Herpesviruses rely on a homodimeric protease for viral capsid maturation. A small molecule, DD2, previously shown to disrupt dimerization of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) by trapping an inactive monomeric conformation and two analogues generated through carboxylate bioisosteric replacement (compounds 2 and 3) were shown to inhibit the associated proteases of all three human herpesvirus (HHV) subfamilies (α, β, and γ). Inhibition data reveal that compound 2 has potency comparable to or better than that of DD2 against the tested proteases. Nuclear magnetic resonance spectroscopy and a new application of the kinetic analysis developed by Zhang and Poorman [Zhang, Z. Y., Poorman, R. A., et al. (1991) J. Biol. Chem. 266, 15591–15594] show DD2, compound 2, and compound 3 inhibit HHV proteases by dimer disruption. All three compounds bind the dimer interface of other HHV proteases in a manner analogous to binding of DD2 to KSHV protease. The determination and analysis of cocrystal structures of both analogues with the KSHV Pr monomer verify and elaborate on the mode of binding for this chemical scaffold, explaining a newly observed critical structure–activity relationship. These results reveal a prototypical chemical scaffold for broad-spectrum allosteric inhibition of human herpesvirus proteases and an approach for the identification of small molecules that allosterically regulate protein activity by targeting protein–protein interactions.
Ubiquitin specific protease 7 (USP7) regulates the protein stability of key cellular regulators in pathways ranging from apoptosis to neuronal development, making it a promising therapeutic target. Here we used an engineered, activated variant of the USP7 catalytic domain to perform structure–activity studies of electrophilic peptidomimetic inhibitors. Employing this USP7 variant, we found that inhibitors with a cyanopyrrolidine warhead unexpectedly promoted a β-elimination reaction of the initial covalent adducts, thereby converting the active-site cysteine residue to dehydroalanine. We determined that this phenomenon is specific for the USP7 catalytic cysteine and that structural features of the inhibitor and protein microenvironment impact elimination rates. Using comprehensive docking studies, we propose that the characteristic conformational dynamics of USP7 allow access to conformations that promote the ligand-induced elimination. Unlike in conventional reversible-covalent inhibition, the compounds described here irreversibly destroy a catalytic residue while simultaneously converting the inhibitor to a nonelectrophilic byproduct. Accordingly, this unexpected finding expands the scope of covalent inhibitor modalities and offers intriguing insights into enzyme–inhibitor dynamics.
Abstract KAT6 paralogs KAT6A (MOZ; MYST3) and KAT6B (MORF; MOZ2; MYST4) are histone acetyltransferase (HAT) enzymes that acetylate histone H3 and thereby epigenetically regulate gene transcription by altering chromatin structure. Dysregulation of KAT6 activity (gene amplification, overexpression, fusion, mutation, etc.) is observed in breast and other cancers. In several subtypes of breast cancer, KAT6A is amplified as part of the 8p11-p12 amplicon and/or is overexpressed in 11 to 15% of overall breast cancer population. Overexpression of KAT6 correlates with worse clinical outcome in ER+/HER2− breast cancers. Here we present the discovery of novel and potent KAT6 inhibitors with robust in vitro and in vivo anti-tumor efficacy and high selectivity against other KAT family members KAT5 and KAT8. Our lead KAT6 inhibitor compounds demonstrate strong anti-proliferative effects in ER positive breast cancer cell lines with KAT6 dysregulation while demonstrating robust target engagement biomarker activity (inhibition of histone H3 acetylation at lysine residue 23). In addition, these compounds exhibit desirable ADME and pharmacokinetic properties including good oral bioavailability in tested species, supporting potential once daily dosing in humans. The lead compounds also demonstrate enhanced in vitro cellular efficacy in breast cancer cell lines when combined with OP-1250, a complete ER antagonist (CERAN), and CDK4/6 inhibitors, illustrating their combination potential. In addition, these lead compounds demonstrate excellent dose-dependent anti-tumor activity resulting in tumor growth inhibition and tumor regression in a KAT6A amplified ER+ breast cancer xenograft model, highlighting their potential as a novel therapy in ER+ breast cancer. Additional nonclinical efficacy testing, and safety profiling of these lead compounds is currently underway to select a clinical candidate. Citation Format: Gopinath S. Palanisamy, Chandregowda Venkateshappa, Megha Goyal, Susanna A. Barratt, Brian R. Hearn, Girish Daginakatte, Aravind A B, Samiulla D S, Kalisankar Bera, Sangeetha S, Leena Khare, Alison D. Parisian, Dirk A. Heerding, Raymond A. Ng, Cyrus L. Harmon, Susanta Samajdar, Murali Ramachandra, David C. Myles. The discovery of potent KAT6 inhibitors that demonstrate anti-tumor activity in preclinical models of ER+ breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A044.