimulated with the proinflammatory cytokine IL-17A. IL-6 and IL-8 cytokine release from the bronchial epithelial cells were measured by immunoassay (Table 4). Again, the ERK5-selective compound AX15836 had no effect on these induced cytokines. In contrast, inhibitors with BRD inhibition activity suppressed inflammatory cytokine response to IL-17A in this cell type.View this table:View inlineView popupTable 4. EC50 values of compounds in reducing cytokines IL-6 and IL-8 produced by bronchial epithelial cells stimulated with IL-17ACellular function of a protein is often studied by reducing its expression via RNA interference. The interpretability of removing the entire protein is typically justified when small molecule inhibitors can demonstrate the same phenotype. In multiple studies (20, 22⇓⇓–25), ERK5 kinase inhibition using XMD8-92 has been used in parallel to siRNA-mediated knockdown of ERK5 to show two lines of supporting evidence for ERK5′s role. Given the lack of cellular effect by selective ERK5 kinase inhibitors, we used siRNA to deplete ERK5 in the HUVECs and BEAS-2B cells to evaluate the role of ERK5 presence. Cells were transfected with siRNA to significantly reduce ERK5 protein expression, as confirmed by Western blots on days 2 and 4 after transfection (Fig. 3 A and B). On day 2 after transfection, cells were stimulated with the respective agonists, and the culture supernatant was collected 2 d later (day 4 after transfection) for cytokine analysis. Indeed, depletion of ERK5 protein resulted in a reduction in IL-6 and IL-8 in both endothelial and epithelial cells (Fig. 3 C and D). These data show that the entire ERK5 protein is necessary for modulating inflammatory response. The contrasting lack of effect by specific small molecule-mediated inhibition of ERK5 kinase activity suggests that a noncatalytic function of ERK5 plays a more important role.Download figureOpen in new tabDownload powerpointFig. 3. ERK5 knockdown suppresses inflammatory cytokine response. HUVEC (A) and BEAS-2B (B) cells were transiently transfected with mock treatment, scrambled siRNA control (siScr), or siRNA against ERK5 (siERK5). Expression of ERK5 and β-actin was monitored by Western blot on day 2 (addition of Pam3CysK4 or IL-17) and day 4 (collection of culture supernatant for cytokine analyses). Immunoassay of IL-6 and IL-8 from treated HUVEC (C) and BEAS-2B (D) cells indicates significant suppression of inflammatory cytokines when ERK5 is depleted. Figures are representative experiments from two independent experiments.We searched for an antiinflammatory effect of AX15836 in additional cellular models of innate and adaptive immunity from both murine and human sources, as listed in Table S1. Inhibition of BRD/acetyl-lysine binding with either JQ1 or I-BET762 resulted in efficacy in several models. In contrast, the ERK5-only inhibitor AX15836 was ineffective against all models tested. We therefore conclude that ERK5 kinase activity does not have a role in cellular immune response.View this table:View inlineView popupTable S1. Comparative efficacy of an ERK5-specific inhibitor and a BRD-specific inhibitor in cell models of innate and adaptive immunityCharacterization of Inhibitors in Cancer Cell Proliferation and Viability.ERK5 and BRDs have been separately studied as targets for oncology. ERK5 was proposed to control multiple processes important for tumorigenesis, including cellular proliferation, survival, invasion, and angiogenesis (43). To reevaluate the role of ERK5 kinase inhibition in cancer cell proliferation, we tested our panel of inhibitors for effects on two cancer cell lines in which ERK5 has been characterized to mediate cell growth and survival. MM.1S multiple myeloma cells express ERK5, which can be activated by IL-6, a growth factor for this cancer type (44). IL-6–induced ERK5 activation was prevented by ERK5 inhibitors (specific ERK5 inhibitor AX15836 and dual ERK5/BRD inhibitor AX15839) (Fig. 4A), confirming target engagement. Overexpression of a dominant negative form of ERK5 was reported to block IL-6–induced MM.1S proliferation (44). We compared IL-6–dependent proliferation in the presence or absence of the highly specific ERK5 inhibitor AX15836 (Fig. 4B), but did not observe a significant effect on cell growth. As such, it seems likely that the reported antiproliferation activity of overexpressing the dominant negative ERK5 mutant in MM.1S cells is the result of non–kinase-related or non–ERK5-related activity of that construct.Download figureOpen in new tabDownload powerpointFig. 4. Multiple myeloma (MM).1S cell growth is not affected by selective ERK5 inhibition. (A) IL-6 activates ERK5 in MM.1S cells, as shown by the appearance of a mobility retarded, auto-phosphorylated ERK5 band (upper arrow) on Western blot. Inhibitors (2 μM) with ERK5 inhibitory activity (AX15836 and AX15839) prevented the induction of p-ERK5. (B) After 3 d of incubation, viable MM.1S cells were quantified in an assay measuring ATP content and expressed as relative luminescence. The ERK5-selective inhibitor AX15836 (1.67 μM) did not significantly reduce IL-6–dependent proliferation relative to the DMSO + IL-6 control. Data graphed in B are the mean ± SD of three independent experiments.We likewise evaluated these inhibitors on the proliferation of the acute myeloid leukemia cell line MV4-11, which expresses the activating internal tandem duplication (ITD) mutation of FLT3 (FLT3-ITD). This driver mutation was reported to constitutively activate ERK5, and inhibition of the upstream kinase MEK5 led to reduced cell proliferation and viability (45). As previously noted in the literature (34), we found reference BRD inhibitors to be effective in this model, with EC50 values of 60 ± 10 and 170 ± 10 nM for JQ1 and I-BET762, respectively (mean ± SD of three experiments). Viability EC50s of the dual ERK5/BRD inhibitors (AX15839, AX15910, and XMD8-92) were less potent and ranged from 1.10 ± 0.25 to 3.28 ± 1.14 μM. Again, however, we observed no effect with the selective ERK5 compounds AX15836 and AX15892 (EC50s 15 μM). Our studies thus demonstrate that highly specific pharmacological inhibition of ERK5 catalytic activity had no effect on cell growth or viability in cancer cell lines previously characterized to be regulated by this kinase. Although xenograft studies might further delineate a more complex role of ERK5 kinase activity, pharmacokinetic characterization of AX15836 (Table S2) did not indicate it to be optimal for in vivo dosing.View this