The antibiotic robenidine exhibits guanabenz-like cytoprotective properties by a mechanism independent of protein phosphatase PP1:PPP1R15A

Abstract

The aminoguanidine compound robenidine is widely used as an antibiotic for the control of coccidiosis, a protozoal infection in poultry and rabbits. Interestingly, robenidine is structurally similar to guanabenz (analogs), which are currently undergoing clinical trials as cytoprotective agents for the management of neurodegenerative diseases. Here we show that robenidine and guanabenz protect cells from a tunicamycin-induced unfolded protein response to a similar degree. Both compounds also reduced the tumor necrosis factor α–induced activation of NF-κB. The cytoprotective effects of guanabenz (analogs) have been explained previously by their ability to maintain eIF2α phosphorylation by allosterically inhibiting protein phosphatase PP1:PPP1R15A. However, using a novel split-luciferase–based protein–protein interaction assay, we demonstrate here that neither robenidine nor guanabenz disrupt the interaction between PPP1R15A and either PP1 or eIF2α in intact cells. Moreover, both drugs also inhibited the unfolded protein response in cells that expressed a nonphosphorylatable mutant (S51A) of eIF2α. Our results identify robenidine as a PP1:PPP1R15A-independent cytoprotective compound that holds potential for the management of protein misfolding–associated diseases.

Introduction

The unfolded protein response (UPR)³ is a cellular reaction to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). This stress response aims to restore homeostasis in the ER by lowering protein translation rates, augmenting the number of folding chaperones, and increasing the degradation of misfolded proteins. This is achieved through different pathways originating from three different ER-stress sensors that are activated by unfolded proteins in the ER (1). One sensor is inositol-responsive enzyme 1 (IRE1), which catalyzes splicing of transcription factor X-box–binding protein 1 (XBP1) mRNA, enabling translation of functional XBP1 and up-regulation of target genes encoding ER chaperones and components of the ER-associated protein degradation pathway (2). A second sensor is activating transcription factor 6 (ATF6), which induces various stress-related genes following its translocation to the Golgi and subsequent proteolytic activation (3). The third sensor is protein kinase R–like endoplasmic reticulum kinase, which phosphorylates the α subunit of eIF2α at Ser-51 to reduce global translation while enhancing the translation of specific stress response genes such as ATF4, CHOP, and PPP1R15A (4). The latter branch of the UPR is also part of the more general integrated stress response (ISR) pathway. Importantly, the UPR and ISR are both regulated by a negative feedback loop that involves the delayed expression of PPP1R15A, also known as growth arrest and DNA damage–induced protein 34 (GADD34) or R15A, which forms a trimeric complex with protein phosphatase 1 (PP1) and G-actin (5, 6). This PP1 holoenzyme counteracts UPR and ISR signaling through dephosphorylation of eIF2α at Ser-51. Inhibition of eIF2α phosphatase(s) is generally considered an attractive therapeutic strategy for prolonging UPR signaling, as it gives cells more time to cope with the detrimental effects of unfolded proteins, a hallmark of several neurodegenerative diseases (7, 8).

Guanabenz is an aminoguanidine-type α₂ adrenoreceptor agonist developed during the 1980s as an anti-hypertension drug but was later identified in a yeast-based screen as a compound with anti-prion activity (9). Subsequently, several groups demonstrated cytoprotective effects of guanabenz (analogs) in murine models of neurodegenerative diseases, such as amyotrophic lateral sclerosis (10), multiple sclerosis (11), and vanishing white matter disease (12). The cytoprotective effects of guanabenz (analogs) have been explained by its ability to maintain eIF2α phosphorylation through inhibition of PP1:R15A/B (13). However, the molecular mechanism underlying the cytoprotective action of guanabenz (analogs) is controversial (14–16). Indeed, the proposed effects of guanabenz (analogs) on PP1:R15 complexes include such diverse mechanisms as disruption of the PP1:R15A interaction (10, 13), interference with the recruitment of eIF2α (17), and targeting of R15B (a constitutively expressed R15 variant) for proteolytic degradation (18). Moreover, guanabenz (analogs) had no effect on the composition or activity of an in vitro reconstituted PP1:R15A:G-actin holoenzyme (16). Furthermore, guanabenz retained its cytoprotective effects in cells and organisms that lacked R15A or only expressed nonphosphorylatable eIF2α (S51A) (15). Despite the uncertainties concerning their mechanism of action, guanabenz (analogs) remain attractive therapeutics. Guanabenz itself has rapidly progressed to clinical trials using a drug-repurposing approach. A phase I clinical trial to evaluate the pharmacokinetics of guanabenz in multiple sclerosis patients has been completed (NCT02423083), and a phase II clinical trial for ALS (EudraCT no. 2014-005367-32) and early-childhood onset vanishing white matter disease (EudraCT no. 2017-001438-25) are ongoing.

