Toll-Like Receptors (TLRs)

Yeast Mycoplasma Histone MALP-2 Bacteria ? Flagellin Pam3CSK1 Bacteria Zymosan MD2

LBP LPS Yeast Viral Viral Bacterial dsRNA ssRNA DNA TLR2 TLR6 TLR10 TLR10 TLR4 TLR4 TLR5 TLR5

TLR2 4 TLR1 2 2 4 CD14 CD36 2 3 TRIF Endocytosis TRAM TIRAP TIRAP TIRAP

MyD88 MyD88 MyD88 MyD88 TLR11 MyD88 MyD88 3

MD2 MyD88 MyD88 MyD88 MyD88 1 1 1 TLR13 1 LPS 1 GRP94

Endosome UNC93B1 R837 R848 ER TLR4 TLR4 TRIL Poly CpG- PRAT4A ODN Progranulin IRAK4 (I:C) TLR3 Cathepsins IRAK1 IRAK2 TLR3 4 TLR12

TRAF6 TLR9 TRIL AP3 TLR9 TRIF

TRAM IRF8 SARM TLR7 TRIF TLR8 AEP LL37 RIP1 TAB2 TAB3 1

TAK1

MyD88 HMGB1 MyD88 5

TRAF3 MyD88 MyD88 1

MyD88 1 MyD88 MKK3/6 MKK4/7 IKKα IKKβ

IKKγ IKBα Degradation TBK1 IKKε P P IRAK4 p38 JNK IKBα MyD88 MyD88 IRAK1 IRAK2 1 Endosome TLR13 p65 p50 TLR13 NFκB IRF3 TRAF6 6 Parasite TRAF3 CREB AP-1 7 TLR11 NLS TLR11 Profilin P MSK1 p65 p50 NFκB IKKα rRNA P Flagellin

P NLS TLR12 TLR12 IRF7

8 S276 p65 p50 Bacteria TLR12

NFκB TLR11 S529/ Nucleus

536 P MyD88

LEGEND P NLS MyD88 AP-1 S276 p65 p50 Adaptor proteins Accessory proteins TIR domains Ligands NFκB IRF3 IRF7 1

CREB S529/

MyD88 536 MyD88 1 MyD88 homodimerization 5 IKK complex formation P 1

2 TIRAP-TLR2 & TIRAP-TLR4 interactions 6 p50 NLS unmasking 3 TIRAP-TLR1/TLR2 & TIRAP-TLR4 interactions 7 p65 Ser276 phosphorylation 4 TIRAP-TLR4 & TRAM-TLR4 interactions 8 p65 Ser529/536 phosphorylation Pro-inflammatory Gene Expression

www.novusbio.com Example: Inhibition of LPS-Mediated TLR4Activationwith VIPER Example: InhibitionofLPS-Mediated contain thetranslocation,butnotinhibitory, sequence. events. Thepeptidesalsohaveacellpermeabletranslocationsequencewhichenablethetopassivelyenterintocell.Control Each inhibitorypeptideemploysaspecificdecoymechanismbasedonitsaminoacidsequencethatblocksthepropagationofdownstreamsignaling What isadecoy?Adecoyrealisticreplicausedaslureorbait. mechanisms. Cell permeablepeptideinhibitorsareresearchtoolsformanipulatingsignaltransductionpathwaysbyblockingsignalingeventsthroughdecoy TLR andNFkBSignalingPeptide Inhibitors VIPER (TLR4) (TLR2 andTLR4) TIRAP (TLR2 andTLR4) TIRAP NFkB p150/p50 (pSer529/536) NFkB p65 (pSer276) NFkB p65 MyD88 IKK-gamma INHIBITORS TLR4 activation(C)whichwasusedtocalculatetheIC inhibitoryeffectonLPS-mediated VIPER hadadose-dependent analyzed usingtheSEAPReporter AssayKit(NBP2-25285). reportercelllines (B),was of NFkBactivationintheNFkB-SEAP for 24h.Secretedalkalinephosphatase(SEAP),anindication Cells werethenstimulatedwith10ng/mlLPS(NBP2-25295) concentrations ofVIPERorcontrol(CP7)peptides(A)for1h. 16h,cellswerepreincubatedwithincreasing cells/well. After (NBP2-26503) wereplatedin96-wellplatesat1x10 stablytransfectedreporter cells TLR4/MD2/CD14 NFkB-SEAP VIPER (D). A. VIPER(TLR4)InhibitorPeptide Set RNTISGNIYSARRRRRRRRR Control peptidewithscrambledsequence: scrambled Protein transductionsequenceforcellpermeability, TIR-TIR interactions Blocksdownstreamsignalingpathwaysdependenton TIR-TIR interactions BindstoTLR4andblocksTLR4-TIRAP andTLR-TRAM sequence. TIRdomain‘decoy’ binding A46vacciniaviralproteinTLR4TIRdomain KYSFKLILAEYRRRRRRRRR VIPER inhibitorypeptidesequence: inflammatory responses downstream signalingpathways,therebyresultingin mediating TLR4signalinginteractionsandactivating TLR4-TIRAP andTLR4-TRAM TIR-TIR interactions:criticalfor sequence, a.k.a.A46orTIRAP/TRAM sequence NBP2-26244 NBP2-26245 NBP2-29331 NBP2-29323 NBP2-29321 NBP2-26505 NBP2-29328 NBP2-26504 CAT. NO. DECOY protein/TIRAP A46 vacciniaviral TIRAP TIRAP p50 unmaskedNLS Ser529/536 site p65 unphosphorylated Ser276 site p65 unphosphorylated MyD88 IKK-alpha/IKK-beta 5

