WO2021007236A1 - Non-native o-glcnac modification of peptide hormones yields potent gpcr agonists with improved serum stability - Google Patents

Abstract

A synthetic peptide hormone comprising a sequence of about.15 to about 50 amino acid residues that are at least about 90% identical to the same length and aligned sequence of a native peptide hormone is disclosed. Each of the synthetic and native peptide sequences exhibits an amphipathic alpha-helical secondary structure, The synthetic peptide hormone includes a mono-O-glyeosylated serine or threonine residue in its sequence that is not present in the native sequence; i.e., as a non-identical residue of the synthetic peptide hormone sequence. A contemplated synthetic peptide hormone has a half-life in human serum at 37°C of about 25 to about 800 percent longer than the native peptide hormone. A composition containing an agonistic effective amount,of an above-described synthetic peptide hormone dissolved or dispersed in a pharmaceutically acceptable carrier and a method of treating a mammal having a GPCR-mediated condition are also disclosed.

Description

Non-native O-G1cNAc Modification of

Peptide Hormones Yields Potent GPCR Agonists with Improved Serum Stability

Description

CROSS-REFERENCE TO RELATED APPLICATION

This international application claims priority to US application Serial No. 62/872,027, filed on July 9, 2019, whose disclosures are

incorporated herein by reference.

STATEMENT REGARDING FEDERALLY

SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under R0G M4114537 awarded by the National

Institutes of Health, The Government has certain rights in the invention.

BACKGROUND ART

G protein-coupled receptors (GPCRs) are cell surface receptors that respond to a wide variety of stimuli, from light, odorants, hormones, and neurotransmitters to proteins and extracellular calcium. GPCRs represent the largest family of signaling proteins targeted by many clinically used drugs. Recent studies shed light on the

conformational changes that accompany GPCR activation and continue through the cellular membrane to alter G protein and arrestin conformations, ultimately resulting signal transduction³⁷.

Glucagon-like peptide-1 (GLP-1) and the parathyroid hormone (PTH) , which respectively help control glucose and calcium levels in the blood are illustrative peptide hormone agonists that bind to

GPCRs . Peptide hormone agonists of GPCRs and other receptors are powerful signaling molecules with high potential as biological tools and therapeutics, but they are typically plagued by instability and short half-lives in vivo.

According to the Oxford Dictionary of Biochemistry and Molecular Biology, A.D. Smith et al, eds. , Oxford University Press, New York, page 308 (1997) a hormone is "any substance formed in very small amounts in one specialized organ or group of cells and carried (sometimes in the bloodstream) to another organ or group of cells, in the same

organism, upon which it has a specific regulatory action; . . ." That same volume defines a peptide hormone as "any peptide with hormonal activity in animals, whether endocrine, neuroendocrine, or paracrine. Such substances form a very diverse group physiologically, and the boundary between peptide hormones and protein hormones is somewhat indistinct. (Id. at page 490.)

Peptide hormones can have amino acid residue sequences of about ten residues such as argipressin or oxytocin, each of which has a sequence of nine amino acid residues, or as many as nearly 250, such as erythropoietin with 165 residues and human chorionic gonadotropin that has a sequence of 237 amino acid residues. Those of interest here have a sequence length of about 15 residues to about 50 residues .

Glucagon-like peptide-1 (GLP-1) is an illustrative peptide hormone that contains a sequence of 30 amino acid residues deriving from the tissue- specific posttranslational processing of the proglucagon gene product. GLP-1 is produced and secreted upon food consumption by intestinal

enteroendocrine L-cells and certain neurons within the nucleus of the solitary tract in the brainstem.

The initial product GLP-1 (1-37) is susceptible to c-terminal proteolytic cleavage and amidation that gives rise to the two truncated and equipotent biologically active forms, GLP-1

(positions 7-36) amide and GLP-1 (positions 7-37) . Aqueous, active GLP-1 contains two a-helices from amino acid residue positions 13-20 and 24-35 that are separated by a linker region.