table:View inlineView popupTable S2. Oral PK parameters of 50 mg/kg AX15836 in CD-1 miceTranscriptome Analysis of Cellular ERK5 Kinase Inhibition.We sought to analyze the effects of selective ERK5 inhibition in comparison to BRD inhibition (either dual ERK/BRD or BRD-only) on genome-wide gene expression. Two cellular models with reported ERK5-regulated signaling were used: Pam3CSK4-stimulated HUVECs (23, 24) as a model of inflammation, and EGF-stimulated HeLa cells (5, 20, 29) as an established cell model of ERK5 regulation. Cells were preincubated with DMSO vehicle, AX15836 (ERK5 inhibitor), AX15839 (dual ERK5/BRD inhibitor), or I-BET762 (BRD inhibitor) and then stimulated with agonist. Cellular responses were verified by immunoassays and Western blots using replicate wells in the same experiment.RNA sequencing (RNA-Seq) of biological triplicates detected at least one transcript for 18,925 genes in HUVEC samples and 17,266 genes in HeLa samples. In both cell types, samples treated with AX15836 showed very few genes to be differentially expressed (Fig. 5A). The total number of genes using the default cutoff (adjusted P ≤ 0.1) for plotting log-intensity ratios (M-values) versus log-intensity averages (A-values) (MA plot) was seven in HUVEC samples and two in HeLa samples. Moreover, the observed maximal fold-changes in expression compared with the DMSO control samples were modest: below 1.6 and 2 for HUVEC and HeLa samples, respectively. Principal component analysis of all samples further confirmed the lack of differential gene expression in samples treated with the ERK5-only inhibitor AX15836. Conversely, cells treated with the dual ERK5/BRD inhibitor AX15839 and those treated with the BRD inhibitor I-BET762 showed a large number of differentially expressed genes (Fig. 5A). The correlation of fold-changes in expression of those genes is shown in Fig. 5B. The majority of the genes showed comparable expression patterns in that they were expressed at either higher or lower levels in each of the treated samples compared with controls, confirming a shared regulatory mechanism between AX15839 and I-BET762.Download figureOpen in new tabDownload powerpointFig. 5. Differential gene expression in HUVECs and HeLa cells treated with AX15836, AX15839, and I-BET762 indicates lack of effect by selective ERK5 inhibition. (A) MA plots for HUVECs (Left) and HeLa cells (Right) treated with AX15836 (836; Top), AX15839 (839; Middle), and I-BET762 (iBET; Bottom). Differentially expressed genes with an adjusted P value (DESeq2) of 0.1 or less are shown in red. (B) Correlation of gene expression profiles in HUVECs (Top) and HeLa cells (Bottom) treated with AX15839 and I-BET762. The log2 FC for compound-treated samples compared with the DMSO control samples are plotted for each gene. Outliers are highlighted in red and include those differentially expressed genes (at least a 1.5× fold-change and an adjusted P value below 0.05 in one of the samples) with a residual outside of three times the SD of all residuals.Looking at individual genes of interest, AX15839 and I-BET762 significantly reduced Pam3CSK4-stimulated HUVEC gene expression of IL6 [log2 fold-change (FC) −0.72, P 0.01 and log2 FC −1.32, P 0.001, respectively] and CXCL8 (log2 FC −0.73, P 0.001 and log2 FC −1.42, P 0.001, respectively), consistent with the observed reductions in IL-6 and IL-8 proteins. SELE (E-selectin) transcripts were also reduced by these compounds (log2 FC −0.47, P 0.001 and log2 FC −0.69, P 0.001, respectively), consistent with the observed reduction in protein expression by flow cytometry. Additionally, both compounds with BRD inhibition (AX15839 and I-BET762) significantly suppressed transcription of other genes involved in inflammation, such as IL7R (IL-7 receptor) (log2 FC −1.84, P 0.001 and log2 FC −2.38, P 0.001, respectively), PTGS2 (COX-2) (log2 FC −1.11, P 0.001 and log2 FC −1.65, P 0.001, respectively), and CSF2 (GM-CSF) (log2 FC −1.02, P 0.001 and log2 FC −1.60, P 0.001, respectively), whereas inhibition of ERK5 kinase alone (AX15836) had no effect. Thus, pharmacological inhibition of ERK5 kinase activity was not able to reduce inflammatory gene expression in endothelial cells, further supporting the concept that the previously observed efficacy in first-generation ERK5 inhibitors was due to an unrecognized inhibition of BRD/acetyl-lysine interaction.We had shown that AX15836 could clearly inhibit the EGF-stimulated, phosphorylated form of ERK5 in HeLa cells, a frequently studied cell model of ERK5 regulation. We thus postulated that if the subsequent transcriptional effects of inhibiting ERK5 catalytic function could be seen, it would be in these cells. However, we found no significant impact of AX15836 treatment. In contrast, the four genes most highly suppressed by both AX15839 and I-BET762 were as follows: HAS2 (hyaluronan synthase 2) (log2 FC −2.53, P 0.001 and log2 FC −3.50, P 0.001, respectively), IL7R (log2 FC −2.08, P 0.001 and log2 FC −2.93, P 0.001, respectively), CXCL8 (log2 FC −1.90, P 0.001 and log2 FC −2.14, P 0.001, respectively), and IL6 (log2 FC −1.73, P 0.001 and log2 FC −2.69, P 0.001, respectively). The transcription of both HAS2 and IL7R have recently been reported to be potently down-regulated by BET BRD inhibition in tumor cell lines and are thought to play key roles in cell growth and survival (46, 47). Consistent with previous observations that BRD inhibitors have differential effects on MYC, we also did not observe a reduction of MYC in HeLa cells (48); however, transcripts for cytokines IL-6 and IL-8, known to be increased in HeLa cells by EGF-mediated signaling (49), were suppressed by BRD inhibition. Our transcriptome profiling indicates that pharmacological inhibition of ERK5 kinase activity has no significant impact on gene transcription in two cell models, suggesting that phenotypes observed from genetic ablation of ERK5 result from kinase signaling-independent mechanisms. Future experiments comparing transcriptome effects of kinase inhibition by small molecules to genetic models of catalytically inactive or auto-phosphorylation site mutant ERK5 could be highly informative.ConclusionThe biological function of ERK5 has been studied by a wide variety of experimental techniques. Genetic approaches in intact animals established a role for ERK5 in vascular and neuronal function. Depletion by siRNA in cell models implicated ERK5 in inflammatory and oncogenic pathways, and overexpression of a kinase-dead ERK5 mutant blocked