50 of

domain A46 TIRbinding TIRAP TIRdomain TIRAP TIRdomain p50 NLS consensus site phosphorylation p65 Ser529/536 ylation consensussite p65 Ser276phosphor- merization domain MyD88 TIRhomodi- consensus binding IKK-alpha/IKK-beta SEQUENCE INHIBITOR B. IKBα Degradation LBP CD14 VIPER LPS p65 domain Binds toTLR4TIR domains TLR2/TLR4 TIR Binds to domains TLR2/TLR4 TIR Binds to unmasking Prevents p50NLS phosphorylated Ser sitebecomes phosphorylated Ser sitebecomes Binds toMyD88 gamma Binds toIKK- IKBα MECHANISM INHIBITOR NFκB

IKKα TRAM MD2 p65 p50

NFκB TLR4 IKKγ MyD88 TLR4 p50 MyD88 IKKβ SEAP TIRAP VIPER BLOCK SIGNALING interactions TRAM-TLR4 TIRAP-TLR4 and interactions not TIRAP-TLR2/TLR6 and TIRAP-TLR4 but TIRAP-TLR2/TLR1 interactions TIRAP-TLR4 TIRAP-TLR2 and translocation p50 nuclear phosphorylation p65 Ser529/536 phosphorylation p65 Ser276 merization MyD88 homodi- formation IKK complex TLR/NFkB SEAPCellLines&Ligands TLR13 TLR12 TLR11 TLR10 TLR9 TLR8 TLR7 TLR6 TLR5 TLR4 TLR3 TLR2 TLR1 Control L SEAPORTER TLR TRIF: adapter-inducing TIR-domain-containing interferonbeta TRAM: TRIF-related adaptormolecule TLR: Toll-like receptor TIRAP: Toll/interleukin-1 receptor(TIR)domaincontaining adaptorprotein TIR: Toll/interleukin-1 receptorhomologydomain andarmadillomotifcontainingprotein SARM: Sterile-alpha PRR: patternrecognitionreceptor PAMP: pathogen-associatedmolecularpatterns NFkB: nuclearfactorkappa-light-chain enhancerofactivatedBcells NFkBessentialmodulatororIKKgamma NEMO: MyD88: myeloiddifferentiationprimaryresponsegene(88) Myddosome: signalingcomplexcontainingMyD88 Innate immunesystem:firstlineofdefense Downstream signaling:responsespropagatedfromreceptoractivation DAMP: molecularpattern damage-associated communicationbetweenmultiplesignalingpathways Cross-talk: Bridging: TLR’s participationtheadaptiveimmuneresponse Adaptive immunesystem:acquireddefense Lingo intheTLRField C. D. Peptide (µm) NBP2-26290 NBP2-26288 NBP2-26289 NBP2-26273 NBP2-26280 NBP2-26279 NBP2-26278 NBP2-26277 NBP2-26503 NBP2-26275 NBP2-26274 NBP2-26260 NOVUS Biologicals & Innate Immunity: The Story Toll’dNOVUS Biologicals&InnateImmunity:TheStory LPS Percent Inhibition SEAP [ng/ml] O’Neill etal.NatRev. 13:453-460(2013). Novus BiologicalsisproudtohavethemostcomprehensiveportfolioofhighlyvalidatedandpublishedTLRproductsincludingantibodies,ligands,inhibitors,engineeredstablecelllines. TLRs havefarreachingrolesbeyondinitialinnateimmuneresponses,includingbridgingandadaptiveimmunityaswelllinkinginflammationdisease. The discoveryofToll sparkedanewageinimmunologyduringthe1990’swhenmammalianhomologues,TLRs,werediscoveredandfoundtobeessentialforinnateimmunity. Itisnowbeingincreasinglyrecognizedthat German for‘weird’becauseflieslackingToll developedinaweirdway. The Toll-like receptor(TLR)story‘Toll’d’ couldbesaidtohavebegunin1985withthediscoveryofproteinsinducingdorsal-ventraldevelopmentduringDrosophilaembryogenesis.Theseweretermed‘Toll’ fromthe 100 120 20 40 60 80 10 20 30 40 50 60 0 0 10 − . . . . . 