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the ‘integrated stress response’ (ISR). eIF2α phosphorylation diminishes protein synthesis, while allowing preferential translation of some mRNAs. Starting with a cell-based screen for inhibitors of PERK signaling, we identified a small molecule, named ISRIB, that potently (IC50 = 5 nM) reverses the effects of eIF2α phosphorylation. ISRIB reduces the viability of cells subjected to PERK-activation by chronic endoplasmic reticulum stress. eIF2α phosphorylation is implicated in memory consolidation. Remarkably, ISRIB-treated mice display significant enhancement in spatial and fear-associated learning. Thus, memory consolidation is inherently limited by the ISR, and ISRIB releases this brake. As such, ISRIB promises to contribute to our understanding and treatment of cognitive disorders. https://doi.org/10.7554/eLife.00498.001 eLife digest The synthesis of proteins is an essential step in many biological processes, including memory, and drugs that inhibit protein synthesis are known to impair memory in rodents. It is thought that the brain needs these proteins to convert short-term memories into long-term memories through a process known as consolidation. A protein called EIF2α has a key role in the regulation of protein synthesis, and has also been implicated in memory. EIF2α can be activated as a result of being phosphorylated by any of four protein kinases: these are in turn activated by processes that subject cells to stress, such as viral infection, UV light or—in the case of a kinase known as PERK—the accumulation of unfolded proteins in a cellular organelle called the endoplasmic reticulum. Activation of EIF2α downregulates most protein synthesis inside the cell, but upregulates the production of a small number of key regulatory molecules: these changes help cells to cope with whatever stressful event they have just experienced. To obtain further insight into the cellular stress response, Sidrauski et al. screened a large library of compounds in search of one that inhibits PERK. They identified a molecule—known as ISRIB—which acts downstream of all four protein kinases by reversing the effects of EIF2α phosphorylation. ISRIB is the first molecule shown to have this effect, and thus represents an important tool for investigating the stress response inside cells. When Sidrauski et al. injected ISRIB into mice, the animals showed improved memory: for example, they learnt to locate a hidden platform in a water maze more rapidly than controls. This suggests that ISRIB could be used to explore the mechanisms that underlie memory consolidation, and possibly even as a memory enhancer. Moreover, given that many tumor cells exploit the cellular stress response to aid their own growth, ISRIB may have potential as a novel chemotherapeutic agent. https://doi.org/10.7554/eLife.00498.002 Introduction In metazoa, diverse stress signals converge at a single phosphorylation event at serine 51 of a common effector, the translation initiation factor eIF2α. This step is carried out by four eIF2α kinases in mammalian cells: PERK, which responds to an accumulation of unfolded proteins in the endoplasmic reticulum (ER), GCN2 to amino acid starvation and UV light, PKR to viral infection, and HRI to heme deficiency. This collection of signaling pathways has been termed the ‘integrated stress response’ (ISR), as they converge on the same molecular event. eIF2α phosphorylation results in an attenuation of translation with consequences that allow cells to cope with the varied stresses (Wek et al., 2006). eIF2 (which is comprised of three subunits, α, β and γ) binds GTP and the initiator Met-tRNA to form the ternary complex (eIF2-GTP-Met-tRNAi), which, in turn, associates with the 40S ribosomal subunit forming the 43S pre-initiation complex (PIC) that scans the 5′UTR of mRNAs to select the initiating AUG codon. Upon phosphorylation of its α-subunit, eIF2 becomes a competitive inhibitor of its guanine nucleotide exchange factor (GEF), eIF2B (Hinnebusch and Lorsch, 2012). The tight and non-productive binding of phosphorylated eIF2 to eIF2B prevents loading of the eIF2 complex with GTP thus blocking ternary complex formation and reducing translation initiation (Krishnamoorthy et al., 2001). Because