PromISR-6, a Guanabenz Analogue, Improves Cellular Survival in an

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PromISR-6, a Guanabenz Analogue, Improves Cellular Survival in an Experimental Model of Huntington's Disease Jeyapriya Rajameenakshi Sundaram, Yilong Wu, Irene Chengjie Lee, Simi Elizabeth George, Monalisa Hota, Sujoy Ghosh, Sashi Kesavapany, Mahmood Ahmed, Eng-King Tan, and Shirish Shenolikar ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00185 • Publication Date (Web): 17 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

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PromISR-6, a Guanabenz Analogue, Improves Cellular Survival in an Experimental Model of Huntington's Disease Jeyapriya Rajameenakshi Sundaram1,3, Yilong Wu2, Irene Chengjie Lee2, #a, Simi Elizabeth George2, Monalisa Hota2, Sujoy Ghosh2, Sashi Kesavapany4, #b, Mahmood Ahmed4, #c, Eng-King Tan1,3 and Shirish Shenolikar1,2, * Programmes in 1Neuroscience and Behavioural Disorders & 2Cardiovascular and Metabolic Disorders, Duke-NUS Medical School, 8 College Road, Singapore 169857; 3National Neuroscience Institute of Singapore, 11 Jalan Tan Tock Seng, Singapore 308433; 4GSK Neural Pathways Discovery Performance Unit, 11 Biopolis way, Singapore 138667. ABSTRACT: Guanabenz (GBZ), an α2-adrenergic agonist, demonstrated off-target effects that restored protein homeostasis and ameliorated pathobiology in experimental models of neurodegenerative disease. However, GBZ did not directly activate the integrated stress response (ISR) and its proposed mode of action remains controversial. Utilizing an iterative in silico screen of over 10,000 GBZ analogues, we analyzed 432 representative compounds for cytotoxicity in Wild-type, PPP1R15A/- and PPP1R15B-/- mouse embryonic fibroblasts. Nine compounds clustering into three functional groups were studied in detail using cell biological and biochemical assays. Our studies demonstrated that PromISR-6 as a potent GBZ analogue that selectively activated ISR, eliciting sustained eIF2α phosphorylation. ISRIB, an ISR inhibitor, counteracted PromISR-6mediated translational inhibition and reduction in intracellular mutant Huntingtin aggregates. Reduced protein synthesis combined with PromISR-6-stimulated autophagic clearance made PromISR-6, the most efficacious GBZ analogue to reduce Huntingtin aggregates and promote survival in a cellular model of Huntington’s disease.

KEYWORDS: Guanabenz, Integrated Stress Response (ISR), Phospho-eIF2α, mRNA Translation, Huntingtin Aggregation, Autophagy

INTRODUCTION Protein quality control mechanisms eroded by aging are further exacerbated by metabolic or oxidative stress triggering the accumulation of intracellular protein aggregates that are hallmarks of many chronic human diseases 1. Thus, neurofibrillary tangles or amyloid plaques are observed in postmortem brains of Alzheimer’s disease (AD) patients, Lewy bodies in Parkinson’s disease (PD), aggregates of mutant Huntingtin in Huntington’s disease (HD) and the RNAbinding proteins, TDP-43 and FUS, in amyotrophic lateral sclerosis (ALS) 2. In these incurable neurodegenerative disorders, the accumulation of misfolded proteins activates the unfolded protein response (UPR), which is transduced by three endoplasmic reticulum (ER)-localized sensors, namely the PERK (Protein Kinase R-like ER kinase or EIF2AK3), the ATF6 (Activating transcription factor 6) and the IRE1(inositol-requiring enzyme 1) that respectively suppress protein synthesis, elevate chaperone expression and activate ER-associated protein degradation (ERAD) to together eliminate misfolded proteins and restore protein homeostasis and cell viability 3. Persistent or unresolved UPR activates apoptosis to eliminate damaged or dysfunctional cells. Mutations in the human PERK gene, which encodes an

eukaryotic translational initiation factor (eIF2α) kinase 4 or the gene encoding PPP1R15B or CReP (constitutive repressor of eIF2α phosphorylation), a subunit of an eIF2α phosphatase 5, 6 have both been linked to diabetes, growth retardation, liver damage and neurological abnormalities. These findings suggested that eIF2α phosphorylation-dephosphorylation and the downstream signaling activated by many environmental stresses, termed the “integrated stress response” (ISR), plays a pivotal role in cell fate and disease. Guanabenz (GBZ; Wytensin), an α2-adrenergic agonist, approved for treatment of high blood pressure7, enhances ongoing ISR8. GBZ, ([2,6, dichlorobenzylidene]amino)guanidine, and Sephin19, a GBZ analogue, ([2-monochlorobenzylidene]amino)guanidine, that lacks α2-adrenergic agonist activity, were postulated to bind PPP1R15A or GADD34 (growth arrest and DNA damageinducible transcript 34), a regulatory subunit of an eIF2α phosphatase to inhibit eIF2α dephosphorylation and enhance ISR signaling. Subsequent studies, however, failed to confirm GBZ binding to GADD3410 or impairment of eIF2α dephosphorylation11, 12 and how GBZ enhances ongoing ISR remains highly controversial. Nevertheless, GBZ provided significant neuroprotection in animal models of prion

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disease13, Macado-Joseph disease14, ALS15 and PD16. This combined with its ability to cross the blood-brain barrier 17 and tolerable side-effects over 40 years of human use18 makes GBZ a very attractive starting point for developing novel therapies for neurodegenerative disease and other protein misfolding disorders. While the weak off-target activity of GBZ and Sephin1 meant that these compounds, by themselves, failed to activate ISR, another GBZ analogue, Raphin1, [(2,3dichlorobenzylidene)amino]guanidine activated ISR and reduced intracellular protein aggregates in HD mice19. Hence, we hypothesized that by evaluating a large number of GBZ analogues, we might identify potent ISR enhancers or activators that would be more effective than GBZ in modulating neurodegenerative disease in preclinical models. Current studies utilized an in silico screen combined with analyses of representative compounds in numerous biological assays to identify N’-(4-diphenylamino) benzylidene cyclopropanecarbohydrazide, termed PromISR-6, which elicited robust ISR, namely increased eIF2α phosphorylation and prolonged translational repression. Moreover, unlike GBZ, Sephin1, Raphin1 and other analogues, PromISR-6 activated autophagy to clear intracellular aggregates and increase survival in neuronal cells expressing aggregationprone mutant Huntingtin (mHtt) protein. RESULTS AND DISCUSSION The prominent role of protein misfolding in human neurodegenerative disease has prompted widespread efforts to identify small molecules that modulated ISR, a key determinant of cell fate20. Thus, ISRIB, an ISR inhibitor, which bind eIF2B and overrides translation repression by P-eIF2α 21, conferred neuroprotection the prion disease mice 22. Remarkably, GBZ, a centrally acting anti-hypertensive drug, whose off-target effects facilitated or enhanced ISR, also provided neuroprotection in several models of neurodegenerative disease23, and because of its excellent safety record in humans, is an attractive entry point for development of novel treatments for these incurable disorders. The enthusiasm around GBZ is highlighted by currently ongoing clinical studies assessing GBZ’s efficacy in ALS24 even without the optimization of off-target proteostatic activity of this drug. Prior structure-activity analyses of GBZ13 focused on two chlorines on the benzene ring with the primary goal of extinguishing its α2-adrenergic agonist activity. However, neither GBZ nor Sephin1, which lacks α2-adrenergic agonist activity, activated ISR and their proposed mode of action as inhibitors of GADD34-containing eIF2α phosphatase has been widely challenged11, 12. Even as advances in molecular biology and genetics have popularized target-based drug discovery, for example, Raphin1 was identified by its binding to recombinant CReP protein19, the limited success of this targetbased approach in identifying first-in-classmedicines25 has led