53 60 30 15 7.5 5.0 2.5 1.0 0.5 0 0 VIPER IC50=9.32µm LPS, 10ng/ml -1 TM CP7 VIPER + + + + + + + + + CELLLINE log[Peptide], (µm) 10 0 10 LIGANDS p D NBP2-26238 CpG ODN R848 NBP2-26231 R848 NBP2-26231 R837 NBP2-26228 MALP-2 NBP2-26219 Flagellin NBP2-25289 LPS NBP2-25295 NBP2-25288 Poly (I:C) Zymosan NBP2-26233 Pam3CSK4 NBP2-25297 MALP-2 NBP2-26219 Histone NBP2-26236 Pam3CSK4 NBP2-25297 1 CP7 VIPER 10 2 pathogens. lar componentslocalizedonthesurfaceof Plasma MembraneTLRs:recognizemolecu- NB100-56566 TLR4 NB100-56563 TLR1 IHC-P: smallintestine IHC-P: colon NBP2-25086 Cell Surface TLR ScreeningPack, NBP2-27243 TLR10 NBP2-24787 TLR5 FLOW: PBMC IHC-P: skin IHC-P: skin Web: www.novusbio.com/canada Email: [email protected] Fax: 905.827.6402 Phone: 855.668.8722 Phone: 905.827.6400 CANADIAN CUSTOMERS Web: www.novusbio.com Email: [email protected] Fax: 303.730.1966 888.506.6887 Phone: 303.730.1950 U.S. ANDINTERNATIONAL CUSTOMERS Plasma Membrane TLRs TLR4 NBP2-24935 IF: TLR7oncolon FLOW: TLR4onsplenocytes NBP2-27165 TLR2 NBP2-26247 Flow AssayKit TLR CellSurface NBP2-25083 TLR ScreeningPack IHC-P: tonsil protein WB: TLR4-Y674 partial FLOW: macrophages NB100-56536 TLR6 (Tyr674) Control TLR7 of DNAandRNAfrompathogens. Endosomal TLRs:mediatetherecognition EUROPEAN CUSTOMERS Email: [email protected] Fax: +4536 927818 Phone: +4536 927817 NOVUS DENMARK Email: [email protected] Fax: +3224012254 Phone: +3224012253 NOVUS BELGIUM Email: [email protected] Fax: +44(0)8719711635 Phone: +44(0)1223426001 NOVUS EUROPE FLOW: TLR9inPBMC WB: varioustissues IHC-P: spleen NBP2-24833 TLR12 NBP2-24729 TLR9 IHC-P: gutlumen FLOW: monocytes NBP2-25085 Intracellular TLR ScreeningPack, NBP2-27332 TLR7 NBP2-24875 TLR3 Endosomal TLRs NBP2-24539 TLR13 IHC-F: tonsil NBP2-26248 Flow AssayKit TLR Intracellular TLR3 NBP2-24904 cell line FLOW: TLR13stable FLOW: splenocytes NB100-56742 TLR11 FLOW: TLR3inPBMC NBP2-24917 TLR8 IHC-P: spleen (Tyr759) Email: [email protected] Fax: +490800 5892679 Phone: +490800 7235208 NOVUS GERMANY Email: [email protected] Fax: +33176774531 Phone: +33176774530 NOVUS FRANCE Email: [email protected] Fax: +358923195449 Phone: +358923195448 NOVUS FINLAND activation, andsubcellularlocalization. tial rolesinTLRfolding, ligandrecognition, TLR AdaptorandAccessoryProteins: essen- WB: spleen H-:gsrctmrIHC-P: brain IHC-P: gastrictumor FLOW: monocytes NBP2-24638 TICAM2 (TRAM)* NBP2-27369 MyD88* NBP1-32407 LPB NBP2-25194 CD14 NB120-13810 TRIF* IHC-P: brain FLOW: Jurkat Accessory Proteins TLR Adaptor*& Email: [email protected] Fax: +312 07168337 Phone: +312 07168336 NOVUS NETHERLANDS Email: [email protected] Fax: +35315060362 Phone: +35315060361 NOVUS IRELAND Email: [email protected] Fax: +390240326340 Phone: +390240326786 NOVUS ITALY NBP2-29625 SARM* WB: spleen NB100-56655 MD2 NB400-144 CD36 IF: HepG2 NB100-56730 TIRAP* WB: Daudi NBP2-24743 UNC93B WB: spleen origins towardstheirdestinations. agate activatedTLRsignalsfromtheir TLR DownstreamSignalingProteins: prop- IHC-P: colon NB100-56699 IRAK NB100-56160 RIPK1 NB100-56583 NFkB p105/p50 NBP1-89544 AP-1 NB100-56724 IKB alpha (2) WB: HeLa (1),NIH3T3 IHC-P: breasttumor ALLN-treated (2) WB: Jurkatcontrol(1), IF: A431 www.novusbio.com Email: [email protected] Fax: +46856619333 Phone: +46856619332 NOVUS SWEDEN Email: [email protected] Fax: +4721034287 Phone: +4721034286 NOVUS NORWAY TLR Downstream (Ser32/36) Signaling WB: tumorcelllines NB100-56712 NFkB p65 NB100-56176 TRAF3 IHC-P: breast NB100-56507 IKB alpha NB100-56509 IKK beta NBP1-47812 IRF3 IHC-P: coloncancer IHC-P: gallbladder IHC-P: ovariancancer