Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth

Article

Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth Graphical Abstract

Authors Andrew Kaplan, Sebastian A. Andrei, Anna van Regteren Altena, ..., Yusuke Higuchi, Christian Ottmann, Alyson E. Fournier

Correspondence [email protected] (A.K.), [email protected] (A.E.F.)

In Brief Kaplan et al. identify a derivative of the small molecule fusicoccin-A (FC-A) that stimulates neurite outgrowth by regulating dozens of protein-protein interactions (PPIs) in the 14-3-3 adaptor protein interactome. This compound, FCNCPC, bidirectionally stabilizes and inhibits 14-3-3 PPIs enriched within the Rap1 signaling network, suggesting new targets for CNS indications.

Highlights d

FC-A is a small molecule that modulates 14-3-3 PPIs and induces neurite outgrowth

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Derivatives with improved potency were identified in a neurite outgrowth screen

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Interactome analysis reveals regulation of 14-3-3 PPIs enriched in Rap1 network

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Derivative FC-NCPC can stabilize or competitively inhibit 143-3 PPIs

Kaplan et al., 2020, Cell Chemical Biology 27, 1–11 June 18, 2020 ª 2020 Elsevier Ltd. https://doi.org/10.1016/j.chembiol.2020.02.010

Please cite this article in press as: Kaplan et al., Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.02.010

Cell Chemical Biology

Article Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth Andrew Kaplan,1,6,7,* Sebastian A. Andrei,3 Anna van Regteren Altena,1 Tristan Simas,1 Sara L. Banerjee,5 Nobuo Kato,2 Nicolas Bisson,5 Yusuke Higuchi,2 Christian Ottmann,3,4 and Alyson E. Fournier1,* 1Department

of Neurology and Neurosurgery, Montre´al Neurological Institute, McGill University, Montre´al, QC, Canada Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka, Japan 3Laboratory of Chemical Biology and Institute for Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, the Netherlands 4Department of Chemistry, University of Duisburg-Essen, Essen, Germany 5De ´ partement de Biologie Mole´culaire, Biochimie Me´dicale et Pathologie, Centre de Recherche sur le Cancer, Universite´ Laval, Que´bec, QC, Canada 6Present address: Yumanity Therapeutics, 40 Guest Street, Boston, MA, USA 7Lead Contact *Correspondence: [email protected] (A.K.), [email protected] (A.E.F.) https://doi.org/10.1016/j.chembiol.2020.02.010 2The

SUMMARY

Targeting protein-protein interactions (PPIs) is a promising approach in the development of drugs for many indications. 14-3-3 proteins are a family of phosphoprotein-binding molecules with critical functions in dozens of cell signaling networks. 143-3s are abundant in the central nervous system, and the small molecule fusicoccin-A (FC-A), a tool compound that can be used to manipulate 14-3-3 PPIs, enhances neurite outgrowth in cultured neurons. New semisynthetic FC-A derivatives with improved binding affinity for 14-3-3 complexes have recently been developed. Here, we use a series of screens that identify these compounds as potent inducers of neurite outgrowth through a polypharmacological mechanism. Using proteomics and X-ray crystallography, we discover that these compounds extensively regulate the 14-3-3 interactome by stabilizing specific PPIs, while disrupting others. These results provide new insights into the development of drugs to target 14-3-3 PPIs, a potential therapeutic strategy for CNS diseases. INTRODUCTION 14-3-3s are a family of cytosolic and nuclear proteins that regulate dozens of signaling pathways by binding to hundreds of ‘‘client’’ proteins, modulating their functions in several different ways, including altering stability, activity, and localization (Kaplan and Fournier, 2017). 14-3-3s bind to client proteins at threonine motifs/threonine motifs via a groove that is highly conserved among seven isoforms expressed in humans (Kaplan et al., 2017a). 14-3-3 proteins are being explored as therapeutic targets in many diseases, including cancer and

neurodegenerative diseases (Kaplan and Fournier, 2017; Stevers et al., 2017b). In the central nervous system (CNS), 14-3-3s are neuroprotective and are critical for proper migration and growth of multiple neuronal populations (Kent et al., 2010; Yam et al., 2012; Shimada et al., 2013; Kaplan et al., 2017b; Lavalley et al., 2016, Mar et al., 2014). Fusicoccanes are a family of small molecules that stabilize 14-3-3 protein-protein interactions (PPIs) by binding to an inter-molecular pocket at the interface of the 14-3-3 groove and the client motif within the groove (Stevers et al., 2017b). These compounds have potential therapeutic applications in many indications due to the extensive involvement of 14-3-3s in cellular functions in health and disease (Molzan et al., 2013, de VriesVan Leeuwen et al., 2013; Stevers et al., 2016; Bier et al., 2016; Stevers et al., 2017a). Fusicoccin-A (FC-A), a member of the fusicoccane family, stimulates neurite outgrowth in cultured neurons (Kaplan et al., 2017b). Here, we use neurite outgrowth screens and identify semisynthetic FC-A derivatives with improved potency. FC-A and derivatives have previously been shown to act as stabilizers of 14-3-3 PPIs. Using proteomics to monitor the global effects of these compounds on the 14-3-3 interactome, we show that that these compounds, in fact, can both stabilize and inhibit 14-3-3 PPIs. Binding assays and co-crystallization with 14-3-3 complexes reveal that these compounds directly stabilize or inhibit the binding of client proteins to 14-3-3 by binding to the same pocket, a dual functionality that has not been described previously. These findings have significance for the design of small molecules to target PPI interfaces and highlight a potential polypharmacological therapeutic strategy to target 14-3-3 PPIs for CNS indications. RESULTS Neurite Outgrowth Screen of FC-A Derivatives To explore structure-activity relationship of FC-A and identify compounds with improved neurite outgrowth potency, we Cell Chemical Biology 27, 1–11, June 18, 2020 ª 2020 Elsevier Ltd. 1

Please cite this article in press as: Kaplan et al., Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.02.010

Figure 1. Neurite Outgrowth Screen of FC-A Derivatives (A) Library of 30 natural and semisynthetic derivatives of the 14-3-3 protein-protein interaction (PPI) modulator fusicoccin-A (FC-A). (B) E18 rat cortical neurons were grown in 96-well plates and treated with compounds in duplicate wells for 24 h. High-content imaging and analysis permitted the automated tracing of neurons for neurite outgrowth analysis. Scale bar, 200 mm. (C) Structures of FC-A 19-amide derivatives and neurite outgrowth fold changes for each compound relative to DMSO control. Compounds FC-NCHC, FCNCPC, and FC-NAc were most potent (n = 5–6 wells from 3 experiments). Shaded area with asterisk denotes significant fold change in neurite outgrowth (p < 0.05, one-way ANOVA, Dunnett’s test). Data are presented as mean + SEM. (D–F) Neurite outgrowth (D), neuritogenesis (E), and branching (F) dose-response curves of FC-A and top 3 hit 19-amide derivatives. (G) Images of E18 rat cortical neurons treated for 2 days with 19-amide derivatives FC-NCPC or FC-NAc. Scale bar, 200 mm.

screened a library of 30 natural and semisynthetic derivatives in a neurite outgrowth assay (Figures 1A and S1). Cortical neurons from embryonic day 18 (E18) rat embryos were plated at low density in 96-well plates and compounds were added at a single 10mM concentration. After 24 h of neurite outgrowth, the neurons were fixed and stained with an antibody against beta III tubulin 2 Cell Chemical Biology 27, 1–11, June 18, 2020

(tuj1), a neuron-specific tubulin isoform that serves as a marker of neuronal morphology. Using a high-content screening system, images were acquired and analyzed for average neurite length per neuron in a fully automated fashion, permitting the analysis of thousands of individual neurons (Figure 1B). Nine compounds with higher potency than FC-A were identified (Figure 1C).

Please cite this article in press as: Kaplan et al., Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.02.010

Figure 2. FC-NCPC Regeneration In Vitro

Stimulates

Axon

(A) E18 rat cortical neurons were grown in microfluidic chambers for 11 days. Axotomy was performed with vacuum aspiration and FC-NCPC was added to both compartments of the chamber. Cultures were fixed and stained 48 h post-axotomy. (B) FC-NCPC significantly stimulates axon regeneration after axotomy (n = 9–10 chambers, p < 0.05, unpaired t test). Data are presented as mean + SEM. (C) Images of regenerating axons. Scale bar, 50 mm.

