Small molecule inhibitors reveal an indispensable scaffolding role of RIPK 2 in NOD 2 signaling

Article19 July 2018Open Access Source DataTransparent process Small molecule inhibitors reveal an indispensable scaffolding role of RIPK2 in NOD2 signaling Matous Hrdinka Matous Hrdinka orcid.org/0000-0002-2981-2825 Nuffield Department of Clinical Medicine, Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK Search for more papers by this author Lisa Schlicher Lisa Schlicher Nuffield Department of Clinical Medicine, Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK Search for more papers by this author Bing Dai Bing Dai Department of Developmental, Molecular & Chemical Biology, Tufts University School of Medicine, Boston, MA, USA Search for more papers by this author Daniel M Pinkas Daniel M Pinkas Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK Search for more papers by this author Joshua C Bufton Joshua C Bufton Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK Search for more papers by this author Sarah Picaud Sarah Picaud Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK Search for more papers by this author Jennifer A Ward Jennifer A Ward Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK Nuffield Department of Clinical Medicine, Target Discovery Institute, University of Oxford, Oxford, UK Search for more papers by this author Catherine Rogers Catherine Rogers Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK Nuffield Department of Clinical Medicine, Target Discovery Institute, University of Oxford, Oxford, UK Search for more papers by this author Chalada Suebsuwong Chalada Suebsuwong Department of Chemistry, University of Houston, Houston, TX, USA Search for more papers by this author Sameer Nikhar Sameer Nikhar Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, TX, USA Search for more papers by this author Gregory D Cuny Gregory D Cuny Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, TX, USA Search for more papers by this author Kilian VM Huber Kilian VM Huber orcid.org/0000-0002-1103-5300 Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK Nuffield Department of Clinical Medicine, Target Discovery Institute, University of Oxford, Oxford, UK Search for more papers by this author Panagis Filippakopoulos Panagis Filippakopoulos Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK Search for more papers by this author Alex N Bullock Alex N Bullock Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK Search for more papers by this author Alexei Degterev Corresponding Author Alexei Degterev [email protected] orcid.org/0000-0002-8240-7132 Department of Developmental, Molecular & Chemical Biology, Tufts University School of Medicine, Boston, MA, USA Search for more papers by this author Mads Gyrd-Hansen Corresponding Author Mads Gyrd-Hansen [email protected] orcid.org/0000-0001-5641-5019 Nuffield Department of Clinical Medicine, Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK Search for more papers by this author Matous Hrdinka Matous Hrdinka orcid.org/0000-0002-2981-2825 Nuffield Department of Clinical Medicine, Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK Search for more papers by this author Lisa Schlicher Lisa Schlicher Nuffield Department of Clinical Medicine, Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK Search for more papers by this author Bing Dai Bing Dai Department of Developmental, Molecular & Chemical Biology, Tufts University School of Medicine, Boston, MA, USA Search for more papers by this author Daniel M Pinkas Daniel M Pinkas Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK Search for more papers by this author Joshua C Bufton Joshua C Bufton Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK Search for more papers by this author Sarah Picaud Sarah Picaud Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK Search for more papers by this author Jennifer A Ward Jennifer A Ward Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK