© 2014 Nature America, Inc. All rights reserved. has has potential to be the most effective approach pharmacological to reversing obesity and related metabolic disorders. effect of inherent glucagon activity. These preclinical studies suggest that, so far, this unimolecular, strategy polypharmaceutical caloric intake and improve glucose control, and GIP action to potentiate the incretin effect and buffer against the diabetogenic efficacy, which results predominantly from synergistic glucagon action to increase energy expenditure, GLP-1 action to reduce constituent activity including genetic knockout, blockade pharmacological and selective chemical knockout, confirmed of contributions each body weight, enhance glycemic control and reverse hepatic steatosis in relevant rodent models. Various models, loss-of-function balanced unimolecular triple agonism proved superior to any existing dual coagonists and monoagonists best-in-class to reduce potency and equally aligned constituent activities at each receptor, all without at cross-reactivity other related receptors. Such polypeptide insulinotropic (GIP) glucose-dependent and glucagon receptors. This triple agonist demonstrates supraphysiological obesity by acting as an agonist at three key peptide hormone metabolically-related receptors: peptide-1 glucagon-like (GLP-1), We report the discovery of a new monomeric peptide that reduces body weight and diabetic in complications rodent models of Matthias H Tschöp Vasily Gelfanov HertelCornelia Correspondence should Correspondence be addressed to B.F. ( Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland. 9 Diabetes Center, Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, University of Alabama at Birmingham, Birmingham, Alabama, USA. College of Medicine, Cincinnati, Ohio, USA. USA. Medicine, Division of Metabolic Diseases,Technische Universität München, Munich, Germany. 1 complement, as well as each oppose, other in the of regulation energy roles that unique with hormones enteroinsular GIP are distinct three GLP-1 Glucagon, of hybrid and hormones. the three single-molecule a through receptors GIP and GLP-1 glucagon, of modulation neous obesity reversing for candidates clinical promising more the as one have of emerged hormones metabolically-related endogenous integrating of actions the complementary peptides multiple molecule loss weight clinical enhanced shown have therapies ceptable safety when used chronically at single molecular targets have exhibited insufficient efficacy or unac elusive mote weight loss and improve metabolic health have largely pro remained effectively that options pharmacological safe and potent need, unmet vast this Despite ineffective. mostly proven has modification perity global health threat and a rapidly increasing burden to economic pros a represent diabetes, 2 type including comorbidities, its and Obesity Sandoval Darleen Joe Chabenne Brian Finan corrects obesity and diabetes in rodents A rationally monomericdesigned peptide triagonist nature medicine nature Received 24 July; accepted 21 October; published online 8 December 2014; Department Department of Medicine, Lunenfeld Tanenbaum Research Institute, Mt. Sinai Hospital, University of Toronto, Toronto, Ontario, Canada. Institute for Diabetes and Obesity, Helmholtz Zentrum München, German Research Center for Health, Environmental Neuherberg, Germany. We sought to explore the synergistic metabolic benefits We of simulta benefits metabolic to sought explore the synergistic 4 Marcadia Marcadia Biotech, Carmel, Indiana, USA. 1 . . Therapeutic intervention is urgently required because lifestyle 2 , partly because many drug interventions historically directed , manydirected because partly historically drug interventions 1 – 3 3 , ,

11 7 10 3 , , Lianshan Zhang , , Paul T Pfluger advance online publication online advance , , Bin Yang in in vivo. , , Sara Belli 5 , , Randy J Seeley 1 , 2 We demonstrate that these individual constituent activities harmonize to govern the overall metabolic 3 , 10 4 , 3 11 , Elena , Sebokova Elena 6 . . However, new multimolecular Research Center, Beijing Hanmi Pharm., Beijing, China. [email protected] , , Nickki Ottaway 1 , 5 2 4 Metabolic Metabolic Diseases Institute, Division of Endocrinology, Department of Internal Medicine, University of Cincinnati , , Timo D Müller , , Kirk M Habegger 5 , , Konrad Bleicher

4– 6 , and single- and , 10 5 , , Karin Conde-Knape 7– , , David L Smiley 1 1 3 , . 2 doi:10.1038/nm.376 , , Diego Perez-Tilve 10 8 , , Katrin Fischer - - - - , , Sabine Uhles ), ), R.D.D. ( but sufficiently distinct to provide exquisite potency and specificity specificity and potency exquisite provide to distinct sufficiently but outcomes. therapeutic All three superior hormones are produce of comparable would size and amino molecule acid composition single a through receptors at three agonism all and aligned simultaneous ble, possi chemically if that, hypothesized we mechanisms, different by GLP-1 complementing independently GIP and glucagon with onists 2 diabetes type uncontrolled with humans in A1C hemoglobin lower to ability as as well the studies, in weight preclinical body and reduced toxicity gastrointestinal diminished efficacy, glycemic enhanced displayed GLP-1R the (GIPR) and GIP receptors Simultaneously, we discovered a high-potency, balanced coagonist for respectively. components, GLP-1 and glucagon the to attributed be weight can body which actions, lower anorectic and to thermogenic coordinated ability through synergistic a exhibited peptide This gon receptors (GLP-1R and respectively) GcgR, into a single peptide assemble balanced, high-potency coagonism for the GLP-1 and gluca homeostasis glucose and [email protected] 3 Department Department of Chemistry, Indiana University, Bloomington, Indiana, 3 9 . Having established the unique efficacy . of Having two the these unique coag established efficacy , , Tao Ma 1 10 7 1 AIT Laboratories, Indianapolis, Indiana, USA. 10 , , , William Riboulet 2 , , Jonathan E Campbell , , Anish Konkar 5 , , Richard D DiMarchi 3 , 6 , , Christoffer Clemmensen 11 14– ) ) or M.H.T. ( These These authors contributed equally to this work. 1 6 . Previously, we reported the ability to to ability the reported we Previously, . [email protected] 10 9 , , Daniel J Drucker 10 . This dual incretin coagonist coagonist . incretin dual This , , Jürgen Funk 10 9 Pharmaceutical Pharmaceutical 3 , & 2

Department Department of s e l c i t r a

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© 2014 Nature America, Inc. All rights reserved. in vitro destroying GLP-1R and GcgR potency. We subsequently tested them sequence core for coagonist GLP-1/glucagon a from derived progressively were candidates triagonist Intermediary other. each to proportion anced bal in potency high maintaining while all receptor, individual each for preference and that convey elements selective the structural eliminate receptor its for ligand each of affinity individual the to maintain was challenge The peptide. sequence-hybridized a of design the ( hormones three the among similarities sequence and structural The selective triagonist GLP-1/GIP/glucagon Discovery of a unimolecular, balanced, high-potency targeting GLP-1, GIP and glucagon receptors. tion to pursue the discovery of unimolecular triagonists simultaneously GcgR agonist alone ( respective single treatments than lower despitepoint a the to hyperglycemic levels glucose propensity blood lower to of able the was agonist simultaneous administration of the GLP-1/GIP coagonist and the GcgR intake than that observed with the coagonist alone ( ( 20.8% by weight body decreased analog glucagon the of dose equimolar an with coagonist monoagonistincretin ( glucose was decreased to a magnitude similar to that induced by either ( analog GLP-1 the with associated with a lower cumulative food intake relative to that observed regard ( body weight by 15.4%, outperforming any of the monoagonists in this ( treatmentthroughout not affect cumulative food intake ( The glucagon analog decreased body weight by 11.1% ( logs lowered tive food intake by more than 50% ( analog decreased body weight by 12.6% ( 6.4% ( induced obese (DIO) mice. The GIP analog decreased body weight by onist a previously validated GLP-1Racylated and GIPR (GLP-1/GIP) coag monoagonists at each receptor with the equimolar physical mixture of acylated of treatment individual daily the compared we Thus, GcgR. potently managed through simultaneous agonism at GLP-1R, GIPR and We wished to determine adiposity whether and glycemia can be more Triple agonism generates synergistic metabolic benefits RESULTS as compared to relevant monoagonists or coagonists. models rodent in diabetes 2 type and obesity diet-induced reversing unparalleled heretofore and apparent an delivered tested we that triagonists various the of iteration final the that found suitable for pharmacokinetics necessary profile at constituenteach receptor, for as optimized well the as being hormone sequences, which were selected to impart the desired activity onist uses distinct amalgamated residues derived from each of the triag The for and GLP-1R,GcgR. potency,GIPR triagonist native balanced receptors. three at these promiscuity balanced and potent with a agonist (triagonist) triple engineer to chemically possible cally hypotheti it render similarities These receptors. individual their for A  All peptide sequences are displayed in Fig. 1 Fig.

Through iterative chemical refinement, we have identified a high- a identified have we refinement, chemical iterative Through s e l c i t r 9 with an appropriately matched, acylated GcgR agonist Fig. 1 d activity at each constituent receptor ( Fig. 1 ), coupled with prior structure-function studies structure-function prior with ), coupled 8 in an iterative manner to introduce GIP agonism without without agonism GIP introduce to manner iterative an in a ) and modestly decreased food intake ( ad libitum– a ). ). This weightbody improvement by the coagonist was Fig. 1 Fig. 1 Fig. 1 Fig. Fig. 1 Fig. fed blood glucose to a similar extent ( Fig. 1 Fig. c ). These observations served as the founda c c a ). Co-administration ofGLP-1/GIP the Co-administration ). ). The GLP-1/GIP coagonist decreased decreased GLP-1/GIPcoagonist The ). b ) without further suppression of food food of suppression further without ) ). Additionally, ). Fig. 1 Fig. 1 Supplementary Supplementary Figure 1 b Fig. 1 ) and increased blood glucose b in vivo in ). Both GIP and GLP-1 ana Supplementary Supplementary Table 1 a ) and reduced cumula study. Ultimately, we ad libitum– ad Fig. 1 Fig. 1 in vivo in Fig. 1 b b 8 ). The GLP-1 , ). Moreover, 9

efficacy in in efficacy , informed , informed fed blood blood fed a 1 7 ) but did Fig. 1 in diet- , , mass c ). ). ). ). ------

receptors ( three the of each at balance and potency solubility, superior shows that residues 39 of molecule single a in results which GLP-1paralog, reptilian-derived a exendin-4, from residues C-terminal–extended agonism mixed supporting also γ a through (C16:0) acid palmitic Lys10 with lipidated site-specifically and stability chemical structure secondary solubility, enhance to functions auxiliary serve also substitutions potency. These GcgR on impact negative the counteract to Asp28 and Leu27 Gln20, Arg17, Glu16, included we GLP-1R and GIPR aminoisobutyric acid substitution also contributes to mixed agonism at this that observed We previously have inactivation. and degradation at position 2 to convey resistance to dipeptidyl peptidase IV–mediated substituted was acid Aminoisobutyric GcgR. and GIPR GLP-1R, for triagonist unimolecular balanced potent, highly first the represents mately succeeded in engineering a highly modified peptide analog that the in is evolution triagonist in are displayed data spectrometry chromatography-mass HPLC and liquid sentative data are in spectrometry summarized GcgR activity at hepatocytes. activity GcgR ( demonstrated potency less 30-fold coagonist GLP-1/GIP the whereas potency, similar with triagonist the and glucagon by induced was production cAMP GcgR express endogenously which In hepatocytes, rat adipocytes. ( potency by GIP, comparable with and the triagonist coagonist the GLP-1/GIP GIPR express abundantly which adipocytes, mouse 3T3-L1 differentiated In cells. beta pancreatic at ( GLP-1 onist induced cAMP production with potency similar to that of native abundantly expresses GLP-1R, the GLP-1/GIP coagonist that and (MIN6) the line triag cell beta pancreatic mouse a In glucagon. and GIP GLP-1, of tissues target conventional represent that lines cell in tion on accumula cAMP receptor, effects the constituent we investigated possesses triagonist the whether Toexplore activity attributed to each targeted receptor The unimolecular triagonist possesses GcgR. and GIPR GLP-1R, for specific highly is triagonist the that glucagon/secretin class of peptide hormones. Therefore, we concluded the of members two receptor, polypeptide cyclase–activating nylate bind to receptor intestinal peptide the or vasoactive the pituitary ade ( receptors triag the onist displayed that binding no to cross-reactive any of show the other screened we binding), ligand native of inhibition (10% result positive a for criteria strict Using assays. binding competitive at the triagonist over 70 receptor different targets in high-throughput screened we Therefore, targets. receptor additional to bind may tide pep the that surmised we molecule, single a within targets receptor tion studies across in studies. different preclinical species the triagonist is suitable for ( monkeys cynomolgus and rats induction) across all three constituent receptors originating from mice, receptors, it has a similar activity profile (based on cyclic AMP (cAMP) onist was designed specifically to possess the requisite activity at human -carboxylate spacer. The acyl moiety promotes albumin binding while Through iterative refinement of the chemical structure, we ulti we structure, chemical the of refinement iterative Through As we were able to introduce balanced agonism for three different different three for agonism balanced introduce to able As were we Supplementary Table 5 Table Supplementary Supplementary Table5 Supplementary Fig. 1e Supplementary Table 4 Table Supplementary advance online publication online advance Supplementary Figure 2 Figure Supplementary – 1 g 9 8 but is to detrimental glucagon activity. Therefore, and . To enhance time-action and and time-action enhance To . Supplementary Table 5 Supplementary Supplementary Table 1 in vivo Supplementary Results Supplementary 1 Supplementary Table 3 Supplementary 9 2 ), confirming full GLP-1R activity activity GLP-1R full confirming ), . Finally, the triagonist features the the features triagonist Finally,the . pharmacological and pharmacological characteriza ), confirming full GIPR activity at activity GIPR full confirming ), 0 , cAMP production was induced induced was production cAMP , ). Notably, the triagonist did not not did triagonist the Notably, ). Supplementary TableSupplementary 2 in vitro and a detailed narrative of narrative a detailed and in vitro in ), thus confirming full full confirming thus ),

). Although the triag and nature medicine nature in vivo in activity at each each at activity . in vivo ). Therefore, ). utility, we we utility,

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© 2014 Nature America, Inc. All rights reserved. with the GIPR antagonist blunted the improvement observed with with observed improvement the blunted antagonist GIPR the with antagonist GIPR validated a with treated DIO to triagonist the administered we activity, confirming ( alone triagonist the with observed tolerance glucose improved the ameliorated antagonist GLP-1R the with Pretreatment antagonist GLP-1R validated a with pretreated mice DIO to onist models. rodent different To in the confirm presence of GLP-1 activity, we the triag administered receptor each at antagonists selective using effects glycemic acute the receptor, we compared cognate each hoc post Tukeyby followed was ANOVA comparisons, both In analog. glucagon the with coadministration its with coagonist incretin dual the comparing ANOVA and injections, compound with vehicle comparing (ANOVA) variance of analysis two-way or one- regular by determined (1 ( kg nmol (2 triagonist the vehicle, with treated ( triagonist. the before antagonist months; 12 (age ( in Data ( a with Lysine X. as denoted is acid Aminoisobutyric triagonist. GLP-1/GIP/glucagon kg (nmol weight body kg per nmol 10 of a dose at all analog; glucagon the with coagonist incretin dual the of coadministration equimolar the or glucagon coagonist, GIP, GLP-1, incretin of dual analogs acylated with ( change weight body 1 Figure nature medicine nature alone ( the triagonist j e h e d a ) Acute effects on glycemia in male STZ mice (age 13 months; months; 13 (age mice STZ male in glycemia on effects ) Acute – To investigate whether the triagonist possesses possesses triagonist the whether Toinvestigate