Robenidine (1,2-bis[(E)-(4-chlorophenyl)methylideneamino]guanidine) is an aminoguanidine that has been used since the early 1970s to prevent coccidian infections in rabbits, chickens, and turkeys (19, 20). Often, animals are fed with robenidine-supplemented pellets (50 mg/kg of food) for their entire life, except for a washout period before slaughter. The chronic and widespread use of robenidine as a food supplement indicates that it is safe and well-tolerated, making it a suitable candidate therapeutic agent for humans. Furthermore, robenidine belongs to the aminoguanidine compound class, which represents a diverse group of bioactive compounds used for the treatment of a broad variety of diseases ranging from bacterial infections to cancer and diabetes, and many of these compounds are approved for human use (21). Despite its long history of use in animals, there have been no in-depth examinations of robenidine as a potential therapeutic agent for the treatment of human diseases. Here we show that robenidine and guanabenz show similar cytoprotective effects in stressed cells. Furthermore, both compounds reduced the expression of UPR and ISR markers in stressed cells. Finally, using mutant cell lines and split-luciferase sensors, we demonstrate that the cytoprotective effects of robenidine do not involve changes in eIF2α phosphorylation or the eIF2α phosphatase PP1:R15A. Our data identify robenidine as a novel aminoguanidine with therapeutic potential for the treatment of protein misfolding diseases.

Results and discussion

Robenidine is a structural and functional analog of guanabenz

Using the PubChem search engine, we noticed structural similarities between robenidine and guanabenz (Fig. 1A). In contrast to guanabenz, robenidine has two substituted phenyl rings attached to its central aminoguanidine scaffold. In addition, these phenyl rings are para-chloro–substituted, whereas the phenyl ring of guanabenz has two ortho-chloro substitutions. Further mining of the PubChem BioAssay database revealed that guanabenz and robenidine were both active in a high-throughput screen against the malaria-causing protozoan Plasmodium falciparum (Fig. 1B) (National Center for Biotechnology Information, PubChem BioAssay Database, AID 504834, https://pubchem.ncbi.nlm.nih.gov/bioassay/504834, accessed July 28, 2018). Several anti-malaria drugs target the apicoplast (a nonphotosynthetic plastid) through interference with its translational machinery (23). Interestingly, a similar mechanism of action was proposed for the cytoprotective properties of guanabenz in mammalian cells undergoing unfolded protein stress (24). These findings prompted us to explore whether robenidine has cytoprotective properties similar to those of guanabenz.

Figure 1.

Structural and bioactive similarities between robenidine and guanabenz. A, chemical structure of guanabenz and robenidine. B, the inhibitory potency (EC₅₀) of guanabenz and robenidine in a quantitative high-throughput screen against P. falciparum apicoplast development. The data were extracted from the PubChem database using the indicated BioAssay assay identification numbers.