Nuffield Department of Clinical Medicine, Target Discovery Institute, University of Oxford, Oxford, UK Search for more papers by this author Catherine Rogers Catherine Rogers Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK Nuffield Department of Clinical Medicine, Target Discovery Institute, University of Oxford, Oxford, UK Search for more papers by this author Chalada Suebsuwong Chalada Suebsuwong Department of Chemistry, University of Houston, Houston, TX, USA Search for more papers by this author Sameer Nikhar Sameer Nikhar Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, TX, USA Search for more papers by this author Gregory D Cuny Gregory D Cuny Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, TX, USA Search for more papers by this author Kilian VM Huber Kilian VM Huber orcid.org/0000-0002-1103-5300 Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK Nuffield Department of Clinical Medicine, Target Discovery Institute, University of Oxford, Oxford, UK Search for more papers by this author Panagis Filippakopoulos Panagis Filippakopoulos Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK Search for more papers by this author Alex N Bullock Alex N Bullock Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK Search for more papers by this author Alexei Degterev Corresponding Author Alexei Degterev [email protected] orcid.org/0000-0002-8240-7132 Department of Developmental, Molecular & Chemical Biology, Tufts University School of Medicine, Boston, MA, USA Search for more papers by this author Mads Gyrd-Hansen Corresponding Author Mads Gyrd-Hansen [email protected] orcid.org/0000-0001-5641-5019 Nuffield Department of Clinical Medicine, Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK Search for more papers by this author Author Information Matous Hrdinka1,7,‡, Lisa Schlicher1,‡, Bing Dai2, Daniel M Pinkas3, Joshua C Bufton3,8, Sarah Picaud3, Jennifer A Ward3,4, Catherine Rogers3,4, Chalada Suebsuwong5,9, Sameer Nikhar6, Gregory D Cuny6, Kilian VM Huber3,4, Panagis Filippakopoulos3, Alex N Bullock3, Alexei Degterev *,2 and Mads Gyrd-Hansen *,1 1Nuffield Department of Clinical Medicine, Ludwig Institute for Cancer Research, University of Oxford, Oxford, UK 2Department of Developmental, Molecular & Chemical Biology, Tufts University School of Medicine, Boston, MA, USA 3Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Oxford, UK 4Nuffield Department of Clinical Medicine, Target Discovery Institute, University of Oxford, Oxford, UK 5Department of Chemistry, University of Houston, Houston, TX, USA 6Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, TX, USA 7Present address: Department of Haematooncology, University Hospital Ostrava, Ostrava-Poruba, Czech Republic 8Present address: Department of Biochemistry, University of Bristol, Bristol, UK 9Present address: Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 617 636 0491; E-mail: [email protected] *Corresponding author. Tel: +44 (0) 1865 617508; E-mail: [email protected] The EMBO Journal (2018)37:e99372https://doi.org/10.15252/embj.201899372 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract RIPK2 mediates inflammatory signaling by the bacteria-sensing receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a proposed strategy to ameliorate NOD-mediated pathologies. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling and show that RIPK2 inhibitors function instead by antagonizing XIAP-binding and XIAP-mediated ubiquitination of RIPK2. We map the XIAP binding site on RIPK2 to the loop between β2 and β3 of the N-lobe of the kinase, which is in close proximity to the ATP-binding pocket. Through characterization of a new series of ATP pocket-binding RIPK2 inhibitors, we identify the molecular features that determine their inhibition of both the RIPK2-XIAP interaction, and of cellular and in vivoNOD2 signaling. Our study exemplifies how targeting of the ATP-binding pocket in RIPK2 can be exploited to interfere with the RIPK2-XIAP