µ Blood glucose (mg/dl) Normalized luminescence ∆body weight (%) g –25 –20 –15 –10 mol kg mol ) Representative dose-response curves for activation of the native hormone and the triagonist at the human GLP-1R ( GLP-1R human the at triagonist the and hormone native the of activation for curves dose-response ) Representative Antagonis

(counts per second) –5 400 600 200 1,000 1,500 2,000 0 500 0 2 1 0 –30

multiple comparison analysis to determine statistical significance. significance. statistical determine to analysis comparison multiple e 0 In vivo In Triagonist – 10 −1 g –15 –6 t ) is the normalized response from three independent experiemnts. ( experiemnts. independent three from response normalized the ) is GLP-1R ** ) or pretreated with the GcgR antagonist before the triagonist. Data in in Data triagonist. the before antagonist GcgR the with pretreated ) or in vivo in Glucose 10 0 demonstration of GLP-1, GIP and glucagon triple agonism through coadministration and unimolecular peptides. ( peptides. unimolecular and coadministration through agonism triple glucagon and GIP GLP-1, of demonstration ** –5 n = 8 per group) treated with vehicle, the triagonist (1 nmol kg nmol (1 triagonist the vehicle, with treated group) = 8 per 15 Time (d) Concentration (nM 10

GLP-1 activity. To confirm the presence of GIP GIP of presence the Toconfirm activity. GLP-1 Time (min) 3 a –4 ), cumulative food intake ( intake food cumulative ), 30 Fig. Fig. 1 advance online publication online advance *** 10 6 5 4 45 antagonist Triagonist +GLP-1R GLP-1R antagonist Triagonist Vehicle –3 i *** 60 ### ), ), thus demonstrating 10 –2 75 i ) Acute effects on intraperitoneal glucose tolerance in male male in tolerance glucose intraperitoneal on effects ) Acute ) Triagonist GLP-1 10 90 –1 105 10 120 b 0 Cumulative food intake −1 9 (g/mouse) . Similarly, pretreatment pretreatment Similarly, . ) or a GIPR antagonist (2 (2 antagonist a GIPR ) or 10 15 i b Blood glucose (mg/dl) 0 5 ) and ) and Antagonist 2 1 0 100 200 300 400 0 in in vivo Glp1r –30 f in vivo in Triagonist ad libitum ad Normalized luminescence

–15 (counts per second) Fig. 1 Fig. 1,000 1,500 2,000 −/− Glucose GIP activity. 500 0 Time (d) activity at activity mice pre mice 0 10 3 n 15 ** –6 = 8 per group) treated with vehicle, the triagonist (1 nmol kg nmol (1 triagonist the vehicle, with treated group) = 8 per h fed blood glucose ( glucose blood fed GIPR ), thus thus ), *** 30 Time (min) 6 5 4 10 −1 –5 45 2 ). ( ). Triagonist +GIPRantagonist GIPR antagonist Triagonist Vehicle 2 Concentration (nM µ - - . 10

mol kg mol d 60 –4 ) Sequences of native GLP-1, GIP and glucagon compared with the the with compared glucagon and GIP GLP-1, native of ) Sequences ment indicative of ment indicative improve metabolic enhanced Todelivers that the triagonist confirm respective dual agonists Metabolic benefits of the triagonist are superior to those of the classical possesses triagonist the that ( alone triagonist the with observed otherwise effect hyperglycemic transient acute, the the inhibited antagonist with GcgR Pretreatment peptide. the of a component glucagon on the with brought by hyperglycemia pretreated induced mice assessed and antagonist GcgR streptozotocin-treated to triagonist the activity GcgR of presence the Toconfirm *** 75 10 h −1 ) Acute effects on intraperitoneal glucose tolerance in male DIO mice mice DIO male in tolerance glucose intraperitoneal on effects ) Acute –3 −1 ) or a GLP-1R antagonist (1 (1 antagonist a GLP-1R ) or 90 a ), or pretreated with the GIPR antagonist before the triagonist. triagonist. the before antagonist GIPR the with pretreated or ), γ – 10 E-C 105 j c c represent mean mean represent –2 ) of male DIO mice (age 9 months; 9 months; (age mice DIO male ) of Blood glucose (mg/dl) ) 16 120 Triagonist GIP 100 120 140 160 180 200 10 acyl attached through the side chain amine is denoted as as denoted is amine chain side the through attached acyl 40 60 80 –1 3 2 1 0 Glp1r 10 0 in in vivo −/− ∆blood glucose (%) j DIO mice (age 12 months; months; 12 (age mice DIO 100 125 150 ± 50 75 s.e.m. * s.e.m. Time (d) –30 Antagonis triple agonism, we compared chronic daily daily chronic we compared agonism, triple *** g –15 Triagonist µ Normalized luminescence - NH mol kg mol (counts per second) P t 0 1,000 1,500 2,000 < 0.05, ** < 0.05, 5 4 2 500 15 0 10 −1 ### in vivo in –6 30 Time (min) GcgR * ), or pretreated with the GLP-1R GLP-1R the with pretreated or ), e P ), GIPR ( GIPR ), n 10 45 < 0.001, as determined by determined as < 0.001, = 8 per group) treated treated group) = 8 per –5 in vivo in P GcgR activity. GcgR 60 < 0.01, *** < 0.01, Concentration (nM Triagonist +GcgRantagonist Gcgr antagonist Triagonist Vehicle −1 10 Fig. 1 Fig. ) or a GcgR antagonist antagonist a GcgR ) or –4 75 n a + acyl-glucagon Acyl-GLP-1/GIP coagonis Acyl-GLP-1/GIP coagonis Acyl-glucagon Acyl-GLP-1 Vehicle Acyl-GIP f – = 8 per group) group) = 8 per ) and GcgR ( GcgR ) and c s e l c i t r a , we administered administered we , 10 90 ) Effects on ) Effects –3 j ), which shows shows which ), 105 10 P 120 –2 < 0.001, < 0.001, ) Triagonist Glucagon 10

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© 2014 Nature America, Inc. All rights reserved. decreased body weight by 14.7%, which is comparable to the coag the to comparable is which 14.7%, by weight body decreased a dose-dependent manner, such that a 33% lower dose of the triagonist by 26.6% after 20 d ( weight body decreased triagonist the whereas by weight 15.7%, body decreased coagonist The mice. DIO in doses equivalent at potency coagonist GLP-1/GIP validated the of extensively compared the metabolic of efficacy the triagonist with that we Thus, action. incretin dual of the that potentiate can that efficacy weight-lowering substantial contributes triagonist the within nent compo We of glucagon dose. the the believe one-twentieth at nearly triag co-agonism the GLP-1/glucagon of with report previous our loss in as onist weight body on effect similar a induce to able ( intake improve ( tolerance glucose markedly to compound only the was triagonist The coagonists. gon and GLP-1/gluca and GIP/glucagon both liraglutide with compared potentiation substantial a 15.1%, by weight body lowered triagonist neal glucose toleranceintraperito ( in improvement concomitant a without occurred loss ( 9.9% by weight body lowered coagonist in the GLP-1/glucagon vehicle DIO whereas mice, to compared tolerance or glucose weight not improve body did onist comparator. coag and At the GIP/glucagon low liraglutide this dose, a benchmark as included GLP-1 analog) lipidated (a mice, liraglutide with DIO in coagonist GIP/glucagon a and coagonist 1/glucagon GLP- a of dose equimolar an with triagonist the compared we ation, related matched and coagonists. As pharmacokinetically a first evalu structurally three the with to treatment triagonist the with treatment group. treatment per 8 mice from Tukeyby followed was ANOVA # mean represent kg nmol (3 coagonist incretin dual the vehicle, with treatment d of 21 the kg nmol (3 coagonist incretin ( score ( tolerance glucose intraperitoneal ( change ( intake food ( change weight ( coagonism. incretin dual with compared benefits metabolic maximizes 2 Figure A  onist at mixed agonists a caused Both dose. a higher similar threefold e a P

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© 2014 Nature America, Inc. All rights reserved. peptides fail as potent mixed agonists; therefore, they do not qualify qualify not do they therefore, agonists; mixed potent as fail peptides with the classical GLP-1R monoagonist exendin-4. Accordingly, these compared even efficacy, metabolic of terms in inferior substantially are peptides these that demonstrate and triagonists purported of the results confirm the imbalanced activity and reduced receptor potency aforementioned parameters ( compared with tolerance vehicle treatment, whereas glucose exendin-4 improved improve all the or body glucose reduce blood or to intake failed food weight, triagonist, our of that than higher times 16 these of administration at compounds of a dose cumulative 50 nmol kg twice-daily mice, render DIO certainly In and activity, triagonists. coagonists not in low-potency extremely best imbalanced at them massively ing were peptides these ( hormones native the with thousandth the activity at least at one of the three receptors compared glucagon agonism tri ple purported with peptides reported recently two with triagonist rodents in liraglutide for h 4–8 of half-life reported ( rodents in h 5 approximately is dose subcutaneous bolus a following triagonist the of half-life average The peptides. two the between are similar species significance. statistical determine to analysis Tukeyby followed was ANOVA comparisons, both In genotypes. between triagonist the of treatment comparing ANOVA *** kg nmol 10 of a dose at day other every administered injections subcutaneous via triagonist the or ( change glucose ( n ( change 3 Figure nature medicine nature caution. with used be should and triagonists as f = 8 per group for each genotype). ( genotype). each for group = 8 per ) in male wild-type ( wild-type male ) in As a point of reference, we compared the the compared we reference, of point a As g d a P ∆body weight (%) ∆body weight (%) ∆body weight (%) < 0.001, determined by ANOVA comparing vehicle with compound injections within each genotype. genotype. each within injections compound with vehicle comparing ANOVA by determined < 0.001, –12 –10 –25 –20 –15 –10 –8 –6 –4 –2 –8 –6 –4 –2 –5 0 2 0 2 0

a The metabolic and glycemic benefits of the triagonist are blunted in in blunted are triagonist the of benefits glycemic and metabolic The 0 8 6 4 2 0 8 6 4 2 0 ), fasted blood glucose change ( change glucose blood fasted ), 2 4 and [ and 24 6 4 2 , 2 h 5 ) and intraperitoneal glucose tolerance ( tolerance glucose intraperitoneal ) and D Bt o tee eotd etds tre YAG- (termed peptides reported these of Both . upeetr Tbe 6 Table Supplementary A

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), fasted blood glucose change ( change glucose blood fasted ), * cause irreversible damage in these preclinical studies. ( recoveredhyperglycemiawas and mild levels, baseline weightnear to received the triagonist for 3 weeks revealed that the mice regained body cological studies. However, 2-week follow-up analysis in DIO mice that by the triagonist may limit the dose escalation in translational and toxi intake ( ( 26.4% byweight body decreased dose higher the and 9.7% byweight body decreased triagonist the of dose lower the weeks, 11 By onist. triag the of doses two with weeks 11 forratsDIO treated we setting, mice. lean these in tested doses at any the of treatment chronic ( intake food or mass, lean weight, body reduce not did triagonist the Additionally, injection. after h 24 at any monitored when hypoglycemia of tested the doses not observe ( mice euglycemic lean, in injection intraperitoneal single-bolus a following glucose blood in decrease a weight- dose-dependent induced The triagonist efficacy. and lowering glucose-lowering considerable the of safety the about concerned still were we mice, DIO in treatment chronic after mass Although we did not observe hypoglycemia or a substantial loss of lean without hypoglycemic risk Chronic treatment with the triagonist safely lowers body weight Glp1r n Supplementary Fig. 6e Supplementary Fig. 6g = 5) or or = 5) To test whether the triagonist has sustained efficacy in a longer-term c −/− ) in wild-type (WT) or or (WT) wild-type ) in i c f Supplementary Fig. 6f , , Blood glucose (mg/dl) Blood glucose (mg/dl) Blood glucose (mg/dl) Gipr Gcgr 200 300 400 100 200 300 400 500 100 200 300 400 500 100 0 0 0 −/− 3 0 3 0 3 0 −/− g Day 10 Day 10 Day 10 and and – ### ### ( * i ) Effects on body weight change ( change weight body on ) Effects n −1 = 7) HFD mice. All mice were treated with vehicle vehicle with treated were mice All mice. HFD = 7) ### *** *** Gcgr 6 0 6 0 6 0 . Data in in . Data Time (min) Time (min) Time (min) ), along with dose-dependent reductions in food , −/− h ### Glp1r *** *** # ), which indicates that the triagonist did not e mice. ( mice. P Supplementary Fig. 6a Fig. Supplementary 9 0 9 0 9 0 ) and (intraperitoneal glucose tolerance tolerance glucose (intraperitoneal ) and < 0.05, < 0.05, a ). This superior body weight loss induced – −/− i represent mean mean represent male HFD mice (age 7 months; 7 months; (age mice HFD male a 0 0 0 – Supplementary Fig. 6b Fig. Supplementary c ### ) Effects on body weight weight body on ) Effects post hoc post 120 120 120 *** P # < 0.001, determined determined < 0.001, multiple comparison comparison multiple ± g s.e.m. * s.e.m. ), fasted blood blood fasted ), s e l c i t r a ). Notably, we did did we Notably, ). Triagonist WT Vehicle Vehicle WT Triagonist Triagonist WT Vehicle Vehicle WT Triagonist Triagonist WT Vehicle Vehicle WT Triagonist P < 0.05, < 0.05, Gcgr Gipr Glp1r