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to the resurgence of cell-based phenotypic screens. These employ a chemical biology approach to identify diseasemodifying bioactive molecules without knowledge of the defined target. Indeed, phenotypic screens are particularly well suited for homeostatic mechanisms, such as protein quality control, where too much or too little pathway modulation can have similar detrimental effects on cell physiology. Screening GBZ Analogues Prior studies8 exploited the pivotal role of ISR in cell death to analyze cytotoxicity of GBZ, an emerging proteostatic modifier in WT, GADD34-/-, which show an attenuated ISR 2628 and CReP-/- MEFs, which display enhanced ISR. Using a similar assay, we noted only modest difference in the sensitivity of WT and CReP-/- MEFs to GBZ (IC50 86.7 and 74.9 µM respectively) although GADD34-/- MEFs were slightly more resistant (IC50 105 µM) to GBZ-mediated cell death (Figure S1A) as previously reported8. Subsequently, we undertook the analysis of 432 GBZ analogues as described in our work plan (Figure S1B). Briefly, commercial compound libraries were subjected to Tanimoto index similarity searches and medical chemistry analyses to identify compounds with >70% structural similarity to the GBZ scaffold as described in Methods. Approximately 100 analogues with the broadest chemical diversity compared to GBZ were analyzed in the aforementioned cytotoxicity assay at two fixed concentrations (25 and 75 µM). Focusing on compounds that displayed higher potency than GBZ against CReP-/- MEFs (at 25 µM), additional 100 analogues were selected retaining the most common structural features of the 10 most potent compounds while excluding if possible the structural features of compounds with reduced activity compared to GBZ (at 75 µM). Through four iterative cycles of machine learning, we interrogated over 10,000 GBZ analogues. Few compounds identified in silico were unavailable, yet others displayed poor solubility in DMSO and/or tissue culture medium and were excluded from this study. Compounds selected from the final screen were resynthesized and reanalyzed over a wide range of concentrations. Dose response curves using WT, GADD34/- and CReP-/- MEFs segregated these compounds into three broad groups (Figure 1). Group 1 (Figure 1A), represented by Compounds 9 and 11, demonstrated 10-fold increased potency (IC50 6-8 µM) over GBZ but did not distinguish between WT, GADD34-/- or CReP-/- MEFs. Group 1 also contained Compound 2 (Figure S2), which was previously identified as the proteostatic modifier, Raphin119, which displayed IC50 of approximately 20 µM for all three cells. Group 2 (Figure 1B), exemplified by Compounds 5 and 10, was marginally more potent than GBZ, but displayed distinct dose-response profiles for the three cells. Group 3 (Figure 1C) was characterized by Compounds 3 and 6 and combined both increased potency and cell selectivity. Compound 3 was the most potent GBZ analogue identified (IC50 3.4 µM against CReP-/- MEFs).


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Figure 1: Cytotoxicity of Guanabenz Analogues in MEFs Panel A shows representative Group 1 compounds, namely Compounds 9 and 11, with their chemical structures and dosedependent cytotoxicity for WT, GADD34-/- and CReP -/- MEFs following 24 hr exposure. Panel B shows Group 2 Compounds 5


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and 10 and Panel C shows Group 3 Compounds 3 and 6. The IC50 values for each compound for each cell type are shown in boxes. Error bars indicate ± s.e.m. All experiments represent n=3. Compound 6, which shared considerable structural similarity with Compound 3, differing only in modification of the guanidine moiety, also retained a preference for CReP-/- cells (IC50 11 µM). By contrast, no GBZ analogues, including Raphin1 or compound 2, a postulated inhibitor of the CReP-containing eIF2α phosphatase, preferentially targeted the GADD34-/MEFs. Based on the critical importance of chlorines and their positioning on the benzene moiety of GBZ for the activation of α2-adrenergic receptor 9, 13, 29, we predicted that none of the nine GBZ analogues identified in our screen activated the α2adrenergic receptor. GBZ Analogues Activated ISR The early events in ISR include the autophosphorylation and activation of PERK, subsequent PERK-catalyzed phosphorylation of eIF2α, the enhanced translation of ATF4 and downstream transcription and translation of CHOP and GADD34 (Figure 2A). This generates a feedback loop whereby the GADD34-associated phosphatase reverses eIF2α phosphorylation and terminated ISR. These individual events were analyzed by immunoblotting MEF lysates following exposure for 3 hr to increasing concentrations of GBZ, Compound 3 and Compound 6, with 1 M thapsigargin (Tg), a widely utilized ISR activator, as a positive control. Tg stimulated ISR, specifically P-PERK, P-eIF2α, ATF4, CHOP and GADD34, very similarly in WT and CReP-/- MEFs. Tg also activated PERK in GADD34-/- MEFs and increased eIF2α phosphorylation, the expression of downstream proteins, namely ATF4 and CHOP, was barely visible in these cells. This was consistent with the previously noted attenuated ISR in GADD34-/- MEFs27, 30 and confirmed the critical requirement of low basal GADD34 levels for translation of mRNAs encoding these ISR proteins. As noted by Tsaytler et al 8, GBZ, even at 25 M, failed to activate ISR (Figure 2B). By contrast, Group 3 Compounds, 3 and 6, activated PERK, enhanced eIF2α phosphorylation and elevated cellular ATF4 and CHOP levels in all three cells (Figure 2B and 2C). Surprisingly, Compounds 3 and 6 were more effective than Tg at enhancing ATF4 expression but the attenuated expression of its downstream target, CHOP, and as anticipated no GADD34, consistent with an attenuated ISR, were noted in GADD34-/- cells compared to WT or CReP-/MEFs. As reported for Compound 2 or Raphin1 19, many Group 1 compounds activated ISR (data not shown). These data argued that the increased potency of Groups 1 and 3 compounds, which represented diverse chemical structures, may uncover the intrinsic ability of the shared benzylidene core to promote ISR. Based on their ability to activate ISR, these compounds were termed PromISRs (or Promoters of ISR).