numerous signaling events. Taken together, these experiments were useful to indicate the range of biological processes that ERK5 could influence. Because ERK5 is a kinase that has been demonstrated to phosphorylate transcription factors, the genetic phenotypes have been interpreted as being the result of removing the catalytic activity. However, the selective ERK5 inhibitors described here lack the antiinflammatory and antiproliferative effects induced by genetic manipulation of ERK5. The simplest explanation for this is that the immune and proliferation effects seen in ERK5 genetic models are due to ablation of nonkinase functions of ERK5. Interestingly, essential noncatalytic functions such as protein scaffolding, allosteric regulation of other enzymes, and DNA binding can be found in all seven major kinase groups (50). Separately, we demonstrate that previously reported ERK5 inhibitors, exemplified by XMD8-92, have off-target activity on an unrelated class of proteins, the BRDs. Our experiments show that BRD inhibition is sufficient to account for the antiinflammatory and antiproliferative cellular responses previously ascribed to ERK5 inhibition via XMD8-92. It appears to be pure happenstance that there is overlap between the phenotypes of BRD inhibition and ERK5 depletion/deletion.Our findings show that ERK5 is a highly unusual MAP kinase. Like the other MAPK members, ERK5 is involved in a number of central biological pathways. In stark contrast, however, the kinase activity of ERK5 appears to be unnecessary for many of its most widely studied functions.Materials and MethodsFor determination of native kinase engagement, compounds were profiled in cell lysates or live cells using the KiNativ chemoproteomics platform (ActivX Biosciences) (27, 28, 51). For flow cytometry, ERK5 auto-phosphorylation, immunoanalyses, and RNA-Seq, cells were treated for 1 h with compounds before agonist stimulation, followed by standard protocols. Experimental details and compound synthetic schemes are provided in SI Materials and Methods. In vivo study protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Agilux Laboratories.SI Materials and MethodsTable S1 and S2 Methods.IL-23–stimulated IL-17A production.Single cell suspensions of spleen were prepared from C57BL/6 mice. Splenocytes were cultured at a density of 1 × 106 cells per well in RPMI 1640 containing 10% (vol/vol) FBS, 2 mM l-glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, and 1 mM Hepes with 3 ng/mL mouse IL-23 (R D Systems/Bio-Techne) in the presence or absence of various concentrations of inhibitors. Following 24 h of incubation at 37 °C under 5% CO2, culture supernatants were collected and analyzed for IL-17A using AlphaLISA (PerkinElmer).Anti-CD40 + IL-4–stimulated IgG production.B cells were purified from single cell suspensions of murine spleens using a B-cell isolation kit (Miltenyi Biotec). More than 95% of the cells were confirmed to be B220+ by flow cytometry. Purified B cells were suspended in RPMI medium 1640 supplemented with 10% (vol/vol) FCS and 1% (vol/vol) StemSure monothioglycerol solution (Wako Pure Chemical Industries) and plated on 96-well plates at the density of 1 × 105 cells per well. The cells were then stimulated with 500 ng/mL anti-CD40 antibody (BD Biosciences) and 10 ng/mL IL-4 (Peprotech) in the presence or absence of test compounds. After a 4-d culture, culture supernatant was collected, and IgG concentration was determined using the mouse IgG ELISA Quantitation Set (Bethyl Laboratories).Anti-CD3/CD28–stimulated IL-2 gene expression and cell proliferation.Jurkat cells were plated in 96-well plates precoated with 5 μg/mL anti-CD3 antibody (clone OKT3; eBioscience) together with 1 μg/mL soluble anti-CD28 antibody (BD Biosciences) at a density of 2 × 105 cells per well in RPMI 1640 containing 10% (vol/vol) FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin in the presence or absence of various concentrations of inhibitors. Following 24 h of incubation at 37 °C under 5% CO2, total RNA from Jurkat cells was isolated using a Qiagen RNeasy mini kit according to the manufacturer’s instructions. RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Quantitative IL2 gene expression analysis was performed by real-time PCR on an AB7500 fast real-time PCR system (Applied Biosystems) using the TaqMan gene expression assay. To instead determine proliferation, cells were quantified using the Cell Counting Kit-8 (Dojindo) after incubation.N-Formyl-Met-Leu-Phe (fMLF)-stimulated respiratory burst.Human promyeloblast HL-60 cells were obtained from ATCC and cultured in Iscove\'s modified Dulbecco\'s medium (IMDM) containing 20% (vol/vol) FBS at 37 °C and 5% CO2. To differentiate HL-60 cells into neutrophil-like cells, HL-60 cells were diluted to 2.5 × 105 cells/mL and treated with 1.5% (vol/vol) DMSO for 7 d. Morphological change was analyzed by microscopy after staining with Diff-Quick. A suspension of 5 × 105 neutrophil-like differentiated HL-60 cells was incubated in 0.5 mL HBSS at 37 °C in the presence of 1 mg/mL cytochrome C for 10 min. Cells were pretreated with various concentrations of inhibitor for 10 min before stimulation. After stimulation with fMLF for 15 min, cells were transferred on ice to stop the reaction. Absorbance of supernatant was measured in a FlexStation III spectrophotometer (Molecular Devices) at 550 nm.Pam3CSK4- or LPS-stimulated TNFα production.Human PBMCs (Astarte Biologics) were seeded at 2 × 106 cells/mL in RPMI 1640 containing 10% (vol/vol) charcoal-dextran–treated, heat-inactivated FBS (Omega Scientific), and 50 μM 2-mercaptoethanol. Cells were pretreated with compound for 1 h and then stimulated with either 10 µg/mL Pam3CSK4 or 0.1 µg/mL lipopolysaccharide (LPS) isolated from Escherichia coli 0111:B4 (EMD Millipore) for 16 h at 37 °C in a humidified atmosphere with 5% CO2. Supernatant was collected and analyzed for TNF-α concentration using a homogenous time-resolved fluorescence (HTRF) kit (Cisbio Bioassays). The HTRF ratio of the fluorescence at 665–620 nm was determined using the Synergy 2 multidetection microplate reader (BioTek Instruments).Animal studies.Male CD-1 mice (∼12 wk old; Envigo) were used in this study. Animals were allowed to acclimate for a minimum of 48 h before the conduction of experiments. All animals were group housed in ventilated cages in the Agilux Laboratories animal facility with regulated temperature from 20 °C to 26 °C and relative humidity at 30–70% with a 12/12-h day/night cycle. Pharmacokinetic