Identification of Potent 19-Amide FC-A Derivatives The top 3 hits from the neurite outgrowth screen were semisynthetic derivatives that have recently been rationally designed and validated to have enhanced binding affinity for 14-3-3, resulting in higher potency PPI stabilization activity (Andrei et al., 2018). These derivatives possess an amide substitution at position 19 (FC-NCHC, N-cyclohexylcarbonyl derivative; FC-NCPC, N-cyclopropylcarbonyl derivative; and FC-NAc, N-acetyl derivative; Figure 1C), which induces the formation of an additional hydrogen bond between the compound and aspartic acid 215 in the back wall of the 14-3-3 groove (Andrei et al., 2018). These compounds were selected for secondary analyses in doseresponse curves. The parent compound 3’deAC-FC-A induced neurite outgrowth with a half maximal effective concentration (EC50) of 20 mM, while the EC50 for FC-NCHC, FC-NCPC, and FC-NAc were all single-digit micromolar, and a maximal effect of a 2.5-fold increase in outgrowth was reached at 25 mM (Figure 1D). At maximal effective concentrations, striking increases in neurite length and complexity are notable, including enhanced neuritogenesis and branching (Figures 1E–1G). FC-A Derivative FC-NCPC Enhances Axon Regeneration In Vitro Because of its improved potency and previous characterization (Andrei et al., 2018), FC-NCPC was selected for further analysis in a secondary assay to assess axon regeneration in an in vitro axotomy model. Cortical neurons were cultured for 11 days in microfluidic chambers with 1-mm microchannels through which axons extend and exit into a discrete compartment (Figure 2A). The 1mm length of the microchannels is prohibitive to the passage of dendrites to the second compartment, thereby permitting selective manipulation of axons (Taylor et al., 2005). The 11-day timescale of the culture also allows the neurons to undergo morphological and functional maturation. Axotomy was performed using vacuum aspiration, producing a rapid shear stress at the plane of the microchannels, resulting in controlled axonal breakage. Immediately after axotomy, FC-NCPC was added to both compartments of the microfluidic chamber. Two days after injury, the cultures were stained with tuj1 and phalloidin to visualize axons and growth cones. Treatment with 25 mM FC-NCPC, a maximal effective concentration in the outgrowth assay, significantly induced axon regeneration after axotomy (Figure 2B). Moreover, the axons adopted the same tortuous growth pattern that was apparent in the neurite outgrowth assays, suggesting a conserved activity after injury of mature cortical neurons (Figure 2C).

Characterization of FC-A and FC-NCPC Effects on 14-33 Interactome FC-A and derivatives have been shown to have PPI stabilization activity toward multiple distinct 14-3-3:client complexes (Stevers et al., 2017b). We took an unbiased approach to identify 14-3-3 clients that are targeted by these compounds by performing proteomic analyses of GST-14-3-33 pull-downs from cortical neuron lysate in the presence of vehicle, or 500 mM FC-A or FC-NCPC, a concentration 503 the neurite outgrowth EC50 to maximize detection of effects on 14-3-3 PPIs. GST-14-33 pull-downs in the presence of vehicle were used to characterize the naive 14-3-3 interactome. GST-14-3-33 with a point mutation (K49E) that prevents binding to clients was used as a control for non-specific protein binding (Figure 3A). Pull-down samples were prepared from three independent replicates and analyzed by mass spectrometry. Samples from wild-type (WT) 14-3-3 pull-downs had a 2.6-fold increase in total peptide count and a 14.4-fold increase in phosphoserine/threonine/ tyrosine peptide count compared with K49E-14-3-3, indicating the selective binding of WT-14-3-3 to a subset of proteins enriched for phosphorylation (Figure 3B). Pull-downs from K49E14-3-3, therefore, served as the background for subsequent SAINT (significance analysis of interactome) (Choi et al., 2011) analysis to identify high-confidence 14-3-3 clients. SAINT analysis with a stringent 0.9 cutoff identified 202, 192, and 202 high-confidence 14-3-3-binding clients in the naive, FC-A, or FC-NCPC interactomes, respectively (Table S1). The combined pool of 14-3-3 clients from all 3 interactomes contained 265 unique proteins. Gene ontology analysis revealed a diverse representation of protein functions, highlighting the pleiotropic activities of 14-3-3 (Figure 3C). We next examined average peptide counts among the three interactomes for each client protein and calculated fold changes in abundance in the FC-A or FC-NCPC interactomes relative to the naive interactome, to measure trends in PPI abundance. Although many PPIs were relatively unchanged, this analysis revealed both increases and decreases in the binding of clients to 14-3-3 in the presence of FC-A or FCNCPC, suggesting that the compounds stabilize certain PPIs and inhibit others. Fold changes were of greater magnitude in the FC-NCPC interactome compared with the FC-A interactome, providing biochemical evidence that FC-NCPC is a more potent stabilizer and inhibitor across a broad range of 14-3-3 PPIs (Figure 3D). Moreover, when examining peptide abundance for individual high-confidence client proteins, trends toward stabilization or inhibition were markedly enhanced by FC-NCPC Cell Chemical Biology 27, 1–11, June 18, 2020 3

Please cite this article in press as: Kaplan et al., Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.02.010

(legend on next page)

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Please cite this article in press as: Kaplan et al., Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.02.010

relative to FC-A, consistent with the idea that FC-NCPC differs in potency, but not client selectivity (Figure 3E). To validate these results, western blots were performed on GST-14-3-3 pulldowns in the presence of a range of FC-NCPC concentrations. These experiments confirmed that two stabilized clients, B-Raf and C-Raf, showed dose-dependent increases and that two inhibited clients, Afadin and Rapgef2, showed decreases in binding to GST-14-3-3, faithfully validating the mass spectrometry results for these clients (Figure 3F). Moreover, pull-downs in the presence of either 10 mM FC-A or FC-NCPC, a concentration near the EC50 of FC-NCPC, confirmed the higher potency PPI inhibition activity of FC-NCPC relative to FC-A, where Rapgef2 binding to GST-14-3-3 was unaffected by FC-A, but was drastically reduced by FC-NCPC (Figure 3G). Together, these results suggest a complex polypharmacological perturbation of the 14-3-3 interactome and suggest a duality in the activity of FC-A and derivatives, which can function as 14-3-3 PPI stabilizers or inhibitors, depending on the client protein. Small-Molecule Screen for Modifiers of FC-NCPCInduced Neurite Outgrowth The stabilization and inhibition of numerous 14-3-3 PPIs suggested that FC-NCPC may function through a polypharmacological mechanism to enhance neurite outgrowth. To identify mechanistically important cell signaling pathways, we analyzed the FC-A and FC-NCPC interactomes to enrich for client proteins with consistent and conserved trends in peptide abundance relative to the naive interactome. Enriched signaling pathways were then obtained by running KEGG pathway analysis on filtered clients. Pathways were then further scored by summing the average fold change magnitudes in PPI abundance of each annotated protein in the FC-NCPC interactome relative to the naive interactome to enrich for pathways that contain highly regulated PPIs. These analyses identified the Rap1 signaling network as a highly enriched pathway for which the binding of many clients to 14-3-3 was either stabilized or inhibited by FCNCPC. Rap1 is a Ras-related small GTPase that is activated by cAMP-dependent GTP exchange factors Epac1/2 and is involved in a multitude of cell signaling functions (Frische and Zwartkruis, 2010). KEGG Mapper (Kanehisa and Sato, 2020) was used to generate a signaling diagram containing regulated PPIs within the Rap1 network. Strikingly, 16 client proteins clustered into this network, both upstream and downstream of a Rap1 node (Figure 4A). We first sought to verify the 14-3-3