Cytoprotective effects of robenidine

The ER stress–induced UPR hampers cell cycle progression and proliferation. Indeed, treatment of HeLa cells with tunicamycin, an inhibitor of protein glycosylation that induces accumulation of misfolded proteins and activates the UPR, caused a dose-dependent decrease in proliferation, as derived from confluency assays with live-cell microscopy (Fig. 2A). This effect of tunicamycin can be rescued by guanabenz (13, 17). Likewise, 5–20 μm robenidine reversed the proliferation defect of tunicamycin-treated HeLa cells in a dose-dependent manner (Fig. 2B). However, at higher concentrations of robenidine, this cytoprotective effect was lost. We selected 15 μm robenidine as an optimal concentration for treatment, as it produced nearly maximal cytoprotective effects but was sufficiently separated from the higher, toxic concentrations. The cytoprotective effect of 15 μm robenidine was detected at relatively narrow tunicamycin concentrations of 400–800 ng/ml (Fig. 2C), similar to what has been reported for guanabenz (15). Guanabenz and robenidine were similarly cytoprotective at a concentration of 15 μm (Fig. 2D).

Figure 2.

Robenidine protects HeLa cells from tunicamycin-induced toxicity. A, proliferation of HeLa cells treated with the indicated concentrations of tunicamycin for 3 days. B, dose-dependent rescue of HeLa cells treated with 400 ng/ml tunicamycin (3 days) by the indicated concentrations of robenidine. The data were analyzed with one-way ANOVA using Dunnett's post hoc test. C, proliferation of HeLa cells treated for 4 days with or without 15 μm robenidine at the indicated concentrations of tunicamycin. Two-way ANOVA indicated that both tunicamycin (F_5,24 = 197.1, p < 0.001) and robenidine (F_1,24 = 130.7, p < 0.001) significantly affected proliferation of the cells. Additionally, there was an interaction of the two factors regarding their effects on proliferation (F_5,24 = 13, p < 0.001). Tukey's post hoc test was used to compare control versus robenidine for every concentration of tunicamycin. D, comparison of the cytoprotective effect of 15 μm guanabenz or robenidine in HeLa cells treated with tunicamycin for 3 days. The data were analyzed with one-way ANOVA using Tukey's post hoc test. E, dose–response relationship of the cytoprotective effect of robenidine or guanabenz on HeLa cells treated with 400 ng/ml tunicamycin for 3 days. Curves were fitted using a four-parameter model with variable slope. For each concentration, the average of three technical repeats is presented. F, isobologram analysis of robenidine (R) and guanabenz (G) combination treatments at the indicated dose ratios. EC₅₀ values of dose–response experiments are plotted. Black open and closed dots represent the mean with 95% confidence intervals of 15:1 and 10:1 combination treatments, respectively. The Loewe additivity line (black line) represents all theoretical robenidine–guanabenz combinations resulting in a 50% cytoprotective effect, assuming that both drugs interact in an additive manner. Datapoints to the left of the curve are synergistic combinations, whereas datapoints to the right are antagonistic. All panels except F show one of at least three independent experiments. A–D show individual datapoints of at least three technical replicates, with bar graphs indicating the mean and error bars the standard deviation (n.s., not significant, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

To evaluate whether drug–drug interactions exist between robenidine and guanabenz, we performed an isobologram analysis (25). For this purpose, we first generated dose–response curves to accurately determine their EC₅₀ values (Fig. 2E). These studies indicated that, on average, guanabenz is 10–15 times more potent than robenidine. Next, the isobologram was drawn by connecting the two axis intersection points (i.e. the EC₅₀ for each drug alone), according to the “Loewe additivity” principle. Robenidine and guanabenz drug combinations were tested at 10:1 and 15:1 ratios, and the corresponding EC₅₀ values of these combination regimens were plotted against the isobologram (Fig. 2F). The 95% confidence interval of the combination treatments overlapped with the Loewe additivity line, indicating that there are no synergistic or antagonistic interactions between robenidine and guanabenz.

Pyrimethamine shares structural features with robenidine, as it also has a central guanidine scaffold and a para-chloro–substituted phenyl ring (Fig. S1A). Moreover, pyrimethamine also displays potent anti-malaria activity (26). However, pyrimethamine was lethal at 10 μm (data not shown) and did not promote survival of tunicamycin-treated HeLa cells at a sublethal dose of 3 μm (Fig. S1B).