studies were performed using fasted mice orally dosed at 50 mg/kg with AX15836 formulated in 2.5% (vol/vol) dimethylacetamide: in 0.3% (wt/vol) carboxymethylcellulose. Blood was sampled for eight time points over 24 h. All of the study protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Agilux Laboratories.Experimental Details.Materials.TLR1/2 agonist peptide Pam3CSK4 was purchased from Enzo Life Sciences. Recombinant human EGF and IL-17A were from EMD Millipore and eBioscience, respectively. Antibodies against ERK5, β-actin, GAPDH, and DyLight 680– and 800–conjugated secondary antibodies were purchased from Cell Signaling Technology. Recombinant human IL-6 and Flt3 ligand, fluorescein-conjugated anti-E-selectin (Clone BBIG-E5), and anti-IgG1 isotype control antibodies were obtained from R D Systems/Bio-Techne. I-BET762, JQ1, and AC220 were purchased from Selleck Chemicals. XMD8-92 was purchased from Tocris/Bio-Techne and synthesized in house as previously described (19). Bortezomib was purchased from LC Laboratories. Commercial compound structures and batch purities (≥99%) were reported by the manufacturers using standard analytical methods (HPLC and NMR).Cell culture and live cell KiNativ.Primary HUVECs were grown in complete EGM-2 media (Lonza) and used at passages 2–3. HeLa cells were kindly provided by Jiing-Dwan Lee (Department of Immunology, Scripps Research Institute, La Jolla, CA) and maintained in Eagles MEM (EMEM) containing 10% (vol/vol) charcoal-dextran treated, heat-inactivated FBS (Omega Scientific) and 1× penicillin/streptomycin/amphotericin B (Lonza). The virally immortalized, normal human bronchial epithelial cell line BEAS-2B was purchased from ATCC and grown in complete bronchial epithelial cell growth medium (BEGM) media (Lonza). The multiple myeloma cell line MM.1S and the FLT3 ITD-driven, acute myeloid leukemia cell line MV4-11 were both obtained from ATCC and maintained in RPMI containing 10% (vol/vol) heat-inactivated FBS and 1× penicillin/streptomycin/amphotericin B. All cells were grown at 37 °C in a humidified atmosphere at 5% CO2.For proliferation studies, cells were plated in maintenance media at 2 × 105 cells/mL and incubated in triplicate with eight-point serial dilution series of compound (starting concentration of 15 μΜ for all compounds except for bortezomib at 1.5 μM) or with DMSO vehicle (0.25% final volume). For MM.1S cells, compound was added 1 h before adding recombinant human IL-6 (R D Systems/Bio-Techne) at 5 nM. After 3 d, the relative number of viable cells was measured via quantitation of ATP using CellTiter-Glo 2.0 reagent (Promega). Luminescence was read on the Synergy 2 multimode reader (BioTek). EC50 values were determined using GraphPad Prism (GraphPad Software), with signal from cells treated with DMSO normalized as 100% and signal from cells treated with maximal concentration of relevant clinical reference inhibitors set as 0%.For determination of cellular kinase engagement, the KiNativ chemoproteomics platform was used. In live cell KiNativ, cells were incubated with compound for 1 h at 37 °C in a humidified atmosphere with 5% CO2, washed, and collected for KiNativ analysis as previously described (27).Detection of E-selectin.HUVECs were pretreated with compound or 0.1% DMSO vehicle for 1 h in complete media and then stimulated with 10 μg/mL Pam3CSK4 for 4 h at 37 °C in a humidified atmosphere with 5% CO2. After detachment, cells were incubated with 10 μg/mL human IgG in flow cytometry staining buffer (FCSB; eBioscience) for 15 min on ice to block nonspecific Fc binding, and then incubated with 2.5 μg/mL fluorescein–anti-E-selectin antibody for 1 h on ice. Cells were analyzed by flow cytometry on the Attune flow cytometer (Applied Biosystems/ThermoFisher Scientific). Pam3CSK4-stimulated, DMSO-treated cells incubated with a fluorescein-labeled, IgG1 isotype control primary antibody were determined to exhibit no change in fluorescein signal relative to nonstimulated, DMSO-treated cells stained with the fluorescein-conjugated anti–E-selectin antibody; therefore, the latter was used as the baseline control. Cells were first gated using forward vs. side scatter, and then 60,000 cells were analyzed for a gain in fluorescein signal relative to unstimulated DMSO-treated cells.ERK5 auto-phosphorylation assay.HeLa cells were pretreated for 1 h with 2 μM compound or DMSO vehicle in maintenance media and stimulated with 50 ng/mL EGF for 15 min at 37 °C. Proteins were separated via SDS/PAGE using 8% (wt/vol) gels (Novex/ThermoFisher Scientific). ERK5 was detected by Western blot analysis using anti-human Erk5 polyclonal antibody and normalized to β-actin. Phosphorylated ERK5 was observable as a slower migrating band. Fluorescent signal from the secondary detection antibodies was detected and quantified using the Odyssey Imaging System (LI-COR Biotechnology).For detection of ERK5 forms in MM.1S cells, the cells were serum starved overnight in RPMI containing 0.5% BSA. After 1-h preincubation with 2 μM compound or DMSO vehicle, cells were stimulated with 5 nM IL-6 for 10 min at 37 °C and then processed as described above.Cytokine immunoanalyses.Cells in their respective growth media were pretreated with DMSO vehicle or compound in duplicate four-point serial dilution series from 10 to 0.08 μM for 1 h, after which cells were left unstimulated or stimulated with either 10 μg/mL Pam3CSK4 (for HUVECs) or 50 ng/mL IL-17A (for BEAS-2B cells). Cells were incubated for 2 d, after which cytokines in the supernatant were determined by ELISA (Life Technologies/ThermoFisher Scientific) or by multiplex immunoassay (Bio-Rad). ELISA absorbance was read at 450 nm using the Wallac 1420 reader (Perkin-Elmer), whereas fluorescence of multiplex magnetic beads was quantified using the Bio-Plex MAGPIX multiplex reader (Bio-Rad). EC50 values were determined using GraphPad Prism software, with cytokine levels from cells treated with DMSO + stimulation normalized as 100% and nonstimulated concentrations set as 0%.Knockdown of ERK5.Two sets of specific siRNA were used to knockdown ERK5 (MAPK7) with similar results: s11149 (Ambion/ ThermoFisher Scientific) and l-003513-00 (Dharmacon/GE Healthcare). Scrambled siRNA duplex SR30004 (Origene) or D-001810-10-05 (Dharmacon/GE Healthcare) were used as negative controls. Transient transfections were carried out using 10 nM siRNA and Lipofectamine RNAiMAX (Invitrogen/ThermFisher Scientific). After 2 d, cells were either used for Western blot analysis