Alongside glucose-dependent insulinotropic peptide (GIP) , GLP-1 is an incretin; thus, it has the ability to decrease blood sugar levels in a glucose- dependent manner by enhancing the secretion of insulin. Beside the insulinotropic effects, GLP-1 has been associated with numerous regulatory and protective effects. Unlike GIP, the action of GLP-1 is preserved in patients with type 2 diabetes and substantial pharmaceutical research has therefore been directed towards the development of GLP-1-based treatment for typ>e 2 diabetes.

Peptide therapeutics have recently garnered significant attention from the pharmaceutical industry due to their specificity, potency, and diminished off-target effects¹. This is particularly true in the case of type-2 diabetes mellitus, where agonists of both the insulin receptor and the glucagon-like peptide-1 receptor (GLP-1R) are well- established clinical targets^2,3. GLP-1R is a GPCR³⁸.

In fact, extensive efforts have focused on the development of small molecule modulators of GLP- 1R with little to no success. However, peptide agonists have proven to be effective as therapeutic agents.

Parathyroid hormone (PTH) is a peptide hormone secreted by the parathyroid glands that regulates the serum calcium through its effects on bone, kidney, and intestine. In a similar manner, the PTH receptor (PTHR1) ³³ has been clinically

targeted by PTH (1-34), marketed by Eli Lilly as

Forteo®, and a peptide analog of the parathyroid hormone-related protein, approved as Tymlos® from Radius Health. PTHR1 is also a GPCR³⁹.

One critical drawback to this strategy is the typically poor pharmacokinetic profiles of peptides in vivo due to proteolytic degradation by endogenous enzymes. For example, GLP-l(7-37) and PTH (1-34) have respective half-lives of only about 2 and 10 minutes in the bloodstream in vivo⁴-³². This rapid degradation is carried out by endogenous proteases, mainly dipeptidyl peptidase-4 (DPP-4) cleavage at alanine 8 of GLP-1, which significantly compromises its potency in vivo and limits its ability to be used as an effective therapeutic agent⁵.

Given the increasing interest in preparing stable peptide and protein therapeutics, several strategies aimed toward engineering stable analogues of GLP-1 have emerged, Thus, unnatural amino acid modifications at the N-terminus of GLP-1, such as

N-pyroglutamyl modification and thioamide

substitution, can block its major degradation pathway through dipeptidyl peptidase (DPP-4) cleavage at alanine 8. However, these analogs can have reduced potency, as the N-terminal segment of GLP-1 is largely responsible for activation and signaling⁶'⁷. Additionally, PEG and lipid modifications introduced into peptides and proteins can often be immunogenic and must be tested on an individual basis . Nature Uses protein oligoglycosylation to increase the serum stability of secreted proteins . However, these extracellular modifications are complex and heterogeneous in structure, making them an impractical solution . Therefore, new approaches to therapeutic peptide stabilization that circumvent some of these issues are still of great interest.

As is disclosed hereinafter, an alternative approach is taken through the unconventional

application of the post-translational modification

O-G1cNAc . O-G1cNAcylation is the addition of the monosaccharide N-acetylglucosamine through a

b-linkage to proteins in the nucleus, cytosol, and mitochondria of metazoans as is shown below^{8, 9}.

O-G1cNAc can have a variety of biological

consequences on its protein substrates, including the modulation of protein-protein interactions and the inhibition of protein aggregation¹⁰'¹¹.

BRIEF SUMMARY OF THE INVENTION

The present invention contemplates a synthetic peptide hormone comprising a sequence of about 15 to about 50 amino acid residues that are at least about 90% identical to the same length and aligned sequence of a naturally-occurring peptide hormone. Each of the synthetic and naturally- occurring (native) peptide sequences exhibiting an amphipathic alpha-helical secondary structure as measured by circular dichroism spectroscopy.