Robenidine inhibits TNFα-induced NF-κB activation

Guanabenz inhibits the TNFα-induced activation of the transcription factor NF-κB (27). However, the concentration of guanabenz (200 μm) that was used in the latter study is toxic and far exceeds the concentrations that are routinely used to study its cytoprotective effects in cultured cells. Therefore, we tested whether robenidine and guanabenz affect NF-κB activation at a more optimal concentration of 15 μm (Fig. 2). To this end, HEK293 cells were transfected with an NF-κB–driven luciferase reporter plasmid and treated with TNFα concentrations of 0.1–10 ng/ml in the absence (CTRL) or presence of 15 μm robenidine (Fig. 3A). Robenidine significantly inhibited NF-κB activation by TNFα concentrations between 0.1 and 0.3 ng/ml, with a maximal effect (inhibition of ∼25%) at 0.3 ng/ml TNFα. A similar level of inhibition was observed with 15 μm guanabenz (Fig. 3B). We ruled out that the observed reduction in bioluminescence was due to loss of viable cells, as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays (Fig. S2).

Figure 3.

Robenidine inhibits NF-κB activation by TNFα. A, HEK293 cells were transfected with an NF-κB–driven luciferase reporter plasmid. After 24 h, the cells were treated for an additional 24 h with vehicle (CTRL) or 15 μm robenidine and the indicated concentrations of TNFα. NF-κB activity was derived from firefly bioluminescence assays and plotted as a percentage of the activity in cells treated with TNFα alone (CTRL). Two-way ANOVA indicated that both TNFα (F_5,231 = 2.6, p = 0.024) and robenidine (F_1,231 = 130.7, p < 0.001) significantly affected the activation of NF-κB. Additionally, there was an interaction of the two factors regarding their effects on NF-κB activation (F_5,231 = 3.3, p = 0.0065). Tukey's post hoc test was used to compare control versus robenidine for every concentration of TNFα. B, the same as in A, but cells were treated with 0.3 ng/ml TNFα with or without the indicated concentrations of robenidine and/or guanabenz. Statistical analysis was performed using one-way ANOVA with Tukey's post hoc test. Datapoints show all technical replicates from three independent experiments, with the means represented by bar graphs and error bars indicating the standard deviation (n.s., not significant, p > 0.05; *, p < 0.05; ***, p < 0.001).

Robenidine does not affect the interaction between R15A and PP1 or eIF2α

The cytoprotective effects of guanabenz (analogs) have been explained by an inhibitory effect on the eIF2α phosphatase PP1:R15A, resulting from disruption of either the PP1:R15A interaction (13) or recruitment of the substrate eIF2α (17). To examine whether guanabenz and robenidine interfere with the binding of PP1 or eIF2α to R15A, we designed NanoBiT split-luciferase sensors that report on PP1:R15A and eIF2α:R15A interactions in live cells (Fig. 4A). We fused the 19-kDa “large bit” (LgBiT) luciferase fragment to the N terminus of PP1 and eIF2α. The “small bit” (SmBiT) luciferase fragment of 11 residues was inserted between the eIF2α-binding PEST repeat region and the PP1-binding KVRF sequence of R15A. This insertion is unlikely to interfere with the activity or structure of R15A, as the protein is almost completely unstructured in its native form. We envisaged that binding of LgBiT-PP1 or LgBiT-eIF2α to R15A-SmBiT would bring the split-luciferase fragments into close proximity, resulting in complementation and active luciferase (Fig. 4B). Indeed, transfection of HEK293T cells with LgBiT-PP1 + R15A-SmBiT or LgBiT-eIF2α + R15A-SmBiT resulted in luminescence, which was largely lost after deletion of the PEST region and mutation of the KVRF sequence to KARA (Fig. 4, C and D). Co-immunoprecipitation analysis confirmed that FLAG-tagged R15A immunoprecipitated endogenous eIF2α and PP1, but this interaction was lost after deletion of the PEST region in combination with a mutation of the PP1-binding site (Fig. 4E). Together, these results indicated that the adopted split-luciferase sensors allowed monitoring of the interaction of R15A with eIF2α and PP1 in live cells. We subsequently used these split-luciferase sensors to test whether guanabenz or robenidine affects the binding of LgBiT-eIF2α or LgBiT-PP1 to R15A-SmBiT in live cells. HEK293 cells transfected with the sensors were treated with 15 μm guanabenz or robenidine for 24 h. However, we observed no reduction in luminescence signal (Fig. 4F). Furthermore, HEK293 cells transfected with a constitutively active split luciferase showed no effect of the compounds on cell viability or luciferase activity. Together, these results indicate that cytoprotective concentrations of guanabenz and robenidine do not measurably affect PP1:R15A or eIF2α:R15A interactions, as measured with transient, cytomegalovirus-driven expression of split-luciferase sensors in live cells.