to confirm ERK5 knockdown or stimulated for cytokine induction as described above. At 4 d after transfection (2 d after stimulation), cells were again analyzed by Western blot to confirm continued knockdown of ERK5, and the culture supernatants were used for immunoanalyses.Preparation of cell samples for RNA-Seq.Cells were preincubated with 0.1% DMSO vehicle, 1 μM AX15836 (ERK5 inhibitor), 5 μM AX15839 (dual ERK5/BRD inhibitor), or 1 μM I-BET762 (BRD inhibitor) for 1 h. HUVECs and HeLa cells were then stimulated with their respective agonists (10 μg/mL Pam3CSK4 or 50 ng/mL EGF) for 5 h at 37 °C. Cells were processed to total RNA using the RNeasy kit (Qiagen). RNA was sent to the Scripps Research Next Generation Sequencing Core Facility for RNA-Seq.One microgram total RNA from each sample was ribodepleted using the Ribo-Zero-rRNA Removal Kit (Epicentre). Sequencing libraries were then prepared from ribodepleted RNA using NEBNext Ultra RNA Library Prep Kit for Illumina following the manufacturer’s recommended protocol and barcoded using standard Illumina TruSeq barcoded adapter sequences. Final libraries were size-selected using Agencourt AMPure XP beads. Purified libraries were pooled and loaded onto an Illumina HiSeq2000 sequencer for 100-base single end sequencing with 7-base index reads.Genome Analyzer Pipeline Software (bcl2fastq ver.2.15.0.4; Illumina) was used to perform image analysis, base calling, and demultiplexing. The program Cutadapt was used to trim the adapter and low base pair called scores. Per-exon gene counts were generated using TopHat2 software (www.ccb.jhu.edu) and the HG19 release of the human genome. Raw count data analysis was performed using the Bioconductor software framework (www.Bioconductor.org) and differential expression statistics were calculated using the DESeq2 package (www.bioconductor.org/packages/release/bioc/html/DESeq2.html). Genes differentially expressed in compound-treated samples vs. DMSO-treated samples were tabulated, and results from both experiment sets were compared with each other using a cutoff of P 0.01.Chemical Syntheses.Materials.Proton NMR (1H NMR) spectra were recorded on a Bruker 400 MHz NMR spectrometer in deuterated solvents using the residual 1H solvent peak as the internal standard. LC/MS (ES) analysis was performed with an Agilent 1260 Infinity Series LC/MSD using ChemStation software equipped with a C18 reverse phase column (Phenomenex Kinetex 5 m XB-C18 50- × 2.10-mm column, or Agilent Poreshell 120 EC-C18 3.0- × 50-mm column) or Agilent 1100 Series LC/MSD using ChemStation software equipped with a C18 reverse phase column (Onyx, monolithic C18 column, 50 × 2.0 mm; Phenomenex), and using a binary system of water and acetonitrile with 0.1% trifluoroacetic acid as a modifier. Flash silica gel column chromatography was carried out on a CombiFlash Rf system (by Teledyne ISCO) or a Biotage SP-4 automated purification system using prepacked silica gel cartridges. HPLC purification was performed by using an Agilent 1200 Series with a C18 reverse phase column [Luna 5 u C18(2)100A, 150 × 21.2 mm, 5 μm; Phenomenex] and using a binary system of water and acetonitrile with 0.1% acetic acid as a modifier.Synthetic protocols.Scheme for the synthesis of AX15836.5-(Methylamino)pyrimidine-2,4(1H,3H)-dione (A3).To a 500-mL three-neck round bottom flask filled with a condenser, 5-bromopyrimidine-2,4(1H,3H)-dione (A1, 20 g, 0.10 mol) and methanamine [A2, 40% (wt/vol) aqueous solution, 160 mL, 1.85 mol] were added. The reaction mixture was stirred and heated at 80 °C for 3.5 h. At 25 °C, the reaction mixture was acidified to pH ∼4.5 with diluted HCl aqueous solution. The generated light yellow precipitates were filtered and washed with water, and then dried in vacuo to provide A3 (10.46 g, 70% yield) as a light yellow solid. ESMS found m/z 142.1 ([M + H+], C5H7N3O2 requires 141.0538).N-(2,4-Dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-N-methyl-2-nitrobenzamide (A4).To a solution of A3 (10.46 g, 74.1 mmol) in tetrahydrofuran (THF) (40 mL), NaOH aqueous solution (2.5 N, 100 mL, 250 mmol) was added at 0 °C. 2-Nitrobenzoyl chloride (12.8 mL, 96.8 mmol) was added slowly. The generated clear brown solution was stirred at 0 °C for 40 min and then at room temperature for 4.5 h. The reaction mixture was acidified by diluted HCl aqueous solution and stored for 3 d. The generated light yellow solid was filtered, and the cake was washed with water and then dried in vacuo to provide A4 (13.47 g, 63% yield) as a light yellow solid. ESMS found m/z 291.0 ([M + H+], C12H10N4O5 requires 290.0651), 313.1 [M + Na+].N-(2,4-Dichloropyrimidin-5-yl)-N-methyl-2-nitrobenzamide (A5).A solution of A4 (7.96 g, 27.4 mmol) and N,N-dimethylaniline (3 mL, 23.7 mmol) in phosphorus oxychloride (POCl3, 135 mL, 1.45 mol) was heated at 100 °C overnight. The solvent was removed by rotavapor, and the residue was dried in vacuo to provide the crude product A5, which was used for the next step reaction without further purification. ESMS found m/z 327.0, 329.0 ([M + H+], C12H8Cl2N4O3 requires 325.9973, 327.9944).2-Chloro-5-methyl-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6(11H)-one (A6).The crude A5 (∼27.4 mmol) was dissolved in acetic acid (100 mL), and then iron (9.1 g, 163 mmol) was added at 25 °C with rigorous stirring. The mixture was heated at 60 °C for 5 h. Water (100 mL) and ethanol (10 mL) were added and the reaction mixture was stirred for 30 min. The precipitates were filtered and extracted between ethyl acetate (EtOAc) and water. The combined EtOAc phase was dried over sodium sulfate and then concentrated to produce the crude A6 (3.25 g, 46% yield for two steps). 1H NMR (400 MHz, CDCl3) δ 8.23 (s, 1H), 7.98 (dd, J = 1.5, 8.0, 1H), 7.45–7.37 (m, 1H), 7.17–7.09 (m, 1H), 6.82 (dd, J = 0.9, 8.1, 1H), 6.71 (s, 1H), 3.51 (s, 3H); ESMS found m/z 261.1 ([M + H+], C12H9ClN4O requires 260.0465).2-Chloro-5-methyl-11-(methylsulfonyl)-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6(11H)-one (A7).NaH [60% (wt/vol), 160.0 mg, 4.00 mmol] was added to a solution of A6 (521.4 mg, 2.00 mmol) in THF (20.0 mL) at 0 °C. The generated orange suspension was stirred at 25 °C for 30 min, and then MeSO2Cl (310.0 μL, 4.00 mmol) was added and the mixture was stirred at 25 °C for 1 h. The reaction was quenched and diluted with H2O. The aqueous phase was extracted with EtOAc. The combined EtOAc phase was washed with saturated (sat.) NaHCO3 and sat. NaCl aqueous solution and then dried over Na2SO4. The dried organic phase was filtered and concentrated by rotavapor. The residue was washed with hexane, and the gum was dried in vacuo to provide the crude A7 as a light yellow solid, which was used for the next step reaction without further purification. 