dependence of FC-NCPC-induced neurite outgrowth using BV02, a small molecule that competitively inhibits client binding to the 14-3-3 groove. Treatment with 10 mM BV02 completely abrogated FC-NCPC-induced neurite outgrowth (Figure 4B), consistent with our previous studies showing that FC-A-dependent neurite outgrowth is inhibited by BV02 or by simultaneous knockdown of multiple 14-3-3 isoforms (Kaplan et al., 2017b). To assess the relative contributions of signaling pathways within the Rap1 network to FC-NCPC-induced neurite outgrowth, we performed a neurite outgrowth screen using a panel of seven small-molecule inhibitors for multiple targets (cAMP, C-Raf, Cdc42, Rac, Erk1/2, Epac1/2-Rap1, and Tiam-Rac) in these pathways (Figure 4C). An inhibitor of HDAC4, another regulated client that did not cluster into the Rap1 pathway, was also included in the screen. Each inhibitor was tested at three concentrations that were selected based on previous studies. Each compound/concentration combination was tested in the presence of vehicle to capture effects on basal neurite outgrowth, or in the presence of a maximal effective concentration of 40 mM FC-NCPC to ascertain effects on FC-NCPCinduced neurite outgrowth. Cell density was also measured to monitor potential toxicity (Figure S2A). Inhibition of Cdc42, Rac, Erk1/2, and Epac1/2-Rap1 all significantly attenuated FC-NCPC-induced outgrowth at non-toxic concentrations. All tested concentrations of the Tiam-Rac inhibitor NSC23766 were toxic and therefore inconclusive with regard to effects on FC-NCPC-induced outgrowth. Inhibition of C-Raf showed a mild trend toward inhibition of FC-NCPC-induced outgrowth, while inhibition of cAMP-dependent signaling with RpCAMPS had no effect on outgrowth. HDAC4 inhibition also failed to affect FC-NCPC-induced outgrowth. These results suggest that FCNCPC functions through multiple pathways within the Rap1 network in a cAMP-independent fashion. Most notably, inhibition of Rap1 activation itself with an Epac1/2 inhibitor, ESI-09, reduced basal neurite outgrowth and profoundly attenuated FC-NCPC-induced outgrowth. Secondary Dose-Response Analyses for Modifiers of FC-NCPC-Induced Neurite Outgrowth Based on the results of the screen, a series of secondary doseresponse analyses were conducted to determine the effects of inhibitors on FC-NCPC-induced outgrowth. Inhibitors were titrated in the presence of vehicle to assess basal effects on outgrowth, or a constant 40-mM concentration of FC-NCPC.

Figure 3. Identification of 14-3-3 PPIs Targeted by FC-A and FC-NCPC (A) Wild-type (WT) or client-binding-deficient K49E mutant GST-tagged 14-3-33 was used for pull-downs from E18 rat cortical neuron lysate. K49E GST-14-3-3 pull-downs served as control for non-specific binding. WT GST-14-3-3 pull-downs were performed in the presence of vehicle, or 500 mM FC-A or FC-NCPC to determine the effect of these compounds on the interaction of 14-3-3 with client proteins. (B) Samples were analyzed by mass spectrometry, revealing an enrichment of phosphorylated peptides in WT GST-14-3-3 pull-downs compared with K49E control. Data are presented as mean + SEM. (C) SAINT (significance analysis of interactome) analysis with 0.9 cutoff revealed a total of 265 unique proteins with high-confidence binding to WT GST-14-3-3 in the presence of vehicle, FC-A, or FC-NCPC. (D) Log2 fold changes in peptide counts for each identified protein with SAINT R 0.9 in FC-A and FC-NCPC interactomes gives a semi-quantitative measure of PPI abundance, revealing both stabilization and inhibition of 14-3-3 PPIs, with FC-NCPC producing effects of greater magnitude. (E) Dotplot representation of sample client proteins whose binding to WT GST-14-3-3 was unchanged, stabilized, or inhibited by FC-A and FC-NCPC. Shading and size indicate peptide abundance (generated using ProHits, n = 3). (F) GST pull-down western blot validation of clients whose binding to 14-3-3 is stabilized (C-Raf and B-Raf) or inhibited (Afadin and Rapgef2) by increasing concentrations of FC-NCPC. (G) Western blots from WT GST-14-3-3 pull-downs in the presence of 10 mM FC-A or FC-NCPC demonstrate higher potency PPI inhibition activity of FC-NCPC.

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Please cite this article in press as: Kaplan et al., Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.02.010

Figure 4. Small-Molecule Inhibitor Screen to Identify Modifiers of FC-NCPC-Induced Neurite Outgrowth (A) KEGG pathway analysis of filtered client proteins revealed an enrichment of FC-NCPC-regulated 14-3-3 PPIs within the Rap1 signaling network (signaling diagram generated using KEGG Mapper). (B) Treatment with 14-3-3 inhibitor BV02 abrogates FC-NCPC-induced neurite outgrowth (n = 3, **p < 0.01, two-way ANOVA, Bonferonni’s test). Data are presented as mean + SEM. (C) Small-molecule inhibitors of signaling pathways within the Rap1 network were used to assess the contribution of various signaling pathways to the neurite outgrowth activity of FC-NCPC. Each inhibitor was tested at three concentrations in combination with either vehicle or FC-NCPC. Bars with a blue # indicate a significant loss in FC-NCPC-induced neurite outgrowth defined by a loss of greater than one order of magnitude in p value. Compound concentrations that caused significant cell loss are indicated with a red # (n = 4). (D) Images from cultures treated with a combination of FC-NCPC and Rap1 inhibitor ESI-09. Scale bar, 100 mm. (E–K) Small-molecule inhibitors of Epac1/2-Rap1 (ESI-09) (E), C-Raf (GW5074) (F), B-Raf (PLX4720) (G), Tiam1-Rac (NSC23766) (H), MEK1/2 (U0126) (I), Erk1/2 (FR180204) (J), or mammalian target of rapamycin (mTOR) (K) were titrated in the presence of either vehicle or a constant 40 mM concentration of FC-NCPC. Non-toxic concentration ranges for each inhibitor were pre-determined and none of the compound/concentration combinations caused significant cell loss (Figure S3) (n = 3–4). Data points marked with # indicate a loss of statistical significance between the vehicle and FC-NCPC condition (p > 0.05, unpaired t test). Data are presented as mean + SEM.

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Please cite this article in press as: Kaplan et al., Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.02.010

Non-toxic concentration ranges for each inhibitor were pre-determined, and measurement of cell density showed no significant cell loss in all tested compound/concentration combinations (Figures S2B–S2H). Attenuation of FC-NCPC-induced growth can be inferred by a reduction in fold change in the presence of FC-NCPC relative to vehicle (i.e., a narrowing of the Dfold change between vehicle and FC-NCPC curves). Intriguingly, while ESI-09 (Epac1/2-Rap1 inhibitor) had a marginal effect on basal growth, it substantially attenuated FC-NCPC-induced growth (Figures 4D and 4E). GW5074 had a marginal effect on basal growth, and mildly attenuated FC-NCPC-induced growth (Figure 4F). PLX4720 (B-Raf inhibitor) modestly enhanced basal growth and had a marginal effect on FC-NCPC-induced growth (Figure 4G). NSC23766 (Tiam-Rac inhibitor), U0126 (MEK1/2 inhibitor), and FR180204 (Erk1/2 inhibitor) all reduced basal growth, and substantially attenuated FC-NCPC-induced growth (Figures 4H–4J). Notably, at the upper ends of the tested concentration ranges, NSC23766 (Tiam-Rac inhibitor) and U0126 (MEK1/2 inhibitor) pushed FC-NCPC-induced growth down to levels at or near the vehicle control. Rapamycin, an inhibitor of mammalian target of rapamycin, another downstream protein in the Rap1 pathway reduced basal growth, but had no effect on FC-NCPC-induced growth (Figure 4K). These results suggest that the neurite outgrowth activity of FC-NCPC is sensitive to inhibitors of multiple cell signaling pathways within the Rap1 network. Structural Analyses of FC-NCPC Effects on 14-3-3 Client Binding To provide more detailed molecular evidence for the proposed FC-A- and FC-NCPC-induced PPI stabilization and inhibition mechanisms on clients within the Rap1 network, biochemical characterization of the 14-3-3 binding capacities of TIAM1, Pak4, Pak6, C-Raf, B-Raf, and Rapgef2 were initiated. Fluorescein-labeled phosphopeptides mimicking the individual phosphorylation sites of these proteins were used to assess their 14-3-3 affinity and susceptibility to either FC-A or FC-NCPC modulation in a fluorescence polarization assay. Phosphorylation sites were extracted from the literature for TIAM1 (pS43, pS60, pS172, and pS231) (Woodcock et al., 2009), PAK4 (pS99 and pS181) (Tinti et al., 2014), PAK6 (pT99, pS113, and pS162) (Tinti et al., 2014), C-Raf (pS233, pS259, and pS621) (Dumaz and Marais, 2003), and B-Raf (pS365 and pS729) (Roskoski, 2010). In the case of Rapgef2 no literature on putative phosphorylation sites could be found, so the 14-3-3Pred online prediction tool was used to predict 14-3-3 binding sites in the Rapgef2 sequence (Madeira et al., 2015). This yielded five interesting phosphorylation sites (pT375, pT445, pT740, pS900, and pS948). Peptides of each of these phosphorylation sites were devised by flanking each site by five amino acids, yielding a small library of 11-mer peptides. Fluorescence polarization assays were then performed in the absence and presence of 10 mM FC-A or FC-NCPC (Figures S3–S5). This screen yielded promising results for the Pak6 pT99 peptide and Rapgef2 pT740 peptide, which recapitulated the PPI stabilization and inhibition captured in the MS analyses with full-length proteins from cell lysate. Dose-response assays were performed for these targets. Because 14-3-3 proteins operate as dimers and often bind their targets bivalently (Obsil and Obsilova, 2011),