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respiratory quotient c 0.70 0.75 0.80 measurable effect on effect measurable body weight loss in HFD lowered fat mass in wild-type mice ( by weight body ( 7.7% decreased The controls. triagonist HFD of treatment these a low-dose in lost was triagonist of the efficacy weight-lowering The in efficacy metabolic its testing by onist of triag component the Weof glucagon the contribution the studied GcgR signaling Improved energy metabolism benefits of tri-agonism depend on intolerance glucose HFD-induced to resistance effect of the triagonist in failed to improve glucose tolerance in these HFD HFD these in tolerance glucose improve to failed 7f Fig. ( efficacy anorectic its lost triagonist the Likewise, mice mice compared with the pair-fed controls ( DIO in triagonist-treated expenditure energy enhanced significantly ( mass lean (21.6%; controls pair-fed in observed that to compared (32.0%) weight reduc body in greater tion a caused triagonist The controls. pair-fed of that to triagonist the of efficacy metabolic the compared we mice, DIO in energy contribute to mechanisms that expenditure the Therefore efficacy. overall supporting thus loss, weight in differences substantial despite triagonist the with treated the those and with coagonist incretin dual treated mice wild-type between intake food in difference integrated the to pharmacology glucagon attributed is which coagonist, GLP-1/glucagon of efficacy coagonist weight-lowering GLP-1/GIP the the to contributor key a not are ture of intolerance glucose from protection inherent and hypoglycemia existing the to attributed partially be may which ∆fat mass (g) –20 –15 –10 We expendi that in changes energy demonstrated have previously –5 0 P < 0.001, determined by ANOVA comparing vehicle with test group group test with vehicle comparing ANOVA by determined < 0.001, ) and lowering effect on fasted blood glucose ( glucose blood fasted on effect lowering and ) ## *** ** ### *** Fig. 4c Fig. post hoc post a i –

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© 2014 Nature America, Inc. All rights reserved. these glycemic improvements were maintained in the high-dose high-dose the in maintained were improvements glycemic these ( control vehicle with compared ance and hemoglobin A1C, and preserved proper islet cytoarchitecture ( efficacy sus tained and onset rapid a with glucose blood fasting and weight body fatty (ZDF) rats. In a dose-dependent manner, the triagonist improved ( islets pancreatic of core the within by alpha as infiltration cell reduced architecture, islet assessed onist in lessening glucose intolerance ( ( doses tested, an comparable effect to that of the GLP-1/GIP coagonist the triagonist protected ( treatment either with altered not was intake ( coagonist the with observed over effect the improvement and showed mice treated The triagonist prevented the excessive weight gain observed in vehicle- coagonist. incretin dual the with compared development diabetes the spontaneous of prevent to triagonist the of capacity the assessed and hyperglycemia, of development the before immediately age, of used We diabetes. to 2 type translate of would models mice rodent obese insulin-resistant in observed onist triag the of efficacy metabolic enhanced the whether Weexplored preexisting hyperglycemia GcgR signaling component of the triagonist does not exacerbate of consequence a intake. caloric simply reduced not is triagonist the weight- of the efficacy that lowering demonstrate data the Collectively, oxidation. fat ( activity during times quotient of feeding ( respiratory lower significantly a had mice treated both with compared curve. time concentration the hoc post of panel left the in data of comparison Statistical significance. statistical determine to Tukeyby followed was ANOVA comparisons, both In doses. equimolar at injections triagonist with coagonist injections. triagonist of dose highest the with vehicle comparing ANOVA by determined kg 1 nmol at triagonist ( mice ( core islet the to invasion ( treatment) of start the from 27 day at (tested tolerance glucose intraperitoneal ( intake ( change ( development. hyperglycemia exacerbate not does activity 5 Figure nature medicine nature ( cessation treatment after weeks 3 group a Fig. Fig. 5 a –

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tics. Notably, the glucagon activity can be selectively fine-tuned with with fine-tuned selectively be can Notably,activity tics. glucagon the glucagon activity might ultimately prove of to titration be more suitable selective a therapeu with triagonists imbalanced of series the that possible is it dosing, long-term for preferable is agonism glucagon aggressive less that testing subsequent in proves it If pharmacology. adverse apparent no with efficacy sustained witnessed we cessation, treatment after analysis follow-up and studies efficacy long-term in not from studies; be however,preclinical accurately these determined can use human chronic for peptides these of index therapeutic The studies. preclinical these in induced and insulin loss circulating of weight reduction of degree high observed the of because warranted is ketoacidosis diabetic on outcomes long-term of assessment ough and GcgR tem, as recent reports suggest direct chronotropic action of GLP-1R sys on of a the including assessment cardiovascular effects thorough warranted, are analyses toxicological Intensive work. development which it is needs to achieved in be established thoroughly subsequent models. knockout of germline use the with associated liabilities the circumvent can reagents of sets its these with limitations, own albeit conditions, wild-type in antagonists selective of use arduous Together the with animals. wild-type in tested subsequently and deleted been has activity component single a which in analogs lar lar pathways may offer a opportunity pharmacological to replicate the that the simultaneous and encompassing modulation of these molecu improvements bolic by surgeries achieved bariatric responses contribute enteroendocrine to the massive and rapid meta adjusted that evident increasingly becoming is It rodents. in cations compli and at its obesity metabolic such low to doses rectify efficacy preclinical such achieve can that date to pharmacotherapy only the and ToGIPR. represents triagonist our this unimolecular knowledge, relevant three different receptor metabolically GLP-1Rtargets: GcgR, [ YAG-glucagon that and triagonists balanced of discovery first Consequently, we believe that the peptidestriagonists. as presented characterized here be represent to qualify the biochemically not do thus and mice DIO in benefits metabolic negligible provide compounds more extensive results presented herein. Here, we confirmed that these our to relative lowering weight body limited their for basis a suggest aligned activityachieve balanceto across failed the threealso receptors. and These 1,000-fold), (nearly triagonist our to and 100-fold) (nearly hormones native the to relative potency in reduced subsequent of 50 nmol kg was no tration more 3% than relative to control vehicle at a dose total the total reported weight loss after three weeks of twice-daily adminis agonism at GLP-1R, GIPR andtriple GcgR concerted of unsupportive and observed have we what to tive molecular triagonists where the body weight lowering was rela paltry deposition. fat excessive with associated diseases liver nondiabetes or diseases neurodegenerative syndrome, Prader-Willi as well as the opportunity to explore nonconventional uses, such as for of the increases likelihood ultimate pharmacology success, molecular inherent their in differ that options several among choose to ability nature of the human condition. In entering the translational stage, the personalized more approachmedicinal to a obesity therapy that the reflects heterogeneous for opportunity the providing 3), position at change acid amino single (a change chemical or structural minimal D A The magnitude of weight loss in a clinical setting and the speed with We report the of discovery a peptide with atbalanced superpotency uni purported of reports previous question into call results Our 2 ]GLP-1/GcG 3 1 agonism . In addition to outcomes, cardiovascular a thor in vitro −1 d −1 2 5 analysis revealed these peptides to be dramatically . Our synthesis of these purported triagonists and triagonists . of Our synthesis purported these do agonism. not relevant offer triple 24 , 25 , 3 2 . In the most recent report s e l c i t r a 3 3 , which suggests , suggests which in vitro results 2 4 and and 2 3 5  0 ------­ ,

© 2014 Nature America, Inc. All rights reserved. reprints/index.htm at online available is information permissions and Reprints version of t The authors declare competing interests:financial details are available in the studies and wrote the manuscript together with B.F. interpreted data. R.D.D. and M.H.T. conceptualized, anddesigned interpreted all in vitro andhistology interpreted data. C.H., A.K. and V.G. anddesigned performed data. J.F. performed liver andhistology interpreted data. S.U. performed pancreas anddesigned performed K.B. anddesigned synthesized compounds. S.U., W.R., C.H., E.S., K.C.-K. and A.K. experiments. J.C. and D.L.S. designed, synthesized and compounds.characterized L.Z. designed and performed designed, supervised data. D.P.-T., P.T.P., K.M.H., J.E.C., D.S., R.J.S., C.C., D.J.D., E.S., A.K. and T.D.M. and led performed co-wrote the manuscript. B.Y. designed, synthesized and compounds,characterized synthesized and compounds,characterized analyzed and interpreted data, and B.F. anddesigned performed Association) and the Canadian Institutes of Health Research (93749). Metabolic (through Diseases the Initiative and Networking Fund of the Helmholtz Helmholtz2009-241592), Alliance ICEMED–Imaging and Curing Environmental Deutsches für Zentrum Diabetesforschung (DZD), EurOCHIP (FP-7-HEALTH- and by grants from the Deutsche Forschungsgesellschaft (DFG; TS226/1-1), Marcadia Biotech, which has acquired been by F. Hoffmann–La Roche Ltd., Hospital) for providing of Medicine) for providing in pharmacokinetic studies. We thank M. Charron Einstein (Albert College C. Rapp, M.S. Gruyer, V. Dall and V. Griesser for bioanalytics; and M. Kapps, C. Flament, P. Schrag, for necropsy and immunohistological procedures; C. Apfel, C. Wohlgesinger studies; pharmacological M. Brecheisen, C. Richardson, G. Branellec and V. Ott we thank A. Roeckel, A. Vandjour and E. Hainaut for assistance during during C. Raver, S. Amburgy, J. Pressler, J. Sorrell, D. Küchler and L. Sehrer for assistance and workseminal in mixed agonist Wepeptides. thank J. Holland, J. Hembree, B. Ward and C. Ouyang for discussions on relationshipschemical structure-activity We thank J. Ford for cell culture maintenance. We thank J. Patterson, J. Day, We thank J. for Levy technical and chemical support of peptide synthesis. o Note: Any Supplementary Information and Source Data files are available in the the ve in available are references associated any and Methods M surgery. bariatric to alternatives of medicinal pursuit the in physiology replicates closely more that pharmacology that for therapeutics protein-based other with combination hormones of nuclear in targeting efficacy the this newly discovered triagonist could be used to and deepen broaden medicines diabetes and obesity for standard ing notion that single-molecule polytherapies are emerging as the gold forward beyond previous attempts of coagonism and reflects the grow of step a represents sizable Ultimately, triagonist the surgeries. bariatric invasiveness and risks surgical consequential the without albeit models, rodent in reports published the to compared when least at ments elicited by the triagonist rival improve those induced bymetabolic bariatricThe surgery, polypharmacy. physiological-mimicking a the bundled multiagonism within this single such embodies molecule altered physiology seen with bariatric surgery. Accordingly, we believe A 1 COMPETING FINANCIAL INTERESTS FINANCIAL COMPETING AUTHOR CONTRIBUTIONS A nline version of the pape 0 cknowledgments et rsion of the pape the of rsion

s e l c i t r h experiments and interpreted data. S.B. led pharmacokinetic studies and in vivo in vivo ods he pape in vitro pharmacological studies. pharmacological At F. Hoffmann–La Roche Ltd., in vivo pharmacology andpharmacology metabolism rodent studies and interpreted l experiments, and analyzed and interpreted data. N.O. designed . r . experiments and interpreted data. K.F. performed r Gipr . r in vivo . Gcgr −/− ′ in vitro, in vivo Asen, Asen, F. Schuler and M. Otteneder for assistance mice. Partial research funding was provided by and −/− mice and Y. (KansaiSeino Power Electric ex ex vivo in vivo analyses in ZDF rats and interpreted experiments and interpreted data. and ex ex vivo 1 1 3 rodent experiments, 1 . It is conceivable that that conceivable is It . or find application in application or find http://www. 3 4 as we search search we as

in vivo nature.com/ in vivo

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Fosgerau, K. W.T.Garvey, Allison, D.B. K.M. Gadde, and present past, drugs: J.P.Anti-obesity Wilding, & M.H. Tschop, R.J., Rodgers, of fall unrelenting The U. Pagotto, & R. Pasquali, V., Vicennati, G., Dalmazi, Di O & J.R. Speakman, Muller, T.D.Muller, Ionut, V., Burch, M., Youdim, A. & Bergman, R.N. Gastrointestinal hormones and bariatrichybrid GIP-oxyntomodulin novel V.A.A Gault, P.R.& Flatt, B.D., V.K.,Kerr, Bhat, N. Panjwani, are receptors GIP Functional M.M. Wolfe, & T.J.Kieffer, M.O., Boylan, R.G., Yip, Ward, B. arr, .. Snoa, .. DAeso DA & ely RJ GP1 n energy and GLP-1 R.J. Seeley, & D.A. D’Alessio, D.A., Sandoval, J.G., Barrera, the for therapies hormone combinatorial Emerging D.J. Drucker, & S.A. Sadry, C. Clemmensen, B. Finan, A. Pocai, Finan, B. Day,J.W. Mukharji, A., Drucker, D.J., Charron, M.J. & Swoap, S.J. Oxyntomodulin increases Oxyntomodulin S.J. Swoap, & M.J. Charron, D.J., Drucker, A., Mukharji, M. Kim, weight body on diet fat high a and aging of Effects D.J. Drucker, & L.A. Scrocchi, Day, J.W. R.W.Gelling, K. Miyawaki, ref. Gault,V.A., Bhat,V.K., Irwin,Flatt,& N. P.R. novelAGLP-1/glucagon hybrid doc. peptide Victoza; for V.A.A. DPP-IV-resistant Report Gault, triple- P.R. & Flatt, S., Vasu, B.D., V.K., Kerr, Bhat, Assessment Agency. Medicines European Patterson, J.T. of Optimization R.D. DiMarchi, V.M.& Gelfanov, M.A., DiMarchi, J.R., Chabenne, K.M. Habegger, K.M. of Habegger, mechanisms and physiology, Pharmacology, D.J. Drucker, & J.E. Campbell, massandprevents diabetes db/dbin mice. (2012). 297–308 study. extension 3 phase placebo-controlled, randomized, a controlled-release phentermine/topiramate in obese and overweight adults (SEQUEL): randomizeda controlled trial(EQUIP). 377 trial. 3 phase placebo-controlled, randomised, a (CONQUER): adults obese and overweight in comorbidities associated and weight on combination topiramate future. obesity. of treatment pharmacological the (2012). 569–573 Sci. FGF21. or exendin-4 with combination in analog leptin optimized an using surgery-inducedweight loss. properties. anti-diabetic and GIP,through acting reducing peptide weight exhibits receptors GLP-1 and glucagon (2013). adipocytes. on present Metab. Mol. (2010). 1322–1331 treatment of obesity and T2DM. and obesity of treatment diet. obesogenic an on maintained (2014). chronically 1422–1427 mice obese in Med. Nat. mice. in obesity humans. and monkeys, Biol. Chem. Nat. nrni hat ae hog te lcgn receptor. glucagon the through rate heart intrinsic pressure. blood of control to secretion 139 receptor peptide-1 glucagon-like in tolerance glucose and DIO-rodents.reductioninweightmaximize 100 mice. knockout receptor glucagon in hyperplasia cell obesity. potentialhigh-fat-fedin mice. with triple-acting agonist activity at GIP, GLP-1 and glucagon receptors and therapeutic insulinotropichigh-fat-fedactionsinmice. acting agonist of GIP, GLP-1 and glucagon receptors with potent glucose-lowering and EMEA/379172/200 endogenousweightcontrolbodybyGLP-1. diabetic in atherosclerosis ApoE male of development attenuate not does but accumulation purposes. medicinal for sequence glucagon native the actions. Endocrinol. action. hormone incretin control. long-term and short-term of Endocrinol. model integrated an balance:

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© 2014 Nature America, Inc. All rights reserved. production was followingassessed the method below. 500 glutathione, mM 2 FBS, 10% Ham’s with supplemented (GIBCO) media F-12 (GCCGCCATCATG) to further aid expression. CHO-K1 cells were cultured in Kozak sequence anartificial as well as onreferencesequences, database based were which monkey, cynomolgus and rat, mouse, from originating receptor plasmids (Life Technologies) containing the native sequence for each individual pcDNA3.1from constructed plasmids expression with transfected transiently Mouse, rat, and cynomolgus monkey receptor activation. (OriginLab). software against concentration of and peptide EC graphed were data Luminescence (PerkinElmer). counter MicroBeta-1450 scintillation a liquid on measured was Luminescence dark. the in min stored 10 and for min 5 for agitated were cells The luciferin. and to lysis lysates the induce to expose cells the to added was (PerkinElmer) reagent substrate luminescence HTS Lite Steady of volume equivalent an incubation, the stop CO 5% and °C 37 at h 5 for cells, incubated and HEK293 co-transfected serum-deprived, the containing Biosciences) plates (BD treated cell-culture to were added 96-well of dilutions the peptides supple Serial (HyClone). (HyClone) (BGS) DMEM serum growth in bovine h (vol/vol) 0.25% 16 with mented for deprived serum 22,000 and of well density per a element at cells seeded response were cAMP Cells a to B-selection). fused (hygromycin (CRE) construct gene reporter luciferase and a (zeocin-selection) cDNA receptor individual each with co-transfected were cells (HEK293) kidney embryonic Human induction. indirectly cAMP that measures assay gene glucagon or reporter GIP, luciferase GLP-1, cell-based the a through activate receptor to ability its for tested individually activation. receptor glucagon GIP,and GLP-1, Human ticated in house by short tandem repeat (STR) DNA profiling. from internal stock at F. Hoffmann-La obtained Roche.were Nonehepatocytes rat and of 3T3-L1, the ATCC.MIN6, cell the lines from obtained were authen on a monthly basis. Parent HEK-293 cells used for lines. Cell trometry. Purified peptides were lyophilized, aliquotted, and stored at 4 °C. Peptidemolecularweights were byconfirmed ESI or MALDI-TOF massspec for purity by analytical reversed-phase HPLC using the conditions listed above. gradient on a Vydac C8 column (2.2 × 25 cm). Preparativebysemi-preparative purified reversed-phaseTFA 0.1%HPLC in anACNwith fractions were analyzed purification. Peptide × 5 cm). (0.46 column or C8 C18 ent on a Zorbax confirmed by analytical reversed-phase HPLC in 0.1% TFA with an ACNin gradi (summarized spectrometry mass (MALDI-TOF) time-of-flight desorption/ionization laser matrix-assisted or (ESI) ionization electrospray by confirmed were lyophilization. weights before molecular Peptide (AcOH) acid acetic 1% and (ACN) acetonitrile 20% least at containing buffer aqueous in dissolved was peptide crude the ether, the of removal After agitation. with TFA/ h 2 with for vol/vol/v) treated (9:0.5:0.5 were TIS/anisole resins peptidyl Completed amines. N-terminal of deprotection for (DMF) piperidine/dimethylformamide 20% and coupling by synthesizer standard using Fmoc DIC/Cl 433A peptide methods on Applied an Biosystems used was (Novabiochem) MBHA resin Rink mmol 0.1 synthesis, peptide ether. neutralization in diethyl For peptide Fmoc-based removed was HF tion. with acid/ treated (HF) were hydrofluoric resins for Peptidyl TFA amines. and amino-terminal coupling of for deprotection DEPBT/DIEA using synthesizer methods peptide Boc 430A standard Biosystems by Applied modified highly a on used was Biotech) (Midwest synthesis, resin (MBHA) peptide 4-methylbenzhydrylamine mmol 0.2 neutralization Boc-based For chemistries. Fmoc-based using methods synthesis synthesis. Peptide ONLINE MET doi: 10.1038/nm.3761 µ g/ml g/ml Geneticin, 100 All cell lines used were confirmed to be mycoplasma free and tested H ODS Peptides were synthesized by solid-phase peptide peptide solid-phase by synthesized were Peptides Following cleavage from the resin, crude extracts were extracts crude resin, the from cleavage Following in vacuo in p µ -cresol (10:0.5 vol/vol) at 0 °C for 1 h with agita with h 1 for °C 0 at vol/vol) (10:0.5 -cresol g/ml g/ml penicillin and 100 n situ in and precipitated the cleaved and deprotected deprotected and cleaved the precipitated and neutralization for both Boc-based and and Boc-based both for neutralization Supplementary Table 1 Table Supplementary 50 values were using Origin calculated 2 in a humidified environment. To environment. humidified a in in vitro µ g/ml g/ml streptomycin. cAMP receptor activity were CHO-K1 cells were Each peptide was was peptide Each ) and character character and ) − HOBt HOBt for - - - - -

experiment. experiment. Data were analyzed using GraphPad Prism. every for parallel in run curve standard on based calculated was production EnVisioncAMP anand using (PerkinElmer), determined was signal rescence 2 Kit (Cisbio) following the manufacturer’s instructions. The time-resolved fluo of lysis buffer and cAMP generated was usingdetermined the cAMP Dynamic addition by stopped was reaction The peptides. the temperature atroomwith and hepatocytes) or the cell monolayer (3T3-L1) was then incubated for 30 min seeded directly into 96-well plates and differentiated. The cell suspension (MIN6 96-well plate at a seeding density of 5000 cells/well. For 3T3-L1 cells, cells were For suspended MIN6 cells and fresh rat hepatocytes, cells were transferred to a 5 min for at 37 incubated °C withwere 5 monolayers mL Cell cell Dissociation cells, solution MIN6 (Gibco)For to PBS. dislodge with the cells. washed were ing 10% FBS. wereCells used 10 days after initiation of differentiation. and 10 DMEM containing with 4 replaced FBS, was 20% days, media 5 20% FBS, 0.5 mM IBMX, 0.4 containingDMEM with differentiate to initiated were cells 3T3-L1 Confluent CO 5% with incubator humidified a in penicillin/streptomycin 1% before the experiment. Mouse 3T3-L1 cells were grown in DMEM/10% FBS and incubator. Rat hepatocytes were thawed from cryopreserved stock immediately 100 1 mM sodium pyruvate,HEPES, 50 mM 10 Laboratories), (PAA FBS 10% with supplemented medium assay. cAMP on a 12-h/12-h light-dark cycle at 22 °C with free access to food and water. and food to access free with °C 22 at cycle light-dark 12-h/12-h a on beginning at protein, 8 weeksfrom orkcal 2 16.4% months and of carbohydrates, age. from DIO kcal mice 25.5% were fat, single- from orkcal group-housed fed a diabetogenic diet (Research Diets), which is a high-sucrose diet with 58% DIO mice. figure legends. mortem analysis. The age and strain of the mice are indicated below and in the from the longitudinal during of blood glucose. The investigators were not blinded during group allocation or rodents were randomized based on body weight, body fat/lean mass, and levels of minimally yield an 80% chance to detect a significant difference in body weight food and water. Group size estimations were based uponto a access power free calculation to with °C 22 at cycle light-dark 12-h/12-h a on group-housed or weight and body fat/lean mass and levels of blood glucose. Rodents were single- obese mice, mice and were randomized lean into the using treatment groups studies based upon For body eight. of size group a reach to available numbers studies utilizing genetically modified animals because there were not sufficient for optimal be to this used, which was determined from previous experiments where we determined For for approaches experimental General receptorspecific binding. of result negative a considered are 25% than less values and binding, receptor tion of control that is greater than 25% is considered a positive result of specific control specific binding) × 100]. In general, results showing a percentage inhibi centage inhibition of control, calculated by [100 − (triagonist bindingspecific / binding [(triagonist specific / control binding) specific × 100], and as also per beled ligand. Results are expressed as percentage specific binding, calculated by unla native, respective a of excess an of non-specific presence the the in determined and binding binding total the between difference the as defined is receptors respective the to binding ligand specific The conditions. incubation 3 Table The exact specifications of each individual assay are detailed in controls positive includingGLP-1R as targets, at(Cerep). 73 andGcgR screen triagonist was profiled in a high-throughput custom-made competitive binding CEREP assay for non-selective receptor binding. P For cAMP production assessment, the medium was removed and the cells cells the and removed was medium the assessment, production cAMP For < 0.05 between the treatment groups. For studies using diabetic rodents, diabetic using studies Forgroups. treatment the between 0.05 < µ in vivo in g/ml g/ml streptomycin and were grown at 37 °C and 5% CO µ in vivo in , which includes receptor source, labeled and unlabeled ligands, and ligands, unlabeled and labeled source, receptor includes which , M rosiglitazone. After 3 days, media was replaced with DMEM contain With all DIO mice, male C57BL/6 mice (Jackson Laboratories) were pharmacological studies, a group size of eight was preferentially preferentially was eight of size group a studies, pharmacological Mouse insulinoma MIN6 cells were cultured in RPMI 1640 1640 RPMI in cultured were cells MIN6 insulinoma Mouse profiling of compounds. No samples or animals were excluded were animals or samplesNo compounds. of profiling in vivo in vivo in µ pharmacological profiling, challenge tests, or port M 2- evaluation. Smaller group sizes were used in the the in used were sizes group Smaller evaluation. µ g/ml g/ml dexamethasone and 5 β -mercaptoethanol, 100 in vivo in pharmacology experiments. pharmacology The cross-reactive binding of nature medicine nature µ µ g/ml g/ml penicillin and g/ml g/ml insulin. After 2 in a humidified Supplementary µ g/ml insulin insulin g/ml 2 at 37 °C. °C. 37 at - - - - -

© 2014 Nature America, Inc. All rights reserved. in the rooms with all tests being performed during the light phase. Access (~21 to°C) foodand relative humidity 55–65%. A 12-h light-dark cycle was maintained fed a special diet (Purina PMI 5008) and housed 1 per cage at room temperature ZDF rats. the during light cycle. performed were tests and injections All composition. body and Rats were randomized and distributed to test groups according to body weight single- were rats DIO housed on The a 12:12-h light-dark study. cycle at 22 long-term °C with free access the to food and water. initiating before weeks 40 forfat, from kcal 58% with diet high-sucrose a is which Diets), (Research diet rats. DIO triagonist. All injections and tests were performed during the light cycle. onist administration (0 min), and at 15, 30, 60, and 120 min after injection of the (TheraSense FreeStyle) before antagonist administration (−15 min), before triag Tail blood glucose concentrations were measured using a handheld glucometer triagonist. the of administration before min antagonist 15 GcgR with toneally to 6 h ofsubjected fasting at onset the ofandintraperi light the cycle injected ad libitum on based wererandomized and insulin the of washoutperiod 72-h a after test challenge glucagon the for used were Mice euglycemia. maintain to weeks 4 with long-acting insulin (made in house) via daily subcutaneous injections for level exceeding 200 mg/dl were considered sufficiently diabetic and were treated weight via a single intraperitoneal injection. After 3 d, mice with a blood glucose libitum.ad water and diet chow standard to access provided and cage per 4 housed were mice. STZ during the light cycle. and were double-housed for the study. injections All and tests were performed by randomized were Mice ies. and water background) were housed 4 per cage and provided access to standard chow diet mice. db/db cycle at 22 °C with free access to food and water. light-dark 12-h/12-h a on group-housed or single- were Mice cycle. light the were performed on mice aged 5 months and injections were administered mentionedduring diabetogenic diet for 6 weeks before initiation of injections. All tests ground) were bred in house and, starting at 12 weeks of age, were fed the afore Gipr cycle at 22 °C with free access to food and water. light-dark 12-h/12-h a on group-housed or single- were Mice cycle. light the were performed on mice aged 5 months and injections were administered mentionedduring diabetogenic diet for 6 weeks before initiation of injections. All tests ground) were bred in house and, starting at 12 weeks of age, were fed the afore Gcgr access to food and water. were single- or group-housed on a 12-h/12-h light-dark cycle at 22 °C with free Mice cycle. light the during administered were injections and months 8 aged mice on performed were tests All injections. of initiation before weeks 12 for background) were bred in house and fed the aforementioned diabetogenic diet Glp1r treatment each for mice of group entire was analyzed. the performed, were analyses If determi required. exclusion was no nation therefore, results; outlier or illness to were due animals test excluded No to composition. body distributed and weight evenly body and to according randomized groups were Mice cycle. light the dur performed ing were tests and injections All ofold. months ages 18 and the months 6 between were and studies pharmacological of weeks initiation 16 before of minimum a for conditions these under maintained were Mice nature medicine nature −/− −/− −/− mice. mice. mice. Nine-week-old male ZDF rats (Charles River Laboratories, USA) were –fed blood glucose and body weight. For the challenge test, mice were Twelve-week-old male Long Evens rats were fed a diabetogenic diabetogenic a fed were rats Evens Long male Twelve-week-old ad libitum Streptozotocin was administered at a dose of 150 mg per kg body body kg per mg 150 of dose a at administered was Streptozotocin Thirteen-month-old male C57BL/6 mice (Jackson Laboratories) Laboratories) (Jackson mice C57BL/6 male Thirteen-month-old Six-week-old male male Six-week-old Male Male Male Male Male Male . . Mice were 9 weeks old when used for the indicated stud Gipr Gcgr Glp1r −/− −/− x vivo ex mice and wild-type littermates (C57BL/6 back (C57BL/6 littermates wild-type and mice mice and wild-type littermates (C57BL/6 back (C57BL/6 littermates wild-type and mice −/− ad libitumad mice and wild-type littermates (C57BL/6 (C57BL/6 littermates wild-type and mice db/db molecular biology/histology/biochemistry biology/histology/biochemistry molecular mice (Jackson Laboratories; C57BL/6 C57BL/6 Laboratories; (Jackson mice –fed blood glucose and body weight, body and glucose blood –fed ------