eIF2B is less abundant than eIF2, phosphorylation of only a small fraction of the total eIF2 has a dramatic impact on eIF2B activity in cells. Paradoxically, under conditions of reduced protein synthesis, a small group of mRNAs that contain upstream open reading frames (uORFs) in their 5′UTR are translationally up-regulated (Hinnebusch, 2005; Jackson et al., 2010). These include mammalian ATF4 (a cAMP element binding [CREB] transcription factor) and CHOP (a pro-apoptotic transcription factor) (Harding et al., 2000; Vattem and Wek, 2004; Palam et al., 2011). ATF4 regulates the expression of many genes involved in metabolism and nutrient uptake and additional transcription factors, such as CHOP, which is under both translational and transcriptional control (Ma et al., 2002). Phosphorylation of eIF2α thus leads to preferential translation of key regulatory molecules and directs diverse changes in the transcriptome of cells upon cellular stress. One of the eIF2α kinases, PERK, lies at the intersection of the ISR and the unfolded protein response (UPR) that maintains homeostasis of protein folding in the ER (Pavitt and Ron, 2012). The UPR is activated by unfolded or misfolded proteins that accumulate in the ER lumen because of an imbalance between protein folding load and protein folding capacity, a condition known as ‘ER stress’. In mammals, the UPR is comprised of three signaling branches mediated by ER-localized transmembrane sensors, PERK, IRE1, and ATF6. These sensor proteins detect the accumulation of unfolded protein in the ER and transmit the information across the ER membrane, initiating unique signaling pathways that converge in the activation of an extensive transcriptional response, which ultimately results in ER expansion (Ron and Walter, 2007). The lumenal stress-sensing domains of PERK and IRE1 are homologous and likely activated in analogous ways by direct binding to unfolded peptides (Gardner and Walter, 2011). This binding event leads to oligomerization and trans-autophosphorylation of their cytosolic kinase domains, and, for PERK, phosphorylation of its only known substrate, eIF2α. In this way, PERK activation results in a quick reduction in the load of newly synthesized proteins that are translocated into the ER-lumen (Harding et al., 2000). Upon ER stress, both the transcription factor XBP1s, produced as the consequence of a non-conventional mRNA splicing reaction initiated by IRE1, and the transcription factor ATF6, produced by proteolysis and release from the ER membrane, collaborate with ATF4 to induce the vast UPR transcriptional response. Transcriptional targets of the UPR include the ER protein folding machinery, the ER-associated degradation machinery, and many other components functioning in the secretory pathway (Walter and Ron, 2011). Although the UPR initially mitigates ER stress and as such confers cytoprotection, persistent and severe ER stress leads to activation of apoptosis that eliminates damaged cells (Shore et al., 2011; Tabas and Ron, 2011). By interrogating a large chemical library for small molecules that block PERK signaling, we identified ISRIB as a potent ISR inhibitor, functioning downstream of all eIF2α kinases. ISRIB proves a powerful tool to explore the consequences of acute inhibition of the ISR in cells and animals. Results Design of cell-based screen for inhibitors of PERK signaling To identify inhibitors of PERK signaling, we engineered a reporter that allows monitoring of PERK activation in living cells. To this end, we constructed a retroviral vector containing the open-reading frame of firefly luciferase fused to the 5′UTR of ATF4 mRNA (Figure 1A), which contains two short open-reading frames (uORFs) that control ATF4 translation in a stress-dependent manner. After infection, we established a HEK293T cell line harboring the stably integrated reporter. We used thapsigargin, a potent ER stressor that inhibits the ER calcium pump, to activate PERK and induce eIF2α phosphorylation. Thapsigargin treatment resulted in a 4.9-fold induction in luciferase activity in a 384 well format with a Z factor of 0.5 (Figure 1B). This format was used to screen 106,281 compounds covering a wide chemical space. We identified 460 hits (0.43%) (Figure 1C), which were further validated in an 8-point dose-response assay using the same reporter. We further triaged the compounds by discarding inhibitors that also affected the IRE1 branch of the UPR using a luciferase-based XBP1 splicing reporter. Less than half (187 hits) of our initial hits proved unique to the PERK branch. We next used an orthogonal secondary screen that employed a different reporter (bi-cistronic ATF4-dGFP-IRES-mCherry) stably integrated into a different cell