Figure 4.

Robenidine does not affect the interaction between R15A and PP1 or eIF2α. A, cartoon representation of the fusions used for the split-luciferase sensors. R15A is shown in beige, PP1 in red, and eIF2α in green. The eIF2α-recruiting PEST domain is shown as gray repeats, and the positions of the SmBiT or LgBiT split-luciferase tags are shown in cyan. The FLAG tag and the PP1-binding KVRF sequence (or the corresponding PP1-binding mutant KARA) are indicated. B, cartoon showing the principle of the split-luciferase sensors. Binding of WT R15A-SmBiT to either LgBiT-eIF2α or LgBiT-PP1 produces an active luciferase through complementation of SmBiT and LgBiT. C, bioluminescent signal of live HEK293 cells transfected with combinations of split-luciferase sensors, as shown in A. All technical replicates of three independent experiments were plotted as a percentage of WT R15:PP1 or R15A:eIF2α sensor signal. Bar graphs indicate the mean and error bars the standard deviation. RLU, relative light unit; M, mutant. D, immunoblot of HEK293 lysates from the experiment shown in C. E, HEK293 cells were transfected with SmBit-tagged WT or mutant R15A variants as shown in A. FLAG traps of cell lysates were analyzed by immunoblotting. The data shown are representative of three independent experiments. F, bioluminescent signal of live HEK293 cells transfected for 48 h with the indicated constructs and treated with guanabenz or robenidine for 24 h. A constitutively active luciferase construct served as a control for interference of the compounds with cell viability or luciferase activity. All technical replicates of three independent experiments were plotted as a percentage of the untreated control, with bar graphs indicating the mean and error bars the standard deviation. For all experiments, statistical analysis was performed using one-way ANOVA with Tukey's post hoc test (n.s., not significant, p > 0.05; ***, p < 0.001).

Robenidine inhibits the UPR and ISR

The cytoprotective effects of guanabenz under conditions of ER stress have been attributed to its inhibitory effect on the UPR and ISR (10). To investigate whether robenidine acts through the same mechanism, we used CHO cells containing reporters for both the ISR (CHOP::GFP) and the UPR branch that involves IRE1 (XBP1s::Turquoise) (15). Flow cytometry analysis disclosed clear induction of both genes following addition of 175 ng/ml tunicamycin for 55 h (Fig. 5A). Cotreatment of these cells with 15 μm robenidine or guanabenz decreased the expression of CHOP and XBP1s by 40–60% (Fig. 5B).

Figure 5.

Robenidine inhibits the UPR and ISR independent of eIF2α phosphorylation. A, representative graph of the two-dimensional plot of the fluorescence signals from WT CHO reporter cells stably transduced with both a CHOP::GFP reporter (reflecting ISR activity) and a XBP1::Turquoise reporter (reflecting IRE1α activity), treated with or without tunicamycin and/or robenidine and analyzed with flow cytometry. Histograms of the distribution of the two reporter signals in the three treatment conditions are plotted on the corresponding axes. B, scatterplots of the median fluorescence signals of WT CHO reporter cells treated for 55 h with the indicated concentrations of tunicamycin, robenidine, and guanabenz and analyzed by flow cytometry. Bar graphs indicate the mean and error bars the standard deviation. C, as in Fig. 4A but with CHO reporter cells expressing eIF2α-S51A. D, as in Fig. 4B, but with CHO reporter cells expressing eIF2α-S51A. For B and D, all technical replicates from four independent experiments were plotted. Statistical analysis was performed using one-way ANOVA with Dunnett's post hoc test (**, p < 0.01; ***, p < 0.001).

