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Mapping of Lipid-‐Binding Proteins and Their Ligandability in Cells Crava%, B. F. et. al. Cell 2015, 161, 1668 Hannah Haley Literature Seminar 5 September 2015 tely 10 Å from the intracellular mem233, located near the midpoint of the xternal and internal negative clusters channels, the amino acids forming the conserved ascan glutamate or aspartate. Lipids have structural (e.g. stabilizing membranes or proteins) or signaling roles e.g. F O (C U Seicosanoids) ORNE LVIIPE IW DS gle most conserved amino acid in Kv Role of Lipids in Physiology and Pathophysiology Structural: Signaling: Box 1 | From phospholipids to eicosanoid signalling O X O O P O O– H O O O O cPLA2 X O Arachidonic acid (AA) O –O 8 14 6 11 H O Lyso-PC OH 1 5-LO PGH2S COX1 or COX2 Conventional NSAIDs O O O– + 5 P COX2 inhibitors e.g. aspirin Prostaglandin, prostacyclin and thromboxane synthases Conversion Inhibition Indirect action PGs, TXs Tissue homeostasis PGs, TXs Leukotriene synthases Leukotrienes PAF Inflammation The production of eicosanoids is initiated by the release of C20-polyunsaturated fatty acids, such |asMolecular arachidonic Reviews Biology Arachidonic acid derived molecules mediate both Nature physiological and Cellacid (AA, C20:4), from phospholipids (X stands for a phospholipid headgroup; see figure) or diacylglycerol (not shown; see 2+ pathophysiological ignaling athways cPLA2 lipase action is the rateBOX 2). This hydrolysis is catalysed by cytosolic phospholipasesA2 (cPLA2).pCa -induced Unusually posiMoned lipids region e. a, Side view of the transmembrane limiting step in eicosanoid formation, and cells lacking cPLA2 are generally devoid of eicosanoids. Liberated fatty acids hypothesized to influence structure are then stereospecifically oxygenated either through the cyclic prostaglandin synthase pathway (prostaglandin H2 epresentation, coloured according to atom synthase (PGH2S), including cyclooxygenase (COX) activity) or through the linear lipoxygenase-dependent pathway funcMon of KcsA n; magenta,and phosphorous) andchannel the channel (5-lipoxygenase; 5-LO), and are thus converted into one of four families of eicosanoids: the prostaglandins (PGs), rom the extracellular side of the membrane prostacyclins, thromboxanes (TXs) and leukotrienes. Eicosanoids short half-life, ranging from R. et. al. Nature 2007, 4at 50, the 376; Wymann, M. P., Schneiter, R. Nhave at. Raev. Mol. Cell Biol. 2008, 9, 1seconds 62 to minutes. Their prime mode of action is mediated by binding n MacKinnon, sphere. The lipid observed Chemical Proteomic Probes to Characterize Lipid-‐Protein Interac>ons ental Figures Probe design based on small molecule-‐protein binding affinity and light-‐induced crosslinking to capture protein Design elements: a) Small molecule to be recognized by protein (“lipid element”) Lipid b) PhotoreacMve element that covalently links Probe small molecule B A C and protein upon UV irradiaMon c) Alkyne to allow late-‐stage conjugaMon to azide tag via Cu-‐ 1. CuAAC catalyzed alkyne-‐azide cycloaddiMon b) photoreactive group lipid c) latent element affinity handle Aa) arachidonoyl (20:4) AA-DA kDa N N 150– 100– N H AEA-DA AEA-DA AA-DA B O OH oleoyl (18:1) O N N 100– O probe targets Fluorescent imaging N H C N N Me OH O N Ngel OEA-DA O H N OH N N arachidonoyl (20:4) kDa 150– A-DA OH Crosslinked palmitoyl (16:0) H N A-DA O-DA S-DA Alkyne affinity handle arachidonoyl (20:4) AEA-DA AA-DA Cells 2. SDS-PAGE 2. Cell lysis Set of lipid probes: Diazirine photocroslinking group rhodamine-azide oleoyl (18:1) kDa 150– O PEA-DA H N Me kDa 150– stearoyl (18:0) N N O-DA 100– A-DA O-DA S-DA Lipid probes Rh N3 1. UV Light 100– H N O Me N N S-DA Crava%, B. F. et. al. Cell 2015, 161, 1668 mentalCharacteriza>on Figuresof Lipid Probe Targets A photoreactive group lipid element Lipid Probe latent affinity handle A B C 1. CuAAC Rh N3 1. UV Light rhodamine-azide 2. SDS-PAGE 2. Cell lysis ve latent affinity handle Crosslinked probe targets kDa 150– 100– 75– 1. UV Light kDa 150– 100–azide Rhodamine 1. CuAAC fluorescent reporter tag N3 Rh75– C kDa 150– Lipid Probe