line (U2OS cells). The read-out of this latter screen was microscopy-based, which allowed us to simultaneously assess acute toxicity by cell counting, further reducing the number of viable hits to 77 (data not shown). As a tertiary screen, we tested compounds for their ability to inhibit ER stress-elicited induction of endogenous ATF4 by Western blot analysis. Twenty-eight compounds passed this test and were analyzed further. Figure 1 Download asset Open asset High-throughput cell-based screen for inhibitors of PERK signaling. (A) Schematic representation of the ATF4 luciferase reporter used in the primary screen. The 5′ UTR of human ATF4 containing the uORFs 1 and 2 was fused to firefly luciferase and inserted into a retroviral expression system. (B) Primary screen optimization. HEK293T stably expressing the ATF4 luciferase reporter were plated in 384-well plates and treated for 6 hr with 100 nM thapsigargin (Tg) or DMSO as a no ER stress control. Luciferase production was measured at the end point after 6 hr (mean ± SD). The Z′ was calculated as 1−(3 [σ Tg + σ DMSO]/[μ Tg–μ DMSO]). (C) Primary screen results. The ATF4 luciferase reporter cell line was treated for 6 hr with 100 nM thapsigargin and library compounds (10 µM). Inhibition of the luciferase activity reporter was calculated as the percent reduction in relative luminescence normalized to thapsigargin treatment (0% inhibition) and the no-ER stress control (100% inhibition). Compounds were considered hits if they lied beyond three standard deviations (SD) from the thapsigargin treatment mean (red line). https://doi.org/10.7554/eLife.00498.003 A symmetric bisglycolamide, ISRIB, is a potent inhibitor of PERK signaling One of the 28 compounds was of particular interest because of its high potency in cells (library compound IC50 = 40 nM). This compound (henceforth referred to as ‘ISRIB’ for Integrated Stress Response inhibitor) is a symmetric bis-glycolamide, containing a central bi-substituted cyclohexane, and can exist as two diastereomers, cis and trans (Figure 2A). We synthesized both isomers and tested their ability to inhibit the ATF4-luciferase reporter (Figure 2B). Trans-ISRIB proved 100-fold more potent (IC50 = 5 nM) than cis-ISRIB (IC50 = 600 nM), indicating that the compound’s interaction with its cellular target is stereospecific. Given the two-order-of-magnitude difference in activity in this assay, the measured activity of cis-ISRIB may be an over-estimate, as we cannot exclude a small contamination with trans-ISRIB, which is far more potent. The lower IC50 of trans-ISRIB relative to the compound in the small molecule library indicates that the library likely contains a mixture of the two stereoisomers. All further experiments in this study were carried out with the synthesized trans-isomer of ISRIB. Figure 2 Download asset Open asset Identification of ISRIB as a potent cell-based inhibitor of PERK signaling. (A) Structures of ISRIB isosteromers. (B) Inhibition of the ATF4 luciferase reporter in HEK293T cells by ISRIB stereoisomers. Inhibition is plotted in relation to the concentration of either the cis or trans isomer of ISRIB. Cells were treated with 2 µg/ml of tunicamycin to induce ER stress and different concentrations of the inhibitors for 7 hr (N = 2, mean ± SD). (C) Effect of ISRIB on production of endogenous ATF4, PERK phosphorylation, and XBP1s production. An immunoblot analysis of PERK, ATF4 and XBP1s in HEK293T cells treated with different ER stress inducers (2.5 µg/ml tunicamycin [Tm] or 100 nM thapsigargin [Tg]) with or without 200 nM ISRIB for 3 hr is shown. The arrowhead marks the XBP1s specific band. (D) Effect of ISRIB on XBP1 mRNA splicing. Taqman assays for XBP1unspliced (XBP1u) and XBP1spliced (XBP1s) on cDNA synthesized from total RNA extracted from U2OS cells treated with 2 µg/ml of tunicamycin in the presence or absence of 200 nM ISRIB for the indicated times are shown. Percent splicing was calculated as the ratio of XBP1s over total XBP1 mRNA (XBP1u + XBP1s) (mean ± SD). https://doi.org/10.7554/eLife.00498.004 ISRIB is PERK-branch specific but does not impair PERK phosphorylation We next determined at which step ISRIB blocks ATF4 production. To this end, we first probed the phosphorylation status of PERK by Western blotting. PERK phosphorylation is indicative of its activation by autophosphorylation and can be recognized by reduced mobility on SDS-polyacrylamide gels. Notably, ISRIB did not inhibit the mobility shift of PERK observed in ER-stressed cells (Figure 2C). Rather, we observed an exaggerated mobility shift, indicative of increased phosphorylation of PERK upon ER stress, induced by either thapsigargin or tunicamycin (an inhibitor of N-linked glycosylation). In