interaction for modulation of NOD signaling. Synopsis Bacteria-sensing NOD receptors promote inflammatory signalling by stimulating XIAP-dependent RIPK2 ubiquitination. New RIPK2 kinase inhibitors act by blocking RIPK2-XIAP binding, thus revealing a strategy to impair NOD signalling independently of RIPK2 kinase activity. RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling. RIPK2 kinase inhibitors block NOD2 signaling by antagonizing RIPK2-XIAP interaction. Arg36 and Arg41 in RIPK2 kinase domain form a basic patch critical for XIAP interaction. RIPK2 inhibitors occluding the deep pocket behind the ATP-binding site display potent inhibition of NOD2 signaling. Introduction Receptor-interacting kinases (RIPKs) are components of innate immune receptor signaling complexes where they become ubiquitinated and contribute to NF-κB-mediated inflammatory signaling and cell death (Hrdinka & Gyrd-Hansen, 2017; Annibaldi & Meier, 2018). The intracellular bacteria-sensing receptors NOD1 and NOD2 (nucleotide-oligomerization domain-containing proteins 1 and 2) stimulate inflammatory signaling by promoting RIPK2 ubiquitination in response to binding of iE-DAP (D-glutamyl-meso-diaminopimelic acid) and MDP (muramyl dipeptide) constituents of bacterial peptidoglycan, respectively (Girardin et al, 2003). NOD1/2 signaling contributes to gastro-intestinal immunity (Philpott et al, 2014), and genetic variants in NOD2 are the strongest susceptibility factors to Crohn's disease—one of the two major inflammatory bowel diseases afflicting millions in Europe and North America alone (Hugot et al, 2001; Ogura et al, 2001; Ananthakrishnan, 2015). Mutations of NOD2 have also been implicated in other auto-inflammatory granulomatous pathologies such as Blau's syndrome and early-onset sarcoidosis (Caso et al, 2015). Stimulation of NOD2 recruits RIPK2 along with several ubiquitin (Ub) ligases, including IAP (Inhibitor of Apoptosis) proteins and LUBAC (linear ubiquitin chain assembly complex) (Hasegawa et al, 2008; Bertrand et al, 2009; Tao et al, 2009; Damgaard et al, 2012; Yang et al, 2013; Watanabe et al, 2014). These ligases, together with deubiquitinases, coordinate the conjugation of Lys63- and Met1-linked Ub chains (Lys63-Ub and Met1-Ub) on RIPK2 to facilitate signal transduction (Hitotsumatsu et al, 2008; Fiil et al, 2013; Draber et al, 2015; Hrdinka et al, 2016). Lys63-Ub and Met1-Ub are central for productive innate immune signaling and transcription of nuclear factor-κB (NF-κB) target genes (Hrdinka & Gyrd-Hansen, 2017). Lys63-Ub is recognized by the TAK1-TAB 2/3 (TGFβ-activated kinase 1; TAK1-binding protein 2/3) kinase complex, and Met1-Ub is bound by the IKK (IκB kinase) complex through the subunit NEMO (NF-κB essential modifier; also known as IKKγ). In turn, the kinase complexes are activated, leading to phosphorylation, ubiquitination, and degradation of the NF-κB inhibitory factor IκBα and activation of MAP kinases (Hrdinka & Gyrd-Hansen, 2017). XIAP (X-linked IAP) is indispensable for NOD2 signaling and familial mutations in XIAP that impact on its function cause severe immunodeficiency with variable clinical presentation, including early-onset chronic colitis in ~20% of afflicted individuals (Bauler et al, 2008; Krieg et al, 2009; Damgaard et al, 2012, 2013; Speckmann et al, 2013; Pedersen et al, 2014). The ubiquitination of RIPK2 by XIAP facilitates recruitment of LUBAC (Damgaard et al, 2012), which in turn conjugates Met1-Ub on RIPK2 (Fiil et al, 2013). Previous data using small molecule inhibitors suggested that catalytic activity of RIPK2 may contribute to XIAP-mediated RIPK2 ubiquitination (Canning et al, 2015; Nachbur et al, 2015). Consequently, the activity of RIPK2 has been implicated in a subset of systemic granulomatous inflammatory diseases (Jun et al, 2013) and, in particular, ablation of Ripk2 or inhibition of RIPK2 by small-molecule kinase inhibitors showed benefits in mouse models of multiple sclerosis (Shaw et al, 2011; Nachbur et al, 2015) and Crohn's disease-like ileitis (Tigno-Aranjuez et al, 2014), positioning RIPK2 as a new target against human inflammatory diseases. However, the molecular basis for the cross-talk between the kinase activity of RIPK2 and its role as a critical ubiquitinated scaffold downstream of NOD1/2 remains enigmatic. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling, show that RIPK2 inhibitors function instead by antagonizing XIAP-binding and XIAP-mediated ubiquitination of RIPK2, and identify structural features of RIPK2 required for XIAP binding and for the design of efficient small molecule inhibitors of NOD1/2-RIPK2-dependent signaling. Our study exemplifies how targeting the ATP-binding pocket in RIPK2 can be exploited to interfere with the RIPK2-XIAP interaction for modulation of NOD signaling. Results RIPK2 kinase activity is dispensable for NOD2 signaling Previous reports showed that tyrosine-kinase inhibitors such as ponatinib, gefitinib, and the RIPK2-selective kinase inhibitors GSK583 and WEHI-345 inhibit cellular responses to the NOD2 agonist MDP (or L18-MDP) by antagonizing RIPK2 function (Tigno-Aranjuez et al, 2010; Canning et al, 2015; Nachbur et al, 2015; Haile et al, 2016). In concordance, ponatinib and GSK583 inhibited the degradation of IκBα and NF-κB-mediated production of the chemokine CXCL8 in a dose-dependent manner in U2OS/NOD2 cells stimulated with L18-MDP (Fig 1A–C). Of note, the U2OS/NOD2 cells used in this study express doxycycline (DOX)-inducible HA-NOD2 and respond to L18-MDP without addition of DOX due to leakiness of the promoter (Fiil et al, 2013). Small molecule kinase inhibitors are categorized into multiple classes, depending on their mode of binding (Roskoski, 2016). This includes type I inhibitors that interact exclusively within the ATP-binding pocket, type II inhibitors that bind both to the ATP, and an additional back pocket created when the activation segment of a kinase adopts an inactive conformation, and type III molecules that bind exclusively to this allosteric back pocket. Curiously, we observed that a subset of known RIPK2 inhibitors belonging to different classes displayed potent (nanomolar) cellular activities, including ponatinib (a type II inhibitor) and GSK583 (an ATP-competitive type I inhibitor), and that these molecules also antagonized NOD2-mediated ubiquitination of RIPK2 (Figs 1C and EV1A; Canning et al, 2015). This implied that the kinase activity of RIPK2 is required for its ubiquitination and, thus, for NOD2 responses. To directly investigate this, we first ablated RIPK2 (RIPK2 KO) by CRISPR-mediated gene editing in U2OS/NOD2 cells (Fig EV1B and C). As expected, degradation of IκBα and production of CXCL8 in response to L18-MDP were completely inhibited in RIPK2 KO cells (Park et al, 2007; Fig 1D and E). Reintroduction of wild-type (WT) RIPK2 restored RIPK2 ubiquitination and CXCL8 production, and partially restored IκBα degradation, confirming that the signaling defect was due to the absence of RIPK2 (Fig 1D and E). Next, RIPK2 KO cells were reconstituted with kinase-dead human RIPK2 variants in which the ATP-binding lysine 47 was substituted for arginine (K47R) or the catalytic aspartate 146 in the "HRD" motif (HHD in human RIPK2) was substituted for asparagine (D146N) (Pellegrini et al, 2017; Figs 1F and G, and EV1D). Strikingly, introduction of both kinase-dead RIPK2 mutants restored NOD2 signaling and CXCL8 production to a similar level as with WT RIPK2 in two independent RIPK2 KO clones, showing that the catalytic function is not needed for RIPK2's role in NOD2-dependent inflammatory signaling (Figs 1G and H, and EV1E). Figure 1. RIPK2 kinase activity is dispensable for NOD2 signaling A, B. Intracellular flow cytometry analysis of CXCL8 in U2OS/NOD2 cells treated with L18-MDP (200 ng/ml, 4 h) and kinase inhibitors ponatinib (A) or GSK583 (B) as indicated. C. Purification of Ub-conjugates using TUBE pulldowns from U2OS/NOD2 