initiation and termination following 6 h of fasting. For assessment of glucose mice andon a C57BL6 background. Fasted blood glucose was measured upon study of size group a with performed were mice wild-type in were measured day every or other every day after the first injection. All studies was administered by single formulated injections. Body weights and food intake indicated doses with the indicated durations. Co-administration of compounds tered by repeated subcutaneous injections in the middle of the light phase at the Rodent pharmacological and metabolism studies. guidelines. local and Animal Care (AAALAC unit no. 001057) and with appropriate federal, state accordance and in and guidelines of Basel-Stadt the Association Office for Veterinary the Assessment Cantonal and Accreditation Swiss ofthe Laboratory by Roche an F.Hoffmann-La to under authorization (ARP) and protocol research Cincinnati, animal of University Munich, Center Helmholtz the of according to the guidelines of the Institutional Animal Care and approval. Use Committee Ethical treatment termination and were subsequently forkilled remaining The analysis. Immediately after treatment cessation, concentrations. glucose fasting and weight body to according ture in conscious animals. ZDF rats were distributed into groups ( punc tail by performed was randomizationfor concentrations glucose blood and water was glucose glucose administration. the after min 120 and 60, 30, 15, 0, −15, −30, at injections subsequent before FreeStyle) (TheraSense glucometer handheld a using by measured were tions Tailintraperitoneal injections. via challenge concentraglucose glucose blood the triagonist (1 nmol per kg body weight) was administered 15 min before the intraperitoneal glucose challenge (2 g of glucose per kg body weight). Vehicle or (1 per group; age 9 months) were pretreated with vehicle or the GLP-1R antagonist comitant antagonism of the GLP-1R, 6-h fasted male C57BL/6 DIO con mice with ( tolerance glucose intraperitoneal on triagonist the of effects the of Acute tolerance test GLP-1Rglucose with antagonist. manufacturers’ instructions. using enzymatic assay kits (Wako). All assays were performed according to the assay kits (Thermo Fisher). Plasma free fatty acids and ketones were measured Plasma cholesterol, triglycerides, ALT, and AST were measured using enzymatic GIP,(Millipore). assay tin, ELISA an by quantified were GLP-1,glucagon and RIA; Linco Research) or by an ELISA assay (Alpco). Plasma FGF21, adiponec insulin was quantified by a radioimmunoassay from Linco (Sensitive 5,000 Rat at Insulin centrifuged ice, on euthanasia using EDTA-coated microvette tubes (Sarstedt), immediately chilled parameters. Blood and top levels of the cage, and activity being expressed as beam breaks. a multidimensional infrared light beam system with beams scanning the bottom sealed cage environment. Home-cage locomotor activity was determined using the into scales of integration by assessments calorimetry indirect the as time same the at h 120 for continuously determined was intake Food weeks. 2 for respiratory quotient and expenditureenergy after an initial treatment regimen every 10 min for a total of 120 h (including 48 h of adaptation) to determine the O Systems). (TSE system calorimetry indirect combined a using assessed were activity home-cage and Energy balance physiology measurements. was measured using nuclear magnetic resonance (EchoMRI). technology Body composition measurements. ment of end experimental points. not blinded to group allocation during the at least 24 h after the last administration of compounds. The investigators were insulin tolerance during chronic treatment, the challenge tests were performed µ mol per kg body weight) via an intraperitoneal injection 30 min before the ad libitum All rodent studies were approved by and performed performed and by approved were studies rodent All Blood was collected after a 6-h fast from tail veins or fromfast after tail 6-h a after collected was Blood . After 2 weeks of acclimatizing, measurement of fasting n 2 n = 4 rats 4 = were monitored for days additional 21 after consumption and CO and consumption = 4 rats group per forwere blindly selected g and 4 °C, and plasma stored at −80 °C. Plasma Plasma °C. −80 at stored plasma and °C, 4 and Whole-body composition (fat and lean mass) in vivo Energy intake, energy expenditure, experiments or to the assess 2 production were measured measured were production Compounds were adminis For the determination ex ex vivo n doi: = 8 per group using using group per 8 = 10.1038/nm.3761 analysis. n = 8/group) ex ex vivo n = 8 ------

© 2014 Nature America, Inc. All rights reserved. ducted with the approval of the local veterinary authority in strict adherence adherence strict in authority veterinary local the of approval the with ducted studies. Triagonistpharmacokinetic not blinded during analysis. was performed using Definiens Architect XD (Definiens AG). Investigators were (Carl Zeiss MicroImaging, Inc.). Quantitative image analysis of islet morphology platform Imager microscope scanning (MetaSystems) a using Z.2 Zeiss a with Fluor). Digital imaging fluorescence microscopy of the pancreas was performed followed10236276001), by fluorescent respective secondary antibodies (Alexa (1:50; Dako; A0565) and 4 out to assess islet anti-insulin morphology: (1:50; Dako; A0564), anti-glucagon following immunofluorescence stainings on tissue The sectionshistology. of liver 4 assess to performed was staining eosin and hematoxylin malin for 24h, dehydrated and subsequently embedded into paraffin. Standard described immunohistochemistry. and Histopathology before (0 min) and at 15, 30, 60, and 120 min after injection. trations were measured by using a handheld glucometer (TheraSense FreeStyle) neally with 0.75 units of insulin per kg body weight. Tail blood glucose concen subjected to 6 h of fasting at the onset of the light cycle and injected intraperito test. tolerance Insulin glucose was Blood monitored using the AccuCheck glucometer system. kg g (2 challenge glucose oral before measured was glucose blood and h) (16 period oral glucose tolerance was assessed in 8 rats per group after an overnight fasting onist (−15 min) in addition to the measurement times listed above. For wereZDF measuredrats, before administration of the antagonist (−30 min) and the triag triagonist with or without respective antagonists, blood glucose concentrations (0 min) and at before 15, 30, 60, FreeStyle) and 120 min (TheraSense after injection. glucometer For the handheld acute effects a of the using measured were toneally with 1 g glucose per kg body weight. Tail blood glucose concentrations to 6 h ofsubjected fasting at onset the ofandintraperi light the cycle injected For challenge. glucose the before min 30 and istration ofexcess triagonist, weredose the administered before15 min agonist admin 1000-fold a antagonists,at antagonists,the using tests tolerance glucose acute body weight for lean mice (20% w/v d-glucose (Sigma) in 0.9% w/v saline). For neally with 1.5 g glucose per kg body weight for DIO mice or 2 g glucose per kg subjected to 6 h of fasting at the onset of the light cycle and injected intraperito test. tolerance Glucose the glucose administration. FreeStyle) before subsequent injections at and 60, 30, 0, 15, after min −15, 120 (TheraSense glucometer handheld a using by measured were concentrations Tail weight). glucose body blood kg per nmol injection (1 triagonist the or vehicle with intraperitoneal the before min 15 injection intraperitoneal an via were pretreated with vehicle or the GcgR antagonist (1 GcgR, 6-h fasted male STZ C57BL/6 lean mice ( the of antagonism concomitant with glycemia on triagonist the of effects the Acute tolerance test antagonist. GcgR glucose with glucose administration. the after min 120 and 60, 30, 15, 0, −15, −30, at injections subsequent before trations were measured by using a handheld glucometer (TheraSense FreeStyle) the glucose challenge via intraperitoneal injections. Tail blood glucose concen orweight) triagonist was the kg administered (2 body nmol15 beforemin per intraperitoneal glucose challenge (1.5 g of glucose per kg body weight). Vehicle (2 per group; age 12 months) were pretreated with vehicle or the GIPR antagonist male fasted 6-h GIPR, the of antagonism comitant con with tolerance glucose intraperitoneal on triagonist the of effects the of antagonist. GIPR with test tolerance glucose Acute doi: µ 10.1038/nm.3761 mol per kg body weight) via an intraperitoneal injection 30 min before the −1 ), and subsequently at 15, 30, 60 and 120 min post-glucose challenge. post-glucose and min and ), subsequently at60 120 30, 15, 35 , 3 6 . In brief, tissue samples were fixed in 10% neutral-buffered for neutral-buffered 10% in sampleswerefixed tissue Inbrief, . For the determination of insulin tolerance, mice were mice tolerance, insulin of determination the For For the determination oftolerance, glucose mice were ′ , 6-diamidino-2-phenylindole (DAPI; 1:1,000; Roche; All pharmacokinetic studies were con were studies pharmacokinetic All n The methodology has been been has methodology The = 8 per group; age 13 months) Glp1r For the determination of µ mol per kg body weight) For the determination determination the For db/db −/− DIO mice ( mice DIO mice, mice were mice mice, µ m were carried n = 8 = ------

Researchers Researchers were not blinded during the investigation. Pharsight). 6.2.0.495, (Build WinNonlin Phoenix software standard industry parameters were determined by Non-Compartmental Analysis (NCA) with the tions in plasma were determined by LC-MS/MS and respective pharmacokinetic frozen immediately. samplesAll were stored at −20 °C. Compound concentra 3000 at centrifugation by min 30 within blood from and placed on ice into EDTA-coated polypropylene tubes. Plasma was prepared postdose by heart puncture, sublingually, or from the brachial vein, respectively, ( monkeys and point), samples from mice ( Blood monkeys. forthe needed was anesthesia no while pureoxygen), in tion Blood from mice and rats was collected under anesthesia (5% isoflurane inhala (45 neck the in injection subcutaneous as compound the with administered were rodents PK colony) were routinely for studies. Nonrandomized pharmacokinetic used male cynomolgus monkeys weighing approximately 10 kg each (Roche monkey humidity, and 12-h light/dark cycle) with free access to food and water. Six adult (Harlan Laboratories) were in housed a controlled environment (temperature, (Charles River), and adult male Wistar rats weighing approximately 250 g each (Harlan Laboratories), adult male DIO mice weighing approximately 60 g each each approximately g weighing 30 mice C57BL/6J Adultmale Basel. Roche at studies PK forperforming permissions animal Roche to according conducted Care Animal Internationalrodentsand nonhumanStudies with (AAALAC). primates were Laboratory of Accreditation and Assessment for Association the of rules the to and protection animal on regulations federal Swiss the to 36. 35. difference in body weight of significant a detect to chance 80% an yield minimally to calculation power a than 0.05 were considered significant. Group size estimations were based upon with Differences groups. treatment between significance tistical (ANOVA) with Tukey variance of analysis two-way or one-way regular a using pattern normal a in analyses. Statistical analysis. Researchers were not blinded during the investigation. receipt of data bioanalytical and transfer into WinNonlin for pharmacokinetic to limited was Use Excel of 2003. Excel Office Microsoft and Inc.) (Pharsight, analysis cokinetic data were analyzed using WinNonlin, version 5.2.1 software pharma tissue and plasma Unaudited flank. the in injection subcutaneous a 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, and 96 h after compound administration via h, 1 min, 30 min, at15 vein/artery femoral the via collected was blood Whole and individually housed in a controlled environment on a colony12-h light/darkhouse in cycle. an from chosen arbitrarily were monkeys cynomolgus adult designed to areavoid or study minimize discomfort, distress, and this pain to animals. in Three procedures possible, Whenever Basel. Roche at studies PK mates were conducted according to animal forpermissions Roche performing Office of Laboratory Animal Welfare. Studies with rodents and nonhuman pri Academy Press, Washington, D.C., National 1996; and the National InstitutesResources, of Health, Animal Laboratory of Institute Animals, Laboratory of (USDA) Agriculture’s of Useand Care forthe Guide the 3); Welfareand 2, Animal 1, Parts CFR Department (9 Act US the with compliance in are tocol studies. pharmacokinetic coagonist GLP-1/GIP

Uhles, S. Uhles, A. Bénardeau, diabetic fatty rat fatty diabetic pancreatic beta-cells 15 rat. fatty diabetic Zucker the in protection organ and control , 164–174 (2013). 164–174 , et al. et µ g kg g Taspoglutide, a novel human once-weekly GLP-1 analogue, protects analogue, GLP-1 Taspoglutide,once-weekly human novel a et al. et −1 in vivo in n Statistical analyses were performed on data distributed distributed data on performed were analyses Statistical ) and nonrandomized monkeys in the flank (15 (15 flank the in and nonrandomized monkeys ) = 2 samples per time point), rats ( Effects of the dual PPAR- dual the of Effects n in vitro post hoc = 3 samples per time point) were collected up to 24 h 24 to up collected werepoint) time per samples 3 = . Diabetes Obes. Metab. Obes. Diabetes P and preserves islet structure and function in the Zucker < 0.05 the between treatment groups. multiple comparison analysis to determine sta α /

γ 13 agonist aleglitazar on glycaemic on aleglitazar agonist All procedures in this pro this in procedures All , 326–336 (2011). 326–336 , g for 5 min at 4 °C and °C 4 at min 5 for n nature medicine nature = 3 samples per time Diabetes Obes. Metab. Obes. Diabetes P values less less values µ g kg g −1 ). ). ------

Integrated triple gut hormone action broadens the therapeutic potential of endocrine biologics for metabolic diseases

Brian Finan1–3*#, Bin Yang3,4*, Nickki Ottaway5, David L. Smiley3, Tao Ma3,6, Christoffer Clemmensen1,2, Joe Chabenne3,7, Lianshan Zhang4, Kirk M. Habegger8, Katrin Fischer1,2, Jonathan E. Campbell9, Darleen Sandoval5, Randy J. Seeley5, Konrad Bleicher10, Sabine Uhles10, William Riboulet10, Jürgen Funk10, Cornelia Hertel10, Sara Belli10, Elena Sebokova10, Karin Conde-Knape10, Anish Konkar10, Daniel J. Drucker9, Vasily Gelfanov3, Paul T. Pfluger1,2,5, Timo D. Müller1,2, Diego Perez-Tilve5, Richard D. DiMarchi3# & Matthias H. Tschöp1,2,5#

Nature Medicine: doi:10.1038/nm.3761 Supplemental Results

Chemical evolution from co-agonism to tri-agonism

The structural and sequence similarities amongst the three hormones (Fig. 1e),

coupled with prior structure-function studies1,2, informed the design of sequence

hybridized peptides of high potency and balanced mixed agonism. The native

hormones share nine conserved amino acids at positions 4, 5, 6, 8, 9, 11, 22, 25, and

26. These residues can be broadly grouped into two regions constituted by amino

acids 4-11 and 22-26. The remaining central residues (amino acids 12-21) and the

proximal terminal amino acids (1-3 and 27-30) exhibit more diversity that imparts the

specificity of receptor interaction with each respective native hormone. Consequently,

the challenge here is to maintain the individual affinity of each ligand for its receptor

while eliminating the structural elements that convey selective preference for each

individual receptor. Or stated differently, the objective here was the identification of a

high affinity, promiscuous peptide for these three receptors.