each case, in the absence of ISRIB, ATF4 and XBP1s were produced upon ER stress induction. In agreement with the behavior of the reporters described above, ISRIB blocked production of endogenous ATF4, whereas XBP1 mRNA splicing (Figure 2D) and XBP1s production persisted (Figure 2C and Figure 3—figure supplement 1). As shown below (Figure 5D), ISRIB also did not affect activation of the ATF6-branch of the UPR. We conclude that ISRIB specifically blocks signaling of the PERK-branch of the UPR. ISRIB-treated cells are resistant to eIF2α phosphorylation Given that PERK phosphorylation was not diminished in ISRIB-treated, ER-stressed cells, we next directly assessed eIF2α phosphorylation. We measured the levels of phosphorylated eIF2α using an antiphospho-eIF2α antibody-based assay to quantify phosphorylation at serine 51 (see ‘Materials and methods’). Upon induction of ER stress by tunicamycin or thapsigargin, phosphorylation of eIF2α increased over time, reaching a fourfold and sevenfold increase after 120 min respectively (Figure 3A). Unexpectedly, ISRIB did not block eIF2α phosphorylation under either of these ER stress-inducing conditions. On the contrary, 120 min after tunicamycin addition, ISRIB further increased the level of eIF2α phosphorylation, approaching that obtained with thapsigargin. ISRIB alone had no effect on eIF2α phosphorylation. These results indicate that ISRIB blocks effects downstream of PERK and eIF2α phosphorylation. Figure 3 with 7 supplements see all Download asset Open asset ISRIB makes cells resistant to eIF2α phosphorylation. (A) ISRIB does not block eIF2α phosphorylation upon ER stress. eIF2α phosphorylation was measured using an alpha-screen Surefire eIF2α p-S51 assay (see ‘Materials and methods’). U2OS cells were plated in 96-well plates and treated with 2 µg/ml tunicamycin or 100 nM thapsigargin in the presence or absence of 100 nM ISRIB for the indicated times or with ISRIB alone for 120 m (N = 4, mean ± SD). See Figure 3—figure supplement 1 for supporting Western blot analysis of eIF2α phosphorylation. (B) ISRIB blocks global translational attenuation observed after eIF2α phosphorylation during ER stress. HEK293T cells were treated with 100 nM thapsigargin and 200 nM ISRIB for either 1 or 3 hr prior to a 20 min pulse with 35S methionine before lysis. Equal amounts of lysate were loaded on an SDS-PAGE gel and quantification of radiolabeled methionine incorporation of lysates was done by gel densitometry (N = 2, SD) using ImageJ. (see Figure 3—figure supplement 2 for SDS-PAGE). (C) Polysome gradient analysis showing the block in global translational attenuation upon addition of ISRIB on ER-stressed cells. MEFs were grown in the presence or absence of 2 µg/ml of tunicamycin with or without 200 nM ISRIB for 1 hr. Cell lysates were loaded on a 10–50% sucrose gradient, centrifuged at 150,000×g for 2.4 hr and absorbance at 254 nm was measured across the gradient (see Figure 3—figure supplement 3 for quantitation of polysome profile). A representative experiment is shown (N = 3). See Figure 3—figure supplement 4 for a close-up of the disome and trisome peaks. (D) Cells treated with ISRIB are resistant to the global translational attenuation exerted by forced expression of eIF2α(S51D). HEK293Trex cells were transduced with a tetracycline inducible phospho-mimetic (S51D) allele of eIF2α. Transgene expression was induced by addition of 25 nM doxycycline for 14 hr in the presence or absence of 200 nM ISRIB. Lysates were collected and analyzed as described in panel (C) (see Figure 3—figure supplement 6 for quantitation of polysome profile). A representative experiment is shown (N = 2). (E) ISRIB does not reverse global translational attenuation exerted through inhibition of CAP-dependent initiation. Wild-type MEFs were treated with 750 nM Torin-1 in the presence or absence of 200 nM ISRIB for 2 hr. Lysates were collected and analyzed as described in panel (C). A representative experiment is shown (N = 2). (F) ISRIB blocks production of ATF4 upon GCN2 or HRI activation. An immunoblot analysis of PERK, ATF4 and total eIF2α in HEK293T cells starved for cysteine and methionine or treated with an HRI activator (6 µM) for 5 hr in the presence or absence of 200 nM ISRIB is shown. Tunicamycin was used as a positive control for induction of ATF4 and the shift in PERK mobility. Under amino acid starvation we consistently observe a partial reduction of ATF4 production by ISRIB by Western blot analysis but observe a complete block in induction of the ATF4 luciferase reporter (see Figure 3—figure supplement 7). https://doi.org/10.7554/eLife.00498.005 One way of explaining why ISRIB blocks ATF4 production yet leaves eIF2α phosphorylation intact