ameliorate neurodegenerative disease. We chose Huntington’s disease (HD), a monogenic disorder associated with misfolding and aggregation of mutant Huntingtin, that is readily modeled in cells. Doxycycline-induced expression of mutant Huntingtin, GFP-mHtt-74Q, in PC12 cells, resulted in readily visible fluorescent puncta or mHtt aggregates at 72 hr (Figure S3A) 31. By comparison, cells expressing GFP-Htt-23Q, representing Huntingtin present in healthy individuals, always displayed diffuse cytoplasmic fluorescence. The number of cells containing GFP-mHtt-74Q aggregates or puncta, quantified by high content imaging, increased over time with more than 80% of cells containing GFP-mHtt-74Q aggregates at 144 hr (Figure S3B). Exposure to 10 M of GBZ or selected Group 1, 2 and 3 analogues showed that GBZ and many Group 1 analogues, such as PromISR-2/Raphin1, had little impact on the accumulation of mHtt aggregates (Figure S3C). This was somewhat unexpected as Krzyzosiak et al 19 reported significant reduction in mHtt aggregates in brains of HD mice dosed with Raphin1 over a protracted period. This likely reflects the difference between our acute and aggressive cellular model (72 hr exposure of mHtt-74Q-expressing cells) and the more chronic animal model (daily oral dosing of mHtt-82Q mice for up to 10 weeks). PromISR-6 and to lesser extent PromISR-7, reduced GFP-mHtt-74Q aggregates. Surprisingly, PromISR-3 (and PromISR-11), significantly increased mHtt aggregation (Figure S3C). This contrasted with Sephin1 which had no effect on mHtt aggregation at all concentrations analyzed (Figure S3D). Comparisons over a wide range of concentrations confirmed that GBZ, up 25 µM, had no impact of GFP-mHTT-74Q aggregation (Figure 3A and 3B), while PromISR-3 increased mutant Htt aggregation, particularly at higher concentrations (Figure 3C). PromISR-6, on the other hand, at all concentrations above 5 µM, significantly reduced mHtt aggregation (Figure 3D). As both PromISR-3 and PromISR-6 activated ISR, these data suggested that ISR activation may be necessary but insufficient to reduce intracellular protein aggregates. Finally, mHtt aggregate-containing PC12 cells showed much greater resistance to PromISR-6-induced cytotoxicity than that seen with PromISR-3 (Figure S4E), albeit that IC50 values for both compounds slightly higher than those seen with MEFs (Figure 1). To discount the possibility that PromISR-6 selectively eliminated the aggregate-containing PC12 cells, we analyzed cell viability in 23Q- and 74Q-mHtt-expressing PC12 cells treated with PromISR-6 (Figure S4). Using the nuclear stain, DAPI (Figure S4B) or the vital stain, Prestoblue (Figure S4C), we observed no significant loss of 74Q-mHtt-expressing PC12 cells to increasing concentrations of PromISR-6. The dose-response curves for PromISR-6-mediated cytotoxicity in nonaggregating 23Q- or aggregate-containing 74Q-mHttexpressing PC12 cells was also similar (Figure S4D).

PromISR-6 Reduced Mutant Huntingtin Aggregation A key mandate for our study was to establish the efficacy of GBZ analogues to reinstate protein homeostasis and


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Figure 2: Activation of Integrated Stress Response (ISR) by Guanabenz and Analogues Panel A shows the ISR pathway, which begins with the autophosphorylation of PERK, subsequent phosphorylation of eIF2α and expression of downstream proteins, ATF4, CHOP and GADD34. Panel B shows the total levels of PERK, P-PERK, eIF2α, P-eIF2α, ATF4, CHOP and GADD34 in WT, CReP-/- and GADD34-/- MEFs by immunoblotting as described in Methods. Thapsigargin (Tg) was used as a positive control and immunoblotting with anti-tubulin established equal protein loading. Representative immunoblots for WT, CReP -/- and GADD34 -/- MEFs treated with vehicle (DMSO), Thapsigargin (Tg-1µM), Guanabenz (GBZ; 10


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and 25 µM), Compound 3 (10 µM) and Compound 6 (10 and 25 µM) for 3 hr are shown (n=3). Panel C shows the quantification of eIF2α phosphorylation relative to total eIF2α and ATF4 normalized to tubulin levels by densitometric scanning using image J and expressed as fold-change relative to DMSO control. All comparisons were made within single experiments or immunoblots. White lines or gaps designate the removal of samples or treatments not relevant to current studies or data such as 3-25, which described cell treatment with 25 M Compound 3, which were compromised by excessive cell death. Error bars indicate ± s.e.m (* p-value ≤ 0.05).

Figure 3: Impact of Guanabenz Analogues on Mutant Huntingtin Aggregation Panel A shows representative images of PC12 cells, expressing GFP-mHtt-74Q induced by doxycycline for 72 hr, in the presence of DMSO (vehicle), Guanabenz (25 µM), PromISR-3 (10 µM) or PromISR-6 (25 µM). Scale bar represents 20 µm. Aggregates of GFPmHtt-74Q were analyzed as described in Methods (n=3). Impact of increasing concentrations of Guanabenz (Panel B), PromISR3 (Panel C) and PromISR-6 (Panel D) on the accumulation of intracellular GFP-mHtt-74Q aggregates is shown in bar graphs as fold-change in cells possessing mHtt aggregates (n=3). Error bars indicate ± s.e.m (*** p-value ≤ 0.001, and * p-value ≤ 0.05). PromISR-6 Elicited Prolonged Protein Synthesis Inhibition in mHtt-expressing PC12 Cells As seen in MEFs (Figure 2), both PromISR-3 and PromISR-6 enhanced P-eIF2α levels in mHtt-expressing PC12 cells (Figure 4A), while GBZ did not change P-eIF2α levels above DMSO control. Utilizing the SUnSET assay 32, we monitored functional consequences of P-eIF2α, namely the inhibition of general protein synthesis, in puromycin-labelled mHttexpressing PC12 cells. Immunoblotting with anti-puromycin established that consistent with its inability to increase PeIF2α, GBZ did not inhibit protein synthesis, which was similar to control DMSO-treated cells. By comparison,

consistent with ISR activation, PromISR-3 and PromISR-6 inhibited protein synthesis in mHtt-expressing PC12 cells (Figure 4B). Cells exposed to PromISR-3 showed robust increase in P-eIF2α at 3 and 6 hr but return to near basal levels at 18 hr (Figure 4C). This was accompanied by substantial recovery in puromycin labeling at 6 and 18 hr. By comparison, PC12 cells treated with PromISR-6 displayed lower but more persistent eIF2α phosphorylation and prolonged inhibition of protein synthesis, which failed to recover at 18 hr (Figure 4D). We speculate that prolonged translational repression by PromISR-6 may be more effective in lowering mHtt levels and slowing or preventing mHtt aggregation.