1H NMR (400 MHz, DMSO-d6) δ 9.07 (s, 1H), 7.82 (dd, J = 1.2, 7.5, 1H), 7.74–7.64 (m, 2H), 7.54 (ddd, J = 2.1, 6.5, 7.8, 1H), 3.79 (s, 3H), 3.56 (s, 3H); ESMS found m/z 339.0 ([M + H+], C13H11ClN4O3S requires 338.0240).Ethyl 3-ethoxy-4-(5-methyl-11-(methylsulfonyl)-6-oxo-6,11-dihydro-5H-benzo[e]pyrimido [5,4-b][1,4]diazepin-2-ylamino)benzoate (A8).A mixture of crude A7 (367 mg, ∼92% purity, ∼1.00 mmol), ethyl 4-amino-3-ethoxybenzoate (251.1 mg, 1.20 mmol), X-Phos (42.0 mg, 0.088 mmol), and K2CO3 (829.3 mg, 6.00 mmol) in tBuOH (10.0 mL) was bubbled with N2 for 20 s. Pd2(dba)3 (54.9 mg, 0.060 mmol) was added and the mixture was bubbled with N2 for additional 20 s. The mixture was then heated at 100 °C under N2 overnight. The reaction mixture was diluted with hexane-EtOAc solution (10:3). The precipitates were filtered and washed with hexane-EtOAc solution (10:3). The combined filtrate was concentrated in vacuo to provide the crude A8 as an orange solid, which was used for the next step reaction without further purification. ESMS found m/z 512.1 ([M + H+], C24H25N5O6S requires 511.1526).3-Ethoxy-4-(5-methyl-11-(methylsulfonyl)-6-oxo-6,11-dihydro-5H-benzo[e] pyrimido[5,4-b][1,4]diazepin-2-ylamino)benzoic acid (A9).A solution of the crude A8 (699.0 mg, ∼73.2% purity, ∼1.000 mmol) in THF (6.05.4 mL), MeOH (2.0 mL), and H2O (2.0 mL) was treated with LiOH hydrate (62.9 mg, 1.50 mmol) at 25 °C overnight. The reaction mixture was concentrated by rotavapor. The residue was diluted with DCM. The DCM phase was extracted with 1 N NaOH aqueous solution. The combined aqueous phase was washed with additional DCM. Then the aqueous phase was acidified with 1 N HCl aqueous solution. The generated precipitates were stirred for 10 min, filtered and washed with H2O, and then dried in vacuo to provide A9 (389.3 mg, 81% yield for 3 steps) as a tan solid. 1H NMR (400 MHz, DMSO-d6) δ 12.70 (br s, 1H), 9.02 (s, 1H), 8.79 (s, 1H), 8.00 (d, J = 8.3, 1H), 7.78 (d, J = 7.8, 1H), 7.65 (s, 1H), 7.63–7.55 (m, 2H), 7.53 (s, 1H), 7.49 (s, 1H), 4.16 (m, 2H), 3.80 (s, 3H), 3.51 (s, 3H), 1.32 (t, J = 6.9, 3H); ESMS found m/z 484.2 ([M + H+], C22H21N5O6S requires 483.1213).2-(2-Ethoxy-4-(4-(4-methylpiperazin-1-yl)piperidine-1-carbonyl)phenylamino)-5-methyl-11-(methylsulfonyl)-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6(11H)-one (A10, AX15836).HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) (356.5 mg, 0.938 mmol) was added to a solution of A9 (377.8 mg, 0.781 mmol), 1-methyl-4-(piperidin-4-yl)piperazine (171.9 mg, 0.938 mmol), and N,N-Diisopropylethylamine (DIPEA) [408.0 μL, 2.34 mmol] in dimethylformamide (DMF) (3.9 mL) at 25 °C. The mixture was stirred at 25 °C for 3 h. The reaction mixture was dried in vacuo. The residue was purified by HPLC to provide the acetic acid salt of A10 (AX15836, 140 mg, 98% purity, 28% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.73 (s, 1H), 7.80–7.75 (m, 1H), 7.72 (d, J = 8.0, 1H), 7.67–7.57 (m, 2H), 7.48 (dd, J = 5.1, 11.1, 1H), 7.03 (d, J = 1.6, 1H), 7.00–6.93 (m, 1H), 4.58–4.26 (m, 1H), 4.16–4.01 (m, 2H), 3.74 (s, 3H), 3.72–3.55 (m, 1H), 3.49 (s, 3H), 3.15–2.88 (m, 1H), 2.86–2.63 (m, 1H), 2.55 (d, J = 0.5, 3H), 2.50 (m, 5H), 2.29 (m, 3H), 2.13 (s, 3H), 1.79 (m, 2H), 1.37 (m, 2H), 1.24 (t, J = 6.9, 3H). 13C NMR (400 MHz, DMSO-d6) δ 169.01, 166.02, 157.48, 156.06, 155.74, 150.69, 140.68, 133.32, 132.97, 132.20, 130.63, 129.05, 128.70, 125.74, 124.63, 123.18, 119.12, 111.33, 64.38, 61.31, 55.60, 48.99, 46.23, 45.42, 40.83, 37.36, 24.44, 14.91; ESMS found m/z 649.3 ([M + H+], C32H40N8O5S requires 648.2842).Scheme for the synthesis of AX15839 and AX15910.N-(4, 6-Dichloropyridin-3-yl)-2-nitro-N-(2-nitrobenzoyl)benzamide (B3).The compound 2-nitrobenzoyl chloride (B2, 11.10 mL, 84.0 mmol) was added slowly to a solution of 5-amino-2, 4-dichloropyridine (B1, 6.520 g, 40.0 mmol) and DIPEA (27.9 mL, 160 mmol) in DCM (100 mL) at 0 °C under N2. The mixture was stirred at 25 °C for 1.5 h. The reaction mixture was concentrated by rotavapor. The residue B3 (brown syrup) was used for B4 preparation without further purification.For characterization, a small amount of crude B3 was diluted with DCM, washed with H2O, sat. NaHCO3 aqueous solution, and sat. NaCl aqueous solution, and then dried over Na2SO4. The dried organic phase was filtered and concentrated. The residue was rinsed with a small amount of DCM and the remaining precipitates were dried in vacuo to provide B3 as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.24 (d, J = 8.3, 2H), 8.08 (d, J = 1.0, 1H), 7.97–7.80 (m, 4H), 7.75 (t, J = 7.8, 2H); ESMS found m/z 461.0 ([M + H+], C19H10Cl2N4O6 requires 459.9977), 483.0 [M + Na+].N-(4, 6-Dichloropyridin-3-yl)-2-nitrobenzamide (B4).A suspension of crude B3 (∼40.0 mmol) in THF (90 mL) and NaOH aqueous solution (∼3.5 N, 72 mL, ∼252 mmol) was stirred rigorously at 25 °C overnight. The reaction mixture was diluted with sat. NaCl aqueous solution. The aqueous phase was extracted with EtOAc. The combined organic phase was washed with sat. NaHCO3 and sat. NaCl solution, and then dried over NaSO4. Filtration and concentration in vacuo provided B4 (11.24 g, 90% yield for two steps) as a pale white solid. 1H NMR (400 MHz, CDCl3) δ 9.42 (br s, 1H), 8.19 (dd, J = 1.0, 8.2, 1H), 7.74 (m, 4H), 7.45 (s, 1H); ESMS found m/z 312.0, 314.0 ([M + H+], C12H7Cl2N3O3 requires 310.9864, 312.9835), 334.0, 336.0 [M + Na+].2-Amino-N-(4, 6-dichloropyridin-3-yl)benzamide (B5).A suspension of B4 (12.48 g, 35.80 mmol) and Fe (4.47 g, 80.0 mmol) in acetic acid (80 mL) was heated at 50 °C with rigorous stirring under N2 for 2 h. Additional Fe (1.12 g, 10 mmol) was added twice during 2 h. The reaction mixture was stirred at 50 °C for additional 1 h. At 25 °C, the extra Fe was removed with a magnetic bar. The reaction mixture was quenched with 1 N NaOH aqueous solution and the aqueous solution was saturated with NaCl. The product was extracted by EtOAc (multiple times and monitored by LCMS). The combined EtOAc phase was washed with sat. NaHCO3 solution and sat. NaCl solution, then dried over Na2SO4. Filtration and concentration in vacuo provided B5 (10.26 g, 91% yield) as a pale white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.07 (br s, 1H), 8.56 (s, 1H), 7.94 (t, J = 1.6, 1H), 7.74 (dd, J = 1.4, 