is by rendering cells insensitive to the effects of this phosphorylation event. In agreement with this notion, ISRIB sustained global translation (as monitored by 35S-methionine incorporation into newly synthesized polypeptides) even in the presence of ER stress (Figure 3B). After thapsigargin treatment, cells experienced a 40% drop in translation, which was abolished by ISRIB. As predicted by this result, extracts prepared from mouse embryonic fibroblasts (MEFs) experiencing ER stress showed a pronounced increase in the 80S monosomes at the expense of polyribosomes (Figure 3C), which was reversed (at least partially) by addition of ISRIB. We chose MEFs for this analysis because they show stronger translational inhibition in response to ER stress than HEK293T cells. ISRIB was the only molecule in our collection of 28 hits that reversed translational attenuation upon ER-stress. Under normal growth conditions, an abundance of 43S pre-initiation complexes (PICs) leads to mRNAs loaded with a small ribosomal subunit in addition to fully assembled ribosomes. The presence of PICs on an mRNA can be detected as ‘halfmer’ peaks on polysome gradients. In the gradients shown in Figure 3C, addition of a PIC to disomes and trisomes was well resolved (enlarged in Figure 3—figure supplement 4). Upon eIF2α phosphorylation in ER-stressed cells, the reduction in PIC resulted in disappearance of the halfmer population. As expected, the disappearance of the halfmer peak upon ER-stress was dependent on eIF2α phosphorylation as no reduction was observed in MEFs that solely express non-phosphorylatable eIF2α(S51A) (Figure 3—figure supplement 5). Importantly, ISRIB partially restored the halfmer population in ER-stressed cells, providing support to the notion that it helps maintain high PIC levels even when eIF2α is phosphorylated (Figure 3—figure supplement 4). These data indicate that ISRIB exerts its function by maintaining elevated ternary complex levels. To further ascertain that cells treated with ISRIB are resistant to the effects of eIF2α phosphorylation, we transduced an inducible phospho-mimetic allele of eIF2α in which serine 51 was changed to an aspartic acid (S51D) into HEK293T cells. Expression of this allele upon doxycycline addition induced translational attenuation (Figure 3D) as seen by an increase in the 80S peak and a decrease in the polysome population. ISRIB rescued translation returning it to the levels observed in non-induced cells. In conclusion, ISRIB restores translation in cells containing either phospho-eIF2α or eIF2α(S51D), thereby excluding any pleiotropic effects that might have been caused by the reagents used to activate ER stress. To rule out that ISRIB exerts non-specific effects on translation independent of eIF2α phosphorylation, we tested whether ISRIB reverses a translational block in CAP-mediated initiation. To this end we used Torin-1, an inhibitor of mTOR that blocks phosphorylation of 4E-BP1 and S6K1, and leads to translational attenuation (Thoreen et al., 2012). Addition of Torin-1 to MEFs led to an increase in the 80S peak and reduction in the polysome population to a similar extent as shown above in cells treated with ER stressors or expressing eIF2α(S51D) (Figure 3E, compare with Figure 3C,D). In contrast to these treatments, addition of ISRIB did not reverse the effect of Torin-1 on translation. Therefore, the ability of ISRIB to block translational attenuation is specific to eIF2α phosphorylation. If ISRIB makes cells insensitive to eIF2α phosphorylation, it should not matter which kinase phosphorylates eIF2α. To test this prediction, we subjected cells to amino acid starvation, which activates the eIF2α kinase GCN2 and leads to ATF4 production. In addition, we used a recently identified small molecule activator to induce eIF2α phosphorylation by activating HRI, another eIF2α kinase (Chen et al., 2011). As expected, ISRIB blocked ATF4 induction after activation of either GCN2 or HRI (Figure 3F). Under both conditions, PERK was not activated as shown by a lack of mobility shift. These data suggest that ISRIB is a bona fide ISR inhibitor that blocks signaling downstream of all eIF2α kinases. Both DDIT3 (the gene encoding CHOP) and PPP1R15A (the gene encoding GADD34) are transcriptional targets of ATF4. Thus, blocking ATF4 accumulation with ISRIB should result in a reduction in the transcriptional induction of the mRNAs encoding these targets. As shown in Figure 4A, GADD34 and CHOP mRNAs accumulated in ER-stressed U2OS cells, and ISRIB significantly reduced their induction. In agreement, we observed no CHOP accumulation after induction of ER stress in ISRIB-treated cells (Figure 4B). Thus ISRIB impairs the transcriptional network