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Figure 4: Guanabenz Analogues Increase eIF2α Phosphorylation and Reduce Protein Synthesis in GFP-mHtt-74Q-expressing PC12 Cells Panel A shows a representative immunoblot of lysates from GFP-mHtt-74Q-expressing PC12 cells, exposed to Guanabenz (25 µM), PromISR-3 (10 µM) or PromISR-6 (25 µM) for various times, using anti-P-eIF2α, anti-eIF2α, and anti-tubulin antibodies. (n=3). Panel B shows a representative anti-puromycin immunoblot of lysates from puromycin-labelled GFP-mHtt-74Q-expressing PC12 cells, exposed to DMSO, Guanabenz or GBZ (25 µM), PromISR-3 (10 µM) or PromISR-6 (25 µM) for periods up to18 hr (n=3). Panel C shows the quantitation of P-eIF2α levels normalized to total eIF2α at 3, 6 and 18 hr expressed as fold-change relative to DMSO control. Panel D shows the quantitation of puromycin labeling normalized to the intensity of Coomassie blue staining in each lane. All comparisons were made within a single experiment or western blot. Error bars indicate ± s.e.m (*** p-value ≤ 0.001, ** pvalue ≤ 0.01 and * p-value ≤ 0.05).


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ISRIB, an ISR Inhibitor, Partially Restores Translational Repression by PromISR-6 and Counteracts PromISR-6mediated Reduction in mHtt Aggregates Puromycin-labelling in 74Q-mHtt-expressing PC12 cells showed that Tg (thapsigargin), a widely used ER stressor, activated PERK to promote eIF2α phosphorylation and translational repression thereby facilitating downstream ISR signaling that resulted in CHOP expression (Figure 5A). Coadministration of ISRIB, an ISR inhibitor, significantly restored protein synthesis and abrogated CHOP expression. Interestingly, as previously noted the combination of Tg and ISRIB paradoxically increased PERK phosphorylation 21 and in

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the PC12 cells also elevated P-eIF2α levels. While ISRIB by itself had no significant effect on protein synthesis (Figures 5B and 5C), the inhibition of puromycin labelling observed in 74Q-mHtt-expressing PC12 cells treated with PromISR-6 was almost completely reversed by the presence of ISRIB. Moreover, ISRIB had no effect on mHtt aggregates, but partially counteracted the dose-dependent decrease in mHtt aggregates seen with PromISR-6 (Figure 5D). These data suggested that ISR activation played a key role in PromISR-6mediated translation repression and reduced mHtt aggregation.

Figure 5: ISRIB Reverses the Inhibition of mRNA Translation and the Reduction of Mutant Huntingtin Aggregation elicited by PromISR-6. Panel A shows a representative anti-puromycin (upper panel) immunoblot of puromycin-labelled GFP-mHtt-74Q-expressing PC12 cells, exposed to DMSO (6 and 12 hours), Thapsigargin (Tg, 0.5 µM), and the combination of Tg and ISRIB (0.2 µM) for periods up to 12 hr. These cell lysates were also subjected to immunoblotting with anti-P-eIF2α, anti-eIF2α, anti-P-PERK, anti-CHOP and anti-tubulin antibodies (lower panels) (n=3). Panel B shows a representative anti-puromycin immunoblot of puromycin-labelled GFP-mHtt-74Q-expressing PC12 cells, exposed to DMSO, ISRIB (0.2 µM), PromISR-6 (25 µM) and combination of ISRIB (0.2 M) and PromISR-6 (25 M) for 3 hr (n=3). Panel C shows the quantitation of anti-puromycin immunoblots as shown panel B by densitometric scanning. The signals were normalized to the intensity of Coomassie blue staining in each lane. Error bars indicate ± s.e.m (* p-value ≤ 0.05 compared to DMSO and # p-value ≤ 0.05 compared to PromISR-6). Panel D shows bar graphs displaying fold-change in GFP-mHtt-74Q-expressing cells possessing aggregates following treatment with DMSO, ISRIB (0.2 M) and increasing concentrations of PromISR-6 with (+) or without (-) 0.2 M ISRIB for 72 hr (n=3). Error bars indicate ± s.e.m (*** pvalue ≤ 0.001 compared to DMSO and ## p-value ≤ 0.01 compared to PromISR- 6).


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PromISR-6 Activated Autophagy in mHtt-expressing PC12 Cells Phosphorylation of eIF2α has been linked to activation of autophagy33, a major mechanism for clearing toxic intracellular protein aggregates in HD and other neurodegenerative disorders34. To assess the ability of GBZ and its analogues to clear preformed mHtt aggregates, we employed an “on-off” assay35. PC12 cells were exposed to doxycycline for 24 hr to express GFP-mHtt-74Q. The number of doxycycline-treated cells possessing mHtt aggregates was confirmed to be substantially higher than control (Ctrl) noninduced cells (Figure 6A). GFP-mHtt-74Q expression was then halted by transferring cells to doxycycline-free medium containing GBZ or analogues and clearance of mHtt aggregates monitored for 24 hr. Rapamycin, a known autophagy activator, significantly reduced GFP-mHtt-74Q aggregates while GBZ and PromISR-3 had no impact on the cellular content of mHtt aggregates. Most importantly, PromISR-6 markedly reduced the number of cells with GFPmHtt-74Q aggregates (Figure 6A). This was independently confirmed by biochemical fractionation. PromISR-6, like Rapamycin, reduced GFP-mHtt-74Q present in SDS-insoluble fraction (Figure 6B) while GBZ and PromISR-3 had no impact on insoluble mHtt aggregates which remained similar to vehicle-treated cells. As direct evidence of autophagy activation, we analyzed the formation of autophagosomes using MDC (Monodansycadaverine) and autophagolysosomes by Lysotracker deep red staining of mHtt-expressing PC12 cells. Compared to GBZ, PromISR-6 dramatically increased MDC staining (Figure 6C) and more modestly elevated Lysotracker (Figure 6D) staining. These data demonstrated that PromISR6 increased the assembly of autophagosomes and possibly also autophagolysosomes to clear intracellular GFP-mHtt-74Q aggregates formed in the 74Q-mHtt-expressing PC12 cells. Increase in the autophagy receptor, p62, and conversion of the autophagosome-binding protein, LC3B I to II, are recognized autophagy markers36. We noted an increase in p62 levels in mHtt-expressing PC12 cells exposed to PromISR-6, well above

that with GBZ or Rapamycin (Figure 6E). However, the conversion of LC3B I to II was surprisingly reduced in PromISR-6-treated cells (Figure 6E). Emerging evidence suggests that increased autophagy flux contributes to rapid turnover of the cleaved LC3B II37. Hence, we analyzed LC3B II levels in cells co-treated with PromISR-6 and Bafilomycin, a known inhibitor of late phase autophagy. The combination of PromISR-6 and Bafilomycin (added in final 4 hr of the experiment) resulted in substantial LC3B II accumulation to levels significantly higher than PromISR-6 alone (Figure 6F). This supported the notion that PromISR-6 increased autophagic flux. Thus, the combination of prolonged protein synthesis inhibition and autophagy activation38 likely accounted for PromISR-6’s unique efficacy to reduce intracellular mHtt aggregates. PromISR-6 Reduces Cell Death Associated with mHtt Aggregation