Me A B C 100– Me +N O 75– rhodamine-azide Me 2. Cell lysis 50– 2. SDS-PAGE 50– Fluorescent gel imaging A-DA O-DA S-DA Cells incubated with probe for 30 min before UV AEA-DA AA-DA B Cells AEA-DA AA-DA igures N 50– O N H CO2 - IdenHficaHon of target proteins kDa 150– N3 100– 75– Me 50– A-DA O-DA S-DA Lipid probes Lipid Probes Differen>ally Label Proteins 2. SDS-PAGE 2. SDS-PAGE 2. Cell 2. lysis Cell lysis AEA-DA O N H OH N N AA-DA A-DA kDakDa 150– 150– D A-DA O-DA A-DA S-DA O-DA S-DA C A-DA O-DA A-DA S-DA O-DA S-DA CC Fluorescent Fluorescent gel imaging gel imaging kDakDa 150– 150– kDakDa 150– 150– 100– 100– 100– 100– 75–75– 75–75– 100– 100– 100– 100– 75– 75– 75–75– 50– 50– 50–50– 50–50– 50–50– 37– 37– 37–37– 37–37– 37–37– 25– 25– 25–25– 25–25– 25–25– O OH N B AA-DA kDakDa 150– 150– Crosslinked Crosslinked probe targets probe targets AEA-DA AA-DA AEA-DA Lipid probes: AA-DA BB AEA-DA AA-DA AEA-DA Cells Cells N O N N N H Me membrane membrane HEK293T HEK293T soluble soluble HEK293T HEK293T membrane membrane HEK293T HEK293T soluble soluble HEK293T HEK293T • AA-‐DA probe labels aLipid-Binding lmost exclusively Figure Chemical Proteomic Probes Mapping Proteins in Cells, to • Related LocaMon of Figure diazirine Figure S1. S1. Chemical Proteomic Probes for for Mapping Lipid-Binding Proteins in Cells, Related to Figure 1 1 effects protein membrane proteins (A) Experimental workflow for gel-based profiling of lipid-binding proteins in mammalian cells. Cells were incubated with lipid probes labeling rofile (A) Experimental workflow for gel-based profiling of lipid-binding proteins mammalian cells. Cells were pincubated with lipid probes for f • May be incorporated into in phospho/ small molecules in native biological systems (Lee and Bogy ! ! crosslinking UV light (10 min, C) and subsequent cell lysis. Probe-labeled proteins were then conjugated a rhodamine-azide (Rhcrosslinking withwith UV light (10 min, 4 C)4and subsequent cell lysis. proteins were then to atoirhodamine-azide (Rh-N 3 • conjugated Protein s UV dSome ependent neutral lipids or Probe-labeled lipidated proteins 2013; Simon et al., 2013; Su etlabeling al., 2013). probes re copper-catalyzed azide-alkyne cycloaddition (CuAAC or ‘‘click’’) chemistry to allow for visualization of probe-labeled targets by SDS-PAGE copper-catalyzed azide-alkyne cycloaddition (CuAAC or ‘‘click’’) chemistry to allow for visualization of probe-labeled targets by SDS-PAGE andanin onprobes innate chemical reactivity with protein residues, wherea • Decide t o u se f a%y a cid a mide scanning. scanning. others exploit andcells. light-induced crosslinking (B) Membrane and soluble protein labeling profiles for the AEA-DA and AA-DA probesbinding (20 mM) affinity in HEK293T Note that the AA-DA prober 1. UV(S-DA) Light acyl chains, as wellrhodamine-azide as photoreactive (diazirine) and alkyne groups. 2. SDS-PAGE 2. Cell (B)lysis AEA-DA and A-DA probes show overlapping but distinct protein interaction profiles in HEK293T cells. Cells were treated with each probe (20 mM) Crosslinked targets for 30 min probe in situ before photocrosslinkingFluorescent and gel imaging analysis of probe-modified proteins as described in Figure S1. (C) Arachidonoyl probe labeling of membrane and soluble proteins depends on UV irradiation of cells. kDa kDa(D) Comparative labeling profiles of kDa lipid probes 150– 150– 150– (20 mM, 30 min) in HEK293T cells. Red and blue arrows mark representative proteins preferentially 100– 100– 100– labeled by arachidonoyl and oleoyl/palmitoyl Preferential 75– 75– probes, respectively. See Figure S1C for profiles of 75– labeling by A-DA, O-DA, and S-DA. AEA-DAC D O N H N OH kDa 150– N OEA-DA H N B 100– OH 75– O C A-DA O-DA S-DA Lipid probes: A-DA O-DA S-DA Cells AEA-DA AA-DA Lipid Probes Differen>ally Label Proteins AEA-DA AA-DA B Lipid probes arachidonoyl probes N N 50– 50– 50– 50– in human cells by gel-based profiling. HEK293T cells were OH 37– 37– probe 37– treated with 37– Preferential O (AA-DA labeling by or AEA-DA; 20 mM, 30 min), irradiated with UV light (10 min, 4! C), and lysed, oleoyl/ N N and the cell proteomes were fractionated