A previous study showed that guanabenz (analogs) retain their cytoprotective activity in cells that express a nonphosphorylatable eIF2α mutant (15). This finding contradicts the proposal that guanabenz's cytoprotective effects are mediated by an increased phosphorylation of eIF2α at Ser-51 (8, 24). To examine whether inhibition of the expression of CHOP and XBP1s by robenidine and guanabenz is mediated by increased eIF2α phosphorylation, we used a variant CHO reporter cell line that expresses nonphosphorylatable eIF2α (S51A). Consistent with ISR-dependent CHOP activation, the activation of the CHOP::GFP reporter was less pronounced in the cell line expressing eIF2α S51A (compare Fig. 5, A and C). Nonetheless, both robenidine and guanabenz inhibited the induction of CHOP::GFP and XBP1s::Turquoise in the mutant cell line upon treatment with tunicamycin (Fig. 5D). These results demonstrate that robenidine, like guanabenz, does not exert its cytoprotective effect by increasing the phosphorylation of eIF2α at Ser-51.

Conclusions

Here we identified robenidine as a new aminoguanidine compound with cytoprotective properties similar to those described previously for the analogs salubrinal (28), guanabenz (13), sephin1 (10), and raphin1 (18). Indeed, robenidine reduced the antiproliferative effects of tunicamycin (Fig. 2), dampened TNFα-induced NF-κB activation (Fig. 3), and inhibited the expression of ISR and UPR markers (Fig. 5). We also found that the cytoprotective effects of robenidine and guanabenz could not be explained by disruption of R15A:PP1 or R15A:eIF2α and were not mediated by changes in the phosphorylation state of eIF2α (Fig. 5). These results therefore demonstrate that the phenotypic effects of these drugs cannot be mediated by inhibition of the R15A–PP1–actin holoenzyme or altered phosphorylation of eIF2α at Ser-51. Similar conclusions were recently drawn for guanabenz and sephin1 (15, 28) but are at variance with reports hinting at inhibitory effects of guanabenz (analogs) on PP1:R15 assembly and its recruitment of eIF2α as a substrate (10, 17).

Other targets of guanabenz (analogs) that have been proposed to underly their cytoprotective function are cholesterol 25-hydroxylase (29), rRNA (30), and lipid membranes (31). It is currently uncertain whether the antibacterial, anti-inflammatory, and cytoprotective properties of robenidine and guanabenz (analogs) arise from the same cellular target(s), but this seems likely, as both compounds had very similar effects at high nanomolar to low micromolar concentrations. Identification of the cellular target(s) of robenidine and guanabenz (analogs) remains an important goal for further exploration, as it will aid the development of analogs with higher potency.

We show here, for the first time, that guanabenz and robenidine significantly inhibit the activation of NF-κB (Fig. 3). However, this effect was rather small (∼25% inhibition) and therefore unlikely to contribute significantly to the cytoprotective properties of these compounds. At present, we cannot exclude that the inhibitory effects of robenidine and guanabenz in the NF-κB and CHOP/XBP1s reporter cell lines (Figs. 3 and 5) are (partially) explained by a general inhibitory effect on protein translation, as the assays rely on the production of luciferase and fluorescent protein for signal readout.

Guanabenz, sephin1, and raphin1 have shown promising results in various murine models of neurodegenerative diseases (10, 11, 18). We speculate that robenidine has similar therapeutic potential, in particular because its widespread industrial use for nearly 50 years as a food additive and coccidostat documents its safety and tolerance, at least in animals. Pharmacokinetic studies in rabbits showed that robenidine is orally available and has a moderate half-life of around 12 h (32). Micromolar serum concentrations are reached with a single oral dose, which are the concentrations needed for cytoprotective effects in vitro (Fig. 2). Our isobolographic analysis suggests that there is no benefit of combining robenidine and guanabenz (analogs). Nonetheless, our studies indicate that robenidine is an attractive candidate for preclinical testing in animal models of neurodegenerative diseases.