Accumulation of toxic protein aggregates contributes to cell death in HD and other neurodegenerative diseases 39. As anticipated, there was marked elevation of the cell death markers, cleaved-caspase 3 and cleaved-PARP (CPARP), in PC12 cells following 7 days of GFP-mHtt-74Q expression. Concomitant exposure to PromISR-6 reduced cellular levels of cleaved caspase 3 and C-PARP in a dosedependent manner (Figure 7A). By comparison, GBZ and PromISR-3, even at the highest concentrations tested, did not alter these two readouts. Monitoring programmed cell death by TUNEL staining confirmed that GBZ, at concentrations up to 25 M, did not reduce cell death in the 74Q-mHtt-expressing PC12 cells. However, PromISR6, at concentrations above 5 M, significantly reduced the number of TUNEL-positive cells (Figure 7B). Thus, consistent with its ability to reduce mHtt aggregates (Figure 3), PromISR-6 promoted cell survival in this HD model.


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Figure 6: PromISR-6 Promotes Autophagic Clearance of mHtt-74Q Aggregates Protein aggregates present after 24 hr in PC12 cells (Ctrl) or cells expressing GFP-mHtt-74Q were monitored for clearance following transfer of cells to doxycycline-free media containing either DMSO (vehicle) or selected GBZ analogues for 24 hr. Panel A shows bar graph of fold-change in cells with aggregates elicited by vehicle (DMSO), Rapamycin (50 nM), Guanabenz, PromISR-3 (labelled 3) and PromISR-6 (labelled 6) (all at 10 µM). *** p-value ≤ 0.001 and * p-value ≤ 0.05). Panel B shows a representative immunoblot for SDS soluble and insoluble fractions from lysates prepared as described in Methods using anti-GFP and anti-tubulin antibodies (n=3). Panel C shows the changes in Monodansycadaverine (MDC) acidified vesicular staining in GFP-mHtt-74Q-expressing PC12 cells exposed to vehicle (DMSO), 10 µM Guanabenz or 10 µM PromISR-6 (*** p-value ≤ 0.001; n=3). Panel D shows fold-change in Lysotracker deep red staining in GFP-mHtt-74Q-expressing PC12 cells with vehicle (DMSO), Guanabenz or PromISR-6 (* p-value ≤ 0.05). Error bars indicate ± s.e.m. Panel E shows a representative immunoblot of lysates from GFP-mHtt-74Q-expressing PC12 cells, exposed to compounds as described above, using anti-p62, anti-LC3B and anti-tubulin antibodies (n=3). All comparisons were made in a single experiment or immunoblot and the white bars or gaps indicated the excision of data not relevant to current work. Panel F shows a representative immunoblot of lysates from GFP-mHtt-74Q-GFP-expressing PC12 cells treated with DMSO, PromISR-6 or 6 (10 µM), Bafilomycin (BaF-10 nM) and combination of BaF and PromISR-6 using anti-LC3B. Equal protein loading was established by immunoblotting with anti-tubulin (n=3).


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Figure 7: Impact of Guanabenz Analogues on Cell Death in GFP-mHtt-74Q- expressing PC12 Cells Panel A shows a representative immunoblot for cleaved-Caspase 3 and cleaved-PARP, two key markers of apoptosis, in GFP-mHtt74Q-expressing PC12 cells exposed to DMSO (vehicle) or increasing concentrations of Guanabenz, PromISR-6 and PromISR-3 for 7 days (n=3). Panel B shows the quantitation of TUNEL staining of fixed PC12 cells expressing GFP-mHtt-74Q treated with DMSO or increasing concentrations of Guanabenz or PromISR-6 for 7 days (n=3). Images were analyzed as described in Methods. Error bars indicate ± s.e.m (*** p-value ≤ 0.001).

Role of PromISR-6-activated PERK and Phosphorylation in Translation Repression

eIF2α

PromISR-6 activated PERK and increased P-eIF2α in WT, CReP-/- and GADD34-/- MEFs (Figure 2). To establish the role for PERK and P-eIF2α in PromISR-6-mediated ISR signaling, we analyzed PERK-/- and eIF2α (S51A) MEFs. As seen in Figure 2B, Tg and PromISR-6, but not GBZ, activated PERK, promoted eIF2α phosphorylation and downstream signaling, namely the expression of ATF4, CHOP and GADD34, in WT MEFs (Figure 8A). With the loss of PERK function, the ISR response in PERK-/- MEFs to both Tg and PromISR-6 significantly dampened (Figure 8A). In contrast to TG which failed to activate ISR in PERK/- MEFs, PromISR-6, despite significantly reduced P-eIF2α levels, modestly elevated ATF4, CHOP and GADD34 levels in these cells (Figures 8A and 8B). By comparison, in AA cells, which contained the knock in mutation, eIF2α

(S51A), while still displaying PERK activation, the downstream ISR signaling by Tg and PromISR-6 was completely abolished (Figures 8C and 8D). These data suggested that PERK was a major eIF2α kinase activated by Tg and PromISR-6. However, in the absence of PERK function, PromISR-6 modestly activated ISR likely utilizing one or more of the three other eIF2α kinases present in mammalian cells. Most importantly, the phosphorylation of eIF2α at serine-51 was essential for ISR signaling by both Tg and PromISR-6. To assess the role of P-eIF2α in the regulation of protein synthesis by PromISR-6, we compared puromycin labelling in S51A (AA) MEFs and WT (or SS) MEFs in response to Tg, GBZ and PromISR-6 (Figure 8E). As shown in PC12 cells (Figure 4), GBZ had no impact on protein synthesis in either WT/SS (Figures 8E and 8F) or S51A (AA) MEFs (Figure 8G and 8H). However, Tg and PromISR-6 resulted in profound reduction in puromycin labelling in SS MEFs at 1 hr, partially recovering over 12 hr (Figure 8E and 8F).


