(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`6 October 2016 (06.10.2016)
`
`WIPOI PCT
`
`\9
`
`(10) International Publication Number
`
`WO 2016/161244 A2
`
`XIE, Jia; 8730 Costa Verde Blvd., Apt. 2519, San Diego,
`CA 92122 (US). STURCHLER, Emmanuel; 2614 26 Ct.,
`Jupiter, FL 33477 (US).
`
`Agents: COOPER, Geoflrey, K. et al.; The Scripps Re-
`search Institute, 10550 North Torrey Pines Road, TPC-8,
`La Jolla, CA 92037 (US).
`
`Designated States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG,
`MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM,
`PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC,
`SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(51)
`
`International Patent Classification:
`A61K38/17(2006.01)
`C12N 15/867 (2006.01)
`
`(21)
`
`International Application Number:
`
`PCT/US201 6/025490
`
`(74)
`
`(22)
`
`International Filing Date:
`
`Filing Language:
`
`Publication Language:
`
`1 April 2016 (01.04.2016)
`
`English
`
`English
`
`(81)
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`(72)
`
`Priority Data:
`62/141,598
`
`1 April 2015 (01.04.2015)
`
`US
`
`Applicant: THE SCRIPPS RESEARCH INSTITUTE
`[US/US]; 10550 North Torrey Pines Road, La Jolla, CA
`92037 (US).
`
`Inventors: LERNER, Richard, A.; 7750 Roseland Drive,
`La Jolla, CA 92037 (US). ZHANG, Hongkai; 4180 Cain-
`ino lslay, San Diego, CA 92122 (US). MC DONALD, Pa-
`tricia; 13839 155th Place N., Jupiter, FL 33478 (US).
`
`(54) Title: METHODS AND COMPOSITIONS RELATED TO GPCR AGONIST POLYPEPTIDES
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ,
`
`[Continued on nextpage]
`
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`
`(57) Abstract: The invention provides combinat-
`orial peptide or polypeptide libraries that can be-
`come membrane tethered once expressed in cells.
`The invention additionally provides methods for
`selecting peptide modulators (e.g., agonists) of
`GPCRs from the combinatorial libraries of the in-
`vention. The invention also provides novel GPCR
`polypeptide modulators, e..g., biased polypeptide
`agonists of the glucagon-like peptide 1 receptor
`(GLP-IR). The invention further provides meth-
`Ods of promoting insulin sensitivity,
`lowering
`h
`-
`b100d glucose, and reducmg body‘Welght as well
`as methods for treating various diseases such as
`diabetes and obesity.
`
`a
`
`c
`
`
`
`W02016/161244A2|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`5
`
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`
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`
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`
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`
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`
`FIG. 1
`
`

`

`WO 2016/161244 A2 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`
`TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, Published:
`TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,
`DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT,
`LU, LV, MC, MK, MT, NL, NO, PL, PT, R0, RS, SE,
`SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA,
`GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG).
`
`— without international search report and to be republished
`upon receipt ofthat report (Rule 48.2(g))
`
`

`

`WO 2016/161244
`
`PCT/U82016/025490
`
`Methods and Compositions Related to GPCR Agonist
`Polypeptides
`
`CROSS-REFERENCE TO RELATED APPLICATIONS
`
`[0001]
`
`The subject patent application claims the benefit of priority to US Provisional
`
`Patent Application Number 62/141,598 (filed April 1, 2015). The full disclosure of the
`
`priority application is incorporated herein by reference in its entirety and for all purposes.
`
`BACKGROUND OF THE INVENTION
`
`[0002]
`
`G-protein-coupled receptors constitute the largest family of cell surface receptor
`
`proteins. Upon activation, GPCRs couple to GTP~binding proteins that can be divided into
`
`four subclasses, Gas-, Gai/o-, Gaq and Gan/13. Gs and Gi/o regulate adenylate cyclases
`
`leading to an increase (Gas) or a decrease (Geri/o) in cAMP production, or to an increase in
`
`intracellular calcium concentration (Guq). Got(12/ 13) activates Rho GEFs, which in tum activate
`
`Rho. GPCRS can also engage B—arrestins. Historically, B-arrestin-l and B-arrestin-Z were
`
`believed to serve an exclusive role in GPCR desensitization. However, it has been shown
`
`that B-arrestins can also function to activate signaling cascades. Many human diseases are
`
`associated with the dysfunction of GPCRs. Thus, GPCRs represent some of the most
`
`attractive therapeutic or molecular targets in the pharmaceutical industry.
`
`[0003]
`
`Insulin regulates the concentration of blood sugar and blood lipid levels through
`
`the promotion of glucose and lipid uptake into cells. Type 2 Diabetes Mellitus ('1‘2DM or
`
`TZD) is a complex metabolic disorder characterized by hyperglycemia arising from a
`
`combination of insufficient insulin secretion together with the development of insulin
`
`resistance. T2D and obesity are closely linked, with obesity accounting for 80—85% ofthe
`
`risk of developing T2D.
`
`lncretin-based therapies represent a promising class of agents for
`
`the treatment of T2D. Incretins including the glucagon—like peptide—1 (GLP-1) and the
`
`glucose-dependent insulinotropic polypeptide (GIP) are endogenous peptide hormones
`
`secreted from the intestine in response to food intake. GLP-1 and GIP exert their action
`
`through G-protein coupled receptor, the GLP-1 and the GIP receptors, respectively. GLP—lR
`
`is expressed in pancreatic B-cells as well as various tissues including liver, smooth muscle,
`
`heart, kidney, gastrointestinal tract, lungs, pituitary, white adipose tissues, and the central
`1
`
`