cells after treatment with L18-MDP (200 ng/ml, 1 h) and ponatinib or GSK583 as indicated. Purified material and lysates were analyzed by immunoblotting, with actin as a loading control. D. Intracellular flow cytometry analysis of CXCL8 following L18-MDP treatment (200 ng/ml, 4 h) of parental U2OS/NOD2 cells and RIPK2 KO cells (clone C5-2) reconstituted or not with RIPK2. E. Purification of Ub-conjugates from parental U2OS/NOD2 cells and RIPK2 KO cells (clone B7-1) reconstituted or not with RIPK2, treated with L18-MDP (200 ng/ml, 1 h). Purified material and lysates were analyzed by immunoblotting. Asterisk indicates a non-specific signal in the TUBE pulldown samples that co-migrates with the signal for unmodified RIPK2. F. Schematic representation of RIPK2. Numbering below schematic refers to amino acid residues in human RIPK2 and indicate domain boundaries. K47 and D146 are catalytic residues for ATP hydrolysis. G. Immunoblot analysis of U2OS/NOD2 RIPK2 KO cells (clone C5-2) reconstituted with RIPK2 variants or empty vector as indicated and stimulated (or not) with L18-MDP (200 ng/ml, 1 h). H. Intracellular flow cytometry analysis of CXCL8 following L18-MDP treatment (200 ng/ml, 4 h) of cells described in (G). Data information: Data in (A, B, D, H) represent the mean ± SEM of 2–4 independent experiments, each performed in duplicate. Statistical significance in (A) and (B) is determined in relation to L18-MDP-stimulated samples without inhibitor. **P < 0.01, n.s., not significant. Two-way ANOVA was used to determine statistical significance. See also Fig EV1. Source data are available online for this figure. Source Data for Figure 1 [embj201899372-sup-0008-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. RIPK2 kinase-dead mutants support NOD2 signaling Purification of Ub-conjugates from THP-1 cells after treatment with L18-MDP (200 ng/ml, 1 h) and ponatinib or GSK583 as indicated. Purified material and lysates were analyzed by immunoblotting. Schematic representation of RIPK2 mRNA and protein with indicated positions of RIPK2 gRNA sequences (CRISPR/Cas9 B and C) and epitopes for RIPK2 antibodies used in this study. E1–E11 indicates RIPK2 exons. Identification RIPK2 knockout U2OS/NOD2 cell clones grown from cell cultures transfected with CRISPR/Cas9 vectors targeting RIPK2. Clones B7-1 and C5-2 were used in the study. Radioactive in vitro RIPK2 kinase assay with RIPK2 variants expressed in U2OS/NOD2 RIPK2 KO cells and purified with anti-HA. The in vitro phosphorylated RIPK2 and universal kinase substrate MBP were separated by SDS–PAGE and exposed to X-ray film. The inputs and precipitated proteins were analyzed by immunoblotting. Intracellular flow cytometry analysis of CXCL8 following L18-MDP treatment (200 ng/ml, 4 h) of U2OS/NOD2 RIPK2 KO cells (clone B7-1) reconstituted with RIPK2 variants or empty vector as indicated. Data information: Data represent the mean ± SEM of at least three independent experiments. *P < 0.05, **P < 0.01, n.s., not significant. Two-way ANOVA was used to determine statistical significance. Source data are available online for this figure. Download figure Download PowerPoint RIPK2 ubiquitination in response to L18-MDP was also not affected by the K47R and D146N mutations, which is surprising since kinase inhibitors blocked RIPK2 ubiquitination (Figs 1C and 2A). Moreover, ponatinib prevented ubiquitination of the kinase-dead RIPK2 variants after NOD2 stimulation and antagonized their capacity to induce NF-κB activation (Fig 2A and B), suggesting that the inhibition of RIPK2 ubiquitination and NOD2 signaling by ponatinib is independent of its inhibition of RIPK2 kinase activity. Although RIPK2 is a high-affinity cellular target of ponatinib, the molecule is a promiscuous kinase inhibitor (Fauster et al, 2015; Najjar et al, 2015; Appendix Fig S1A; Dataset EV1). To determine whether ponatinib's inhibitory activity was a result of its binding to RIPK2, we substituted the threonine 95 "gatekeeper" residue with a bulky tryptophan (T95W) to prevent ponatinib's binding to the RIPK2 ATP-binding pocket (Fig 2C). We first confirmed that the T95W mutation indeed ablated the binding of ponatinib to RIPK2 in cells at the concentrations used to inhibit signaling using a nano-bioluminescence resonance energy transfer (nanoBRET) assay (Vasta et al, 2018) and cellular thermal shift assay (CETSA; Jafari et al, 2014), which measure cellular target engagement. Use of our recently reported ponatinib-derived kinase tracer SGC-590001 (Vasta et al, 2018) in conjunction with nanoLuc-RIPK2 showed that the T95W mutation prevented detectable interaction at inhibitor concentrations up to more than 100 nM (Fig 2D). In accordance with this and with previous findings (Canning et al, 2015), ponatinib (300 nM) induced a substantial thermal shift indicative of binding to WT RIPK2 (and K47R RIPK2), but this was not observed for the RIPK2 T95W and K47R+T95W mutants (Appendix Fig S1B). Introduction of the T95W mutation, either alone or in combination with kinase-dead RIPK2 substitutions, largely abolished the inhibitory effect of ponatinib on RIPK2-induced NF-κB activation (Fig 2B) and on NOD2 signaling as determined by RIPK2 ubiquitination, IκBα degradation, phosphorylation of NF-κB (p65), and production of CXCL8 (Fig 2E and F). Together, these observations show that ponatinib inhibits NOD2 signaling through its binding to RIPK2 but not by inhibiting the kinase activity of RIPK2. Figure 2. Ponatinib antagonizes NOD2 signaling through binding to RIPK2 but independently of its kinase activity Purification of Ub-conjugates from U2OS/NOD2 RIPK2 KO cells (clone C5-2) reconstituted with RIPK2 variants or vector as indicated and treated with L18-MDP (200 ng/ml, 1 h) and/or ponatinib. Purified material and lysates were analyzed by immunoblotting. Asterisk indicates a non-specific signal in the TUBE pulldown samples that co-migrates with the signal for unmodified RIPK2. NF-κB activity in lysates of HEK293FT cells transfected with dual luciferase NF-κB reporters and HA-RIPK2, and treated with DMSO or ponatinib (200 nM, 24 h) as indicated. Relative luciferase activity in ponatinib-treated samples is shown relative to the activity in the corresponding HA-RIPK2 transfected sample not treated with inhibitor. Structure of the RIPK2 kinase domain in complex with ponatinib (green) (PDB ID: 4C8B). Sticks are shown for catalytic residues Lys47 (blue) and Asp146 (red), and the gatekeeper residue Thr95 (yellow). NanoBRET assay in HEK293 cells transiently transfected with the NanoLuc-RIPK2. Cells were treated with serial dilutions of SGC-590001 probe and incubated with ponatinib (1 μM) or DMSO as a control for 3 h before measurement of BRET ratios. Gray box indicates range of ponatinib concentrations used in signaling experiments in this study. Purification of Ub-conjugates from U2OS/NOD2 RIPK2 KO cells reconstituted with RIPK2 variants or vector as indicated and treated with L18-MDP (200 ng/ml, 1 h) and/or ponatinib (100 nM). Purified material and lysates were analyzed by immunoblotting. Intracellular flow cytometry analysis of CXCL8 of U2OS/NOD2 RIPK2 KO cells reconstituted retrovirally with RIPK2 variants or vector as indicated and treated with L18-MDP (200 ng/ml, 4 h) and ponatinib (50 nM). Values represent CXCL8-positive cells relative to L18-MDP treatment for each RIPK2 variant or empty vector. Data information: Data in (B, D, F) represent the mean ± SEM of 3–4 independent experiments, each performed in duplicate. **P < 0.01, n.s., not significant. Two-way ANOVA was used to determine statistical significance. See also Appendix Fig S1. Source data are available online for this figure. Source Data for Figure 2 [embj201899372-sup-0009-SDataFig2.pdf] Download figure Download PowerPoint Development of a new series of potent small molecule RIPK2 inhibitors Ponatinib displays highly promiscuous inhibitory activity (Fauster et al, 2015; Najjar et al, 2015), and