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In the absence of eIF2α phosphorylation, Tg resembled GBZ and had no effect on puromycin labelling in the S51A (AA) MEFs. However, PromISR-6 still moderately inhibited protein synthesis in the AA MEFs, with noticeable recovery by 12 hr. These data suggested that unlike Tg, which suppressed protein synthesis exclusively via eIF2α phosphorylation, PromISR-6 utilized both PeIF2α-dependent and -independent mechanisms to suppress protein synthesis in MEFs. Analyses of cell viability of WT (SS) and S51A (AA) MEFs in the presence of increasing concentrations of GBZ, PromISR-3 and PromISR-6 (Figure S5) emphasized, as previously seen with various stresses 40-42, that AA cells were more sensitive to these compounds than WT or SS MEFs with PromISR-6 displaying the widest margin. While these data highlighted the cytoprotective effects of ISR, they also suggested that cell death mediated in S51A (AA) cells by GBZ and its analogues may be mediated by non ISR mechanisms. PromISR-6 Does Not Activate ATF6 and IRE1 Arms of UPR Signaling PERK is maintained in an inactive state by binding of the ER luminal chaperone, Bip or GRP78. Accumulation of misfolded proteins in the ER competes Bip away from PERK, allowing PERK dimerization, transphosphorylation and activation as an eIF2α kinase43. The ability of PromISR6 and other GBZ analogues to activate PERK raised the possibility that the compounds acted pleiotropically to promote protein misfolding, thereby activating UPR. Hence, we analyzed the genes expressed downstream of IRE1 (Figure 9A-D) and ATF6, the two other arms of UPR signaling, in WT MEFs exposed to GBZ or PromISR-6. Early steps following IRE1 activation, include the enhanced splicing of Xbp1 mRNA, and subsequent transcription of the Xbp1-responsive genes, P58IPK and EDEM. We also analyzed the transcription of ATF6 responsive genes, XBP1 and BIP 44, 45 (Figure 9E-G). The data established that in addition to ISR, Tg also activated the ATF6- and IRE1mediated pathways (Figure 9). By comparison, GBZ and

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PromISR-6 did not activate these UPR pathways. Prior studies also showed that Raphin1 did not alter cellular Bip levels 19. The data showed that PromISR-6 (and other potent GBZ analogues) selectively activated ISR or the PERK/P-eIF2α/ATF4 pathway but not the IRE1 or ATF6 arms of UPR signaling. In conclusion, the current study represents to date the most extensive analysis of ISR modulation by GBZ analogues. Employing an unbiased chemical biology approach, we showed that analogues with potencies higher than GBZ, despite possessing diverse chemical structures, enhanced eIF2α phosphorylation and inhibited general protein synthesis. This was, however, insufficient to reduce intracellular mHtt aggregates and promote cell survival in a cellular HD model, where most analogues failed to modulate these parameters. PromISR-6 was far superior to GBZ, Sephin1 and Raphin1 in modifying HD and was characterized by prolonged inhibition of protein synthesis, utilizing both P-eIF2α-dependent and –independent mechanisms, and activation of autophagy, which together reduced mHtt aggregates and increased cell survival. Animal studies to confirm that PromISR-6 shared the desirable drug-like properties of GBZ, such as oral availability, central nervous system penetration and tolerable side-effects, are warranted before considering further medicinal chemistry to fine-tune the PromISR-6 template. Availability of two structurally related chemical templates, namely active PromISR-6 and inactive PromISR-3, whose very differing biological activities were underscored by genome-wide expression profiling (Figure S6), also open the way for initiating target discovery 46, that could also enable an independent target-based drug development programme for novel first-in-class medicines for HD and other neurodegenerative diseases.


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Figure 8: Role of PERK and P-eIF2α in PromISR-6 mediated Translational Repression Panel A shows ISR signaling analyzed in PERK-/- with genetically matched WT while Panel C shows and eIF2α-S51A (AA) MEFs with matched WT or SS MEFs in response to vehicle (DMSO), 1µM thapsigargin (Tg), 25 µM Guanabenz (GBZ) and 25 µM PromISR6 (labelled 6) for 3 hr using immunoblotting as described in Methods (n=3). The quantitation of P-eIF2α relative to total eIF2α and ATF4 levels relative to tubulin is shown for WT and PERK KO cells is shown in Panel B while that for WT (SS) and S51A (AA) MEFs is shown in Panel D. These data were acquired by densitometric scanning using image J with phospho-eIF2α normalized to total eIF2α and ATF4 signals to tubulin and expressed as fold-change relative to DMSO control. Error bars indicate ± s.e.m (* p-value


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≤ 0.05 and ** p-value ≤ 0.01). Panels E and G show representative anti-puromycin immunoblots of puromycin-labelled MEFs (SS panel E) and AA-panel G) exposed to DMSO, thapsigargin (Tg-1µM), Guanabenz (25 µM) and PromISR-6 (25 µM) for periods up to12 hr (n=3). Quantitation of puromycin labeling in panels E and G is shown in panels F and H respectively. Immunosignals were normalized to the intensity of Coomassie blue staining in each lane. Error bars indicate ± s.e.m (** p-value ≤ 0.01 and * p-value ≤ 0.05).

Figure 9: PromISR-6 Does Not Activate IRE-1 and ATF6 Arms of UPR Signaling Panel A shows a brief schematic of IRE-1 signaling which is initiated by XBP-1 mRNA splicing and subsequently activates the transcription of p58IPK and EDEM. These events were monitored in WT MEFs (Panel B) treated with Tg (0.5 µM), GBZ (10 and 25 µM) and PromISR-6 (labeled as 6) (10 and 25 µM) with XBP-1 splicing shown in panel B. The bar graphs show fold-changes in expression of the Xbp1 target genes, P58IPK (Panel C) and EDEM (Panel D). Panel E shows a simplified schematic of ATF6 signaling with bar graphs showing the fold-changes in two ATF6-regulated genes, XBP-1 (Panel F) and BIP (Panel G). The data represent the sum of at least three independent experiments.


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ACS Chemical Neuroscience Guanabenz Analogues

MATERIALS AND METHODS Cell lines Wild-type (WT) and GADD34-/- Mouse embryonic fibroblasts (MEFs) were generated from mice obtained from Mutant Mouse Regional Resource Centre (MMRRC) 47. CReP-/- MEFs 48 were provided by David Ron, Cambridge Institute for Medical Research, United Kingdom. PERK-/- 49 and genetically matched WT MEFs were obtained from ATCC (CRL 2976). WT and mutant (eIF2α-S51A) MEFs 40 were provided by Randal. J. Kaufman, Sanford Burnham Prebys Medical Discovery Institute, USA. All MEFs were cultured in Dulbecco's modified Eagle medium (DMEM; 11995 Invitrogen/Life Technologies) containing 10% fetal bovine serum (FBS; HyClone/GE Healthcare), 100 U/ml penicillinstreptomycin (Gibco/Life Technologies), 1X minimal essential medium-nonessential amino acids (MEM-NEAA) (Gibco/Life Technologies), and 55 μM β-mercaptoethanol (Sigma) at 37°C in a 5% CO2 incubator. PC12 cells displaying inducible expression of GFP-tagged Huntingtin (exon-1 fragment) with 23Q- or 74Q-encoding CAG repeats 31 were provided by David Rubinsztein, Cambridge Institute for Medical Research, United Kingdom. PC12 cells were cultured in DMEM (11995 Invitrogen/Life Technologies) supplemented with 10% horse serum (HS; Life Technologies), 5% Tet system approved FBS (Clontech), 100 U/ml penicillin-streptomycin (Gibco/Life Technologies), 75 μg/ml Hygromycin (Invitrogen), 50 μg/ml Geneticin (G418; Gibco/Life Technologies) at 37°C in a 5% CO2 incubator. All culture flasks, dishes and plates were coated with Rat collagen (Gibco, 1:20 in sterile Mili-Q water) overnight and washed with sterile Mili-Q water and culture media before seeding cells. Huntingtin expression was initiated by addition of 1μg/ml doxycycline (Clontech).