`

`WO 2016/161244
`
`PCT/USZOl6/025490
`
`nervous system. GLP-l lowers postprandial glucose excursion by potentiating glucose-
`
`stimulated insulin secretion from pancreatic [3-eells and has recently been shown to promote
`
`B-cell survival in rodents.
`
`In addition, GLP—l exerts extrapancreatic actions such as
`
`promoting gastric emptying, weight loss, intestinal growth and increasing insulin sensitivity
`
`in peripheral tissues.
`
`[0004]
`
`A need exists in the art for better and more robust means for identifying agonists
`
`or activators displaying novel pharmacology of various GPCRs. There is also need in the alt
`
`for new compounds and methods for treating diseases associated with insulin deficiency and
`
`high blood glucose. The instant invention addresses these and other currently unmet needs
`
`in the art.
`
`SUMMARY OF THE INVENTION
`
`[0005]
`
`In one aspect, the invention provides isolated or recombinant polypeptides that
`
`contain a first randomized peptide fused at its C-terminus to a second peptide capable of
`
`binding to the cxtraceliular domain of glucagon-like peptide 1 receptor (GLP-IR). In these
`
`polypeptides, the randomized peptide typically has an amino acid sequence ot‘XXXXXXX
`
`(SEQ ID N011), CXXXXXCXX (SEQ ID N022) or CXXXXCXXX (SEQ ID NO:3),
`
`wherein X is any amino acid residue. In various embodiments, these polypeptides can
`
`activate glucagon-like peptide 1 receptor (GLP—lR).
`
`In some embodiments, the randomized
`
`peptide contains an amino acid sequence that is substantially identical to a sequence selected
`
`from SEQ ID NOs:4—16 and 22—3 1 , or an active variant or fragment thereof. In some
`
`embodiments, the randomized peptide contains an amino acid sequence that is identical to a
`
`sequence selected from SEQ ID NOsz4-16 and 22-31, except for conservative substitution at
`
`one or more residues.
`
`In some embodiments, the randomized peptide contains an amino acid
`
`sequence shown in SEQ ID NO:4 (ACCIDSVCVI), SEQ ID NO:5 (VCPDCQV), SEQ ID
`
`NO:6 (ACSYMIDCVL), SEQ ID N028 (ELVDNAV), SEQ ID NO:22 (CCIDSVCVI), or
`
`SEQ ID NO:23 (CSYMIDCVL).
`
`[0006]
`
`In some polypeptides of the invention, the second peptide capable of binding to
`
`glucagon-like peptide 1 receptor contains an amino acid sequence that is substantially
`
`identical to at least 8 contiguous amino acid residues of Ex4 (9-39) (SQE ID N0217), Ex4
`
`(9-30) (SEQ ID N013 8), or human GLP-l (7-37) peptide (SEQ ID NO:18).
`
`In some
`
`embodiments, the second peptide capable of binding to GLP-IR contains a sequence that is
`
`2
`
`