8.0, 1H), 7.25 (ddd, J = 1.5, 7.1, 8.4, 1H), 6.78 (dd, J = 0.9, 8.3, 1H), 6.67–6.58 (m, 1H), 6.53 (br s, 2H); ESMS found m/z 282.1, 284.0 ([M + H+], C12H9Cl2N3O requires 281.0123, 283.0093), 304.0, 306.0 [M + Na+].3-Chloro-5H-benzo[e]pyrido[3,4-b][1,4]diazepin-10(11H)-one (B6).A suspension of B5 (10.263 g, 36.38 mmol) in NMP (80.0 mL) was heated at 200 °C under N2 for 4 h. At 25 °C, a diluted HCl aqueous solution (0.33 N, 240 mL) was added. The generated suspension was stirred at 25 °C for 1 h. The precipitates were filtered and washed with H2O and then dried in vacuo to provide B6 (8.623 g, 96% yield) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 8.76 (s, 1H), 7.85 (s, 1H), 7.80–7.68 (m, 1H), 7.47–7.32 (m, 1H), 6.96 (m, 3H); ESMS found m/z 246.1 ([M + H+], C12H8ClN3O requires 245.0356), 268.0 [M + Na+].3-Chloro-5,11-dimethyl-5H-benzo[e]pyrido[3,4-b][1,4]diazepin-10(11H)-one (B7).NaH [60% (wt/vol), 2.15 g, 53.9 mmol] was added portion-wise to a suspension of B6 (5.513 g, 22.4 mmol) and MeI (3.36 mL, 53.9 mmol) in anhydrous DMF (67.3 mL) at 0 °C under N2. The reaction mixture was then stirred at 25 °C under N2 overnight. At 0 °C, the diluted HCl aqueous solution (0.25 N) was added slowly to the generated suspension reaction mixture. The mixture was stirred at 25 °C for 1 h. Hexanes were added and the mixture was stirred at 25 °C for an additional 1 h. The generated precipitates were filtered and washed with H2O and then dried in vacuo to provide B7 (5.145 g, 84% yield) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.36 (s, 1H), 7.65 (dd, J = 1.7, 7.7, 1H), 7.55–7.44 (m, 1H), 7.34 (s, 1H), 7.23–7.11 (m, 2H), 3.45 (s, 3H), 3.31 (s, 3H); ESMS found m/z 274.1 ([M + H+], C14H12ClN3O requires 273.0669), 296.1 [M + Na+].3-((1r,4r)-4-Hydroxycyclohexylamino)-5,11-dimethyl-5H-benzo[e]pyrido[3,4-b][1,4]diazepin-10(11H)-one (B8, AX15839).A mixture of B7 (4.13 g, 15.1 mmol), trans4-aminocyclohexanol (2.09 g, 18.1 mmol), Pd2(dba)3 (691 mg, 0.755 mmol), tBuBrettPhos (732 mg, 1.51 mmol), and tBuONa (5.08 g, 52.9 mmol) in 1,4-dioxane (150 mL) was bubbled with N2 at 25 °C and then stirred at 100 °C for 1 h. After cooling to room temperature, the reaction mixture was filtered through a Celite pad, and the solvent was removed under reduced pressure. After purification by silica gel column chromatography [Biotage Ultra 100 g, toluene to 15% (vol/vol) ethanol-toluene], the residue was suspended with ethyl acetate. The precipitate was collected by filtration and dried in vacuo to afford B8 (2.30 g, 95% purity, 43% yield) as a pale yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 1.11–1.20 (4H, m), 1.80–1.88 (4H, s), 3.17 (3H, s), 3.37 (3H, s), 3.38 (1H, brs), 3.57–3.58 (1H, m), 4.50 (1H, d, J = 4.4 Hz), 6.18 (1H, s), 6.34 (1H, d, J = 8.0 Hz), 7.11 (1H, t, J = 8.0 Hz), 7.17 (1H, d, J = 8.0 Hz), 7.45 (1H, t, J = 8.0 Hz), 7.60 (1H, d, J = 8.0 Hz), 7.92 (1H, s); 13C NMR (101 MHz, DMSO-d6) δ 167.95, 157.12, 156.33, 151.78, 142.88, 132.53, 131.82, 127.50, 123.79, 123.51, 117.31, 96.42, 68.87, 49.08, 38.14, 36.75, 34.50, 34.46, 31.05, 30.96; HRESIMS found m/z 353.19729 ([M+H+], C20H24N4O2 requires 352.1899).Ethyl 4-(5,11-dimethyl-10-oxo-10,11-dihydro-5H-benzo[e]pyrido[3,4-b][1,4]diazepin-3-ylamino)-3-ethoxybenzoate (B9).A mixture of B7 (4.927 g, 18.0 mmol), ethyl 4-amino-3-ethoxybenzoate (4.520 g, 21.6 mmol), X-Phos (755.1 mg, 1.58 mmol), and K2CO3 (14.93 g, 108.0 mmol) in tBuOH (90 mL) was bubbled with N2 for 30 s. Pd2(dba)3 (494.5 mg, 0.540 mmol) was added, and the mixture was bubbled with N2 for additional 1 min. The suspension was then heated at 100 °C (flushed with condenser) under N2 for 23 h. The reaction mixture was diluted with EtOAc at 25 °C. The suspension was filtered through a Celite filter column, and the precipitates were washed with EtOAc. The filtrate was washed with 0.5 N HCl aqueous solution and sat. NaCl aqueous solution and then dried over Na2SO4. The organic solution was filtered and concentrated by rotavapor. The residue was diluted with CH3CN, and the filtrate was concentrated and dried in vacuo to provide the crude B9 as a yellow solid, which was used for next step reaction without further purification. ESMS found m/z 447.2 ([M+H+], C25H26N4O4 requires 446.1954), 469.1 [M + Na+].4-(5,11-Dimethyl-10-oxo-10,11-dihydro-5H-benzo[e]pyrido[3,4-b][1,4]diazepin-3-ylamino)-3-ethoxybenzoic acid (B10).The crude B9 (∼78.5% pure, 10.00 g, ∼17.6 mmol) was dissolved in THF (52.7 mL), MeOH (17.6 mL), and H2O (17.6 mL). LiOH monohydrate (2.066 g, 49.2 mmol) was added, and the mixture was stirred at 25 °C for 4 h. Additional LiOH monohydrate (1.033 g, 24.6 mmol) and H2O (10 mL) were added, and the mixture was stirred at 25 °C for additional 1.5 h. The reaction mixture was concentrated by rotavapor. The residue was diluted with 0.5 N NaOH aqueous solution. The basic aqueous phase was washed with ether then acidified with 3 N HCl solution. The generated precipitates were filtered and washed with H2O, rinsed with small amount of EtOAc, and then dried in vacuo to provide B10 (6.071 g, 81% yield for two steps) as a tan solid. 1H NMR (400 MHz, DMSO-d6) δ 9.65–9.27 (br s, 1H), 8.14 (s, 1H), 8.00 (br s, 1H), 7.68 (dd, J = 1.7, 7.7, 1H), 7.64–7.44 (m, 3H), 7.29 (d, J = 8.1, 1H), 7.20 (t, J = 7.5, 1H), 7.05 (s, 1H), 4.17 (q, J = 7.0, 2H), 3.45 (s, 3H), 3.33 (s, 3H), 1.33 (t, J = 6.9, 3H); ESMS m/z: 419.1 [M + H+]. ESMS found m/z 419.1 ([M+H+], C23H22N4O4 requires 418.1641).3-(2-Ethoxy-4-(4-(pyrrolidin-1-yl)piperidine-1-carbonyl)phenylamino)-5,11-dimethyl-5H-benzo[e]pyrido[3,4-b][1,4]diazepin-10(11H)-one (B11, AX15910).To a solution of B10 (2.40 g, 5.74 mmol) and 4-pyrrolidin-1-ylpiperidine (1.062 g, 6.88 mmol) in DMF (30 mL), DIPEA (4.00 mL, 22.9 mmol) and HATU (3.053 g, 8.03 mmol) were added at 25 °C. The reaction mixture was stirred at 25 °C for 2 h and then concentrated by lyophilization. Water was added to the residue, and the mixture was stirred at 25 °C for 30 min. The generated precipitates were filtered and purified by prep HPLC. Lyophilization of the pure product fractions provided B11 (AX15910, 2.15 g, 95% purity, 68% yield) as a white powder. 