Intermediary tri-agonist candidates were built from a glucagon-based core

sequence with residues incorporated from GLP-1 that were previously shown to

impart balanced and potent co-agonism at GLP-1R and GcgR1. This starting chimeric

peptide features specific GLP-1 residues in the C-terminal portion at positions 17, 18,

20, 21, 23, and 24 (sequences of intermediate analogs displayed in Supplementary

Figure 1 and mass spectrometry data is summarized in Supplementary Table 1). A

series of peptide analogs was progressed in an iterative manner to introduce GIP

agonism without destroying GLP-1R and GcgR potency. Each peptide was assessed

for potency and maximal activity in a highly sensitive cell-based reporter gene assay

that measured cAMP induction where one of the three human receptors was over-

expressed in HEK293 cells (Table 1). In initial attempts to gain GIP activity, GIP-

Nature Medicine: doi:10.1038/nm.3761 specific N-terminal amino acids were individually and selectively introduced into

peptide analogs of the parent, chimeric peptide. However, these GIP-derived

substitutions, including Tyr1 and Ile7, which are well-characterized to be essential for

native GIP activity3, demonstrated little improvement in GIP potency (data not

shown). Separately, Glu3 substitution into the parent chimeric peptide, had noticeably

enriched GIP character, but this resulted in a substantial reduction in potency at GcgR

compared to the starting peptide (Table 1, peptide 9). Each of the three individual

substitutions resulted in a concomitant loss of activity at the two other receptors, and

in particular GcgR potency seemed most sensitive with the Glu3 substitution being

especially destructive, which is consistent with published reports4,5. This

demonstrated that imparting sufficient GIP activity would not be trivial and suggested

that extensive sequence modifications were essential to introduce the requisite triple

agonism we desired. With the eventual intent of using these peptides for in vivo

study, we considered the prospect of using site-specific lipidation to extend duration

of biological action by promoting plasma albumin binding. As we have shown

previously, site-specific lipidation can also serve as a chemical tool to enhance

secondary structure and broaden biological activity6. We introduced a lysine at

residue ten in the parent peptide to which a palmitic acid (C16:0), which was

amidated through a single glutamic acid coupled at its gamma carboxylate (γE

spacer). The suspected ability of the lipidation to stabilize secondary structure in a

non-covalent manner that is analogous to what the lactam bond provides. One last

change was the inversion of serine stereochemistry (d-Ser) at position two in order to

render the analog resistant to dipeptidyl peptidase IV (DPP-IV)-mediated degradation,

which is the endogenous enzyme responsible for N-terminal truncation of the first two

amino acids of GLP-1, GIP, and glucagon. Secondly, this substitution at position two

Nature Medicine: doi:10.1038/nm.3761 also serves to preserve the potency at GcgR. This single peptide (Table 1, peptide 10)

had a receptor activity profile similar to the starting peptide, but did not install any

appreciable gain in GIPR agonism.

We had previously observed that enhanced alpha helical content is beneficial

for inducing mixed agonism of GLP-1R and GcgR1. To determine if enhancing

helicity likewise imparts GIPR agonism, further stabilization of the backbone helix

within the aforementioned lipidated peptide (Table 1, peptide 10) was achieved with

employment of an aminoisobutyric acid (Aib) substitution at position 16. Additionally

and with the eventual intent for using these compounds in vivo, Aib was also

employed at position two in order to convey resistance to DPP-IV inactivation in an

analogous fashion as d-Ser. We have previously observed that Aib2 also contributes to

mixed agonism at GLP-1R and GIPR2, however this substitution can be detrimental to

glucagon activity. Therefore, a series of glucagon-specific residues were introduced to

counter-act this anticipated loss in GcgR potency, which included Arg17, Gln20, and

Asp28, of which the latter substitution also enhances aqueous solubility in neutral pH

buffers7. However, these cumulative substitutions (Table 1, peptide 11), despite

preserving GcgR potency, did not introduce appreciable GIP activity when compared

to the initial lipidated analog (Table 1, peptide 11).

In a parallel modification to the lipidated analog (Table 1, peptide 10), we

included Aib2, but we retained Glu16 instead of substitution with Aib16. Glu16 likewise

stabilizes the alpha helix through a non-covalent intra-helical interaction with Lys20

albeit to a lesser degree than Aib16, as in peptide 11. Glu16 also provokes mixed

agonism at GLP-1R and GcgR, and was thus retained to counterbalance the

detrimental effects of Aib2 on GcgR activity. We also included Leu27, which is

specific to native glucagon, in an additional attempt to boost glucagon activity. But

Nature Medicine: doi:10.1038/nm.3761 again much like the aforementioned previous attempts, these cumulative substitutions

failed to enhance GIPR activity despite enhancing balanced, mixed agonism at GLP-

1R and GcgR (Table 1, peptide 12). Each of these separate yet collective changes

highlighted in peptide 11 and peptide 12 was investigated as a means to retain

glucagon potency yet enhance GIP potency. Despite these failures in gaining GIP

activity, these modifications led to the discovery of a high potency GLP-1/glucagon

co-agonist with lipidation suitable for use in vivo and of enhanced solubility and

chemical stability relative to the native hormones (Table 1, peptide 12). Nonetheless,

the primary objective of balanced tri-agonism was no closer to a reality than when we

started since peptide 12 is reduced in GIP potency relative to the other two

constituent activities by approximately one thousand-fold.

The structure-activity relationship (SAR) of position 2 was interrogated as we

have previously reported the constructive interactions at this site with the central

region of the peptide to change bioactivity8. To make a peptide backbone suitable for

position 2 SAR without a subsequent loss of glucagon potency, a peptide scaffold was

generated using several of the previously employed changes in the middle and C-

terminal regions of peptide 11 and peptide 12, including Glu16, Arg17, Gln20, Leu27,

and Asp28, all of which also serve auxiliary functions to enhance solubility and

chemical stability. Additionally, in an attempt to selectively enhance GIP and

glucagon activity, we introduced Asp21 and Val23, which are each specific to native

GIP and glucagon. Mixed agonism at GLP-1R and GcgR was preserved without an

enrichment of agonism at GIPR (Table 1, peptide 13). Since the second amino acid

influences selective activity at each constitutive receptor target, and also because the

residues in the native sequences of GLP-1, GIP, and glucagon (alanine and serine) are

both susceptible to in vivo proteolysis by DPP-IV9,10, we chose amino acid

Nature Medicine: doi:10.1038/nm.3761 substitutions that retain a slightly different side chain composition, but one that was

resistant to enzymatic cleavage. However, substitution with Aib2, dSer2, Gly2,

sarcosine (Sar2), or dAla2 did not appreciably change any of the constitutive receptor

activity profiles (Table 1, peptides 13-17) such that unimolecular tri-agonism still

remained elusive.

In discovering the dual GLP-1/GIP co-agonist, we observed that the terminal

ends of the peptide need to be coordinately optimized to achieve high potency dual

incretin receptor agonism2. Consequently, we inserted the three C-terminal residues of

one of the endogenous forms of GLP-1, which included Gly29, Arg30, and Gly31, into

the dSer2 containing peptide 14 to generate an analog of 31-amino acid length (Table

1, peptide 18). These elongating substitutions impart a subtle gain in GIP activity that

inspired further C-terminal extension of the peptide. Application of the C-terminal-

extended (Cex) residues from exendin-4 to generate a 39-residue analog (Table 1,

peptide 19) resulted in enhanced GIP activity and represents a breakthrough in the

SAR to realize substantially higher GIP potency than the starting point peptide, or any

of the aforementioned intermediate analogs. However, the GIP activity in peptide 19

was unbalanced relative to its GLP-1 and glucagon counterparts, with an EC50 at

GIPR that is of ~30-fold less relative potency (Table 1, peptide 19). Substitution with

Aib2 corrected this relative GIP imbalance to provide a unimolecular analog with a

length identical to exendin-4, that is of exquisite potency and balance at each of the

three receptors (Table 1, peptide 20). This peptide represents the first highly potent,

balanced unimolecular triple agonist at GLP-1R, GIPR, and GcgR. Furthermore, this

single molecule hybrid also possesses optimized chemical stability and

pharmacokinetics due to site-specific acylation.

Nature Medicine: doi:10.1038/nm.3761 References

1 Day, J. W. et al. A new glucagon and GLP-1 co-agonist eliminates obesity in rodents. Nature chemical biology 5, 749-757, doi:10.1038/nchembio.209 (2009). 2 Finan, B. et al. Unimolecular dual incretins maximize metabolic benefits in rodents, monkeys, and humans. Science translational medicine 5, 209ra151, doi:10.1126/scitranslmed.3007218 (2013). 3 Moon, M. J. et al. Tyr1 and Ile7 of glucose-dependent insulinotropic polypeptide (GIP) confer differential ligand selectivity toward GIP and glucagon-like peptide-1 receptors. Molecules and cells 30, 149-154, doi:10.1007/s10059-010-0100-5 (2010). 4 Runge, S., Wulff, B. S., Madsen, K., Brauner-Osborne, H. & Knudsen, L. B. Different domains of the glucagon and glucagon-like peptide-1 receptors provide the critical determinants of ligand selectivity. British journal of pharmacology 138, 787-794, doi:10.1038/sj.bjp.0705120 (2003). 5 Pocai, A. et al. Glucagon-like peptide 1/glucagon receptor dual agonism reverses obesity in mice. Diabetes 58, 2258-2266, doi:10.2337/db09- 0278 (2009). 6 Ward, B., Ottaway N., Perez-Tilve D., Ma D., Gelfanov VM.,Tschop MH., and DiMarchi RD. Structural Changes Associated with Peptide Lipidation Broaden Biological Function. Molecular metabolism, doi:10.1016/j.molmet.2013.08.008 (2013). 7 Chabenne, J. R., DiMarchi, M. A., Gelfanov, V. M. & DiMarchi, R. D. Optimization of the native glucagon sequence for medicinal purposes. Journal of diabetes science and technology 4, 1322-1331 (2010). 8 Patterson, J. T., Day, J. W., Gelfanov, V. M. & DiMarchi, R. D. Functional association of the N-terminal residues with the central region in glucagon-related peptides. Journal of peptide science : an official publication of the European Peptide Society 17, 659-666, doi:10.1002/psc.1385 (2011). 9 Pospisilik, J. A. et al. Metabolism of glucagon by dipeptidyl peptidase IV (CD26). Regulatory peptides 96, 133-141 (2001). 10 Kieffer, T. J., McIntosh, C. H. & Pederson, R. A. Degradation of glucose- dependent insulinotropic polypeptide and truncated glucagon-like peptide 1 in vitro and in vivo by dipeptidyl peptidase IV. Endocrinology 136, 3585-3596 (1995).

Nature Medicine: doi:10.1038/nm.3761 GLP-1 Receptor GIP Receptor Glucagon Receptor Peptide # Analog EC50 (nM) STDev Relative % EC50 (nM) STDev Relative % EC50 (nM) STDev Relative % 1 GLP-1 0.028 0.002 100 1513.825 78.950 0 2449.288 106.460 0 2 GLP-1 (Aib2 E16 Cex K40-C16 acyl) "Acyl-GLP-1" 0.015 0.001 187 122.883 10.668 0 162.402 29.771 0 3 GIP 1567.012 65.780 0 0.020 0.004 100 417.480 10.370 0 2 40 4 GIP (Aib Cex K -C16 acyl) "Acyl-GIP" 6.330 2.690 0 0.012 0.001 168 2.920 0.840 1 5 Glucagon 3.100 0.494 1 1538.109 329.020 0 0.032 0.006 100 2 10 20 6 Glucagon (Aib K -γEγE-C16 acyl Aib ) "Acyl-glucagon" 0.479 0.068 6 22.650 6.100 0 0.005 0.001 625 7 "GLP-1/GIP co-agonist" 0.005 0.001 516 0.003 0.001 691 1.286 0.103 2 GIP SAR 16 17 18 20 21 23 24 8 Glucagon (E Q A K E I A )-NH2 0.022 0.002 127 6.258 1.278 0 0.031 0.009 103 3 9 Peptide 8 (E )-NH2 0.030 0.003 93 0.867 0.099 2 5.225 0.441 1 2 10 10 Peptide 8 (dS K -γE-C16 acyl)-NH2 0.039 0.005 72 2.691 0.416 1 0.041 0.002 78 2 16 17 20 21 24 28 11 Peptide 10 (Aib Aib R Q D Q D )-NH2 0.015 0.003 187 2.194 0.329 1 0.028 0.009 114 2 16 27 28 12 Peptide 10 (Aib E L D )-NH 2 0.003 0.001 933 3.545 0.156 1 0.003 0.001 1067 Position 2 SAR 2 17 20 21 23 24 27 28 13 Peptide 10 (Aib R Q D V Q L D )-NH2 0.068 0.008 41 2.032 0.117 1 0.005 0.001 640 2 14 Peptide 13 (dS )-NH 2 0.003 0.001 933 1.654 0.164 1 0.006 0.001 533 2 15 Peptide 13 (G )-NH2 0.093 0.013 30 1.103 0.200 2 0.005 0.001 640 2 16 Peptide 13 (Sar )-NH 2 0.115 0.014 24 5.828 0.334 0 0.016 0.003 200 2 17 Peptide 13 (dA )-NH 2 0.040 0.005 70 1.646 0.298 1 0.009 0.001 356 29 30 31 18 Peptide 14 (G R G )-NH2 0.002 0.001 1400 0.812 0.101 2 0.004 0.001 800 29 19 Peptide 14 (G Cex)-NH2 0.002 0.001 1400 0.085 0.009 24 0.003 0.001 1067 2 20 Peptide 19 (Aib )-NH2 "Tri-agonist" 0.003 0.001 933 0.003 0.001 673 0.004 0.001 800 Position 3 SAR 3 21 Peptide 20 (hSer )-NH2 0.003 0.001 933 0.004 0.001 505 0.016 0.002 200 3 22 Peptide 20 (nVal )-NH2 0.003 0.001 933 0.002 0.001 1010 0.025 0.004 128 3 23 Peptide 20 (V )-NH2 0.006 0.001 467 0.005 0.001 404 0.026 0.004 123 3 24 Peptide 20 (nLeu )-NH 2 0.003 0.001 933 0.003 0.001 673 0.033 0.003 97 3 25 Peptide 20 (Dap(Ac) )-NH2 0.002 0.001 1400 0.010 0.002 202 0.043 0.006 74 3 26 Peptide 20 (Met(O) )-NH2 "Imbalanced tri-agonist" 0.003 0.001 933 0.018 0.002 112 0.089 0.007 36 3 27 Peptide 20 (E )-NH2 "Matched co-agonist" 0.004 0.001 700 0.002 0.001 1010 1.420 0.090 2 FLATT 28 YAG-Glucagon 5.484 0.423* 1 1128.970 57.430* 0 3.270 0.109* 1 29 [DA2]GLP-1/GcG 100.480 0.411* 0 17.110 0.920* 0 5.139 0.184* 1 RECIPROCAL 30 GLP-1/glucagon 0.004 0.001 700 0.133 0.084 15 0.007 0.002 457 31 GIP/glucagon 0.568 0.192 5 0.004 0.001 505 0.008 0.003 400

Supplementary Table 1. In vitro human receptor activity profile of peptides. EC50 values represent the effective peptide concentrations (nM) that stimulate half-maximal activation at the human GLP-1, GIP, and glucagon receptors. STDev values represent the standard deviation of the calculated EC50 from each separate experiment. A minimum of three separate experiments was performed for each peptide at each respective receptor. Those peptides denoted with an asterisk (*) were only tested once at each respective receptor. Relative % activity at each receptor = (native ligand EC50/analog EC50) x 100. Continuing down from peptide 10, the peptides feature the same sequence of the peptide number denoted in the analog box with the subsequent modification contained within the parentheses. All sequences are found in Supplementary Figure 1.