Preferably, each of those peptide sequences is free of covalent intrachain linkage, The synthetic peptide hormone includes a mono-0-glycosylated serine or threonine residue in its sequence as a non- identical residue of the synthetic peptide hormone sequence; i.e. , the naturally-occurring sequence does not include that mono-O-glycosylated residue, The synthetic peptide hormone containing the mono-O- glycosylated serine or threonine residue has a half- life in human serum at 37°C of about 25 to about 800 percent longer than the naturally-occurring peptide hormone.

A composition containing an agonistic effective amount of an above-described synthetic peptide hormone dissolved or dispersed in a

pharmaceutically acceptable carrier is also

contemplated.

A method of treating a mammalian G protein- coupled receptor-mediated [GPCR-mediated] condition is also contemplated. Such conditions typically arise due to a lack or ineffectiveness of an

appropriate endogenous peptide hormone .

That method comprises administering an agonistic effective amount of an above-described synthetic peptide hormone dissolved or dispersed in a pharmaceutically acceptable carrier to that mammal having that GPCR-mediated condition, This

contemplated treatment can be repeated as needed to maintain the treated mammal . One more specific aspect of the invention comprises a GLF-1 derivative peptide. The sequences of the two native GLF-1 peptide hormones are shown immediately below,

GLF-1 (7-37) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

(SEQ ID NO: 6) ; and

GPL-1 (7-36) amide HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂

(SEQ ID NO: 7) .

A contemplated synthetic GLP-1 peptide hormone sequence is shown below as Formula 1 and SEQ ID NO: 1, from left to right and in the direction from amino-terminus to carboxy-terminus,

R⁷ R⁸N-HX₁EGTFTSDX2SX₃YLEGQAAKEFIAHLVR (1 where

X₁ = -NH- (CH₂)m-CR¹R²-C (O) - or A (Ala) ;

X₂ = -NH- (CH₂) _n-CR³R⁴-C (O) - or V (Val) ;

X₃ = -NH-CH [ (CHQ) -O- (Sac) ] -C (O) -;

X4 = G (Gly) , G-NR⁵R⁶ or -NR⁵R⁶;

m = 0 or 1;

n = 0 or 1;

Q = H or methyl;

R¹ and R² are the same or different substituents that are hydrogen (H) or C₁-C₃ hydrocarbyl, with the proviso that only one of R¹ and R² is hydrogen, or R¹ and R² together with the depicted carbon to which they are bonded form a 5-7-membered ring;

R³ and are the same or different substituents that are hydrogen (H) or C₁-C hydrocarbyl, or and R⁴ together with the depicted carbon to which they are bonded form a 5-7-membered ring;

R⁵, R⁶, R7 and R⁸ are the same or different and are H, C₁-C₁₈ hydrocarbyl, C₁-C₁₈ acyl, C₁-C₁₈ hydrocarbyIsulfonyl, or either or both of R⁵ and R⁶ and and R⁸ together with their depicted nitrogen atoms forms a 5-7-membered ring that optionally contains 1 or 2 additional hetero atoms that

independently are nitrogen, oxygen or sulfur, with the proviso that only one of and R⁶ and R⁷ and R⁸ includes a hydrocarbyl, acyl or sulfonyl group having more than six carbon atoms; and

Sac is a glycosidically-linked C₅-C₆ saccharide, deoxysaccharide or 2- (N-acetylamino) C₅-C₆ saccharide or deoxysaccharide. Preferred saccharides are

G1cNAc, GalNAc, ribose, deoxyribose, mannose, glucose and galactose.

A contemplated peptide of Formula 1 has been found to be particularly specific, potent, and exhibit diminished off-target effects as an agonist of the glucagon-like peptide-1 receptor (GLP-1R) . GLP-1 is an agonist for each of GLP-1R and the insulin receptor (IR) , both of which are well- established clinical targets²'³. In addition, such a peptide also exhibits enhanced stability to

hydrolysis by dipeptidyl peptidase-4 (DPP-4) and other endogenous proteases compared to GLP-1 itself.