palmitoyl probes into membrane and soluble components O N N A-DA 25– 25– 25– 25– by centrifugation prior to conjugation to N Me a fluorescent reporter tag (Rh-N3) using H CuAAC (Figure S1A). Analysis of probe targets by SDS-PAGE and in-gel fluomembrane soluble membrane soluble rescence scanning revealed distinct H O-DA Me N HEK293T HEK293T HEK293T protein-labeling profiles for each HEK293T probe (Figure S1B). The AA-DA probe showed N N O Figure S1. Chemical Proteomic Probes for Mapping Lipid-Binding Proteins in Cells, Related to Figure 1 almost exclusive labeling of membrane • Polyunsaturated a rachidonoyl p robes ( AEA-‐DA, A -‐DA) d emonstrate more (A) Experimental gel-basedproteins, profiling ofwhich lipid-binding proteins inwas mammalian cells. Cells of were incubated w mall molecules in native biological systems workflow (Lee andforBogyo, we suspected a consequence rapid extensive p rotein l abeling t han m onounsaturated ( OEA-‐DA, O -‐DA) o r s aturated ! withSome UV lightprobes (10 min,rely 4 C) and subsequent cell lysis.probe Probe-labeled proteins were then conjugated S-DA 013; Simon et al., 2013; H Su crosslinking et al.,Me 2013). sequestration of this into membranes through its meta-to a rhoda probes (PEA-‐DA, -‐DA) N copper-catalyzed azide-alkyne cycloaddition (CuAACSor ‘‘click’’) chemistry to allow for visualization of probe-labeled targets b n innate chemical reactivity with protein residues, whereas bolic incorporation into phospho/neutral-lipids or into lipidated scanning. N O thers exploit binding affinity andNlight-induced crosslinking proteins, has been noted for other fatty acidlipid probes (Haber• reChoose to map for pas roteins that interact with arachidonoyl probes (B) Membrane and soluble protein labeling profiles the AEA-DA and AA-DA probes (20 mM) in HEK293T cells. Note that ctions to capture proteins (Heal et al., 2011). The latter group kant et al., 2013; Tate et al., 2015). In contrast, the AEA-DA probe PEA-DA H N Iden>fica>on of Protein Targets with SILAC and LC-‐MS/MS SILAC Ratio 15.0 light/heavy A 10.0 ratio 20.0 UltraCentrifuge Membrane CuACC Biotin-azide Streptavidin enrichment MS1 intensity LC/LC-MS/MS Protein ID and quantification AEA-DA vs AEA-DA 5.0 15.0 3.0 C On-bead trypsin digest C 0.0 10.0 0 Soluble No UV A-DA O-DA S-DA No UV A-DA O-DA S-DA Protein number 20.0 Mix MS1 intensity 0 200 600 Protein number 5.0 3.0 0.0 400 800 0 200 400 600 800 Protein number Protein number D A-DA vs A-DA 15.0 60 10.0 40 30 UV Bas proteins labeled AEA-DA A-DA • Lipid probe No protein targets defined in UV A-DA vs A-DA dependent manner (SILAC raMo ≥ 3.0 in 20probe-‐versus-‐no UV) and not enriched in probe vs. probe control (SILAC raMo < 2.0) 0 20 40 60 80 Light: 10 A-DA Heavy: 1) A-DA 442 (No UV) 2) A-DA H ** ** 20 5.0 3.0 0 0.0 Membrane Soluble 0 200 400 600 800 40 Protein number AEA-DA A-DA OMIM Disease Hematological Renal Developmental 20.0 Reproduction Immune Ratio of light/heavy Psychiatric 15.0 Neurological peaks definesCardiovascular SILAC ratio Cancer 10.0 Metabolic A-DA Selective 0.0 A O-DA/S-DA Selective 3.0 Crosslinked probe targets Light Heavy • SILAC Ratio i) ± UV Light ii) Cell Lysis and 13C6, 15N4arginine enriched 5.0 SNX9 TMED2 IPO7 ACAT2 Comparison heavy cell groups: HMGCS1 a) Same condiMons as light cells (probe-‐versus-‐probe control) NUCB1 20.0 b) Same probe as light cells but no UV (probe-‐versus-‐no UV) No UV PITRM1 AEA-DAKIAA0664 vs AEA-D c) Other lipid probe (OEA-‐DA, O-‐DA, PEA-‐DA, S -‐DA) NUCB2 15.0 200 400 600 800 ALDH1A2 FDFT1 HADHA 10.0 FDPS RTN4IP1 PSMB6 O 5.0 AEA-DA No UV O N NDUFB9 N A-DA 3.0 OH NAMPT A-DA vs N A-DA N Me PMPCA H 0.0 H DCTN2 0 200 400 600 800 N SRPRB N SNX1 AEA-DA A-DA 100 NDUFS2 ZADH2 No UV 20.0 No UV OCIAD1 80 % of Proteins Heavy Cells 10.0 15N -lysine 6, 2 SILAC Ratio Light Cells • Light cells are control and treated with arachidonoyl probe and UV • Heavy cells are comparison A-DA Targets X = OEA,PEA, O or S 13C SILAC Ratio A(EA)-DA or X-DA ILAC Ratio A(EA)-DA AEA-DA vs AEA-DA 15.0 % of Proteins in situ treatment (30 min) SILAC Ratio Stable-‐isotope labeling bBy amino C-‐tandem 20.0 acids in