In Silico Screen for GBZ Analogues Two complimentary approaches were utilized to explore the structural features of the GBZ molecule, namely Tanimoto Index based similarity search 50 and traditional medicinal chemistry to rationally vary functional groups on the GBZ scaffold. During the Tanimoto exercise, we filtered search hits with >70% similarity, followed by prioritization based on descending similarity. Quick visual inspection of hit clusters was used to select equal numbers of molecules covering the range of 70% to 99.9% similarity. Additionally, the in silico screen output was subjected to a rational medicinal chemistry analogue selection process, looking for modifications of the key functional elements: guanidine head group, double bond link to phenyl group and substituents attached to the phenyl moiety. For each element, we aimed to incorporate minimal structural changes as well as significant modifications through elimination or replacement with the overall goal of identifying alternative (“better”) chemical templates, as well understand the minimal pharmacophoric features necessary for the desired biological activity. Output from the above workflows was combined, and the final compound set for screening was confirmed after checking for their commercial availability.

Guanabenz (GBZ; Tocris), Sephin1 (Tocris) and other GBZ analogues, obtained in powder form, were dissolved in DMSO (Sigma) as 25 mM stock solutions and stored in small volumes in amber tubes at -20oC. Compounds were added to culture media at varying concentrations with the final DMSO content of the medium being 0.1% for compound concentrations up to 25 μM and 0.4% for 100 μM. CellTiter-Glo Luminescent Cell Viability Assay Cells (5,000/well), seeded in 96 well plates (white, 655098, Greiner bio-one), were treated with DMSO (Vehicle) or GBZ and analogues over a wide range of concentrations. CellTiterGlo luminescent cell viability assay reagent (100μl; Promega) was added to each well along with 100 μl culture medium according to manufacturer's instructions. Luminescence was monitored using Tecan Infinite M200 microplate reader and percent viability was calculated for cells exposed to GBZ or analogues relative to the vehicle (DMSO). SUnSET Analysis of Protein Synthesis MEFs (4 X 105) were seeded in 60 mm dishes and treated with DMSO, 1 μM thapsigargin (Tg; Tocris) or GBZ analogues in the presence or absence of 10 μg/ml puromycin (Sigma) for the final 30 minutes. Cells were washed with 1X PBS, scrapped in ice-cold lysis buffer, 50 mM Tris-HCl (pH 7.0), 150 mM NaCl, 1% Triton-X-100, 1 mM EDTA, 1.5 mM MgCl2 and 5% glycerol, supplemented with complete mini-protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche) and incubated on ice for 20 minutes. PC12 cells (3 X 105), either non-induced (control) or induced (with 1μg/ml doxycycline) were treated with DMSO, 300 nM Rapamycin (Sigma), ISRIB (Tocris, 200 nM) or GBZ analogues in the presence or absence of 10 μg/ml puromycin (Sigma) for the final 30 minutes and lysed using RIPA buffer, 50 mM TrisHCl (pH 7.0), 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and 1 mM EDTA, supplemented with complete mini-protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche). Western Immunoblot Analyses Protein concentration of cell lysates was analyzed following centrifugation at 15,000 xg for 20 minutes using Bradford method and used to ensure equal protein loading. Proteins were separated by SDS-PAGE 30, 51. After electrophoretic transfer to PVDF membranes, immunoblotting used the following antibodies with dilutions indicated : anti-eIF2α (sc11386; Santa Cruz Biotechnology; 1:1000), anti-phospho-eIF2α (Ser52) (44728G; Invitrogen; 1:1000), anti-PERK (CS3192; Cell Signaling Technologies; 1:1,000), anti-phospho-PERK (Thr980) (CS3179; Cell Signaling Technologies; 1:1,000), antiATF4 (CS11815; Cell Signaling Technologies; 1:1,000), antiCHOP (CS2895; Cell Signaling Technologies; 1:1,000), antiGADD34 (C-19; SC-825; Santa Cruz Biotechnology; 1:700), anti-poly(ADP-ribose) polymerase (PARP, cleaved and total) (CS9532S; Cell Signaling Technologies; 1:1,000), anti-caspase-3 (cleaved and total) (CS9662; Cell Signaling Technologies; 1:1,000), anti-Puromycin (3RH11; EQ0001; Kerafast 1:2,000), anti-SQSTM1/p62 (CS5114; Cell Signaling Technologies; 1:700), anti-LC3B (LC3B I and II) (CS12741; Cell Signaling


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Technologies; 1:1,000) and anti-tubulin (T5168; Sigma; 1:10,000). Cell Biological Analysis of Fluorescent Huntingtin Aggregates PC12 cells expressing GFP-mHtt-74Q or GFP-mHtt-23Q were seeded (7,000 cells/well) in glass bottom 96 well plates (Thermo Scientific) coated with rat collagen and mHtt expression induced by doxycycline (1 μg/ml, Clontech) for varying periods in the presence or absence of a range of concentrations of GBZ analogues for 72 hr. Cells were washed with 1X PBS, fixed with 4% Formaldehyde/PBS (Calbiochem), permeabilized with 0.1% Triton-X-100/PBS (Bio-Rad) and stained with DAPI (Invitrogen, 1:1000 in PBS). Finally, cells washed twice with 1X PBS were viewed using green (FITCGFP) and blue (DAPI) filters from 10-12 random areas in each well at 20X magnification using Operetta high content imaging system (PerkinElmer). Images were analyzed using Columbus (high content imaging software 2.7.1, PerkinElmer). DAPI staining indicated the total number of cells. Cells possessing GFP-mHtt-74Q aggregates were calculated as percentage relative to total number of cells. Cells expressing GFP-mHtt-23Q were used as controls. Biochemical Analysis of Huntingtin Aggregates GFP-mHtt-74Q was induced in PC12 cells (3 X 105) in 60 mm dishes following doxycycline addition for 24 hr and then, terminated by adding fresh media lacking doxycycline but containing either DMSO or 300 nM Rapamycin (Sigma) or GBZ analogues for 24 hr. Cells were lysed in RIPA buffer, supplemented with complete mini-protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche). Soluble and insoluble fractions were separated by centrifugation at 15,000 x g for 20 minutes. The insoluble pellets were washed twice with RIPA buffer at 15,000 x g for 15 minutes and resuspended in 15 µl of formic acid (Sigma, 695076) at 37oC for 3 hours. These suspensions were dried at RT overnight, resuspended with 20 µl of 2X Laemmli Sample buffer (Bio-Rad, 1610737) and boiled at 95oC for 5 minutes. Proteins from soluble and insoluble fractions were separated by SDS-PAGE and immunoblotted with anti-GFP antibody (1:2,000 RT for 2 hours; Santa Cruz Biotechnology, SC9996). Analysis of Autophagasomes PC12 cells expressing GFP-mHtt-74Q were seeded (7,000 cells/well) in 96 well plates coated with rat collagen and mHtt expression induced by 1μg/ml doxycycline for 24 hr. Expression of GFP-mHtt-74Q was terminated by replacing cells in doxycycline-free medium containing DMSO or GBZ analogues 6 for 24 hr. Non induced cells were used as controls. Cells were stained with monodansycadaverine (MDC), a fluorescent compound that detects autophagic vacuoles 52 as per manufacturer’s instructions using Autophagy staining Kit (Cayman chemical 600140). Cell staining was analyzed using Tecan Infinite M200 Microplate reader at 335 nm (excitation) and 512 nm (emission) wavelengths. Analysis of Acidic Autophagolysosomes PC12 cells expressing GFP-mHtt-74Q were seeded (7,000 cells/well) in 96 well plates coated with rat collagen and mHtt expression was induced by adding 1μg/ml doxycycline for 24