A composition containing an IR- and/or GLP- 1R- agonistic effective amount of a peptide of

Formula 1 dissolved or dispersed in a

pharmaceutically acceptable carrier is also

contemplated. A method of treating a mammalian G protein- coupled receptor-mediated [GPCR-mediated] such as an IR- or GLP-lR-mediated condition like type 2 diabetes is also contemplated, That method comprises

administering an IR- or GLP-lR- agonistic effective amount of a peptide of Formula 1 dissolved or dispersed in a pharmaceutically acceptable carrier to that mammal having a GLP-lR-mediated condition such as type 2 diabetes. This contemplated treatment can be repeated as needed to maintain the treated mammal.

Another specific contemplated aspect of this invention is a parathyroid hormone (PTH) derivative peptide whose sequence is shown below, as Formula 2 and Seq. ID. No.: 7, from left to right and in the direction from amino-terminus to carboxy- terminus.

R¹R²N-SVSEIQLMHNLGKX₁LNSX₂ERVX₃WLRX₄KLQDVHNX₅ (2) wherein

-X₁- = -NH-CH [ (CH₃) _n (CHp) O- (Sac) ] -C (O) - or H

(His) ;

-X₂- = -NH-CH [(CH₃)_n (CHp) O- (Sac) ]-C(0)- or M

(Met) ;

-X3- = -NH-CH [ (CH3)_n (CHp) O- (Sac) ]-C(0) - or E

(Glu) ;

-X4- = -NH-CH [(CH₃)_n (CHp) O- (Sac) ]-C(0)- or K

(Lys) ;

-X5 = F (Phe) or F-NR³R⁴, with the proviso that only one of -X₁-, -X₂-, -X₃- and -X₄- is other than the stated amino acid residue; i.e., His, Met, Glu and Lys, or only one O-glycosylated [O-(Sac)] serine or threonine residue is present in the peptide; n = 0 or 1 and p = 1 or 2, with the proviso that when n = 1, p - 1, and when n = 0, p = 2;

R¹, R², R³ and R⁴ are the same or different and are H, C₁-C₁₈ hydrocarbyl, C₁-C₁₈ acyl

(hydrocarobyl) , C₁-C₁₈ hydrocarbylsulfonyl, or either

Or both of and and ^R3 and R⁴ together with their depicted nitrogen atoms forms a 5-7-membered ring that optionally contains 1 or 2 additional hetero atoms that independently are nitrogen, oxygen or sulfur, with the proviso that only one of R¹, R², R^ and R⁴ includes a hydrocarbyl, acyl or sulfonyl group having a chain of carbon atoms that contains more than six carbon atoms; and

Sac is a glycosidically-linked C₅-C₆ saccharide, deoxysaccharide or 2- (N-acetylamino) C₅-C₆

saccharide . Preferred saccharides are G1cNAc,

GalNAc, ribose, deoxyribose, mannose, glucose and galactose .

A composition containing a PTHR1 agonistic effective amount of a peptide of Formula 2 dissolved or dispersed in a pharmaceutically acceptable carrier is also contemplated.

A method of treating a mammalian GPCR- mediated condition such as a PTHR1-mediated condition like osteoporosis is also contemplated. That method comprises administering a PTHRI agonistic effective amount of a peptide of Formula 2 dissolved or dispersed in a pharmaceutically acceptable carrier to that mammal having osteoporosis . This contemplated treatment can be repeated as needed to maintain the treated mammal.

Definitions The word "hydrocarbyl" is used herein as a short hand term for a non-÷aromatic group that includes straight and branched chain aliphatic as well as alicyclic groups or radicals that contain only carbon and hydrogen_* Thus, alkyl, alkenyl and alkynyl groups are contemplated, whereas aromatic hydrocarbons such as phenyl are not included for this term. Usual chemical suffix nomenclature is followed when using the word "hydrocarbyl" except that the usual practice of removing the terminal "yl" and adding an appropriate suffix is not always followed because of the possible similarity of a resulting name to one or more substituents .