cell culture A-DAM vs.S (LC-‐MS/MS) A-DA vs. No UV(SILAC) and LD A 361-4.0 3170 LMNB1 SLC25A5 SLC25A4 ALG1 SLC25A3 SDHA PDIA6 GANAB SAR1B UBA52 SLC25A6 1000 SLC1A5 RUVBL1 SLC25A11 FKBP4 GOLIM4 ATP5O IRS4 4.0 AEA-DA A-DA Classifica>on of Iden>fied Protein T0 argets 200 AEA-DA Ta E N A O P 20 3.0 N A O P B % of Pr i) 80 ± UV Light ** ii) Cell Lysis 15.0 % of Proteins SILAC Ra AEA-DA vs AEA-DA mRNA Pro MED25 SLC25A44A FECH Electron T HEATR3 40 0 PITRM1 In 5.0 Host-Virus 442 361 317 TNPO3 3.0 Lipid Me OTUB1 20 COPS6 Protein T 0.0 SCARB2 0 200 400 600targets 800 1000 Crosslinked probe RRM2 0 NUDT1 803 678 Membrane Soluble SNX2 NUCB2 40 AHSA1 AEA-DA A-DA mRNA Processing ARF4 23 10 5 Protein Classes CACYBP Apoptosis 44 AEA-DA vs. AEA-DA vs. 30 UltraNUCB1 Electron Transport Other NAMPT Centrifuge PSMB6 Enzymes Host-Virus Interaction 20 TOMM22 263 Transporters FAH Lipid Metabolism 420 Light: Chaperones KIAA0664 Heavy: Protein Transport MED25 PFKL 10 Receptors SLC25A44 0 20 40 60 80 100 120 PCK2 60 10 0.0 400 600 8 M e ito ch us on dr ia C el lM E em R br an e G o Ly l so gi so AEA-DA Selective m e Protein number sm 10.0 N N N H 40 Me op AEA-DA A-DA 20 10 442 361 317 la op yt C 803 678 C uc l N Cancer LRRC59 cells). For instances where multiple isoforms of a given protein are en Heavy: 1) AEA-DA (No UV) Metabolic PTGR2 -4.0 values 0 for the4.0 Figure 2. Mapping Protein Targets of Lipid Probes by Quantitative Proteomics (H)SSR4 Heatmap showing the relative protein enrichment AE 2) AEA-DA Protein ID and quantification 0 20 40 60 80 100 120 LOG TRAM1 3) OEA-DA 0 20 40 60prob 2R (A) Heavy/light SILAC ratio plots for total proteins identified in experiments comparing the labeling profiles of lipid probe itself with (AEA-DA) or without UV irradiation (no UV) as controls, CCDC47 4) PEA-DA R =of Number of Targets S1HMGCS1 for complete list oflight lipidcells probetreated targets.with probe without UV irradiation) or the equivalent probe (both heavy and 20 mM the same p Number ofLight/Heavy Targets s N m M ucl e ito ch us on dr ia C el lM E em R br an e G Ly olg so i so m e 0 Protein Transport A/PEA-DA Selective % of Proteins AEA-DA A-DA 30 Adaptors MS1 intensity yt Protein number 40 409 FECH C 600 800 1000 Membrane Soluble 5.0 3.0 HEATR3 Number ofChannels Targets 0.0 678Soluble RPN1 Membrane0 803 PITRM1 LRRC59 8 0 200 400 600 TNPO3 Cell PTGR2 OTUB1 CuACC Proteins d ifferenHally e nriched b y Mitochon acetyl-CoA F G membrane SSR4 COPS6 Protein number Protein class distribuHon 10Biotin-azide SCARB2 TRAM1 two 23 probes (from HEK293T (human) FASN (6.6) RRM2 2. Mapping Protein Targets of Lipid Probes by CCDC47 Figure 5 Protein Classes CPT1A Quanti (7.9) ACSL 44 and N FATP NUDT1 Streptavidin euro2a (mouse) cells)(A)SNX2 HMGCS1 FA-C FA-CoA FA FA Heavy/light SILAC ratio plots for total proteins identified in experime OMIM Disease enrichment ALDH3A2 Other NUCB2 (18) GPAT SLC25A20 SCARB1 COPS3and probe heavy AHSA1 without UV irradiation) or the equivalent probe (both(20) Biological Enzymes Process (20) (20) On-bead POR E ARF4 Hematological threshold ratio values (R3-fold in no UV experiments) for designation 263 trypsin digest MTDH LPA Transporters CACYBP 420 ILVBL and (B) Venn diagram of shared Renal and unique protein targets of AEA-DA NUCB1 Chaperones mRNA Processing PAP NAMPT TBL2 Developmental (C–F) Analysis of lipid probe targets based on (C) presence (membrane PSMB6 LC/LC-MS/MS SSR1 Receptors Reproduction Apoptosis DAG TOMM22 ELOVL2 subcellular distribution; (E) involvement in specific biological process Adaptors Immune 409 FAH SRPRB PNPLA2 (20) Electron Transport DGAT tations. presented as the mean percentage of total pro KIAA0664Data in (C) are Lipid Probe Targets Psychiatric SPTLC1 Channels ABHD5 (20) PFKL (Nolipid UV probe Ratio) targets (red) TAG Host-Virus Interaction (G) Diagram highlighting in major fattyLSS acid m Neurological PCK2 Light Cardiovascular parentheses next to gene names (data shown are for the A-DA probe i Heavy Lipid Metabolism RPN1 Light: AEA-DA AEA-DA Targets 0400 200 317 la 20 361 OEA-DA/PEA-DA Selective O 442 10.0 AEA-DA Targets 60 A-DA MS1 intensity % of Proteins ** ** SILAC Ratio N 80 H No UV AEA-DA OEA-DA PEA-DA 100 s N m u M c ito leu ch s on dr ia C el AEA-DA Selective lM E em R br an e G Ly olg so i so m e OH No UVN AEA-DA H A-DA A-DA vs A-DA N No UV AEA-DA OEA-DA PEA-DA O AEA-DA % of Proteins C yt op la IdenMfied protein targets include many with known links to lipid biology (eg enzymes and lipid carriers involved in fa%y acid uptake, transport, biosynthesis, and catabolism) but also m20.0 any without prior links No UV Protein number A-DA vs A-DA 200 400 600 800 Biological Process E 15.0 Protein number BD AEA-DA F G Mix A-DA ALDH3A2 threshold ratio values (R3-fold in no for designation of lipid probe targets (also see Figure S2). CellUV experiments)COPS3 Known or predicted Biological Process POR acetyl-CoA links and to dNeuro2a isease cells G (B) Venn diagram Protein of shared and unique protein targets AEA-DA andMitochondria A-DA probesProtein in HEK293T FA membranein biological involvement pofrocesses MTDH subcellular distribuHon ILVBL 10 InFASN Situ (6.6) Drug Profiling with Lipid Probes (C–F) Analysis of lipid probe targets based on (C) presence 23 mRNA Processing TBL2 (membrane) or absence (soluble) of known/predicted tran Lipid-‐Interac>on Proteome Enriched in Known Drug Targets A Non-DrugBank Targets Lipid Probe Targets Proton pump (ATP4A) Cathepsin D (CTSD) Dihydrofolate reductase (DHFR) Epoxide hydrolase 2 (EPHX2) Lanosterol synthase (LSS) Leukotriene A-4 hydrolase (LTA4H) Monocarboxylate transporter 1 (SLC16A1) Multidrug resistance protein 1 (ABCB1) Nicotinamide phosphoribosyltransferase (NAMPT) Prostaglandin-endoperoxide synthase 1 (PTGS1) Progesterone receptor component 1 (PGRMC1) Sterol O-acyltransferase 1 (SOAT1) Chaperones (4%) Transporters Receptors (2%) 22% 280 (25%) Classes considered ligandable 71% 29% 800 Others 45% Enzymes 27% DrugBank Targets Non-DrugBank Targets Number of Proteins A 840 (75%) Aladin (AAAS) B-cell receptor-associated protein 31 (BCAP31) Cleft lip and palate transmembrane protein 1 (CLPTM1) ER lumen protein-retaining receptor 1 (KDELR1) HEAT repeat-contain protein 3 (HEATR3) Mitochondrial carrier homolog 1/2 (MTCH1/2) Nuclear pore complex protein (NUP205) Nucleobindin 1/2 (NUCB1/2) OCIA domain-containing protein 1 (OCIAD1) Protein QIL1 (QIL1) Transmembrane protein 97 (TMEM97) Transportin-1 (TNPO1) Considered not Neuro2a cells ligandable Light A-DA A-DA PTGS1 (Neuro2a) Heavy A-DA (NoUV) A-DA B 600 82% 18% PTGS2 (A549) DMSO DMSO DMSO Light DMSO DMSO DMSO A(EA)-DA DrugBank MeO 2S MS1 intensity C • B25% DMSO of the idenHfied Competitor lipid interacHon proteome enriched in Non-DrugBank drug targets, while DMSO 12% of Flurbi total hRofe uman pDMSO roteome is dRofe rugged 400 Flurbi Heavy MS1 intensity Me • Suggests lipid probes may preferenHally interact with proteins that can bind other small molecule ligands 200 PTGS1 CO 2H • Hypothesize that lipid probes can provide methods to determine drug target engagement and selecHvity 0 e O bl e F lu Competed target br i) UV Light ii) Cell Lysis O So Heavy Cells an Light Cells Rofecoxib SILAC Ratio 15 1.4PTGS1 and PTGS2 Inhibitors for M em (±)-Flurbiprofen Prostaglandin biosyntheMc enzymes PTGS1 and PTGS2 are inhibititor) (PTGS2 (PTGS1/2 inhibitor) lipid probe targets: Digest LC-MS/MS Analysis MS1 intensity Non-competed target Light SILAC Ratio Heavy Competed target 15 1.4 A-DA (±)-Flurbiprofen Heavy A-DA (NoUV) Neuro2a cells 5.0 4.0 PTGS1 3.0PTGS2 2.0 8.0 6.0 Neuro2a cells 3.0 2.0 1.0 0.0 A-DA A-DA Rofecoxib AKR1B8 PTGS1 1.0 0 SILAC Ratio 100 200 3.2 300 0.0 0 1.1 100 are known: 1.3 1.1 3.5 4.6 A549 cells SILAC Ratio Enrich Light D SILAC Ratio (DMSO/Competitor) PTGS1 “Click” MS1 intensity ank MS1 intensity Light A-DA A-DA Mix Heavy A-DA (NoUV) A-DA D A549 cells MS1 intensity B C Neuro2a Lipid probe targetscells 3.9 1.4 SILAC Ratio 200 20.0 Me No UV (±)-Flurbiprofen A-DA vs A-DA A549 cells CO H 15.0 10.0 5.0 3.0 0.0 2 5.0 4.0 PTGS2 F 3.0 4.0 2.0 2.0 (±)-‐Flurbiprofin 0.0 0 O 5.0 0 1.0 200 400 600 PTGS1/2 inhibitor 300 MeO 2S Rofecoxib A549 cells 3.0 1.0 800 Protein number 100 200 300 400 0.0 0 PTGS2 O Rofecoxib PTGS2 inhibitor 100 200 300 400 