clinical development of GSK583 was halted (Haile et al, 2016), raising a need for new classes of RIPK2 inhibitors. We have developed a new chemical series of RIPK2 inhibitors, termed CSLP. A subset of these inhibitors, exemplified by CSLP37 and CSLP43, displayed excellent potency in the NOD2/HEKBlue reporter assay, measuring NF-κB activation in response to L18-MDP [Table 1; full details of the synthesis and structure–activity relationship (SAR) will be reported separately (CS, BD, DMP, ALD, LL, MH, LS, MGH, MHU, ANB, AD, GDC, manuscript in preparation)]. However, correlation analysis of an entire panel of CSLP analogs revealed a startling disparity between inhibition of RIPK2 kinase activity in vitro and suppression of the NOD2/RIPK2 pathway in cells. Specifically, while many CSLP inhibitors displayed comparably potent activity against RIPK2 kinase activity in vitro, only a small subset of these compounds, i.e., CSLP37/43, provided potent suppression of cellular NOD2/RIPK2-dependent responses as determined by L18-MDP-induced CXCL8 production in U2OS/NOD2 cells and NF-κB activation in NOD2/HEKBlue cells (Figs 3A and B, and EV2A; Table 1). Of note, CSLP37/43 and other CSLP inhibitors did not have measurable toxicity in the cell lines used in this study (Fig EV2B). Additionally, CSLP37 and CSLP43 showed no inhibitory activity against the two closest mammalian homologs of RIPK2, RIPK1, and RIPK3 (Fig EV2C). Table 1. In vitro and cellular activities of CSLP analogs Compound ID X R1 R2 R3 IC50 (nM) In vitro kinase Cellular activity RIPK2 ADPGIo HEKBlue NOD2 nanoBRET RIPK2 binding nanoBRET residence time, min CSLP43 NH2 OMe OMe -NHSO2nPr 19.9 ± 0.8 1.3 ± 0.4 10.1 ± 3.8 106.9 CSLP37 NH2 F OMe -NHSO2nPr 16.3 ± 4.6 26.3 ± 3.7 36.3 ± 20.2 27.1 CSLP18 NH2 H OMe -NHSO2nPr 31.6 ± 8.7 476.0 ± 96.7 577.6 ± 34.1 66.6 CSLP38 NH2 F H -NHSO2nPr 39.1 ± 1.5 740.3 ± 60.7 166.8 ± 19.0 106.5 CSLP48 NH2 H OH -NHSO2nPr 53.5 ± 5.7 > 5,000 1,231.3 ± 344.5 126.7 CSLP53 Me F OMe -NHSO2nPr 1,414.5 ± 311.6 2,556.5 ± 252.8 > 10,000 ND CSLP55 NH2 F OMe OMe 39.1 ± 3.9 595.1 ± 69.6 194.6 ± 47.1 6.8 WEHI-345 37.3 ± 1.3 3,370.7 ± 382.8 521.2 ± 171.6 7.5 Compounds were tested against recombinant RIPK2 kinase using ADPGlo assay, L18-MDP-induced NF-κB reporter assay (HEKBlue assay), nanoBRET cellular RIPK2 target engagement assay (in HEKBlue cells), and nanoBRET cellular RIPK2 residence time assay (in HEKBLue cells). ND—not determined due to very poor binding of CSLP53 to RIPK2 in nanoBRET assay. For each inhibitor, at least three titrations were performed and data were used to calculate average IC50 and SD values. Details of each assays are described in the Appendix Supplementary Methods. Chemical structure of the CSLP scaffold and WEHI-345 is shown in above table. Figure 3. CSLP series of RIPK2 inhibitors reveal molecular determinants for NOD2 pathway inhibition Comparison of inhibitory activity of CSLP compounds on in vitro RIPK2 kinase activity (ADPGlo)) and NOD2 signaling in cells (HEKBlue). Compounds characterized further in this study are indicated in red. Intracellular flow cytometry analysis of CXCL8 in U2OS/NOD2 cells treated with L18-MDP (200 ng/ml, 4 h) and CSLP inhibitors as indicated. Data represent the mean of three independent experiments. Chemical structure of CSLP compounds (18, 37, 43) that differ only in R1 group. Structure of RIPK2 kinase domain in complex with CSLP18 (orange) (PDB ID 6FU5). Sticks are shown for catalytic residues Lys47 and Asp146 (in DFG motif), Glu66 forming a salt bridge to Lys47 in active Glu-in conformation, and residues involved in binding of CSLP inhibitors as described in the text. Spacefill rendering of RIPK2 kinase domain structure with CSLP18 (top) and models with CSLP37 (bottom left) and CSLP43 (bottom right). Dark gray represents areas occupied by RIPK2; white areas indicate empty spaces in CSLP binding pocket. Dotted white circle indicates cavity occupied by R1 group of CSLP37/43. Dotted black box indica