Where a specific aliphatic hydrocarbyl substituent group is intended, that group is recited; i.e. , C₁-C₃ alkyl, methyl or ethyl or iso-propyl. Exemplary hydrocarbyl groups contain a chain of 1 to 3 carbon atoms, and preferably 1 or 2 carbon atoms .

A particularly preferred hydrocarbyl group is an alkyl group. As a consequence, a generalized, but more preferred substituent can be recited by replacing the descriptor "hydrocarbyl" with "alkyl" in any of the substituent groups enumerated herein.

A hydrocarbyl group can be straight chained, branched chain or cyclic. Hydrocarbyl groups with six or fewer carbon atoms are used below for illustrative purposes with the understanding that a skilled worker will be well aware of the substituents with larger numbers of carbon atoms, particularly the straight and branched chained substituents .

Examples of alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl and the like. Examples of suitable alkenyl radicals include ethenyl (vinyl) , 2-propenyl, 3-propenyl, 1,4- pentadienyl, 1, 4-butadienyl, 1-butenyl, 2-butenyl, 3-butenyl, hexenyl, hexadienyl and the like.

Examples of alkynyl radicals include ethynyl,

2-propynyl, 3-propynyl, 1-butynyl, 2-butynyl,

3-butynyl, and the like.

As a skilled worker will understand, a substituent that cannot exist such as a alkenyl or alkynyl group are not intended to be encompassed by the word "hydrocarbyl", although such substituents with two or more carbon atoms are intended, as is a Ci alkyl or methyl group.

A contemplated hydrocarbyl group can also include a 5- to 7-membered hydrocarbyl ring.

Illustrative of such rings are cyclopentyl,

cyclohexyl and cycloheptyl, as well as

2-cyclopentenyl, 2- and 3-cyclohexenyl, and 2-, 3- and 4-cycloheptenyl groups.

A (C₁-C₁₈)hydrocarboyl group is a straight, branched chain or cyclic acyl hydrocarbyl residue that can contain one to through seven carbon atoms. Illustrative (C₁-C₁₈) hydrocarboy1 groups include formyl, acetyl, propionyl, benzoyl, acryloyl, methacryloyl, cyclopentylcarbonyl, hexanoyl, lauroyl, stearoyl and the like.

C₅-C₆ monosaccharides, their 2-deoxy derivatives and the 2-N-acetyl (NAc) derivatives of both types are well known in the in the chemical and biochemical arts . Exemplary C₅-C₆ monosaccharides include allose, altrose, fructose, glucose,

galactose, gulose, iodose, talose, mannose, xylose, ribose, fucose, arabinose, and lyxose. G1cNAc,

GalNAc, ribose, deoxyribose, mannose, glucose and galactose are preferred, whereas glucose (Glc) and galactose (Gal) and their 2-NAc derivatives are more preferred monosaccharides for use herein.

The terms "physiologically acceptable" and "pharmaceutically acceptable" in their various grammatical forms refer to any non-toxic cation or anion or combination thereof commonly used in the pharmaceutical industry, which can be prepared by methods known in the art. A contemplated cation and/or anion to provide ionic neutrality to an acid or basic amino acid residue side group, or can provide a water-soluble salt such as sodium chloride to raise osmolality above that of water, or a buffer such as dipotassium phosphate. Preferably, the cation salts are sodium, potassium, calcium and ammonium in either the mono or dibasic salt form.