Prostaglandin-endoperoxide synthase 1 (PTGS1) Monocarboxylate transporter 1 (SLC16A1) Progesterone receptor component (PGRMC1) Multidrug resistance1protein 1 (ABCB1) Nicotinamide phosphoribosyltransferase (NAMPT) Sterol O-acyltransferase 1 (SOAT1) Others 45% Protein QIL1 (QIL1) Nuclear pore complex protein (NUP205) Enzymes Nucleobindin 1/2 (NUCB1/2) Transmembrane protein OCIA domain-containing protein 1 (OCIAD1) (TNPO1) 27% Transportin-1 Non-DrugBank Targets Protein QIL1 (QIL1) Chaperones (4%) Aladin (AAAS)protein 97 (TMEM97) Receptors (2%) Transmembrane Transporters B-cell receptor-associated protein 31 (BCAP31) Transportin-1 (TNPO1) Cleft lip and palate transmembrane protein 1 (CLPTM1) ER lumen protein-retaining receptor 1 (KDELR1) 22% HEAT repeat-contain protein 3 (HEATR3) carrier homolog 1/2 (MTCH1/2) Others PTGS1Mitochondrial PTGS2 (A549) (Neuro2a) Nuclear pore complex protein (NUP205) 45% Enzymes Light Nucleobindin 1/2 (NUCB1/2) DMSO DMSO DMSO DMSO DMSO DMSO OCIA domain-containing protein 1 (OCIAD1) 27% DMSO Flurbi Rofe DMSO Flurbi Rofe Heavy Protein QIL1 (QIL1) MeO 2S Transmembrane protein 97 (TMEM97) Transportin-1 (TNPO1) Compe>>on Between Lipid Probe and Drug For Protein A DrugBank Targets Non-DrugBank Targets Engagement 280 840 CompeMtors : (25%) C C B Light Cells Light Cells B (±)-‐Flurbiprofin (±)-Flurbiprofen F PTGS1/2 (PTGS1/2 inhibitor)inhibitor C A(EA)-DA Enrich Mix Digest LC-MS/MS Analysis Enrich MS1 intensity Light Heavy MS1 intensity Non-competed Competed target target Lipid probe targets “Click” CO 2H D Competed target (±)-Flurbiprofen Neuro2a cells 5.0 SILAC Ratio SILAC Ratio SILAC Ratio (DMSO/Competitor) (DMSO/Competitor) (DMSO/Competitor) ii) Cell“Click” Lysis (±)-Flurbiprofen MeO 2S inhibitor) (PTGS1/2 Me Heavy Cells Mix i) UV Light Lipid probe targets 2 F Lipid probe targets Light Cells CO H MeO2 S CO H Competed target Competed Competitor target DMSO Me 2 DrugBank CO 2H F Targets Non-DrugBank Targets Heavy i) UV Light Cells i) UV Light ii) Cell Lysis Me Me Heavy Cells ii) Cell Lysis (75%) D F O O 8.0 O O 2.0 5.0 1.0 O O Rofecoxib Neuro2a cells O Rofecoxib Neuro2a cells PTGS2 inhibitor 3.0 2.0 1.0 D0.0 0.0 4.0 PTGS1 (±)-Flurbiprofen 0 0 100 200 300 O PTGS1 8.0 5.0 lipid probe DM DM PTGS2 (A549) PTGS1 (Neuro2a) DMSO DMSO DMSO Light Rofecoxib Flurbi 1.3 DMSO 3.9 Rofe Heavy SILAC Ratio 1.4 (PTGS2 inhibititor) Rofecoxib (±)-Flurbiprofen AKR1B8 6.0(PTGS2 inhibititor) 4.0(PTGS1/2 PTGS1 inhibitor) (±)-Flurbiprofen 3.0 DMSO DMSO DMSO DMSO Flurbi Rofe Light Heavy Rofecoxib (PTGS2 inhibititor) MeO 2S 97 PTGS1 (Neuro2a) MS1 intensity CompeMMon experiment: MS1 intensity DrugBank Targets Non-DrugBank Targets MS1 intensity Lipid Probe Targets Prostaglandin-endoperoxide synthase 1 (PTGS1) Proton1pump (ATP4A) Progesterone receptor component (PGRMC1) Cathepsin D (CTSD) Sterol O-acyltransferase 1 (SOAT1) Dihydrofolate reductase (DHFR) Epoxide hydrolase 2 (EPHX2) Lanosterol synthase (LSS) Leukotriene A-4 hydrolase (LTA4H) Monocarboxylate transporter 1 (SLC16A1) Multidrug resistance protein 1 (ABCB1) Competitor Competitor(NAMPT) DMSO Nicotinamide DMSO phosphoribosyltransferase Prostaglandin-endoperoxide synthase 1 (PTGS1) A(EA)-DA A(EA)-DA Progesterone receptor component 1 (PGRMC1) Sterol O-acyltransferase 1 (SOAT1) lipid probe SILAC RatioNOT1.4 (±)-Flurbiprofen competed byA549 flurbicellscompeted by rofe 5.0 DMSO DMSO DMSO DMSO 3.5 Rofe Flurbi 4.6 1.1 lipid probe 3.9 1.3 Rofecoxib competed 1 flurbi A549bycells and rofe CompeHHon ith known inhibitor 4.0 PTGS2 consistent w 4.0 (±)-Flurbiprofen Rofecoxib PTGS2 selecHvity 3.0 3.0 A549 Neuro2a 3.9 1.4 1.3 1.1 cells 3.5 4.6 SILAC Ratio cells AKR1B8 2.0 1.0 5.0 2.0 1.0 5.0 6.0 (±)-Flurbiprofen Rofecoxib Rofecoxib 0.0 0.0 4.0 PTGS2 4.0 100 200 300 0 100 A549 200 cells 300 400 0 100 A549 200 cells 300 400 Neuro2a cells Neuro2a cells Protein 3.0 Protein number Protein number Protein number ProteinAnalysis ID and quantification 3.0 3.0 LC-MS/MS 3.0 number “Click” 5.0 8.0 5.0 5.0 Enrich AKR1B8 Non-competed Competed 2.0 6.0 4.0 PTGS1 2.0 2.0 2.0 4.0 PTGS2 4.0 target targetDigest PTGS1 PTGS2 Figure 4. The Lipid-Interaction