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hr before terminating GFP-mHtt-74Q expression by replacing cells in doxycycline-free medium containing DMSO or GBZ analogues for 24 hr. Non-induced cells were used as controls. Finally, cells were placed in fresh medium containing 25 nM Lysotracker Deep Red (Life technologies), which stains acidic organelles, including autophagolysosomes 53 for 15 min at 370C. Cells were washed, fixed and stained with DAPI. Images in the red channel (592 nm) were taken from 10-12 different areas in individual wells at 20X magnification using Operetta high content imaging system. Cells positive for Lysotracker Deep Red staining (200 units above of basal staining in control cells) were counted using Columbus high content image analysis software. Analysis of Programmed Cell Death PC12 cells expressing GFP-mHtt-74Q were seeded (3,000 cells/well) in 96 well plates coated with rat collagen and mHtt expression induced by adding doxycycline (4μg/ml) in the presence of DMSO or varying concentrations of GBZ analogues for 5 days. Non-induced cells were used as controls. Cells were washed with 1X PBS, fixed with 4% Formaldehyde/PBS and permeabilized with 0.1% Triton-X100/PBS (60 µl per well, 2 min on ice). TUNEL staining using In Situ Cell Death Detection Kit, TMR red (Sigma) was performed according to manufacturer’s instructions. Cells were also stained with DAPI and images taken using red (590 nm) and blue (DAPI) filters from 10-12 random areas in each well using Operetta high content imaging system (PerkinElmer). Images were analysed using Columbus (high content imaging software 2.7.1, PerkinElmer). DAPI identified the total number of cells and TUNEL staining were calculated as a percentage of total. Analysis of Gene Expression Total RNA was extracted from WT MEFs using the NucleoSpin RNA isolation kit (MN740955.250) as per manufacturer’s instructions. 1µg of RNA was used to synthesize the cDNA using iScript cDNA Synthesis Kit (BioRad) and quantitative real-time PCR (RT-PCR) performed on a CFX96 Touch RealTime PCR Detection System (Bio-Rad) using SsoFast EvaGreen Supermix (Bio-Rad). The results were normalized to GAPDH using the ΔΔCT method. The primer pairs used : mouse XBP-1 Forward: 5’-GAA CCA GGA GTT AAG AAC ACG-3’, mouse XBP-1 Reverse: 5’-AGG CAA CAG TGT CAG AGT CC-3’, mouse BIP Forward: 5’-TTC AGC CAA TTA TCA GCA AAC TCT-3’, mouse BIP Reverse: 5’-TTT TCT GAT GTA TCC TCT TCA CCA GT-3’, mouse EDEM Forward: 5’-CTA CCT GCG AAG AGG CCG-3’, mouse EDEM Reverse: 5’GTT CAT GAG CTG CCC ACT GA-3’, mouse P58IPK Forward: 5’-GGC GCT GAG TGT GGA GTA AAT-3’, mouse P58IPK Reverse: 5’-GCG TGA AAC TGT GAT AAG GCG-3’ and mouse GAPDH Forward: 5’-CTT CAC CAT GGA GAA GGC-3’ mouse GAPDH Reverse: 5’-GGC ATG GAC TGT GGT CAT GAG-3’. XBP1 splicing was analyzed as published 30. Statistical Analyses

All data were expressed as mean ± standard error (s.e.m). Statistical significance was determined using nonlinear regression log (agonist) vs. normalized response-variable slope for dose response study in MEF in Figures 1, S1, S2, S4 (D & E) and S5. One-way ANOVA (Dunnett’s multiple


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comparison test) was used for quantitations in Figure 2, 3, 5, 6, 8, 9 Supp.Figure S3D and S4B & C. Two-way ANOVA (Dunnett’s multiple comparison test) was used for quantitations in Figure 4, 7, Supp. Figure S3B and unpaired T-test was used in Figure S3C. P < 0.05 was defined as statistical significance.

ASSOCIATED CONTENT Supporting Information. The Supporting information is available free of charge on the ACS Publication website. PDF contains Supplemental methods, Supplemental Figures supporting the main figures and the relevant references.

AUTHOR INFORMATION Corresponding Author * Shirish Shenolikar, Programmes in Cardiovascular and Metabolic Disorders, and Neuroscience and Behavioural Disorders, Duke-NUS Medical School, 8 College Road, Singapore 169857. Phone – 65 6516 2588; Fax – 65 6221 8625; Email: [email protected].

Present Addresses #a Medical Education, Research & Evaluation Department, Duke-NUS Medical School, Singapore 169857; #b NTU Institute of Health Technologies, Nanyang Technological University, 50 Nanyang Drive, Singapore 637553; #c School of Physical and Mathematical Sciences, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798.

Author Contributions Conceptualization: S.S, M.A, S.K, J.R.S, and I.C.J.L; Methodology: S.S, and J.R.S; Investigation: J.R.S, Y.W, S.E.G, M.H and S.G; Writing – Original Draft: S.S and J.R.S; WritingReview & Editing: S.S, J.R.S, and I.C.J.L; Funding Acquisition: S.S & T.E.K; Supervision: S.S and T.E.K; Project Administration: S.E.G

Notes The authors declare no competing interests

ACKNOWLEDGMENT This work was supported by GSK Academic Centre of Excellence Award –RL2012-029 (to SS) and support from NMRC Translational Clinical Research (TCR) Flagship Award - NMRC/TCR/013-NNI/2014 (to SS and TEK).

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PromISR-6, a Guanabenz Analogue, Improves Cellular Survival in an

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