The preferred anions are halogens such as chloride and bromide, and C₁-C₆ hydrocarbyl carboxylates . The reader is directed to Berge, J. Pharm. Sci. 1977 68 (1) : 1-19 for lists of commonly used physiologically (or pharmaceutically) acceptable acids and bases that form physiologically acceptable salts with

pharmaceutical compounds and buffers.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure,

Fig. 1A - Fig. IF illustrate the cell-based characterization of the exemplary GLP-1 peptides prepared herein. Fig.lA shows that neither

O-G1cNAcylation at serine residue 18 alone nor

O-G1cNAcylation plus 2-aminoisobutyric acid (AIB) substitution affects the EC₅₀ of cAMP production when compared to unmodified GLP-1. Fig. 1B shows that O-G1cNAcylation of GLP-1 alone does not alter Ca²⁺ accumulation, but in combination with AIB

substitution at residue 4 in Peptides G3 and G4 results in biased agonism. Fig. 1C and Fig. 1D illustrate that O-G1cNAcylation alone or in

combination with AIB substitution does not affect kinase signaling, Fig. 1E and Fig. 1F illustrate that b-Arrestin-l (IE) or b-Arrestin-2 (1F)

recruitment to GLP-1R is unaffected by

O-G1cNAcylation but is reduced by O-G1cNAcylation in combination with two AIB substitutions, resulting in biased agonism. All studies were performed in CHO cells expressing the human GLP-1R.

Fig. 2A and Fig. 2B illustrate that position 18 O-G1cNAcylation stabilizes GLP-1 from proteolysis and improves glucose clearance in vivo in human serum. Fig. 2A shows that Q-G1cNAc

modification improves the stability of GLP-1. The indicated peptides were incubated in triplicate with human serum for 24 hours . The stability of each peptide was measured using RP-HPLC after the

indicated lengths of time in serum. Fig. 2B

O-G1cNAcylation of GLP-1 improves glucose clearance in a glucose tolerance test, Lean mice (n = 5) were subjected to a single intraperitoneal co-injection of vehicle (saline) or the indicated concentrations of peptide with glucose challenge, Blood glucose levels were then measured after the indicated lengths of time. Error bars represent ± s.e.m., and statistical significance was calculated by one-way ANOVA followed by Dunnett's post-test.

Fig. 3 shows the circular dichroism (CD) spectrum of the GLP-1 peptide and also spectra of each of the synthesized peptides . Examination of the five spectra shows that each is substantially identical to the others thereby indicating that the presence of the O-G1cNAc moiety O-bonded to the position 18 serine residue does not alter the GLP-1 secondary

Structure. The CD spectra were collected for freshly dissolved samples of the indicated peptides at 50 mM concentration.

Fig. 4 shows a conformational model of the full length GLP-1R-G1 complex and the P2-PTH1R complex . Fig. 4A shows an overview of the GLP-1R-G1 molecular surface. Fig. 4B shows a close-up showing stabilizing interactions of the O-G1cNAc moiety. In both panels, GLP- 1R is shown as grey, with certain regions additionally delineated. The G1 peptide is represented as ribbon, with the O-G1cNAc moiety highlighted as transparent spheres. Intermolecular hydrogen bonds and salt bridges are shown by dotted lines. The P2-PTH1R complex is represented as molecular surface in Fig. 4C. Fig. 4D depicts a close-up showing predicted stabilizing interactions of the O-G1cNAc moiety. P2 peptide is represented as ribbon, where the O-G1cNAc moiety is highlighted as transparent spheres.

Fig. 5A - Fig. 5E illustrate an analysis of

O-G1cNAcylated PTH-analogs . Thus, Fig. 4A and Fig.

4B show cAMP production by O-G1cNAcylated PTH when compared to unmodified PTH in cells that either overexpress (Fig. 5A, GP-2.3) or endogenously express (Fig. 5B, SGS-72) PTHR1 (n =4 for each peptide) .