Proteome Is3.0 Rich in Drug Targets 3.0 3.0 LC-MS/MS Analysis 3.0 1.0 1.0 1.0 1.0 (A) CategorizationNon-competed of lipid probe targets based on distribution in DrugBank (left pie chart) and further analysis of non-DrugBank targets by protein classe Competed Light 2.0 2.0 2.0 2.0 target target PTGS1 0.0 considered ligandable (e.g., enzymes, receptors,0.0 transporters) or not (others). Heavy 0.0 0.0 1.0 0 1.0300 1.0200 1.0 100 cells 300 with0vehicle 0 100 200 100 200or300 400 ligand (B) Scheme for in situ competitive profiling of ligands using lipid probes. Isotopically light and heavy are treated (DMSO) competitor 0 Light 0.0 0.0 Heavy 0.0 0.0 enriched, and digested fo Protein number Protein number 0 respectively, along with a lipid probe for 30 min. Cells are then UV irradiated and lysed, and light and heavy lysates are combined, Protein ID and quantification 100 200 300 0 100 200 300 0 100 200 300 400 Protein 0 100number 200 300 400 LC-MS/MS analysis. Ligand are designated as proteins show light/heavy ratios of R3.0. Protein that number Protein number Protein number Protein number Protein ID and targets quantification MS1 intensity MS1 intensity Mix Digest MS1 intensity MS1 intensity B 840 (75%) (25%) (C) Chemical structures of the dual PTGS1/2-inhibitor (±)-flurbiprofen and PTGS2-selective inhibitor rofecoxib and representative peptide MS1 chromatogram • cells, PTGS enzymes are among that the (±)-flurbiprofen most competed -‐DA competes target proteins, ndicaHng good selecHvity for PTGS1 and PTGS2 in Neuro2a and A549 respectively, showing (25AmM) A-DA (5imM) labeling of both PTGS1 and PTGS2 . The Lipid-Interaction Proteome IsIs Rich Drug Targets Figure 4. The Lipid-Interaction Proteome Rich inin Drug Targets • AKR1B8 i s m ouse o rtholog o f h uman a ldo-‐keto r eductase w hich i s m odified/inhibited by prostaglandins whereas rofecoxib (25 mM) selectively competes PTGS2 labeling. (A) Categorization of lipid probe targets basedon on distribution in DrugBank (left pie chart) and further of non-DrugBank by protein class orization of lipid probe targets based distribution in DrugBank (left pie chart)analysis and further analysistargets of non-DrugBan Lipid Probes as Screening Tool for Discovery of New Ligands • Nucleobindin protein NUCB1 known to interact with PTGS1 and PTGS2 and enhance PTGS2-‐mediated prostaglandin synthesis, but not before known to bind small molecule ligands • Hypothesized to play role in cellular lipid metabolism A B A O Me 2N A B FI-‐AEA FluoPol Probe + NMe C B 2 C C CO2- O N H N H O Me D D D O 2N O med chem N HN NH 2 H initial HTS hit G 16,000 compounds screened for compeHHve binding to NUCB1 relaHve to FI-‐AEA probe G G Fluorescence polarizaHon by arachidonoyl but Fdecreased F not palmitoyl compeHtor lipids Increase in fluorescence polarizaHon upon FI-‐AEA E bEinding Eto purified recombinant NFUCB1 H R1 O 2N R2 O N R3 21 compounds R4 R1 O N R2 N N Me MJN228 (R1 = Ph; R 2 = H) KML110 (R1 = Ph; R 2 = Me) KML181 (R1 = H; R 2 = H), inactive control OpHmizaHon of iniHal screen hit to generate more potent NUCB1 binding ligands H H MJN228 Competes Arachidonoyl Probe for NUCB1 Binding G H NUC1B ligands: O 2N R1 O N R2 A N N Me MJN228 (R1 = Ph; R 2 = H) KML110 (R1 = Ph; R 2 = Me) KML181 (R1 = H; R 2 = H), inactive control Adapting Lipid Probes for HTS to Discover NUCB1 Ligands CompeHHve binding of opHmized ligands to purified NUCB1 relaHve to FI-‐AEA probe e of Fl-AEA probe. on of the Fl-AEA probe (0.5 mM) with recombinant human NUCB1 (1.0 mM) produced a strong FluoPol signal that was significantly suppressed by the lipid AA (20 mM; Z0 = 0.54). A C lipids AEA, 2-AG, and AA, but not palmitoyl lipids PEA, 2-palmitoyl tration-dependent suppression of the NUCB1-FluoPol signal by arachidonoyl D B PG), or palmitic acid (PA). Error bars represent SD (n = 5). See Figure S6A for profiling of additional lipids. of 16,000 compounds identified small molecules that inhibited the NUCB1-FluoPol signal by 20% or greater (dotted black line). e of confirmed HTS hit 1 and positions modified for medicinal chemistry optimization. See Figures S6B–S6D for summary of medicinal