`

`WO 2016/161244
`
`PCT/U82016/025490
`
`substantially identical to SEQ ID NO:17 or SEQ ID NO:18.
`
`In some embodiments, the
`
`second peptide capable of binding to GLP-IR contains a sequence shown in SQE ID NO: 17
`
`or SEQ ID NO:18, except for conservative substitution at one or more residues.
`
`In some
`
`embodiments, the second peptide capable of binding to glucagon-like peptide 1 receptor
`
`contains a sequence shown in GGDLS KQMEE EAVRL FIEWL KNGGP SSGAP PPS
`
`(SEQ ID NO:21). Some of the polypeptides of the invention are G protein biased. Some
`
`polypeptides of the invention contain a sequence that is substantially identical to a sequence
`
`selected from the group consisting of SEQ ID NOs:32-3 7.
`
`In some of these embodiments,
`
`the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID
`
`NOsz32-37.
`
`In some embodiments, the GLP—lR agonist polypeptides of the invention
`
`further contain an Fc-domain fused at its N-terminus or C-terminus. Some of these
`
`polypeptides contain an Fc-domain that is fused at the C-terminus of SEQ ID NO:36, e.g.,
`
`the polypeptide shown in SEQ ID NO:45. Some of these polypeptides can additionally
`
`include a signal peptide at the N-terminus.
`
`[0007]
`
`In another aspect, the invention provides methods for identifying an agonist of a
`
`G protein-coupled receptor (GPCR). These methods entail (1) introducing a vector
`
`expressing a reporter gene under the control a cAMP response element (CAMP) into a
`
`population ofhost cells expressing the GPCR, (2) expressing in the host cells a
`
`combinatorial library of membrane tethered candidate polypeptides to produce a population
`
`of host cells co—expressing a candidate polypeptide and the GPCR, (3) identifying a host cell
`
`that has increased expression level of the reporter gene relative to a control cell not
`
`expressing any candidate polypeptide, and (4) determining the identity of the candidate
`
`polypeptide expressed in the identified host cell; thereby identifying an agonist of the G
`
`protein—coupled receptor.
`
`In some methods, each candidate polypeptide is fused at its C—
`
`terminus to a linker and a transmembrane protein domain. In some methods, the employed
`
`transmembrane protein domain is the PDGFR transmembrane domain.
`
`In some methods,
`
`the GPCR is expressed from an expression vector in the host cells. For example, the vector
`can be a lentiviral vector.
`
`[0008]
`
`Some of the methods of the invention employ a reporter gene that encodes a GFP
`
`protein.
`
`In these methods, the reporter gene can be introduced into the host cell via a
`
`lentiviral vector. In some embodiments, the employed host cell is the HEK 293T cell.
`
`In
`
`some embodiments, the employed GPCR is human GLP—IR.
`
`In some methods of the
`
`

`

`WO 2016/161244
`
`PCT/USZOl6/025490
`
`invention, each candidate polypeptide contains a first randomized peptide that is fused at its
`
`C-terminus to a second peptide capable of binding to the extracellular domain of glucagon-
`
`like peptide 1 receptor (GLP-lR), and the randomized peptide contains an amino acid
`
`sequence of XXXXXXX (SEQ ID NO: 1), CXXXXXCXX (SEQ ID NO:2) or
`
`CXXXXCXXX (SEQ ID N023), wherein X is any amino acid residue. In some ofthese
`
`methods, the second peptide contains an amino acid sequence that is substantially identical
`
`to Ex4 (9-39) (SQE ID NO:17), Ex4 (9-30) (SEQ ID N0z38), human GLP-l (7-37) peptide
`
`(SEQ ID NO:l8), or an active variant thereof.
`
`In some methods, the transmembrane protein
`
`domain and the second peptide are connected via a linker peptide. For example, a linker
`
`peptide containing two or more tandem repeats of GGGGS (SEQ ID N0243) can be
`
`employed in the methods.
`
`[0009]
`
`In some methods of the invention, each candidate polypeptide further contains a
`
`signal peptide at the N—terminus. For example, the methods can employ a signal peptide
`
`containing the III-2 signal sequence.
`
`In some of these methods, the signal peptide can
`
`contain an amino acid sequence of MYRMQLLSCIALSLALVTNS (SEQ ID N0:44).
`
`[0010]
`
`In another aspect, the invention provides methods for activating a signaling
`
`pathway mediated by the glucagon—like peptide 1 receptor (GLP-lR) in a cell. These
`
`methods involve contacting the cell with an effective amount of a polypeptide containing a
`
`first randomized peptide that is fused at its C-terminus to a second peptide capable of
`
`binding to the extracellular domain of glucagon-like peptide 1 receptor (GLP-lR).
`
`In these
`
`methods, the randomized peptide contains an amino acid sequence of XXXXXXX (SEQ ID
`
`NO: 1), CXXXXXCXX (SEQ ID N02) or CXXXXCXXX (SEQ ID N03), wherein X is
`
`any amino acid residue. In some methods, the second peptide contains a sequence that is
`
`substantially identical to Ex4 (9-39) (SQE ID NO:17), Ex4 (9—30) (SEQ ID NO:38), human
`
`GLP-l (7-37) peptide (SEQ ID N0:18), or an active variant thereof.
`
`In some embodiments,
`
`the randomized peptide contains an amino acid sequence as shown in SEQ ID N0122, SEQ
`
`ID N015, or SEQ ID N018.
`
`In some methods, the employed polypeptide contains an amino
`
`acid sequence as shown in SEQ ID N0z36, SEQ ID N0134, or SEQ ID N0:32.
`
`[0011]
`
`In some methods, the signaling pathway to be activated results in insulin
`
`biosynthesis and release. In some embodiments, the activated cell is a pancreatic beta cell.
`
`For example, the methods can be directed to activating insulin biosynthesis and release in
`
`pancreatic beta cells present in a subject.
`
`In some of these embodiments, the subject has or
`
`4
`
`