governed by ATF4 during the ISR. Figure 4 Download asset Open asset ISRIB impairs induction of the transcriptional network controlled by ATF4. (A) ER-Stress dependent induction of CHOP and GADD34 mRNA is impaired in cells treated with ISRIB. qPCR analysis of total RNA extracted from U2OS cells treated with 2 µg/ml of tunicamycin in the presence or absence of 200 nM ISRIB for the indicated times. mRNA levels for each sample were normalized to GAPDH (N = 4, mean ± SD). p values are derived from a one-tail Student’s t-test for unpaired samples. Statistical significance: CHOP, *p=0.0006; GADD34, *p=0.0008. (B) ISRIB blocks CHOP production during ER stress. An immunofluorescence analysis of U2OS cells treated with 100 nM thapsigargin for 2 or 4 hr in the presence or absence of 200 nM ISRIB is shown. A secondary Alexa Dye 488 anti-mouse antibody and rhodamine-phalloidin were used to visualize CHOP and actin, respectively. https://doi.org/10.7554/eLife.00498.013 ISRIB impairs adaptation to ER stress As previously shown, cells homozygous for non-phosphorylatable eIF2α, eIF2α(S51A), are unable to cope with ER stress properly, leading to reduced viability (Lu et al., 2004). This indicates that events downstream of eIF2α phosphorylation are required to resolve the stress. As shown in Figure 5A, ISRIB treatment of wild-type cells had similar consequences. Importantly, addition of ISRIB alone did not affect cell viability, as judged by the number of colonies that form after acute treatment. By contrast, ISRIB addition caused a strong synergistic effect on ER-stressed cells, reducing colony number and size significantly more than ER-stress alone. This reduction in cell survival resulted from activation of apoptosis as the activity of the executioner caspases 3 and/or 7 was significantly induced under these conditions (Figure 5B; Salvesen and Ashkenazi, 2011). Figure 5 with 2 supplements see all Download asset Open asset ISRIB impairs adaptation to ER-stress prolonging activation of the UPR sensors. (A) ISRIB sensitizes cells to acute ER stress. HEK293T cells were subjected with an acute dose of tunicamycin (2 µg/ml), ISRIB (200 nM) or a combination of both for 24 hr. The treated cells were equally diluted to a concentration that would allow single cell clonal expansion and re-seeded onto six-well plates in a threefold dilution series. Clonal colonies were visualized by Crystal Violet stain. (B) ISRIB synergizes with ER stress to activate caspase 3/7. Hela cells were plated in 96-well plates and treated with 5 µg/ml of tunicamycin or 500 nM thapsigargin with or without 25 nM ISRIB for the indicated times. Caspase3/7 activation was measured using Cellplayer kinetic caspase 3/7 reagent and cells were imaged in an IncuCyte system. Green object count/mm2 representing caspace-3/7 activation was measured at 2 hr intervals (See Figure 5—figure supplement 1 for endpoint quantitation of % cells with activated caspase 3/7). (C) IRE1 oligomers are sustained on ER-stressed cells treated with ISRIB. Confocal microscopy micrographs of HEK293Trex cells carrying an inducible GFP-tagged IRE1 allele were treated with 10 nM doxycycline for 24 hr to induce the transgene, followed by treatment with 5 µg/ml of tunicamycin in the presence or absence of 200 nM ISRIB for the indicated times. (See Figure 5—figure supplement 2 for corresponding XBP1 mRNA splicing data). (D) ATF6 cleavage is sustained in ER-stressed cells treated with ISRIB. Immunoblot analysis of ATF6 processing in HEK293Trex cells carrying an inducible FLAG epitope-tagged ATF6. Cells were treated with 50 nM doxycycline for 18 hr to induce the transgene followed by treatment with 100 nM thapsigargin in the presence or absence of 200 nM ISRIB for the indicated times. Total eIF2α is used as a loading control. https://doi.org/10.7554/eLife.00498.014 The notion that ER stress remains unmitigated in ISRIB-treated cells is supported by sustained activation of all three UPR sensors. First, as shown in Figure 2C, PERK was hyper-phosphorylated. Second, cells expressing an IRE1-GFP fusion protein showed prolonged foci formation (Figure 5C), indicative of IRE1 oligomerization. Third, we observed prolonged ER stress-induced proteolytic processing of ATF6 (Figure 5D). Importantly, in the absence of ER stress ISRIB treatment alone did not induce any of these sensors (Figure 3—figure supplement 1; Figure 5C and data not shown). ISRIB increases long-term memory in rodents eIF2α+/S51A (Eif2s1+/S51A) heterozygote mice display enhanced memory, while induction of the eIF2α kinase PKR in brain pyramidal cells impairs memory (Costa-Mattioli et al., 2007; Jiang et al., 2010). Based on these observations, we wondered whether treatment of mice with ISRIB