chemistry MJN228 selecHvely n of NUCB1 ligands. inhibits AEA-‐DA ration-dependent blockade of AEA-DA (5 mM) labeling of purified, recombinant NUCB1 (0.25 mM) doped into HEK293T lysates (0.75 mg/ml) by HTS probe binding to 00 mM). NUCB1 over ~400 Structures and competition profiling results (G) and IC50 curves and values (H) for NUCB1 ligands MJN228 and KML110 and the inactive control other probe targets KML181. and (H) represent mean values ± SD from at least three independent experiments. InhibiHon of AEA-‐DA probe labeling of NUCB1 in D 6H) and did not new ligands 1 withBan IC50 value ofcells 3.3by mM (Figure o disrupt other arachidonoyl probe-protein interactions E (25 mM) produced substantial (!3- to 5-fold) reductions in lipid probe enrichment of NUCB1, whereas KML181 had no effect Metabolic Effects of NUCB1-‐ligand Interac>on A NUC1B ligands: O 2N C R1 C O N R2 N N Me MJN228 (R1 = Ph; R 2 = H) KML110 (R1 = Ph; R 2 = Me) KML181 (R1 = H; R 2 = H), inactive control • Site on NUCB1 of both ADA-‐DA probe and ligand MJN228 Ebinding mapped to PTGS1/2 binding domain D B • Suggests common region for NUC1B lipid-‐protein and protein-‐protein interacHons IdenMfying metabolic consequences of NUCB1-‐MJN228 interacMon: D E G F Cell treatment with MJN228 leads to elevated levels of N-‐acyl ethanolamines (NAEs) and N-‐acyl taurines (NATs), two classes of fady acid amides NAEs and NATs are both metabolized by the enzyme fady acid amide hydrolase (FAAH), but neither MJN228 or KML110 Ginhibit FAAH (PF-‐7845 is known FAAH inhibitor) Metabolic Effects of NUCB1-‐ligand Interac>on D NUC1B ligands: O 2N D D E E R1 O N R2 E N N Me MJN228 (R1 = Ph; R 2 = H) KML110 (R1 = Ph; R 2 = Me) KML181 (R1 = H; R 2 = H), inactive control NUCB1 G ligands G control compounds FAAH inhibitor Cell treatment with MJN228 and KML110 leads to elevated levels of N-‐acyl ethanolamines G NUCB1 ligands also inhibit the oxidaHve metabolism of exogenously added arachidonylethanolamine control NUCB1 Engagement and Effects Lipid Metabolism Effects of NUCB1 Ligands shRNA knockdown Lipid Metabolism of NUCB1 Ligands eptide MS1 chromatograms of AEA-DA probe labeling in Neuro2a cells by MJN228 and KML110, tograms showing blockade of showing AEA-DA labeling of endogenous NUCB1ofinendogenous Neuro2a cellsNUCB1 by MJN228 and KML110, Knocking down expression of probe Nblockade UC1B leads to elevated levels of fady acid amides NUCB1 ligands control PTGS2 inhibitor on performed with MJN228 (10 mM) with and the AEA-DA (5 mM). forexperiment in situ competition experiment performed MJN228 (10probe mM) and the AEA-DA probe (5 mM). nd Lipid Metabolism Effects of NUCB1 Ligands nent MJN228-sensitive, AEA-DA-modified NUCB1 peptide (aa 53–68) in Neuro2a tification of a prominent MJN228-sensitive, AEA-DA-modified NUCB1 peptide cells. (aa 53–68) in Neuro2a cells. Data collecMvely uggests that Nshow UCB1 pendogenous lays i(10 ndirect role iinn Neuro2a facilitaMng fby a%y acid a(NAEs mide metabolism, e.g. matograms showing blockade of sAEA-DA labeling of NUCB1 cells MJN228 and KML110, als that profiling Neuro2a cells treated with MJN228 (10 mM) elevated fatty acid amides (NAEsfatty and NATs) compared to cells bolite reveals that Neuro2a cellsprobe treated with MJN228 mM) show elevated acid amides and NATs) compared to cells serving See s i5also ntracellular to dTable eliver S5. lipids to fa%y acid amide hydrolase (FAAH) (p 0.0001, (10 n = mM) 5 per(pcondition). S5.carrier or <KML181 < 0.0001, na= perTable condition). See also A Global Map of Lipid-Binding Proteins and T Ligandability in Cells Proteins and Their Globally M apping Lipid-‐Binding Ligandability Graphical Abstract Authors Micah J. Niphakis, Kenn Armand B. Cognetta III, Fabiana Piscitelli, Hugh Benjamin F. Cravatt Correspondence [email protected] (M [email protected] (B. In Brief A chemical proteomics a a global lipid-protein inte provides evidence for th druggability of lipid-bind cells. Highlights Proteomic probes allow mapping of cellular targets of small molecules and discovery of new small molecule d Chemical probes provide a global protein ligands map of lipid-binding