`

`WO 2016/161244
`
`PCT/USZOl6/025490
`
`is at risk of developing a metabolic disorder characterized by high blood glucose and low
`
`insulin levels, e.g., type 2 diabetes, obesity, non—alcoholic fatty liver disease (NAFLD), non-
`
`alcoholic steatohepatitis (NASH) and atherosclerosis.
`
`[0012]
`
`In still another aspect, the invention provides methods for increasing insulin
`
`level, insulin sensitivity, and decreasing blood glucose level, or for treating a disorder
`
`characterized by high blood glucose and low insulin levels in a subject. These methods
`
`entail administering to the subject an effective amount of a polypeptide containing a first
`
`randomized peptide that is fused at its C-terminus to a second peptide capable of binding to
`
`the extracellular domain of glucagon—like peptide 1 receptor (GLP-IR). In various
`
`embodiments, the randomized peptide in the employed polypeptide contains an amino acid
`
`sequence of XXXXXXX (SEQ ID NO:1), CXXXXXCXX (SEQ ID NO:2) or
`
`CXXXXCXXX (SEQ ID N023), wherein X is any amino acid residue.
`
`In some ofthese
`
`methods, the second peptide contains an amino acid sequence that is substantially identical
`
`to EX4 (9—39) (SQE ID NO:17), Ex4 (9—30) (SEQ ID N0238), human GLP—l (7-37) peptide
`
`(SEQ ID NO:18), or an active variant thereof. In some methods, the randomized peptide
`
`contains an amino acid sequence as shown in SEQ ID NO:22, SEQ ID NO:5, or SEQ ID
`
`NO:8.
`
`In some methods, the employed polypeptide contains an amino acid sequence as
`
`shown in SEQ ID N0236, SEQ ID NO:34, or SEQ ID NO:32. Some of these methods are
`
`directed to increasing insulin level and decreasing blood glucose in a subject who has or is at
`
`risk of developing diabetes, obesity, non—alcoholic fatty liver disease (NAFLD), non-
`
`alcoholic steatohepatitis (NASH), atherosclerosis, Parkinson’s disease, Alzheimer’s Disease
`
`or drug addiction.
`
`[0013]
`
`The invention also provides the use of a polypeptide containing a first
`
`randomized peptide fused at its C—terminus to a second peptide capable of binding to the
`
`extracellular domain of glucagon—like peptide I receptor (GLP-IR) in the preparation of a
`
`pharmaceutical composition for treating diabetes or other disorders characterized by high
`
`blood glucose and low insulin levels. The randomized peptide typically contains an amino
`
`acid sequence of XXXXXXX (SEQ ID NO:1), CXXXXXCXX (SEQ ID NO:2) or
`
`CXXXXCXXX (SEQ ID NO:3), wherein X is any amino acid residue.
`
`In some
`
`embodiments, the second peptide contains an amino acid sequence that is substantialiy
`
`identical to Ex4 (9-39) (SQE ID NO:17), Ex4 (9—30) (SEQ ID N023 8), human GLP—l (7-37)
`
`peptide (SEQ ID NO: 18), or an active variant thereof. In some embodiments, the
`
`