Experimental procedures

Materials

The following antibodies were used: FLAG (Sigma-Aldrich, F1804), β-actin (GeneTex, GTX26276), PP1 (made in-house (22)), eIF2α (Thermo Fisher Scientific, AHO0802). Secondary HRP-tagged antibodies were purchased from Agilent-Dako. Guanabenz was purchased from TCI Europe (G0456), robenidine from Acros Organics NV (459400010), tunicamycin from SanBio BV (11445), human TNFα from Sigma-Aldrich (SRP3177), coelenterazine from Carl Roth GmbH (4094.3), D-luciferin from SanBio BV (14682-50), and pyrimethamine from Acros Organics NV (461200010). Split-luciferase sensors were made using ligation-independent cloning techniques. LgBiT was fused via a 15-amino-acid flexible linker (GSSGGGGSGGGGSSG) to the N terminus of full-length PP1α or eIF2α. SmBiT was inserted at residue 537 of R15A with a five-residue flexible linker (GSTSG) at both ends. A constitutively active NanoBiT luciferase that served as a control consisted of LgBiT fused to SmBiT via a 15-amino-acid flexible linker.

PubChem database searching

The guanabenz entry was accessed (https://pubchem.ncbi.nlm.nih.gov/) under PubChem CID 5702063. Robenidine (CID 9570438) was identified through the “related record” search function, focusing on substituted aminoguanidines. High-throughput assays that tested both compounds were identified manually under the “BioAssay results” section. Subsequently, the BioAssay assay identification numbers were used to query the obtained potency for each compound. MarvinSketch was used for drawing and exporting chemical structures (MarvinSketch, 17.21.0, ChemAxon).

Cytoprotection experiments

HeLa cells were plated at ∼10% confluency in transparent 96-well plates. Three technical replicates were used per condition. Treatment with compounds started at the time of seeding. All compounds were dissolved in DMSO. The final concentration of DMSO was the same under all conditions of each experiment and never exceeded 0.5%. Proliferation was measured using an IncuCyte ZOOM system and analyzed using IncuCyte ZOOM software (Essen BioScience).

Isobologram analysis

Cytoprotection experiments were performed as described above. The EC₅₀ isobole was generated by performing three dose–response experiments for both robenidine and guanabenz. From these data, the average EC₅₀ values for both drugs were calculated and used as intercepts of the isobologram. Next, dose–response experiments were performed for the indicated robenidine–guanabenz combination ratios, and the calculated EC₅₀ values were plotted individually on the isobolograph. Calculation of EC₅₀ values was performed using the four-parameter model with variable slope with GraphPad Prism V5.

NF-κB reporter assay

HEK293 cells were transfected with an NF-κB–driven firefly luciferase reporter plasmid (Addgene, 14886) for 24 h. Subsequently, the cells were seeded in white opaque 96-well plates at ∼50% confluency and treated with the indicated concentrations of TNFα, guanabenz, and/or robenidine. A minimum of four technical replicates were used per condition. The final concentration of DMSO was identical under all experimental conditions and never exceeded 0.5%. One day after seeding and treatment of the cells, firefly activity was measured on a Luminoskan Ascent (Thermo Scientific) after replacement of the cell culture medium with homemade firefly assay buffer (0.5% Triton X-100, 50 mm Tris (pH 7.4), 5 mm MgCl₂, 20 μm pyrophosphate, 500 μm ATP, and 150 μg/ml D-luciferin). As firefly luciferase exhibits flash-type kinetics, the signal was measured in the stable phase, which typically occurred 3–4 min after incubation with firefly assay buffer. To account for possible variations in cell proliferation between the different conditions, a parallel plate of HEK293 cells was seeded and treated identically to the firefly assay plate. At the time of luciferase measurement, cell proliferation was measured in the parallel plate using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. For this, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dissolved in Tris buffer (pH 7.4) was added at a final concentration of 0.5 mg/ml to the assay medium and incubated overnight. Formazan crystals were dissolved in DMSO and transferred to a transparent 96-well plate, and the absorbance was measured at 550 nm using a BioTek ELx808 microplate reader.