assays were performed with a Pak6 bisphosphorylated peptide spanning both pT99 and pS113 sites. The pT99-pS113 Pak6 peptide was dose dependently stabilized by FC-A and FC-NCPC (Figures 5A and 5B). Moreover, FC-NCPC was clearly the stronger stabilizer and saturated at a 5-fold increase in affinity, while FC-A showed a stabilizing effect up to 3.5-fold at 100 mM (Figures 5 A and 5B). For Rapgef2, FC-NCPC yielded a dose-dependent inhibitory effect of up to 3-fold, whereas FC-A did not show any activity, recapitulating the FC-NCPC-selective effect observed in the 14-3-3 pull-down assays at low- to mid-micromolar concentrations (Figures 5D and 5E). As these results confirmed the stabilizing and inhibitory effect of FC-NCPC, we next sought to solve the binary crystal structures of Pak6 pT99 and Rapgef2 pT740 binding to 14-3-3, and their ternary structures with FC-NCPC. The two binary structures show the phosphorylated residues binding to the phosphatebinding groove of 14-3-3 as expected. Four additional amino acids of Pak6 were resolved in the electron density (Figure 5C; see Table S2 for crystallographic statistics). Fusicoccanes are known to bind to a conserved hydrophobic pocket at the far end of the 14-3-3 binding groove. Fusicoccane amide derivatives, such as FC-NCPC, bind in the same pocket and use their amide functionality to form a strong hydrogen bond with Asp215 of the 14-3-3 protein, increasing their binding affinity and inducing a small helix shift in the protein (Andrei et al., 2018). Upon soaking with FC-NCPC, binding of the compound was clearly visible in the expected pocket and a shift in helix 9 of 14-3-3 was indeed observed (Figure 5C). The conformation of the peptide changed slightly to accommodate stabilizer binding, most notably the residues beyond the valine on the +1 position relative to the phosphate move away from the fusicoccane binding site (Figure 5C, arrow). The +1 valine itself is seen making a favorable hydrophobic contact with one of the cyclopentane rings of FC-NCPC, providing a structural rationale as to why Pak6 binding is enhanced. This observation is in line with previous reports where a valine residue on the +1 position can provide an opportunity for fusioccane-mediated PPI stabilization (Bier et al., 2016). For Rapgef2, three amino acids were well resolved but, upon soaking of FC-NCPC, the visible electron density was reduced to only the phosphate-bearing residue, suggesting that the peptide is competed out upon binding of FC-NCPC (Figure 5F). Contrary to Pak6, Rapgef2 features a serine residue on the +1 position. The polar nature of this serine results in an unfavorable interaction with the hydrophobic FC-NCPC cyclopentane ring, effectively decreasing its affinity for 14-3-3 in the presence of FC-NCPC. From either FC-NCPC structures it is not evident that the small shift in 14-3-3 helix 9 induced by the compound has an effect on the binding of the peptides. These structures corroborate that binding of FC-NCPC is not compatible in the 14-3-3:Rapgef2 pT740 complex, but is permitted and has a stabilizing effect on 14-3-3:Pak6 pT99. Altogether, these results provide compelling evidence for a dual function of FC-NCPC as both a 14-3-3 PPI stabilizer and inhibitor. DISCUSSION Traditional target-based drug development approaches rely heavily on the selection and validation of a single target with critical biological importance. Polypharmacology, the use of Cell Chemical Biology 27, 1–11, June 18, 2020 7

Please cite this article in press as: Kaplan et al., Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.02.010

Figure 5. Molecular Characterization of the Interaction between PAK6 and RapGef2 and 14-3-3 (A) Fluorescence polarization assay showing dose-dependent stabilization of the PAK6 pT99-pS113 interaction with 14-3-3 by FC-A. (B) Dose-dependent stabilization of the PAK6 pT99-pS113 interaction with 14-3-3 by FC-NCPC. (C) Crystal structure of the binary complex of PAK6 pT99 bound to 14-3-3s (top) and the ternary complex with FC-NCPC (bottom). The 14-3-3 protein is shown in gray, the PAK6 peptide in yellow and FC-NCPC in green. The 2Fo Fc electron density map is contoured at 1s in a dark blue mesh. Movement of helix 9 and the PAK6 peptide are indicated with arrows. (D) Fluorescence polarization assay showing dose-dependent inhibition of the RapGef2 pT740 interaction with 14-3-3 by FC-A. (E) Dose-dependent inhibition of the RapGef2 pT740 interaction with 14-3-3 by FC-NCPC. (F) Crystal structure of the binary complex of RapGef2 pT740 bound to 14-3-3s (top) and the ternary complex with FC-NCPC (bottom). The 14-3-3 protein is shown in gray, the RapGef2 peptide in violet, and FC-NCPC in green. The 2Fo Fc electron density map is contoured at 1s in a dark blue mesh. Movement of helix 9 is indicated with an arrow. Error bars indicate the SEM of one triplicate experiment in the titration curves and the SEM of three independent triplicate experiments in the bar charts. The fluorescent probe concentration (10 nM) is indicated with a dashed line.

drugs that act on multiple distinct targets, is a promising approach for complex diseases without a known genetic basis, where hitting a single target is unlikely to be efficacious. Here, we discovered and characterized a unique polypharmacological approach based on a small molecule that bidirectionally stabilizes and inhibits PPIs between the master regulator 14-3-3 proteins and an array of functionally diverse client proteins in multiple cell signaling pathways. 8 Cell Chemical Biology 27, 1–11, June 18, 2020

The dual function of FC-NCPC as a more potent inhibitor and stabilizer of 14-3-3 PPIs, compared with FC-A, stems from its stronger binding affinity for apo-14-3-3 (Andrei et al., 2018). Amino acid sequences downstream of the phosphorylation site in each client protein motif are probable determinants of whether FC-NCPC functions as a competitive inhibitor or a stabilizer. Our structural analyses of 14-3-3 binding to motifs within Pak6 and Rapgef2 confirm the dual activity of FC-NCPC as both a direct

Please cite this article in press as: Kaplan et al., Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.02.010