`

`WO 2016/161244
`
`PCT/USZOl6/025490
`
`randomized peptide contains an amino acid sequence as shown in SEQ ID NO:22, SEQ ID
`
`N05, or SEQ ID N028.
`
`In some embodiments, the employed polypeptide contains an
`
`amino acid sequence as shown in SEQ ID NO:36, SEQ ID NO:34, or SEQ ID NO:32.
`
`[0014]
`
`In a related aspect, the invention provides isolated or recombinant
`
`polynucleotides that encode a polypeptide containing a randomized peptide fused at its C—
`
`terminus to a second peptide capable of binding to the extracellular domain of glucagon-like
`
`peptide 1 receptor (GLP—lR). The randomized peptide in the encoded polypeptide typically
`
`contains an amino acid sequence of XXXXXXX (SEQ ID N021), CXXXXXCXX (SEQ ID
`
`NO:2) or CXXXXCXXX (SEQ ID N013), wherein X is any amino acid residue. In some
`
`embodiments, the second peptide contains a sequence that is substantially identical to Ex4
`
`(9-39) (SQE ID NO: 17), Ex4 (9-30) (SEQ ID NO:38), human GLP-l (7—37) peptide (SEQ
`
`ID N0:l8), or an active variant thereof.
`
`In some embodiments, the randomized peptide
`
`contains an amino acid sequence as shown in SEQ ID N022, SEQ ID NO:5, or SEQ ID
`
`NO:8. Some polynucleotides of the invention encode a polypeptide containing an amino
`
`acid sequence as shown in SEQ ID NO:36, SEQ ID NO:34, or SEQ ID NO:32.
`
`[0015]
`
`In some embodiments, the polypeptide encoded by the polynucleotide further
`
`contains a transmembrane protein domain at the C-terminus of the second peptide. For
`
`example, the encoded polypeptide can contain the PDGFR transmembrane domain. In some
`
`embodiments, the transmembrane protein and the second peptide in the encoded polypeptide
`
`are connected via a linker peptide. For example, a linker peptide containing two or more
`
`tandem repeats of GGGGS (SEQ ID NO:43) can be employed. In some embodiments, the
`
`encoded polypeptide can further contain a signal peptide at the N-terminus. For example,
`
`the IL—2 signal sequence can be used.
`
`In some specific embodiments, the employed signal
`
`peptide contains a sequence of MYRMQLLSCIALSIALVTNS (SEQ ID NO:44).
`
`[0016]
`
`In some related aspects, the invention additionally provides fusion polypeptides
`
`encoded by the isolated or recombinant polynucleotides, as well as expression vectors
`
`harboring the isolated or recombinant polynucleotide of the invention.
`
`[0017]
`
`A further understanding of the nature and advantages of the present invention
`
`may be realized by reference to the remaining portions of the specification and claims.
`
`DESCRIPTION OF THE DRAWINGS
`
`[0018]
`
`Figure 1 shows autocrine-based system for selection of agonists from large
`
`6
`
`