Nature Medicine: doi:10.1038/nm.3761 Peptide # Analog Name Theoretical+Mass Observed+Mass 1 GLP-1 3355.71 3357.1 2 GLP-1 (Aib2 E16 Cex K40-C16 acyl) "Acyl-GLP-1" 4429.06 4429.72 3 GIP 4855.46 4856.00 2 40 4 GIP (Aib Cex K -C16 acyl) "Acyl-GIP" 4606.28 4606.37 5 Glucagon 3482.79 3483.40 2 10 20 6 Glucagon (Aib K -γEγE-C16 acyl Aib ) "Acyl-glucagon" 3868.42 3868.49 7 "GLP-1/GIP co-agonist" 4473.11 4473.14 GIP SAR 16 17 18 20 21 23 24 8 Glucagon (E Q A K E I A )-NH2 3381.73 3382.19 3 9 Peptide 8 (E )-NH2 3383.69 3384.10 2 10 10 Peptide 8 (dS K -γE-C16 acyl)-NH2 3714.25 3713.00 2 16 17 20 21 24 28 11 Peptide 10 (Aib Aib R Q D Q D )-NH2 3726.27 3727.40 2 16 27 28 12 Peptide 10 (Aib E L D )-NH 2 3695.23 3694.50 Position 2 SAR 2 17 20 21 23 24 27 28 13 Peptide 10 (Aib R Q D V Q L D )-NH2 3727.25 3726.00 2 14 Peptide 13 (dS )-NH 2 3754.22 3753.00 2 15 Peptide 13 (G )-NH2 3721.90 3720.50 2 16 Peptide 13 (Sar )-NH 2 3735.91 3735.00 2 17 Peptide 13 (dA )-NH 2 3738.22 3738.00 29 30 31 18 Peptide 14 (G R G )-NH2 3924.39 3924.16 29 19 Peptide 14 (G Cex)-NH2 4546.03 4546.00 2 20 Peptide 19 (Aib )-NH2 "Tri-agonist" 4543.08 4544.26 Position 3 SAR 3 21 Peptide 20 (hSer )-NH2 4516.05 4515.50 3 22 Peptide 20 (nVal )-NH2 4528.11 4530.80 3 23 Peptide 20 (V )-NH2 4528.11 4529.60 3 24 Peptide 20 (nLeu )-NH 2 4528.11 4528.00 3 25 Peptide 20 (Dap(Ac) )-NH2 4543.08 4543.00 3 26 Peptide 20 (Met(O) )-NH2 "Imbalanced tri-agonist" 4576.17 4577.47 3 27 Peptide 20 (E )-NH2 "Matched co-agonist" 4544.06 4545.05 FLATT 28 YAG-Glucagon 3259.54 3259.56 2 29 [DA ]GLP-1/GcG 3556.01 3556.03 RECIPROCAL 30 GLP-1/glucagon 3921.43 3922.39 31 GIP/glucagon 4484.18 4484.00

Supplementary Table 2. Mass profiles for peptide analogs. Theoretical and observed masses of each analog. Peptide molecular weights were determined electrospray ionization (ESI) or matrix-assisted laser desorption/ionization time-of- flight (MALDI-TOF) mass spectrometry, and character was confirmed by analytical reversed-phase high performance liquid chromatography (HPLC).

Nature Medicine: doi:10.1038/nm.3761

GLP1R GIPR GCGR

Species Peptide EC504[nM] EC504[nM] EC504[nM] Mean SD Mean SD Mean SD GLP$1/GIP(co$agonist 0.280 0.030 0.071 0.020 10.100 1.830 Mouse Tri$agonist((peptide(20) 0.070 0.030 0.060 0.020 0.040 0.020 Native(Ligand 0.080 0.005 0.050 0.010 0.008 0.004

GLP$1/GIP(co$agonist 0.124 0.080 0.040 0.016 20.317 11.390 Rat Tri$agonist((peptide(20) 0.060 0.030 0.030 0.010 0.090 0.030 Native(Ligand 0.050 0.005 0.010 0.007 0.032 0.016

GLP$1/GIP(co$agonist 0.117 0.070 0.109 0.105 28.585 2.425 Cyno Tri$agonist((peptide(20) 0.060 0.010 0.080 0.030 0.090 0.020 Native(Ligand 0.080 0.040 0.030 0.010 0.190 0.030

Supplementary Table 3. cAMP production in CHO cells individually expressing recombinant mouse-, rat-, or cynomolgus monkey-derived GLP-1R, GIPR, or GcgR. EC50 values represent the effective peptide concentrations (nM) that stimulate half-maximal activation, as assessed by cAMP accumulation, at the mouse, rat, and cyno GLP-1, GIP, and glucagon receptors. SD values represent the standard deviation of the calculated EC50 from each separate experiment. A minimum of three separate experiments was performed for each peptide at each respective receptor from each species.

Nature Medicine: doi:10.1038/nm.3761

Receptor Non-Specific Ligand Radioligand Cell Souce Receptor Source Incubation Time (min) Incubation Temp (°C) Specific Binding % STDev % Inhibition of Control

A1 Adenosine Receptor DPCPX (1µM) DPCPX (1nM) CHO Recombinant 60 RT 96.3 5.7 3.8 A2A Adenosine Receptor NECA (10µM) CGS (6nM) HEK-293 Recombinant 120 RT 112.2 11.2 -12.2 A3 Adenosine Receptor IB-MECA (1µM) IB-MECA (0.15nM) HEK-293 Recombinant 120 RT 127.2 7.7 -27.2 Alpha 1 Adrenergic Receptor Prazosin (0.5µM) Prazosin (0.25nM) Rat Cortex Endogenous 60 RT 105.7 2.5 -5.7 Alpha 2 Adrenergic Receptor (-) Epinepherine (100µM) RX 8211002 (0.5nM) Rat Cortex Endogenous 60 RT 109.1 28.6 -9.1 Beta 1 Adrenergic Receptor Alprenolol (50µM) CGP 12177 (0.3nM) HEK-293 Recombinant 60 RT 92.6 8.4 7.5 Beta 2 Adrenergic Receptor Alprenolol (50µM) CGP 12177 (0.3nM) CHO Recombinant 120 RT 106.3 5.6 -6.3 Norepinepherine Transporter Desipramine (1µM) Nisoxetine (1nM) CHO Recombinant 120 4 111.5 6.3 -11.5 Angiotensin II Receptor Type 1 Angiotensin II (10µM) Sar1, Ile8-Angiotensin II (0.05nM) HEK-293 Recombinant 120 37 135.2 9.1 -35.2 Angiotensin II Receptor Type 2 Angiotensin II (10µM) CGP 42112A (0.01nM) HEK-293 Recombinant 240 37 101.0 5.2 -0.9 Central Benzodiazepine Receptor Diazepam (3µM) Flunitrazepam (0.4nM) Rat Cortex Endogenous 60 4 124.9 8.7 -24.9 Peripheral Benzodiazepine Receptor PK 11195 (10µM) PK 11195 (0.2nM) Rat Heart Endogenous 15 RT 108.1 7.7 -8.1 Bombesin Receptor Bombesin (1µM) Tyr4-Bombesin (0.01nM) Rat Cortex Endogenous 60 RT 105.2 1.7 -5.2 Bradykinin Receptor B2 Bradykinin (1µM) Bradykinin (0.3nM) CHO Recombinant 60 RT 101.6 3.9 -1.6 Calcitonin Receptor-Like Receptor CGRPa (1µM) CGRPa (0.03nM) CHO Recombinant 90 RT 112.5 9.3 -12.5 L-Type Calcium Channel D 600 (10µM) D 888 (3nM) Rat Cortex Endogenous 120 RT 117.8 0.7 -17.8 Small Conductance Calcium-Activated Potassium Channel Apamin (100nM) Apamin (0.007nM) Rat Cortex Endogenous 60 4 98.2 11.2 1.8 Cannabinoid Receptor Type 1 WIN 552312-2 (10µM) CP559400.5nM) CHO Recombinant 120 37 111.4 6.4 -11.4 Cholecystokinin A Receptor CCK (1µM) CCK (0.08nM) CHO Recombinant 60 RT 111.9 4.8 -11.9 Cholecystokinin B Receptor CCK (1µM) CCK (0.08nM) CHO Recombinant 60 RT 120.4 9.3 -20.4 Dopamine Receptor D1 SCH 23390 (1µM) SCH 23390 (0.3nM) CHO Recombinant 60 RT 111.2 0.4 -11.2 Dopamine Receptor D2 (+) Butaclamol (10µM) Methyl-spiperone (0.3nM) HEK-293 Recombinant 60 RT 94.0 3.1 6.0 Dopamine Receptor D3 (+) Butaclamol (10µM) Methyl-spiperone (0.3nM) CHO Recombinant 60 RT 113.8 4.9 -13.8 Dopamine Receptor D4 (+) Butaclamol (10µM) Methyl-spiperone (0.3nM) CHO Recombinant 60 RT 110.0 6.8 -10.0 Dopamine Receptor D5 SCH 23390 (10µM) SCH 23390 (0.3nM) GH4 Recombinant 60 RT 109.1 8.1 -9.1 Endothelin Receptor Type A Endothelin-1 (100µM) Endothelin-1 (0.03nM) CHO Recombinant 120 37 117.3 12.0 -17.3 Endothelin Receptor Type B Endothelin-1 (0.1µM) Endothelin-1 (0.03nM) CHO Recombinant 120 37 103.0 12.3 -3.0 Dopamine Transporter BTCP (10µM) BTCP (4.5nM) CHO Recombinant 120 4 103.1 0.6 -3.1 GABA Receptor GABA (100µM) GABA (10nM) Rat Cortex Endogenous 60 RT 103.5 6.6 -3.5 GABA-Gated Chloride Channel Picrotoxinin (20µM) TBPS (3nM) Rat Cortex Endogenous 120 RT 94.3 4.5 5.8 Galanin Receptor 1 Galanin (1µM) Galanin (0.1nM) HEK-293 Recombinant 60 RT 115.6 3.5 -15.6 Galanin Receptor 2 Galanin (1µM) Galanin (0.05nM) CHO Recombinant 120 RT 99.8 1.8 0.2 Glucocorticoid Receptor Triamcinolone (10µM) Dexamethasone (1.5nM) IM-9 Endogenous 360 4 100.5 3.1 -0.5 Glucagon Receptor Glucagon (1µM) Glucagon (0.025nM) CHO Recombinant 120 RT 23.3 5.9 76.7 Glucagon-Like Peptide 1 Receptor GLP-1 (1µM) GLP-1 (0.025nM) BTC6 Endogenous 120 37 -5.6 4.7 105.6 Platelet-Derived Growth Factor Receptor PDGF BB (10nM) PDGF (0.3nM) Balb/c 3T3 Endogenous 180 4 104.8 1.7 -4.8 Interleukin 8 Receptor IL-8 (30nM) IL-8 (0.025nM) HEK-293 Recombinant 60 RT 101.3 0.0 -1.3 C-C Chemokine Receptor Type 1 MIP-1a (100nM) MIP-1a (0.01nM) HEK-293 Recombinant 120 RT 109.7 6.0 -9.7 Cluster of Differentiation 120 TNFa (10nM) TNFa (0.1nM) U-937 Endogenous 120 4 102.2 2.3 -2.2 Histamine H1 Receptor Pyrilamine (1µM) Pyrilamine (1nM)) HEK-293 Recombinant 60 RT 98.1 11.0 1.9 Histamine H2 Receptor Tiotidine (100µM) APT (0.075nM) CHO Recombinant 120 RT 120.4 7.1 -20.4 Melanocortin 4 Receptor NDP-aMSH (1µM) NDP-aMSH (0.05nM) CHO Recombinant 120 37 109.0 3.3 -8.9 Melatonin Receptor Type 1A Melatonin (1µM) 2-iodomelatonin (0.01nM) CHO Recombinant 60 RT 113.3 8.1 -13.3 Muscarinic Acetylcholine Receptor M1 Atropine (1µM) Pirenzepine (2nM) CHO Recombinant 60 RT 133.1 5.0 -33.1 Muscarinic Acetylcholine Receptor M2 Atropine (1µM) AF-DX 384 (2nM) CHO Recombinant 60 RT 110.1 6.2 -10.1 Muscarinic Acetylcholine Receptor M3 Atropine (1µM) 4-DAMP (0.2nM) CHO Recombinant 60 RT 103.2 3.8 -3.2 Muscarinic Acetylcholine Receptor M4 Atropine (1µM) 4-DAMP (0.2nM) CHO Recombinant 60 RT 117.7 13.1 -17.7 Muscarinic Acetylcholine Receptor M5 Atropine (1µM) 4-DAMP (0.3nM) CHO Recombinant 60 RT 108.7 8.2 -8.7 Tachykinin Receptor NK1 Sar9, Met(O2)11 -Substance P (1µM) BH-Substance P (0.15nM) U-373MG Endogenous 60 RT 105.4 0.8 -5.4 Tachykinin Receptor NK2 Nleu10-Neurokinin A (4-10) (300nM) Neurokinin A (0.1nM) CHO Recombinant 60 RT 108.4 10.2 -8.4 Tachykinin Receptor NK3 SB 222200 (10µM) SR 142801 (0.4nM) CHO Recombinant 120 RT 105.6 4.5 -5.6 Neuropeptide Y Receptor Y1 NPY (1µM) Peptide YY (0.025nM) SK-N-MC Endogenous 120 37 103.6 1.1 -3.6 Neuropeptide Y Receptor Y2 NPY (1µM) Peptide YY (0.015nM) KAN-TS Endogenous 60 37 113.3 7.1 -13.3 Neurotensin Receptor 1 Neurotensin (1µM) Tyr3-Neurotensin (0.05nM) CHO Recombinant 60 4 108.5 1.7 -8.5 N-Methyl-D-Aspartate Receptor MK 801 (10µM) TCP (10nM) Rat Cortex Endogenous 120 37 103.0 10.0 -3.0 Delta Opioid Receptor Naltrexone (10µM) DADLE (0.5nM) CHO Recombinant 120 RT 101.0 2.6 -0.9 Kappa Opioid Receptor Naloxone (10µM) U 69593 (1nM) CHO Recombinant 60 RT 109.2 8.1 -9.2 Mu Opioid Receptor Naloxone (10µM) DAMGO (0.5nM) HEK-293 Recombinant 120 RT 100.4 5.0 -0.3 Nociceptin Receptor Nociceptin (1µM) Nociceptin (0.2nM) HEK-293 Recombinant 60 RT 99.7 1.8 0.3 Pituitary Adenylate Cyclase-Activating Polypeptide Type 1 Receptor PACAP (1-27) (100nM) PACAP (1-27) (100nM) CHO Recombinant 120 RT 112.8 7.6 -12.8 Peroxisome Proliferator-Activated Receptor Gamma Rosiglitazone (10µM) Rosiglitazone (5nM) E. coli Recombinant 120 4 94.5 7.1 5.6 Voltage-Gated Potassium Channel a-Dendrotoxin (50nM) a-Dendrotoxin (0.01nM) Rat Cortex Endogenous 60 RT 112.2 1.0 -12.2