PPI inhibitor and stabilizer. Previously identified bioactivities of FC and derivatives, including anti-cancer activity, have been attributed solely to 14-3-3 PPI stabilization (Miyake et al., 2015; Ohkanda et al., 2018). Our results suggest that 14-3-3 PPI inhibition may also constitute an important mechanism of action for these compounds, with relevance for the future development of compounds to target 14-3-3 PPIs. To understand the mechanisms underlying its biological activity in neurons, we used a combination of proteomic and bioinformatic analyses and found an enrichment of FC-NCPC-regulated 14-3-3 PPIs in a Rap1 signaling network. Rap1 is a small GTPase involved in many cellular processes, notably cell polarity and adhesion (Frische and Zwartkruis, 2010). Most of the targets we identified lie downstream of a Rap1 node; however, FC-NCPC also inhibits 14-3-3 binding to Rapgef2, an upstream GTP exchange factor (GEF) that activates Rap1. Rap1 is also activated by cAMP-dependent GEFs, Epac 1/2, which have been implicated in the prooutgrowth effects of cAMP (Murray and Shewan, 2008). Rapgef2 itself has also previously been implicated in cAMP-dependent neurite outgrowth (Emery et al., 2013). Interestingly, we found that antagonism of cAMP signaling with RpCAMPS failed to affect FC-NCPC-induced growth, yet inhibition of Rap1 activity with the Epac1/2 inhibitor ESI-09 substantially attenuated FC-NCPCinduced growth (Figure 4E). Whether Rapgef2 binds cAMP is controversial (Liao et al., 1999) and it is possible that FC-NCPC stimulates Rap1 activity through a cAMP-independent mechanism via 14-3-3-dependent regulation of Rapgef2, or simply that a certain basal level of active Rap1 is important for the activity of FC-NCPC toward other downstream targets. It is also possible that FC-A and derivatives engage other targets. For instance, FC-A has been shown to perturb tyrosine kinases in a cell-free kinase activity screen (Bury et al., 2013). However, multiple lines of evidence suggest that in neurons FC-induced growth occurs through 14-3-3-dependent mechanisms, as 14-3-3 knockdowns impair FC-A-induced neurite outgrowth (Kaplan et al., 2017b) and small-molecule inhibition of 14-3-3s abrogates FC-NCPCand FC-A-induced neurite outgrowth. However, the functional significance of 14-3-3 binding to many of the client proteins identified in this study remains unknown. Using a variety of tool compounds, we discovered that FC-NCPC stimulates neurite outgrowth through a polypharmacological mechanism involving multiple signaling pathways, many of which are also important for basal growth. It is conceivable that some of these pathways may be directly targeted by FC-NCPC, but also that basal levels of signaling are required for FC-NCPC activity, but not directly induced by FC-NCPC. Besides representing a new polypharmacological approach, these findings also shed light on the complex biology underlying neuron growth. FC-Arelated molecules can be viewed not only as potential drugs, but also as chemical probes which can be used for target-discovery and as general tool compounds to understand 14-3-3 biology. SIGNIFICANCE We have identified a semisynthetic FC-A derivative with improved neurite outgrowth potency. Using proteomics, we have discovered that FC-A and derivatives can stabilize as well as inhibit dozens of 14-3-3 PPIs, a unique polypharmacological activity that could be used in drug development

or as a tool to study 14-3-3 biology. We have validated this phenomenon using X-ray crystallography and binding assays to show that 14-3-3 binding to phospho-sites in peptides derived from some of these targets can lead to stabilization or competitive inhibition of complex formation. We further found that the neurite outgrowth activity of this compound depends on multiple signaling pathways within a Rap1 signaling network. These results suggest new pharmacological strategies and new targets for CNS diseases. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

d d

KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS B Cortical Neuron Culture B Protein Expression for Crystallization METHOD DETAILS B Neurite Outgrowth Assays B High-Content Analysis B Semisynthetic FC Derivatives B Small Molecule Inhibitors B Antibodies B Immunoblotting B GST-14-3-3 Protein Production and Purification B Pull-Down Assays B Mass Spectrometry Analysis B Mass Spectrometry Data Analysis B Phosphopeptides B Protein Expression B Fluorescence Polarization B Crystallization QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND CODE AVAILABILITY

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. chembiol.2020.02.010. ACKNOWLEDGMENTS This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and ERA-NET-FRQS to A.E.F. and ECHO-STIP grant 717.014.001 from the Netherlands Organization for Scientific Research (NWO) to C.O. A.K. was supported by the Ann and Richard Sievers Neuroscience Award at the Montreal Neurological Institute and a fellowship from the Fonds de Recherche du Que´bec – Sante´. N.B. holds a Canada Research Chair in Cancer Proteomics (Tier 2). S.L.B. is supported by a PROTEO scholarship. Mass spectrometry was performed at the CHU de Que´bec – Universite´ Laval Proteomics Platform. We thank Erika Wee at the McGill University Advanced BioImaging Facility (ABIF) and Isabel Rambaldi and Brandon Jansonius for technical assistance. We also thank Chloe Song for insight and discussion. AUTHOR CONTRIBUTIONS Conceptualization, A.K. and A.E.F.; Experiments, A.K., S.A.A., A.v.R.A., T.S., S.L.B., and Y.H.; Writing, A.K., S.A.A., C.O., and A.E.F.; Supervision, A.K., A.E.F., N.B., N.K., and C.O.

Cell Chemical Biology 27, 1–11, June 18, 2020 9

Please cite this article in press as: Kaplan et al., Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.02.010

DECLARATION OF INTERESTS The authors declare no competing interests. Received: November 30, 2019 Revised: February 5, 2020 Accepted: February 28, 2020 Published: March 26, 2020 REFERENCES Adams, P.D., Afonine, P.V., Bunkoczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., et al. (2010). Phenix: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221. Andrei, S.A., de Vink, P., Sijbesma, E., Han, L., Brunsveld, L., Kato, N., Ottmann, C., and Higuchi, Y. (2018). Rationally designed semisynthetic natural product analogues for stabilization of 14-3-3 protein-protein interactions. Angew. Chem. Int. Ed. 57, 13470–13474.

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Cell Chemical Biology 27, 1–11, June 18, 2020 11

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STAR+METHODS KEY RESOURCES TABLE

REAGENT

SOURCE

IDENTIFIER

Antibodies Rabbit monoclonal anti-B-Raf

Abcam

Cat#ab33899; RRID: AB_725762

Rabbit polyclonal anti-l + S Afadin

Abcam

Cat#ab11338; RRID: AB_297944

Rabbit polyclonal anti-Raf1

Abcam

Cat#ab137435

Rabbit polyclonal anti-RAPGEF2

Abcam

Cat#ab112093; RRID: AB_10861528

Mouse monoclonal anti-betaIII tubulin (clone Tuj1)

Biolegend

Cat# 801201 or 801202; RRID: AB_2313773 or RRID: AB_10063408

Alexa 568 phalloidin

Thermo Fisher

Cat#A12380

Alexa 488 anti-rabbit

Thermo Fisher

Cat#A11034; RRID: AB_2576217

Alexa 568 anti-rabbit

Thermo Fisher

Cat#A11011; RRID: AB_143157

Alexa 568 anti-mouse

Thermo Fisher

Cat#A11031; RRID: AB_144696

Alexa 488 anti-mouse

Thermo Fisher

Cat#A11029; RRID: AB_138404

RpCAMPS

Tocris

CAS:151837-09-1; Cat#1337/1

ESI-09

Tocris

CAS:263707-16-0; Cat#4773

NSC23766

Tocris

CAS:1177865-17-6; Cat#2161

EHT1864

Tocris

CAS:754240-09-0; Cat#3872

Y27632

Tocris

CAS:129830-38-2; Cat#1254

ZCL278

Calbiochem

CAS:587841-73-4

GW5074

Tocris

Cat#1381

FR180204

Tocris

CAS:865362-74-9; Cat#3706

LMK235

Tocris

CAS:1418033-25-6; Cat#4830/10

Rapamycin

Abcam

CAS:53123-88-9; Cat#ab120224

PLX4720

Abcam

CAS:918505-84-7; Cat#ab141362

U0126

Tocris

CAS:109511-58-2; Cat#1144

BV02

Sigma

CAS:292870-53-2

Glutathione Beads

G-Biosciences

Cat#786-310

Benzonase Nuclease

Milipore

Cat#71206-3

Fusicoccin-A

Nobuo Kato lab, Osaka University

N/A

Fusicoccin-A semi-synthetic analogues

Nobuo Kato lab, Osaka Univeristy

N/A

PAK6 pT99 + 14-3-3 Structure

This Paper

PDB 6QDR

PAK6 pT99 + 14-3-3 + FC-NCPC Structure

This Paper

PDB 6QDS

Chemicals, Peptides, and Recombinant Proteins

Deposited Data

RapGef2 pT740 + 14-3-3 Structure

This Paper

PDB 6QDT

RapGef2 pT740 + 14-3-3 + FC-NCPC Structure

This Paper

PDB 6QDU

Proteomics data

This paper

ProteomeXchange, PXD017317 and 10.6019/PXD017317

Primary cortical neurons – E18/19 Sprague Dawley rat

Charles River Labs

N/A

NiCo21(DE3) cells

New England Biolabs

Cat #C2529H

Experimental Models: Cell Lines

(Continued on next page)