`

`WO 2016/161244
`
`PCT/USZOl6/025490
`
`combinatorial peptide libraries. a, Schematic representation of membrane tethered exendin—4.
`
`b, FACS analysis of mCherry and GFP expression 2 of days after transduction of HEK293—
`
`GLP-lR-GFP cells with the membrane tethered exendin-4 displaying different linker size. c,
`
`Schematic representation of the autocrinc based selection ofcombinatorial peptide library.
`
`The lentivirus peptide library is prepared from lentiviral plasmids (step 1). The CRE—
`
`responsive GLP-lR reporter cell line is transduced with lentiviral library (step 2). GFP
`
`expressing cells are sorted (step 3) and peptide encoding genes are amplified from genomic
`
`DNA of sorted cells to make the library for the next selection round(step 4). After iterative
`
`rounds of selection, enriched peptide sequences are analyzed by deep sequencing(step 5). d,
`
`Enrichment of GFP positive cells during three rounds of FACS selection.
`
`[0019]
`
`Figure 2 shows in vitro pharmacological characterization of agonist peptide P5.
`
`a-b, Concentration response curves for P5- and Ex4-induced increase in CAMP production in
`
`CHO cells expressing the human GLP-lR (a) or in HEK293 expressing the mouse GLP-lR
`
`(b). 0, Concentration response curves for P5- and Exe4-induced calcium mobilization in
`
`CHO cells expressing the human GLP-lR. d—e, Concentration response curves for P5- and
`
`Exe4-induced B—arrestin-l (d) and B—arrestin-Z (e) recruitment in CHO cells expressing the
`
`human GLP-lR. f, Biased factors ([3) from an equiactive comparison indicate bias for P5.
`
`The data are mean d: SEM of typical experiment that was performed three times.
`
`[0020]
`
`Figure 3 shows acute P5 injection lowers glucose in diabetic mice. a-i,
`
`Intraperitoneal glucose tolerance (“insulin disposal”) test; a single intraperitoneal injection of
`
`saline, Ex4 or P5, co—injected with an intraperitoneal glucose challenge (n=5). Glucose
`
`tolerance in lean mice treated with 10 ug/kg (a), 1 ug/kg (b) or 0.1ug/kg peptides (c), in
`
`GLP—1~R KO treated with 1 ug/kg peptides (d), in ob/ob mice treated with 1 ug/kg peptides
`
`(e) and in DIO mice treated with l rig/kg peptides (l). g—i, Plasma insulin levels was
`
`analyzed after a single intraperitoneal co-injection of saline, 1 ug/kg of Ex4 or 1 pg/kg of P5
`
`G—protein biased agonist with glucose challenge (n=5) in lean mice (g), in ob/ob mice (h)
`
`and in DIO mice (i). Data are mean :I: s.e.m. Statistic by two—tailed t—test: *P < 0.05; **P <
`
`0.01, comparing saline to peptide injection; #P < 0.05; ##P < 0.01, comparing Ex4 to P5
`
`injection. AUC, area under the curve.
`
`[0021]
`
`Figure 4 shows that chronic administration of the G-protein biased GLP-lR
`
`agonist peptide P5 improves glycemic status in diabetic mice. a-g, four week of treatment of
`
`DIO male mice with daily dose of Ex4 and P5 biased agonist. Effect on body weight (a),
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`intraperitoneal glucose tolerance (b), ad-libitum-fed plasma glucose level (0), insulin plasma
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`level (d), HbAIC (e), islet morphology (f) and GIP plasma level (g) after daily subcutaneous
`
`injections of saline, Ex4 and P5 G-protein biased agonist atl ug/kg (lighter shade) or 10
`
`ug/kg (darker shade) . Data are mean i s.e.m. Statistic by two—tailed t—test: *P < 0.05; **P <
`
`0.01 , comparing saline to peptide injection. AUC, area under the curve.
`
`[0022]
`
`Figure 5 shows additional in vitro pharmacological characterization of Peptide P5
`
`selectivity. a-b, Concentration response curves for Ex9—induced decrease in CAMP
`
`production in presence of an E090 of P5 in CHO cells expressing the human GLP-lR (a) or
`
`in HEK293 expressing the mouse GLP-lR (b).
`
`c, Concentration response curves for
`
`glucagon— P5— and Ex4-induced calcium mobilization in CHO cells expressing the human
`
`glucagon receptor. The data are mean :t SEM of typical experiment that was performed three
`
`times.
`
`[0023]
`
`Figure 6 displays additional results indicating effect of acute P5 injection on
`
`glycemia and insulin level in ob/ob mice. a—i, Acute treatment of male mice with P5 G-
`
`protein biased agonist. a—c, Effect on intraperitoneal glucose tolerance and plasma insulin
`
`levels following a single intraperitoneal co-injection with glucose challenge (n=5) of saline,
`
`Ex4 and P5 G-protein biased agonist. Glucose tolerance in ob/ob mice treated with 10 [Lg/kg
`
`(a), 0.1 [Lg/kg (b) (n=5). 0, Plasma insulin level in ob/ob mice were analyzed after a single