Flow cytometry with CHO reporter cell lines

CHO cells were plated in 6-well plates at ∼20% confluency and treated with the indicated concentration of compounds for 55 h. Three technical replicates were included per condition. The final concentration of DMSO (<0.5%) was the same under all conditions. Cell culture medium and compounds were refreshed halfway through the incubation period to prevent medium evaporation and stressing the cells. Subsequently, cells were washed, harvested in PBS, and used for flow cytometry. GFP and Turquoise expression were measured on a BD FACSCanto II HTS system using the following filters: GFP: excitation, 488 nm; emission, 500–560 nm; Turquoise: excitation, 405 nm; emission, 400–500 nm. At least 10,000 cells were recorded per sample. Dead cells were excluded based on the scatter profile of a calibration sample containing 50% dead and 50% live cells. Data were processed using FlowJo software (Tree Star, Inc.).

NanoBiT split-luciferase assay

HEK293 cells were plated at ∼20% confluency and transfected with the indicated split-luciferase sensors for 48 h. To achieve sufficient sensor activity, sensors were cloned in vectors containing the cytomegalovirus promoter and not in the weaker TK promoter-driven vectors of the NanoBiT kit. Subsequently, the cells were harvested and resuspended in PBS. The luciferase substrate coelenterazine was added to the cell suspension at a final concentration of 25 μm, and cells were dispensed at ∼10,000 cells/well in white 96-well plates. Luciferase activity was measured on a Luminoskan Ascent (Thermo Scientific). When the cells were treated with compounds, they were reseeded in opaque 96-well plates after the 48-h transfection period. A minimum of three technical replicates were used per condition. To correct for effects of the compounds on cell proliferation or expression of the split-luciferase sensors, a third batch of cells, expressing a constitutively active NanoBiT luciferase (see “Materials”), was seeded and treated in parallel.

Cell culture

HEK293 cells (ATCC) were cultured in Dulbecco's modified Eagle's medium with high glucose (4,500 mg/liter glucose, Sigma-Aldrich) containing 10% fetal calf serum (Sigma-Aldrich), 100 units/ml penicillin, and 100 μg/ml streptomycin. HeLa cells (ATCC) were cultured in the same medium but with 1,000 mg/liter glucose. Transfections were performed with jetPRIME (Polyplus). All cell lines were mycoplasma-free.

Biochemical techniques

HEK293 cells were lysed with lysis buffer (50 mm Tris-HCl (pH 7.4), 0.01% saponin, 150 mm NaCl, and 10% glycerol) supplemented with protease inhibitors (0.5 mm phenylmethanesulfonyl fluoride, 0.5 mm benzamidine, and 5 μm leupeptin). Subsequently, lysates were sonicated with three 10-s cycles at high intensity and cleared by centrifugation at 10,000 × g. For FLAG traps, cleared cell lysates were incubated with 20 μl of FLAG affinity beads (Sigma, catalog no. P4333) for 2–3 h at 4 °C. The beads were then washed once with PBS and subjected to immunoblotting. SDS-PAGE gel electrophoresis was performed with 10% or 4%–12% BisTris (NuPAGE, Invitrogen). Immunoblots were visualized using ECL reagent (PerkinElmer Life Sciences) in an ImageQuant LAS4000 imaging system (GE Healthcare). The signals were quantified using ImageJ (National Institutes of Health).

Author contributions

Z. C. and M. B. conceptualization; Z. C., M. J., and A. C.-C. resources; Z. C. data curation; Z. C. software; Z. C. formal analysis; Z. C. validation; Z. C. investigation; Z. C. visualization; Z. C., M. J., and A. C.-C. methodology; Z. C. writing-original draft; Z. C., M. J., A. C.-C., and M. B. writing-review and editing; M. B. supervision; M. B. funding acquisition; M. B. project administration.