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Continued REAGENT

SOURCE

IDENTIFIER

pGEX-6P1- GST-14-3-3e WT

Gift from Dr. Philippe P. Roux, Universite´ de Montre´al

N/A

pGEX-6P1- GST-14-3-3e K49E

Gift from Dr. Philippe P. Roux, Universite´ de Montre´al

N/A

MetaXpress – Automated Neurite Outgrowth Module

Molecular Devices

N/A

Prism 6

GraphPad

RRID: SCR_002798

Scaffold version 4.8.4

Proteome Software Inc

http://www.proteomesoftware.com/ products/scaffold/

XCalibur software version 3.0.63

Thermo Scientific

https://www.thermofisher.com/store/ products/OPTON-30965#/OPTON-30965

Thermo Proteome Discoverer version 1.4.0.288

Thermo Scientific

https://www.thermofisher.com/ca/en/ home/industrial/mass-spectrometry/liquidchromatography-mass-spectrometry-lcms/lc-ms-software/multi-omics-dataanalysis/proteome-discoverersoftware.html

Recombinant DNA

Software and Algorithms

Mascot version 2.5.1

Matrix Science

http://www.matrixscience.com/

Protein Prophet algorithm

Nesvizhskii, Keller, Kolker & Aebersold, 2003

http://proteinprophet.sourceforge.net/ index.html

SAINTexpress

Teo et al., 2014

http://saint-apms.sourceforge.net/ Main.html

CRAPome

Mellacheruvu et al., 2013

https://www.crapome.org/

xia2 DIALS x-ray diffraction processing

https://xia2.github.io/index.html

AIMLESS x-ray data merging algorithm

Winn et al., 2011

http://www.ccp4.ac.uk/html/aimless.html

PHASER algorithm for molecular modeling

McCoy et al., 2007; Winn et al., 2011

http://www.ccp4.ac.uk/html/phaser.html

Phenix.Refine algorithm for structure refinement

Liebschner et al., 2019

https://www.phenix-online.org/

Crystallographic Object-Oriented Toolkit (COOT)

Emsley et al., 2010

https://www2.mrc-lmb.cam.ac.uk/ personal/pemsley/coot/

MolProbity structure validation suite

Williams et al., 2018

http://molprobity.biochem.duke.edu/

PyMol

Schro¨dinger

https://pymol.org/

KEGG Mapper

Kanehisa and Sato, 2020

https://www.genome.jp/kegg/mapper.html

ImageJ

NIH

RRID: SCR_003070

DotPlot from ProHits-viz

Knight et al., 2017

http://prohitstools.mshri.on.ca/

Molecular Devices

N/A

Other ImageXpress – Automated High Content Imaging System UltiMate 3000 nanoRSLC

Dionex/Thermo

Cat#ULTIM3000RSLCNANO

Orbitrap Fusion mass spectrometer + nanoelectrospray

Thermo Scientific

N/A

EmulsiFlex-C3 homogenizer

Avestin

N/A

Amicon Ultra-4 and Ultra-5 1000 DA MWCO spin silter

Amicon

Cat#UFC801008; FC901008

Tecan Infinite F500 plate reader

Tecan

N/A

Dectris Pilatus3 X 200K-A detector

Dectris

N/A

384-well Low Volume Black Round Bottom Polystyrene NBS Microplate

Corning

Cat#:4515

Cell Chemical Biology 27, 1–11.e1–e6, June 18, 2020 e2

Please cite this article in press as: Kaplan et al., Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.02.010

LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Andrew Kaplan ([email protected]). All unique/stable reagents generated in this study are available from the Lead Contact without restriction. EXPERIMENTAL MODEL AND SUBJECT DETAILS Cortical Neuron Culture All studies were approved by the McGill University Animal Care and Use Committee. For cortical neuron cultures, cortices were dissected from male and female E18 Sprague Dawley rats in cold Leibovitz L15 medium (Thermo Fisher). Isolated cortices were incubated in 0.25% trypsin-EDTA for 30min at 37 C and dissociated into a single cell suspension by gentle trituration in DMEM supplemented with 10%FBS. Neurons were seeded in culture dishes coated with 100mg/mL poly-l-lysine (PLL) and washed 3 times with PBS. Culture medium consisted of Neurobasal medium supplemented with 2% B27, 1% N2, 1% penicillin/streptomycin and 2mM L-glutamine. Protein Expression for Crystallization NiCo21(DE3) cells (New England Biolabs) were used for expression of proteins for crystallization studies. Colonies were grown on LB agar plates + 100 mg/mL ampicillin overnight at 37 C. One colony was picked and precultured in 25 mL sterile LB medium + 100 mg/mL ampicillin overnight at 37 C. 2 TB medium + 100 mg/mL ampicillin was then inoculated with the preculture and incubated at 37 C until an OD600 of 1.0 was reached. Protein expression was then induced with 0.4 mM IPTG and the cells incubated overnight at 18 C. Cells were then harvested by ultracentrifugation at 20000 rpm at 4 C, flash frozen in liquid nitrogen and stored at -80 C. METHOD DETAILS Neurite Outgrowth Assays Cortical neurons were seeded in 96-well plates, 7000 cells per well. For the FC-A derivative screen, the medium was replaced with pre-diluted compounds at 10mM final concentration, 0.1% DMSO, 2hr after plating. Each compound was tested in duplicate wells. For the small molecule inhibitor screen, the neurons were treated with each inhibitor at 3 indicated concentrations at a constant 0.2% final DMSO concentration across all wells and the neurons were fixed and stained after 48hr. Each compound/concentration combination was tested in duplicate wells. For dose-response curves, compounds were serially diluted and DMSO concentrations were constant across all concentrations and never exceeded 0.2%. Neurons were fixed with 4% paraformaldehyde/20% sucrose, permeabilized with 0.2% Triton-X, blocked with 5% BSA, and stained with anti-bIII tubulin (1:1000, BioLegend) for 1hr at room temperature, Hoechst 33342 (1:10000, Sigma) and fluorescent secondary antibodies (1:1000, Thermo Fisher) for 1hr at room temperature. High-Content Analysis Automated image acquisition of bIII tubulin and Hoechst stain was performed using the ImageXpress high-content imaging system (Molecular Devices). Automated neurite outgrowth analysis and cell counts were performed using the neurite outgrowth module of the MetaXpress software (Molecular Devices). Images were collected analyzed in the McGill University Life Sciences Complex Advanced BioImaging Facility (ABIF). Semisynthetic FC Derivatives FC-A was purified from phomopsis amygdali cultures and used as starting material for the synthesis of derivatives. Detailed synthetic procedure and molecular characterization of 19-amide FC-A derivatives is provided in the previous report (Andrei et al., 2018). Small Molecule Inhibitors The following small molecule inhibitors were used in neurite outgrowth assays: RpCAMPS (1337/1, Tocris), ESI-09 (4773, Tocris), NSC23766 (2161, Tocris), EHT1864 (3872, Tocris), Y27632 (1254, Tocris), ZCL278 (CAS 587841-73-4, Calbiochem), GW5074 (1381, Tocris), FR180204 (3706, Tocris), LMK235 (4830/10, TOCRIS), Rapamycin (ab120224, Abcam), PLX4720 (ab141362, Abcam), U0126 (1144, Tocris). Antibodies The following primary antibodies were used for western blotting: B-Raf (1:300, Abcam, 33899), Afadin (1:1000, Abcam, ab11338), C-Raf (1:500, Abcam, ab137435), Rapgef2 (1:100, Abcam, ab112093). Immunoblotting Lysates were separated by SDS-PAGE on 4-20% gradient gels (Bio-Rad) and transferred to PVDF membranes. Membranes were incubated in Ponceau stain for 5min and de-stained in water. Following Ponceau stain, membranes were blocked with 5% milk e3 Cell Chemical Biology 27, 1–11.e1–e6, June 18, 2020

Please cite this article in press as: Kaplan et al., Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.02.010