`
`intraperitoneal co—injection of saline, 10 ug/kg of Ex4 or 10 ug/kg of P5 G-protein biased
`
`agonist with glucose challenge (n=5). Data are mean i s.e.m. Statistic by two—tailed t-test:
`
`*P < 0.05; **P < 0.01, comparing saline to peptide injection; #P < 0.05; ##P < 0.01,
`
`comparing Ex4 to P5 injection. AUC, area under the curve.
`
`[0024]
`
`Figure 7 displays additional results indicating that chronic administration of
`
`Peptide P5 improves glycemic status in diabetic mice independently of body weight and fat
`
`mass. a-b Four week of treatment of DIO male mice with daily dose of Ex4 and P5 biased
`
`agonist. MRI analyses of fat (a) and body weight (b) following daily subcutaneous injections
`
`of saline, Ex4 and P5 G—protein biased agonist at 10 ug/kg (n=8). Data are mean :: s.e.m.
`
`Statistic by two-tailed t-test: *P < 0.05; **P < 0.01, comparing saline to peptide injection.
`
`[0025]
`
`Figure 8 displays additional results indicating effect of chronic administration of
`
`Peptide P5 on metabolic hormones in DIO mice. Four week of treatment of DIO male mice
`
`with daily dose of Ex4 and P5 biased agonist. Effects on c—peptide plasma level (a) and
`
`glucagon plasma level (b) (n=8) after daily subcutaneous injections of saline, Ex4 and P5 G—
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`protein biased agonist at 10 [Lg/kg. Data are mean i s.e.m. Statistic by two-tailed t—test: *P <
`
`0.05; **P < 0.01, comparing saline to peptide injection.
`
`[0026]
`
`Figure 9 displays additional results indicating effect of chronic administration of
`
`P5 G—protein biased agonist on hepatoxicity and steatosis in DIO mice. Four week of
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`treatment of DIO male mice with daily dose of Ex4 and P5 biased agonist. Effects on plasma
`
`ALP (a), plasma ALT (b), plasma AST (0) and hepatic steatosis (n=8) following daily
`
`subcutaneous injections of saline (control), Ex4 and P5 G—protein biased agonist. Data are
`
`mean i s.e.m. Statistic by two—tailed t—test: *I’ < 0.05; **P < 0.01, comparing saline to
`
`peptide injection.
`
`[0027]
`
`Figure 10 shows schematically the construction of the HEK293-GLP—1R—GFP
`
`reporter cell line.
`
`[0028]
`
`Figure 11 shows G—protein bias of other agonist peptides in activating GLP-lR
`
`signaling. (A) Classical paradigm of signal transduction by GPCR. The agonist binding to
`
`the receptor stimulates heterotrimeric G proteins and is rapidly phosphorylated by G protein-
`
`coupled receptor kinases (GRKs that recruit B—arrestin to desensitize the receptor; CRE—
`
`responsive GLP-lR reporter cell line (B) and Tango GLP—lR-Bia U208 cell line (C) are
`
`used to monitor the CAMP increase and B-arrestin recruitment after GPCR activation.
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`[0029]
`
`Figure 12 shows in vitro characterization of the long acting G-protein biased
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`agonist fusion polypeptide PS-Fc. (a) Schematic representation of PS-Fc (b) Concentration
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`response curves for P5— and PS-Fc—induced increase in CAMP production in CHO cells
`
`expressing the human GLP—lR.
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`(0) Concentration response curves for P5, and Ex4—induced
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`B-arrestin—Z recruitment in CHO cells expressing the human GLP-lR.
`
`[0030]
`
`Figure 13 shows that GLP—lR agonist fusion polypeptide PS-Fc displays
`
`enhanced pharmacodynamics. (a) Glucose tolerance tests performed either directly following
`
`a single injection of P5 (20nmol/kg) or PS-fc (5 nmol/kg) (day 0) or two and seven days later.
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`(b) Effect ofa single injection of P5~fc (50 nmol/kg) on fed blood glucose level.
`
`[0031]
`
`Figure 14 shows that P5-Fc decreases body weight and fat mass in diabetic mice.
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`(a) Effect of once—weekly administration of PS—Fc on body weight in DIO mice.
`
`(b) Effect
`
`of once-weekly administration of P5 -Fc on fat mass, fluid mass and lean mass and in DIO
`
`mice ( after injection 6). n=5; *, p<0.05; **, p<0.01.
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`[0032]
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`Figure 15 shows that P5-Fc decreases food intake in diabetic mice. (a) and daily
`
`food intake (b). P5~Fc significantly decreases food intake for 2—3 days post injection. 11:5; *,
`
`p<0.05; **, p<0.01.
`
`[0033]
`