1H NMR (400 MHz, CDCl3) δ 8.11–8.01 (m, 2H), 7.83 (dd, J = 1.7, 7.7, 1H), 7.45–7.36 (m, 1H), 7.12 (dd, J = 3.1, 10.7, 2H), 7.06–6.96 (m, 3H), 6.53 (s, 1H), 4.13 (q, J = 7.0, 2H), 3.57 (s, 3H), 3.31 (s, 3H), 2.93 (s, 2H), 2.75 (s, 4H), 2.47 (s, 1H), 2.02 (s, 1H), 2.02–1.92 (m, 2H), 1.87 (s, 4H), 1.65 (s, 2H), 1.47 (t, J = 7.0, 3H); 13C NMR (101 MHz, DMSO-d6) δ 169.47, 167.90, 156.31, 153.96, 151.73, 147.11, 142.17, 132.81, 132.09, 132.00, 128.59, 127.17, 126.78, 123.70, 119.78, 117.90, 117.48, 111.03, 100.84, 64.48, 61.20, 51.27, 40.38, 38.16, 23.38, 17.20, 15.06; ESMS found m/z 555.3 ([M+H+], C32H38N6O3 requires 554.3005), 1131.6 [2M + Na+].Scheme of synthesis of AX15892.Ethyl 3-[(2-chloro-5-nitro-4-pyridyl)amino]pyridine-2-carboxylate (C3).A mixture of 2,4-dichloro-5-nitro-pyridine (C1, 1.0 g, 5.2 mmol), ethyl 3-aminopyridine-2-carboxylate (C2, 0.86 g, 5.2 mmol), Cs2CO3 (3.38 g, 10.4 mmol), and 2,2′-diphenylphosphino-1,1′-binaphthyl (0.19 g, 0.31 mmol) in anhydrous 1,4-dioxane (133 mL) was degassed with N2 at 25 °C, and then Pd2(dba)3 (0.19 g, 0.21 mmol) was added. The mixture was degassed again and heated at 70 °C overnight. The reaction was filtered through Celite and washed with DCM. The filtrate was concentrated, and the resulting residue was purified by silica gel flash chromatography using EtOAc/hexane as eluent solution. The product containing fractions were concentrated to provide C3 (0.653 g, 39% yield) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 11.48 (s, 1H), 9.18 (s, 1H), 8.71–8.60 (m, 1H), 8.02 (d, J = 8.4, 1H), 7.64 (dd, J = 4.5, 8.4, 1H), 7.27 (s, 1H), 4.58 (q, J = 7.1, 2H), 1.50 (t, J = 7.1, 3H); ESMS found m/z 323.1 ([M + H+], C13H11ClN4O4 requires 322.0469).7-Chloro-5H-dipyrido[3,4-b:3′,2’-e][1,4]diazepin-11(10H)-one (C4).To a solution of C3 (653 mg, 2.02 mmol) in AcOH (10 mL), Fe powder (560 mg, 10.1 mmol) was added at 25 °C. The mixture was then heated at 70 °C overnight. The Fe was removed with a magnetic stirring rod. The reaction mixture was concentrated and dried in vacuo to provide C4 (0.50 g, 84% yield) as an off-white solid, which was used for next step reaction without further purification. ESMS found m/z 247.1 ([M + H+], C11H7ClN4O requires 246.0308), 269.0 [M + Na+], 515.0 [2M + Na+].2-Chloro-5,11-dimethyl-5H-benzo[e]pyrimido[5,4-b][1,4]diazepin-6(11H)-one (C5).To a suspension of C4 (0.50 g, 2.0 mmol) and MeI (0.30 mL, 4.8 mmol) in anhydrous DMF (10.0 mL), NaH was added [60% (wt/vol), 0.19 g, 4.8 mmol] at 0 °C under N2. The reaction mixture was stirred at 0 °C for 10 min and then warmed at 25 °C overnight. The reaction was quenched with 0.5 N HCl aqueous solution at 0 °C and stirred at 25 °C for additional 1 h. The precipitate was filtered, washed with 1 N HCl aqueous solution, H2O, and hexanes, and then dried in vacuo to provide C3 as a solid (0.44 g, 80% yield). ESMS found m/z 275.1 ([M + H+], C13H11ClN4O requires 274.0621).7-((2-Ethoxy-4-(4-(4-methylpiperazin-1-yl)piperidine-1-carbonyl)phenyl)amino)-5,10-dimethyl-5H-dipyrido[3,4-b:3′,2\'-e][1,4]diazepin-11(10H)-one (C6, AX15892).A mixture of C5 (25 mg, 0.091 mmol), C6 (34.7 mg, 0.100 mmol), K2CO3 (37.7 mg, 0.273 mmol), Pd2(dba)3 (5.0 mg, 0.0055 mmol), and X-Phos (3.8 mg, 0.0080 mmol) in tButanol (1 mL) was purged with nitrogen gas several times, and then heated in a sealed vial at 100 °C overnight. At 25 °C, the reaction mixture was filtered through a short Celite column and washed with methanol. The filtrate was concentrated and the residue was purified by HPLC to obtain product C7 (AX15892, 2.07 mg, 95% purity, 3.9% yield) as a white powder. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.27–1.43 (m, 2 H) 1.39 (t, J = 6.95 Hz, 3 H) 1.70–1.82 (m, 2 H) 1.86 (s, 4 H) 2.12 (s, 3 H) 2.18–2.36 (m, 4 H) 2.37–2.48 (m, 4 H) 3.27 (s, 3 H) 3.46 (s, 3 H) 4.14 (q, J = 6.82 Hz, 2 H) 6.91 (dd, J = 8.21, 1.64 Hz, 1 H) 6.98 (d, J = 1.77 Hz, 1 H) 7.03 (s, 1 H) 7.48 (dd, J = 8.46, 4.42 Hz, 1 H) 7.72 (dd, J = 8.59, 1.01 Hz, 1 H) 8.21 (s, 1 H) 8.24 (s, 1 H) 8.32 (d, J = 8.34 Hz, 1 H) 8.37 (dd, J = 4.42, 1.14 Hz, 1 H); ESMS found m/z 585.0 ([M + H+], C32H40N8O3 requires 584.3223).AcknowledgmentsWe thank our colleague and friend, the late Dr. Kevin Shreder, for invaluable contributions. We thank Kai Nakamura, Lan Pham, Ray Li, and Julia Ayers for chemical syntheses and Maria Sykes and Heidi Brown for assistance on live cell KiNativ.Footnotes↵1To whom correspondence may be addressed. Email: emmel{at}activx.com or jonr{at}activx.com.Author contributions: E.C.K.L., J.I., and J.S.R. designed research; E.C.K.L. and C.M.A. performed research; Y.H., S.S., and B.L. contributed new reagents/analytic tools; E.C.K.L., C.M.A., T.K.N., and H.W. analyzed data; and E.C.K.L., H.W., J.I., Y.H., J.W.K., and J.S.R. wrote the paper.Conflict of interest statement: E.C.K.L., C.M.A., T.K.N., H.W., Y.H., S.S., B.L., J.W.K., and J.S.R., employees of ActivX Biosciences, a wholly owned subsidiary of Kyorin Pharmaceutical Co., Ltd., and J.I., an employee of Kyorin Pharmaceutical Co., Ltd., have commercial interests in the development of ERK5 and BRD inhibitors.This article is a PNAS Direct Submission.Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE86577).This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1609019113/-/DCSupplemental. 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We do not capture any email address.CAPTCHAThis question is for testing whether or not you are a human visitor and to prevent automated spam submissions. ERK5 kinase activity is not required Emme C. K. Lin, Christopher M. Amantea, Tyzoon K. Nomanbhoy, Helge Weissig, Junichi Ishiyama, Yi Hu, Shyama Sidique, Bei Li, John W. Kozarich, Jonathan S. Rosenblum Proceedings of the National Academy of Sciences Oct 2016, 113 (42) 11865-11870; DOI: 10.1073/pnas.1609019113 ERK5 kinase activity is not required Emme C. K. Lin, Christopher M. Amantea, Tyzoon K. Nomanbhoy, Helge Weissig, Junichi Ishiyama, Yi Hu, Shyama Sidique, Bei Li, John W. Kozarich, Jonathan S. Rosenblum Proceedings of the National Academy of Sciences Oct 2016, 113 (42) 11865-11870; DOI: 10.1073/pnas.1609019113 Sign up for the PNAS Highlights newsletter to get in-depth stories of science sent to your inbox twice a month: Relatively clean snow and ice in the Indus River Basin during the COVID-19 pandemic may have reduced meltwater in 2020, compared with the 20-year average. Atmospheric and climate conditions could have created a cloud greenhouse effect to warm Mars and support liquid surface water. Researchers report a safety guideline to limit airborne transmission of COVID-19 that goes beyond the six-foot social distancing guideline. Interventions include using rice husks, manipulating paddy water and soil, and genetic changes that could stop arsenic from reaching the grain. Going beyond conventional approaches, researchers are using carefully cultured bacterial communities to improve sewage treatment.