TBS-T for 1hr, probed with primary antibody for 1hr at room temperature or overnight at 4 C and then probed with HRP-conjugated secondary antibodies (Jackson) for 1hr at room temperature. Signal was developed using ECL detection system (Pierce). GST-14-3-3 Protein Production and Purification GST-14-3-33 WT and K49E mutant constructs in pGEX-6P1 vectors were gifts from Dr. Philippe P. Roux, Universite´ de Montre´al. Expression of GST-14-3-33 WT and K49E were induced in cultures of BL21 E. coli with 0.5mM IPTG for 3-4hrs at 37 C. Bacteria were pelleted by centrifugation and resuspended in ice cold PBS supplemented with protease inhibitors (complete protease inhibitor cocktail, Roche). Bacteria were sonicated 4X 30sec at 30% amplitude, with 30sec rests on ice. Triton-X was then added at a final concentration of 1% and lysates were rotated at 4 C for 30min. Cell debris was removed by centrifugation and GST-fusion proteins were then isolated from the supernatant by rotation with glutathione beads for 2hr at 4 C. Beads were washed 3X with cold PBS and protein concentration was determined by DC protein assay (Bio-Rad). Pull-Down Assays E18 rat cortical neurons (7DIV) were lysed in ice cold lysis buffer composed of 100mM NaCl, 50mM Tris-HCl, 5mM EDTA, 2mM MgCl2, 10% glycerol and 1% NP-40 supplemented with protease inhibitors (complete protease inhibitor cocktail, Roche) and phosphatase inhibitors (10mM sodium fluoride and 1mM sodium orthovanadate). Lysates were clarified by centrifugation and protein concentration was determined by DC protein assay (Bio-Rad). Lysates were pre-cleared with glutathione beads for 30min at 4 C with rotation. Beads were removed by centrifugation and then 20mg of GST-14-3-33 WT or K49E coupled to beads were added to 1.8mg of cortical neuron lysate for each reaction. FC-A or FC-NCPC or vehicle (ethanol) were added at the indicated concentrations to each reaction (final 1.25% ethanol concentration across all reactions). Pull-downs were performed for 2hr at 4 C with rotation. For preparation of samples for mass spectrometry, beads were washed 3X with lysis buffer, followed by 2 washes with 50mM Tris-HCl. Proteins were eluted with 50mM phosphoric acid, 3X 10min on ice with occasional gentle agitation. Beads were removed by centrifugation and supernatants were stored at -80 C. For western blot validation, beads were washed 3X with lysis buffer after pull-downs and then boiled in 2X sample buffer. Mass Spectrometry Analysis For mass spectrometry, eluted proteins were processed for on-beads tryptic digestion as described(Beigbeder et al., 2016) and peptides were desalted using StageTips(Rappsilber et al., 2007). Peptide identification was carried out on an Orbitrap Fusion mass spectrometer equipped with a nanoelectrospray ion source (Thermo Scientific) and coupled to an UltiMate 3000 nanoRSLC (Dionex/Thermo). Data dependent acquisition of mass spectra was performed using XCalibur software version 3.0.63 (Thermo Scientific). Full scan mass spectra (350 to 1800 m/z) were acquired in the orbitrap using an automatic gain control target of 4e5, a maximum injection time of 50 ms and a resolution of 120 000. Selected ions were isolated using the quadrupole analyzer in a window of 1.6 m/z and fragmented by higher energy collision-induced dissociation (HCD) with 35% of collision energy. The resulting fragments were detected by the linear ion trap at a rapid scan rate. Dynamic exclusion of previously fragmented peptides was set for a period of 20 s and a tolerance of 10 ppm. All MS/MS peak lists were generated using Thermo Proteome Discoverer version 1.4.0.288 (Thermo Scientific). MGF sample files were then analyzed using Mascot (Matrix Science, London, UK; version 2.5.1). Mascot was set up to search Uniprot Rattus norvegicus database (April 2018 release, 31636 entries) supplemented with ‘‘common contaminants’’ from the Global Proteome Machine (GPM, thegpm.org, July 2017 release) assuming the digestion enzyme trypsin. They were searched with a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 10 ppm. Carbamidomethyl of cysteine was specified as a fixed modification. Deamidated of asparagine and glutamine, oxidation of methionine and phospho of serine, threonine and tyrosine were specified in Mascot as variable modifications. Two miscleavages were allowed. Mass Spectrometry Data Analysis Scaffold (version 4.8.4, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 89.0% probability to achieve an FDR less than 1.0% by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability to achieve an FDR less than 1.0% and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm(Nesvizhskii et al., 2003). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. MS data were analyzed with SAINTexpress, a simplified version of the Significance Analysis of INTeractome method(Teo et al., 2014) via the CRAPome website(Mellacheruvu et al., 2013). SAINT probabilities were calculated using MS data from control pull-downs from K49E-14-3-3 beads. SAINT output was filtered with cut-off of 0.9. Figures utilized the DotPlot, a protein-protein interaction data visualization tool(Knight et al., 2017). Phosphopeptides All phosphopeptides were obtained through GenScript. Of the selected 14-3-3 binding phosphorylation sites, peptides were ordered with 5 amino acids on either side of the putative phosphorylation site. All peptides had an amide C-terminus and either an acetylated (For crystallography) or FITC-6-aminohexanoic acid-labeled (FITC-Ahx-)(For fluorescence polarization) N-terminus. The following sequences were used:

Cell Chemical Biology 27, 1–11.e1–e6, June 18, 2020 e4

Please cite this article in press as: Kaplan et al., Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.02.010