`Figure 16 shows effect of P5 and PS—Fc on reducing re-feeding of fasting mice.
`
`Male C57BL/6 mice (12—14 wk; 28—30g) fasted for 16-18h were injected ip with P5, P5-Fc
`
`and Ex4. Cumulative F1 was measured at 1, 2, 4, 8, 24 and 31 h post-injection. n=3; *,
`
`p<0.05
`
`[0034]
`
`Figure 17 shows anti-hyperglycemia efficacy ofPS-Fc in treating diabetic mice.
`
`(a) Effect of once—weekly administration of PS—Fc on glucose tolerance in response to
`
`glucose challenge (one week after injection 5 and before injection 6). (b) Effect of once—
`
`weekly administration of PS—Fc on fed plasma glucose levels (one week after injection 5 and
`
`before injection 6) and (c) on HbAlc (after injection 5). n=5; *, p<0.05; **, p<0.01.
`
`[0035]
`
`Figure 18 shows that PS—Fc enhances insulin sensitivity in diabetic mice. (a)
`
`Effect of once—weekly administration of PS-Fc on glucose level in response to insulin
`
`injection (0.75 U/kg) (one week after injection 4 and before injection 5). (b) Data are
`
`represented as a % of basal blood glucose level for each individual group. n=5; *, p<0.05;
`
`**, p<0.01.
`
`[0036]
`
`Figure 19 shows that P5-Fc induces intestinal growth. Effect of once-weekly
`
`administration of P5—Fc on (a) small bowel (SB) length, and (b) large bowcl (LB) length,
`
`n=5; *p<0.05, **, p<0.01.
`
`[0037]
`
`Figure 20 shows thatvchronic administration of a G-protein biased agonist
`
`improves hepatic steatosis in DIO mice.
`
`[0038]
`
`Figure 21 shows that chronic administration of the G-protein biased agonist
`
`modulates adipogcncsis and insulin sensitivity in DIO mice.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`1.
`
`Overview
`
`[0039]
`
`The invention is predicated in part on the development by the present inventors of
`
`an autocrine-based system for selection of agonists from large intracellular combinatorial
`
`peptide libraries. As detailed herein, this system allows one out of about 108 different
`
`peptides and a receptor to be co—localized in the plasma membrane of cells. When the co—
`
`localized peptide activates the neighboring receptor a fluorescent signal is generated such
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`that each cell becomes a reporter unto itself. The system was validated by selection of highly
`
`potent agonists for the GLP—l GPCR receptor that activated signaling pathways and induced
`
`insulin secretion and lowering of blood glucose in mice. Unlike the balanced agonists,
`
`endogenous GLP—l or the synthetic peptide Exendin4 that activate both Goa—protein and B—
`
`arrestin signaling pathways, the selected molecules were biased in that they activated both
`
`Gocs and Gocq but not the B—arrestin mediated signal transduction pathways. Such studies
`
`open the way to select new agonists for GPCRs and other receptors. Agonists selected by
`
`these methods are useful for de~convolution of signal transduction pathways and discovery
`
`of new mechanisms of action.
`
`[0040]
`
`As exemplified herein, the inventors examined the glucagon-like peptide-l (GLP-
`
`1) system. GLP-l carboxamide is an important incretin hormone that activates the GPCR
`
`GLP-1 receptor (GLP-l-R) resulting in a lowering of blood glucose levels in a glucose-
`
`dependent manner. When the endogenous GLP-l binds to the receptor, a complex network
`
`of downstream signaling pathways are activated that include, amongst others, G—proteins, [3-
`
`arrestin and a variety of kinases. Specifically, the inventors constructed combinatorial
`
`peptide libraries and expressed them in reporter cells such that each cell expresses the GLP—
`
`IR and a different peptide that are co-localized on the plasma membrane.
`
`In one library, a
`
`sequence of 7 randomized seven amino acids was coupled to the Exentin—4 C—terminus
`
`(amino acids 9-3 9) as an anchor sequence. Exendin-4 from the Gila monster lizard
`
`Heloderma suspectum was chosen as the anchor sequence so as not to be confused by the
`
`agonist activity of the C-terminal GLP-l domain. When the amino terminus of exendin—4 is
`
`truncated by the first eight amino acid residues (Ex9—39), it acts as an antagonist of the GLP—
`
`lR. This autocrine selection system is linked to a fluorescent signal such that receptor
`
`activation can be monitored and selected in the cell sorter.
`
`[0041]
`
`Some of the GLP-lR agonists identified by the inventors are G-protein biased
`
`(e.g., peptide P5).
`
`It is known that GLP—lR coupling to G—protein upon activation can lead
`
`to an increase in intracellular 3’,5’ cyclic AMP (CAMP) and elevation of intracellular Ca2+
`
`concentrat