Prostaglandin E2 Receptor PGE2 (10µM) PGE2 (3nM) HEK-293 Recombinant 120 RT 102.7 8.5 -2.7

Prostaglandin E4 Receptor PGE2 (10µM) PGE2 (0.5nM) HEK-293 Recombinant 120 RT 106.1 0.6 -6.1 Prostacyclin Receptor Iloprost (10µM) Iloprost (6nM) HEK-293 Recombinant 60 RT 118.2 9.3 -18.2 Purinergic Receptor dATPaS (10µM) dATPaS (10nM) Rat Cortex Endogenous 60 RT 91.2 5.2 8.8 Purinergic Receptor P2X a,B-MeATP (10µM) a,B-MeATP (3nM) Rat Bladder Endogenous 120 4 101.6 0.9 -1.6

Serotonin Receptor HT 1A 8-OH-DPAT (10µM) 8-OH-DPAT (0.3nM) HEK-293 Recombinant 60 RT 106.7 0.3 -6.7

Serotonin Receptor HT 1B Serotonin (10µM) CYP (0.1nM) / isoproterenol (30µM) Rat Cortex Endogenous 120 37 107.8 10.1 -7.7

Serotonin Receptor HT 2A Ketanserin (1µM) Ketanserin (0.5nM) HEK-293 Recombinant 60 RT 104.5 4.7 -4.5

Serotonin Receptor HT 2B (±) DOI (1µM) (±) DOI (0.2nM) CHO Recombinant 60 RT 109.9 11.6 -9.9

Serotonin Receptor HT 2C RS 102221 (10µM) Mesulergine (1nM) HEK-293 Recombinant 120 37 104.4 4.0 -4.3

Serotonin Receptor HT 3 MDL 72222 (10µM) BRL 43694 (0.5nM) CHO Recombinant 120 RT 100.7 1.8 -0.7

Serotonin Receptor HT 5a Serotonin (100µM) LSD (1.5nM) HEK-293 Recombinant 120 37 102.3 3.1 -2.3

Serotonin Receptor HT 6 Serotonin (100µM) LSD (2nM) CHO Recombinant 120 37 103.3 4.0 -3.3

Serotonin Receptor HT 7 Serotonin (10µM) LSD (4nM) CHO Recombinant 120 RT 118.4 7.4 -18.4 Serotonin Transporter Imipramine (10µM) Imipramine (2nM) CHO Recombinant 60 RT 109.1 6.6 -9.1 Sigma Receptor Haloperidol (10µM) DTG (10nM) Jurkat Endogenous 120 RT 105.4 1.6 -5.4 Somatostatin Receptor Somatostatin (300nM) Tyr11 -Somatostatin (0.05nM) AtT-20 Endogenous 60 37 111.3 10.0 -11.3 Vasoactive Intestinal Polypeptide Receptor 1 VIP (1µM) VIP (0.04nM) CHO Recombinant 60 RT 94.8 0.6 5.3 Arginine Vasopressin Receptor 1A AVP (1µM) AVP (0.3nM) CHO Recombinant 60 RT 95.1 2.8 5.0

Supplementary Table 4. In vitro receptor binding profile of the tri-agonist at off- target receptors. The GLP-1/GP/glucagon tri-agonist was screened for non-selective binding at 79 receptors or ion channels using customized high-throughput competitive binding assays. Results are expressed as % specific binding, calculated by [(tri- agonist specific binding / control specific binding) x 100], and also as % inhibition of control, calculated by [100 – (tri-agonist specific binding / control specific binding) x 100]. In general, results showing a % inhibition of control that is greater than 25% is considered a positive result of specific receptor binding and values less than 25% are considered a negative result of specific receptor binding. For each individual assay at the respective receptors, the ligand used to measure non-specific binding, the radioligand utilized to measure displacement from the receptor, the source of the receptor, and assay conditions are listed in the table. The tri-agonist was tested at a concentration of 1 µM and the non-specific ligands and radioligands were tested at the concentrations depicted in the table. A minimum of two separate experiments was performed for each respective receptor.

Nature Medicine: doi:10.1038/nm.3761 Cells GLP(1 GIP Glucagon GLP(1/GIP2Co(agonist Tri(agonist2(Peptide220) MIN6 0.45)±)0.09 16.87)±)4.00 31.29)±)7.33 1.13)±)0.56 0.81)±)0.08 Rat)hepatocytes N/A N/A 0.65)±)0.55 21.32)±)8.97 0.59)±)0.29 3T3>L1)adipocytes N/A 1.41)±)0.30 N/A 0.53)±)0.32 4.55)±)5.61

Supplementary Table 5. Effects of the tri-agonist on cAMP production in cultured pancreatic β cells, hepatocytes, and adipocytes. Values represent mean EC50 ± standard deviation (nM) of cAMP production in the respective cell lines in response to a 30 min stimulation with the indicated peptides. Human GLP-1, GIP, and glucagon were used. A minimum of three separate experiments was performed for each peptide in each cell line.

Nature Medicine: doi:10.1038/nm.3761

Tri-agonist / peptide 20 Species C57BL/6J Mice DIO Mice Rats Dogs Monkeys Dose (mg/kg) / (nmol/kg) 0.045 / 10 0.045 / 10 0.045 / 10 ND 0.015 / 3

Cmax (ng/ml) 195 636 34 ND 112

tmax (h) 4 2 2 ND 4

t1/2 (h) ~5 ~4 ~6 ND ~5 GLP-1/GIP co-agonist Species C57BL/6J Mice DIO Mice Rats Dogs Monkeys Dose (mg/kg) / (nmol/kg) 0.15 / 3 ND 1 / 222 0.045 / 10 0.045 / 10

Cmax (ng/ml) 383 ND 480 90 55

tmax (h) 4 ND 4 4 6 t1/2 (h) ~12 ND ~8 ~6 ~5

Supplementary Table 6. Pharmacokinetic comparison of the GLP-1/GIP co- agonist and the tri-agonist in different species. The pharmacokinetic parameters of the tri-agonist and the dual incretin co-agonist were determined following a subcutaneous injection of the peptides at the indicated doses in the different species. The concentration of the peptides in plasma was determined by LC-MS/MS and the pharmacokinetic analyses were determined by non-compartmental analysis with WinNonLin. Cmax, maximal plasma concentration; tmax, time for maximal concentration, t1/2, elimination half-life.

Nature Medicine: doi:10.1038/nm.3761

Supplementary Figure 1. Sequences of peptide analogs. Amino acid sequences of intermediate peptide analogs with the iterative residue substitutions from the preceding analog highlighted in blue. Aminoisobutyric acid is denoted as X. Lysine with a γE-C16 acyl attached through the side chain amine is denoted as underlined K. Sarcosine is denoted as #. D-enantiomers of select amino acids are italicized.

Nature Medicine: doi:10.1038/nm.3761

Supplementary Figure 2. HPLC and mass spectrometry. HPLC traces of the GLP- 1/GIP co-agonist using a (a) basic buffer system and a (b) acidic buffer system. (c) LC-MS data of the GLP-1/GIP co-agonist. HPLC traces of the tri-agonist using a (d) basic buffer system and a (e) acidic buffer system. (f) LC-MS data of the tri-agonist.

Nature Medicine: doi:10.1038/nm.3761

Supplementary Figure 3. Comparison to the tri-agonist to GLP-1/ glucagon and GIP/glucagon co-agonists. Effects on (a) body weight change, (b) glucose tolerance on day 16, (c) final body composition, and (d) cumulative food intake of male DIO mice treated with vehicle (black squares), liraglutide (gray circles), the GLP- 1/GIP/glucagon tri-agonist (orange diamonds), a matched GLP-1/glucagon co-agonist (blue triangles), or a matched GIP/glucagon co-agonist (red circles). All mice were treated by daily subcutaneous injections at a dose of 3 nmoles kg–1. Data in (a–d) represent means ± s.e.m. *P < 0.05, ** P < 0.01, *** P < 0.001, determined by ANOVA comparing vehicle to compound injections, and ##P < 0.01, ### P < 0.001, determined ANOVA comparing tri-agonist injections to co-agonist injections. In both comparisons, ANOVA was followed by Tukey post hoc multiple comparison analysis to determine statistical significance.

Nature Medicine: doi:10.1038/nm.3761

Supplementary Figure 4. Plasma analysis of DIO mice treated mice (complement to Figure 2). Effects on (a) plasma levels of acetaminophen 25 min. after oral gavage and 40 min. after injection of tri-agonist (3 nmoles per kg body weight). Effects on (f) blood glucose following a single bolus injection of compounds. Effects on plasma levels of (c) leptin (d) adiponectin, (e) free fatty acids, (f) triglycerides, (g) ketone bodies, (h) ALT, (i) AST, (j) total GIP, (k) glucagon, and (l) total GLP-1 of male DIO mice treated with vehicle (black), a dual incretin co-agonist (purple; 3 nmoles kg–1), or a single molecular GLP-1/GIP/glucagon tri-agonist at 1 nmoles kg–1 (yellow) or 3 nmoles kg–1 (orange). All mice were treated by daily subcutaneous injections. Data in (a–l) represent means ± s.e.m. *P < 0.05, determined by ANOVA comparing vehicle to compound injections, and #P < 0.05, determined ANOVA comparing dual incretin co-agonist to tri-agonist injections. In both comparisons, ANOVA was followed by Tukey post hoc multiple comparison analysis to determine statistical significance.

Nature Medicine: doi:10.1038/nm.3761

Supplementary Figure 5. Comparison to other purported triple agonists. Effects on (a) body weight change, (b) cumulative food intake, (c) fasted blood glucose, and (d) glucose tolerance of male DIO mice treated with vehicle (black squares), two 2 different purported GLP-1/GIP/glucagon triple agonists: DA -GLP-1/GcG (olive triangles) and YAG-glucagon (brown triangles), or exendin-4 (red circles). All mice were treated by twice daily subcutaneous injections (separated by 8 hours) at a cumulative dose of 50 nmoles kg–1 day–1 (2 x 25 nmoles kg–1). Data in (a–d) represent means ± s.e.m. *P < 0.05, ** P < 0.01, *** P < 0.001, determined by ANOVA followed by Tukey post hoc multiple comparison analysis comparing vehicle to compound injections.

Nature Medicine: doi:10.1038/nm.3761

Supplementary Figure 6. Lack of acute hypoglycemia or long-term adverse effects with tri-agonist treatment. Effects on (a) acute fasted blood glucose, (b) body weight change, (c) lean mass, and (d) cumulative food intake of lean male C57Bl/6 DIO mice (n = 8 per group; age 6 months) treated with vehicle (black squares) or increasing daily doses of the tri-agonist at 1 (olive diamonds), 3 (yellow diamonds), 5 (orange diamonds), or 10 nmoles kg–1 (brown diamonds). Effects on (e)

Nature Medicine: doi:10.1038/nm.3761 body weight change and (f) cumulative food intake of DIO Long Evans rats treated 3- times per week with vehicle (black squares) or the tri-agonist at 1 or 3 nmoles kg–1 (yellow diamonds and orange diamonds, respectively). Effects on (g) body weight regain and (h) ad libitum-fed blood glucose of DIO male mice two weeks after treatment cessation. All mice were treated daily for 3 weeks with vehicle (black squares), a dual incretin co-agonist (purple triangles; 3 nmoles kg–1), or a single molecular the tri-agonist at 1 nmoles kg–1 (yellow diamonds) or 3 nmoles kg–1 (orange diamonds). Data in (a–h) represent means ± s.e.m. *P < 0.05, ** P < 0.01, *** P < 0.001, determined by ANOVA followed by Tukey post hoc multiple comparison analysis comparing vehicle to tri-agonist injections.

Nature Medicine: doi:10.1038/nm.3761

Supplementary Figure 7. The metabolic benefits of the tri-agonist are blunted in Glp1r–/–, Gipr–/–, and Gcgr–/– mice (complement to Figure 3). Effects on (a) fat mass change and (b) cumulative food intake in wild-type or Glp1r–/– male DIO mice. Effects on (c) body weight change and (d) cumulative food intake in wild-type or Gipr–/– male HFD mice. Effects on (e) body weight change and (f) cumulative food intake in wild-type or Gcgr–/– male HFD mice. All mice were treated every other day with vehicle (wt: black squares; ko: gray squares) or the tri-agonist (wt: orange diamonds; ko: yellow diamonds) at a dose of 10 nmoles kg–1. Data in (a–f) represent means ± s.e.m. *P < 0.05, ** P < 0.01, *** P < 0.001, determined by ANOVA comparing vehicle to compound injections within each genotype, and #P < 0.05, ##P < 0.01, ### P < 0.001, determined ANOVA comparing treatment of the tri-agonist between genotypes. In both comparisons, ANOVA was followed by Tukey post hoc multiple comparison analysis to determine statistical significance.

Nature Medicine: doi:10.1038/nm.3761

Supplementary Figure 8. Unimolecular GcgR, GLP-1R, and GIP triple agonism prevent hyperglycemia in ZDF rats. Effects on (a) body weight progression, (b) fasted blood glucose, (c) intraperitoneal glucose tolerance, (d) HbA1c, (e) islet cytoarchitecture and immunohistochemistry for insulin (green), glucagon (red), and Dapi staining (blue) following 6-weeks of treatment with escalating doses of the tri- agonist in male ZDF rats (age 9 weeks at start of study). Effects on (d) HbA1c, (e) islet cytoarchitecture and immunohistochemistry, and (f) body weight regain following 3 weeks of compound wash-out and 9 weeks after treatment initiation. Data in (a–f) represent means ± s.e.m. *P < 0.05, ** P < 0.01, *** P < 0.001, determined one- or two-way ANOVA followed by Tukey post hoc multiple comparison analysis to determine statistical significance comparing vehicle to tri-agonist injections.

Nature Medicine: doi:10.1038/nm.3761