Peptide Name

Labeled Sequence

Acetylated Sequence

TIAM1 pS43

FITC-Ahx-RTRHApSSGKVI-CONH2

Ac-RTRHApSSGKVI- CONH2

TIAM1 pS60

FITC-Ahx-STRSSpSTPSIP-CONH2

Ac-STRSSpSTPSIP- CONH2

TIAM1 pS172

FITC-Ahx-KKRSKpSADIWR-CONH2

Ac-KKRSKpSADIWR- CONH2

TIAM1 pS231

FITC-Ahx-CQRANpSLGDLY-CONH2

Ac-QRANpSLGDLY- CONH2

PAK4&6 pS474

FITC-Ahx-VPRRKpSLVGTP-CONH2

Ac-VPRRKpSLVGTP- CONH2

PAK4 pS162

FITC-Ahx-EKRPKpSSREGS-CONH2

Ac-EKRPKpSSREGS- CONH2

PAK4 pS99

FITC-Ahx-VTRSNpSLRRDS-CONH2

Ac-VTRSNpSLRRDS- CONH2

PAK4 pS181

FITC-Ahx-DKRPLpSGPDVG-CONH2

Ac-DKRPLpSGPDVG- CONH2

PAK6 pT99

FITC-Ahx-VISSNpTLRGRS-CONH2

Ac-VISSNpTLRGRS- CONH2

PAK6 pS113

FITC-Ahx-RRRAQpSLGLLG-CONH2

Ac-RRRAQpSLGLLG- CONH2

PAK6 pS231

FITC-Ahx-EARPQpSCLVGS-CONH2

Ac-EARPQpSCLVGS- CONH2

PAK6 pT251

FITC-Ahx-SPSPKpTRESSL-CONH2

Ac-SPSPKpTRESSL- CONH2

PAK6 pT99+pS113

FITC-AhxVISSNpTLRGRSPTSRRRAQpSLGLLGCONH2

AcVISSNpTLRGRSPTSRRRAQpSLGLLGCONH2

CRaf pS233

FITC-Ahx-SQHRYpSTPHAF-CONH2

Ac-SQHRYpSTPHAF- CONH2

CRaf pS259

FITC-Ahx-RQRSTpSTPNVH-CONH2

Ac-RQRSTpSTPNVH- CONH2

CRaf pS621

FITC-Ahx-INRSApSEPSLH-CONH2

Ac-INRSApSEPSLH- CONH2

BRaf pS365

FITC-Ahx-RDRSSpSAPNVH-CONH2

Ac-RDRSSpSAPNVH- CONH2

BRaf pS729

FITC-Ahx-IHRSApSEPSLN-CONH2

Ac-IHRSApSEPSLN- CONH2

RapGef2 pS573

FITC-Ahx-GTLSSpSNPDLE-CONH2

Ac-GTLSSpSNPDLE- CONH2

RapGef2 pS572

FITC-Ahx-GGTLSpSSNPDL-CONH2

Ac-GGTLSpSSNPDL- CONH2

RapGef2 pT375

FITC-Ahx-KRRLMpTHLSIT-CONH2

Ac-KRRLMpTHLSIT- CONH2

RapGef2 pT445

FITC-Ahx-ILRNNpTSCANL-CONH2

Ac-ILRNNpTSCANL- CONH2

RapGef2 pT740

FITC-Ahx-KLRSKpTSCANL-CONH2

Ac-KLRSKpTSCANL- CONH2

RapGef2 pS900

FITC-Ahx-VGRMApSVNMDP-CONH2

Ac-VGRMApSVNMDP- CONH2

RapGef2 pS948

FITC-Ahx-RVRRSpSFLNAK-CONH2

Ac-RVRRSpSFLNAK- CONH2

Protein Expression The 14-3-3sDC protein for crystallization was expressed and purified according to the following general protocol. Plasmids were transformed into NiCo21(DE3) cells (New England Biolabs) according to the manufacturers protocol. Prior to purification, cell pellets were thawed and resuspended in 10 mL/g pellet lysis buffer (25 mM Tris, pH = 8.0, 150 mM NaCl, 5% v/v glycerol, 10 mM imidazole, 4 mM BME and 1 mM PMSF). The cells were then lysed twice by homogenization using an EmulsiFlex-C3 homogenizer. The lysate was incubated with benzonase (Merck Millipore) for 15 minutes and then centrifuged at 20000g for 15 minutes. The supernatant applied in overnight circulation at 4 C to a 5 mL HisTrap column pre-equilibrated with 20 CV lysis buffer. The column was then washed with 20 CV wash buffer (25 mM Tris, pH = 8.0, 300 mM NaCl, 5% v/v glycerol, 25 mM imidazole and 4 mM BME) and the protein eluted with 40 mL elution buffer (20 mM HEPES, pH 8.0, 100 mM NaCl, 5% v/v glycerol, 250 mM imidazole and 4 mM BME). The protein was then pipetted into a SpectrumLabs Spectra/Por 10000 Da MWCO dialysis bag together with 1:500 mg/mg TEV protease and dialysed overnight at 4 C against dialysis buffer (25 mM HEPES, pH = 8.0, 100 mM NaCl, 4 mM BME, 2 mM MgCl2). The protein was then applied to a 5 mL HisTrap column, pre-equilibrated with 20 CV dialysis buffer. The flowtrough was captured and concentrated to 50 mg/mL using 10000 Da MWCO Amicon spinfilters. The concentrated protein was then applied to a HiLoad superdex 75 16/60 SEC column using an A¨kta FPLC apparatus. The fractions containing protein were then pooled, concentrated to 50 mg/mL protein, flash frozen in liquid nitrogen and stored at -80 C until further use. The 14-3-3sz protein for fluorescence polarization was expressed and purified according to the following general protocol. Plasmids were transformed into NiCo21(DE3) cells (New England Biolabs) according to the manufacturers protocol. Colonies were grown on LB agar plates + 100 mg/mL ampicillin overnight at 37 C. One colony was picked and precultured in 25 mL sterile LB medium + 100 mg/mL ampicillin overnight at 37 C. 2 TB medium + 100 mg/mL ampicillin was then inoculated with the preculture and incubated at 37 C until an OD600 of 1.0 was reached. Protein expression was then induced with 0.4 mM IPTG and the cells incubated overnight at 18 C. Cells were then harvested by ultracentrifugation at 20000 rpm at 4 C, flash frozen in liquid nitrogen and stored at -80 C. Prior to purification, the cell pellets were thawed and resuspended in 10 mL/g pellet lysis buffer (25 mM Tris, pH = 8.0, 150 mM NaCl, 5% v/v glycerol, 10 mM imidazole, 4 mM BME and 1 mM PMSF). The cells were then lysed twice by homogenization using an EmulsiFlex-C3 homogenizer. The lysate was incubated with benzonase (Merck Millipore) for 15 minutes and then centrifuged at 20000g for 15 minutes. The supernatant applied in overnight circulation at 4 C to a 5 mL HisTrap column pre-equilibrated with 20 CV lysis buffer. The column was then washed with 20 CV wash buffer (25 mM Tris, pH = 8.0, 300 mM NaCl, 5% v/v glycerol, e5 Cell Chemical Biology 27, 1–11.e1–e6, June 18, 2020

Please cite this article in press as: Kaplan et al., Polypharmacological Perturbation of the 14-3-3 Adaptor Protein Interactome Stimulates Neurite Outgrowth, Cell Chemical Biology (2020), https://doi.org/10.1016/j.chembiol.2020.02.010

25 mM imidazole and 4 mM BME) and the protein eluted with 40 mL elution buffer (20 mM HEPES, pH 8.0, 100 mM NaCl, 5% v/v glycerol, 250 mM imidazole and 4 mM BME). The protein was then pipetted into a SpectrumLabs Spectra/Por 10000 Da MWCO dialysis bag and dialysed overnight at 4 C against dialysis buffer (25 mM HEPES, pH = 8.0, 100 mM NaCl, 4 mM BME, 2 mM MgCl2). The protein was then concentrated to 50 mg/mL using 10000 Da MWCO Amicon spinfilters, flash frozen in liquid nitrogen and stored at -80 C until further use. Fluorescence Polarization FP assays were performed in 10 mM HEPES, pH 7.4, 150 mM NaCl, 1.0 mg/mL BSA and 0.01% v/v Tween-20 buffer. Fluorescently labeled peptides were dissolved to 10 nM in FP buffer as a mastermix. The desired compounds were then added from DMSO stock as required, to a final DMSO content of 1%. This solution was used to fill Corning Low-binding Black Round Bottom 384-well plates (Corning #4514) with a final volume of 10 mL per well. A two-fold dilution series was then performed with the 14-3-3 proteins, starting from 274 mM for 14-3-3z. Fluorescence polarization was then measured in a Tecan Infinite F500 platereader, using 485 (20) nm excitation and 535 (20) nm emission filters. The obtained anisotropy values were then plotted and fitted against a 4-parameter one site binding model in GraphPad Prism. Crystallization Crystals of the binary complex of RapGef2_pT740 and PAK6_pT99 and 14-3-3sDC were grown by mixing 10 mg/mL 14-3-3sDC in a molar ratio of 1 : 2 with the appropriate acetylated peptide in 10 mM HEPES pH 7.4, 150 mM NaCl and 2 mM BME and incubating overnight at 277 K. The formed complex was then set up for crystallization by mixing 1:1 with 0.095 M HEPES pH 7.1, 28% PEG400, 0.19 M CaCl2 and 5% glycerol at 277 K. Crystals grew within a week. Compound 3b was then soaked into the binary crystals by placing a grain of solid 3b into the drops containing binary crystals. Unsoaked crystals were fished directly and soaked crystals after a week of incubation and flash-cooled in liquid nitrogen. X-ray diffraction data was collected using an in-house Rigaku Micromax-003 sealed tube X-ray source and a Dectris Pilatus 200K detector at 100K. The data was indexed and integrated using xia2 DIALS(Winter et al., 2013) and scaled and merged using Aimless(Evans and Murshudov, 2013; Karplus and Diederichs, 2012) Phasing was done by molecular replacement using Phaser(McCoy et al., 2007; Winn et al., 2011) and 3P1O as a starting model and was followed by iterative rounds of refinement and manual model building using Phenix.Refine(Adams et al., 2010; Liebschner et al., 2019) and Coot(Emsley and Cowtan, 2004; Emsley et al., 2010) respectively. Model validation was performed using MolProbity(Chen et al., 2010; Williams et al., 2018) prior to PDB submission. Figures were created using PyMol. QUANTIFICATION AND STATISTICAL ANALYSIS All statistical tests were performed with GraphPad Prism 6. As indicated in figure legends, the following statistical tests were used: unpaired t-test, one-way ANOVA with Dunnett, Bonferroni test, Two-way ANOVA with Dunnett or Bonferroni post-test. Sample sizes are indicated in figure legends and significance was defined as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. NS=not significant. DATA AND CODE AVAILABILITY Proteomics data generated in this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD017317 and 10.6019/PXD017317. The x-ray crystal structures generated in this study have been deposited to the Protein Data Bank (PDB codes: 6QDR, 6QDS, 6QDT, 6QDU).

Cell Chemical Biology 27, 1–11.e1–e6, June 18, 2020 e6