would affect memory. ISRIB showed favorable properties in pharmacokinetic profiling experiments indicating sufficient bioavailability for in vivo studies. ISRIB displayed a half-life in plasma of 8 hr (Figure 6A) and readily crossed the blood-brain barrier, quickly equilibrating with the central nervous system (Figure 6B). After a single intraperitoneal injection, we detected ISRIB in the brain of mice at concentrations several fold higher than its IC50 (24 hr after injection, the ISRIB concentration in the brain was approximately 60 nM). To explore ISRIB's effects on memory, we injected mice intraperitoneally with ISRIB and tested hippocampus-dependent spatial learning. To this end, we trained mice in a Morris water maze, in which animals learn to associate visual cues with the location of a submerged hidden platform. Because we were looking for memory enhancement, we used a weak training protocol. As shown in Figure 6C, ISRIB-treated mice reached the hidden platform significantly faster (escape latency after 5 days of training = 16.4 ± 4.8 s) compared to vehicle treated controls (68.1 ± 20 s, p<0.05). The difference was already pronounced by days 3 and 4. In agreement with these results, ISRIB-treated mice significantly preferred the target quadrant in a ‘probe test’ conducted at the end of the training sessions
The syntheses and biological evaluation of six epothilone D analogues are reported. These side-chain variants of the (E)-9,10-didehydroepothilone scaffold contain C-15 thiazole appendages that are derived from bromomethyl ketone intermediates. Although each of these analogues is less cytotoxic than the parent (E)-9,10-didehydroepothilone D, three maintain IC50 values in the double-digit nanomolar range against both susceptible and resistant cell lines.
<div>Abstract<p>The goal was to develop and characterize a companion diagnostic for the releasable PEG<sub>40kDa</sub>∼SN-38 oncology drug, PLX038, that would identify tumors susceptible to high accumulation of PLX038. PEG conjugates of the zirconium ligand desferroxamine B (DFB) of similar size and charge to PLX038 were prepared that contained one or four DFB, as well as one that contained three SN-38 moieties and one DFB. Uptake and associated kinetic parameters of the <sup>89</sup>Zr-labeled nanocarriers were determined in tumor and normal tissues in mice using μPET/CT imaging. The data were fit to physiologically based pharmacokinetic models to simulate the mass-time profiles of distribution of conjugates in the tissues of interest. The time–activity curves for normal tissues showed high levels at the earliest time of measurement due to vascularization, followed by a monophasic loss. In tumors, levels were initially lower than in normal tissues but increased to 9% to 14% of injected dose over several days. The efflux half-life in tumors was very long, approximately 400 hours, and tumor levels remained at about 10% injected dose 9 days after injection. Compared with diagnostic liposomes, the PEG nanocarriers have a longer serum half-life, are retained in tumors at higher levels, remain there longer, and afford higher tumor exposure. The small PEG<sub>40kDa</sub> nanocarriers studied here show properties for passive targeting of tumors that are superior than most nanoparticles and might be effective probes to identify tumors susceptible to similar size therapeutic nanocarriers such as PLX038.</p></div>
The earlier discovery of the Antarctic ozone "hole" and current scientific evidence indicate that CFC emissions into the atmosphere deplete the ozone layer and present a long-term threat to the quality of human life. The items of most concern, from an ozone protection standpoint, are the long-lived, fully halogenated compounds—halons, CFCs, and chlorocarbons. Scientific information indicates that most, if not all, of the chlorine or bromine content of these compounds is transported to the stratosphere, where it has the potential to destroy ozone. Furthermore, these compounds remain in the atmosphere for an extended number of years, providing a significant background chlorine concentration. According to DuPont.2 an 85 percent reduction in global CFC emissions from 1986 levels is necessary just to maintain current atmospheric levels of chlorine from these compounds. The refrigerants used within environmental test chambers have been included among those identified as ozone depleting. Specifically, these are CFC-12 and CFC-502. The 1987 Montreal Protocol was revised in June of 1990. Further regulations on CFC products are contained within the Clean Air Bill that is being debated in the Fall of 1990. Restrictions pertaining to CFC-13 are being proposed.
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