Fig. 5C shows that O-G1cNAc modification improves the stability of PTH. Unmodified PTH or P2 (n = 3) were incubated with human serum for 48 hours at 37°C. The stability of each peptide was measured using RP-HPLC after the indicated lengths of time, Fig. 5D shows that O-G1cNAcylation of PTH exhibits similar Ca²⁺ mobilization compared to unmodified PTH in vivo. Mice (n = 5) were subjected to a single subcutaneous injection of vehicle or the indicated concentrations of PTH or PTH peptide P2 . Blood. Ca²⁺ concentrations were then measured after the indicated lengths of time. Fig 5E shows that ionized Ca²* area under the curve (AUC) calculated from the data in Fig. 5D over 8 hours with a baseline of 1.25 mM. Error bars represent is.e.m., and statistical significance of differences in AUC between vehicle and peptides was calculated using one-way ANOVA with a Dunnett post- test. Results with PTH and P2 were not

statistically-significantly different from each other .

Fig. 6A - Fig. 6F show the circular dichroism (CD) spectrum of the PTH peptide and also spectra of each of the synthesized, G1cNAcylated (P1, P2, P3 and P4) peptides, Examination of the five spectra shows that the spectra for PTH and P2 are similar and different from the other spectra, thereby indicating that the presence of the O-G1cNAc moiety O-bonded to the position synthetically-added serine residue does not alter the PTH secondary structure, whereas the other substitution do change the structure.

The CD spectra were collected for freshly dissolved samples of the indicated peptides at 50 mM

concentration.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention contemplates a synthetic peptide hormone comprising a sequence of about 15 to about 50 amino acid residues that are at least about 90% sequentially identical to the same length aligned sequence of a naturally-occurring peptide hormone . Each of the synthetic and naturally-occurring peptide sequences exhibit an amphipathic alpha-helical secondary structure .

Preferably, each of those peptide sequences is free of a covalent intrachain linkage . The synthetic peptide hormone includes a mono-O-glycosylated serine or threonine residue in its sequence as a non- identical residue compared to the naturally-occurring (native) peptide hormone sequence; i.e. , the

naturally-occurring sequence does not include that glycosylated residue. The synthetic peptide hormone containing the mono-O-glycosylated serine or

threonine residue has a half-life in human serum at 37°C of about 25 to about 800 percent longer than the naturally-occurring peptide hormone.

The synthetic and naturally-occurring peptide hormones are agonists of a G protein-coupled receptor (GPCR) . A GPCR for which a synthetic and naturally-occurring peptide hormone is an agonist mediates a mammal's bodily function via the G protein to which it is coupled and the protein (s) associated with that particular G protein as is well known.

Exemplary particular naturally-occurring (native) peptide hormones agonists of G protein receptors are discussed hereinafter.

Turning to the synthetic peptide hormone and its corresponding native peptide hormone, it is seen that the two sequences have same number of amino acid residues in their linear sequences, and are therefore of the same length, The number of amino acid residues in such a sequence is about 15 to about

50. More preferably, that length is about 20 to about 45 residues, and most preferably about 25 to about 40 amino acid residues. Not only do the two peptide sequences have the same sequence length, the synthetic peptide hormone is at least about 90% sequentially identical to the aligned sequence of a naturally-occurring peptide hormone . Thus, if the native peptide hormone contains a sequence of 50 residues, a contemplated synthetic peptide hormone can contain up a sequence of 50 residues including to about 5 residues, including the mono-O-glycosylated serine or

threonine, that are at a different position in the sequence and/or of a different identity at a

specified sequence position as compared to the naturally-occurring peptide hormone sequence .

Because there are no partial amino acid residues, the "about 90%" utilized herein is to permit the

"rounding up to a whole residue where the 90% calculation provides for a partial residue. Thus, for example, a contemplated synthetic peptide hormone whose corresponding native peptide hormone has a sequence of about 34 residues can contain three to four non-native residues including the mono-O- glycosylated serine or threonine.

It is to be understood that a mono-O- glycosylated serine or threonine residue utilizes the hydroxyl group of the serine or threonine to form a glycosidic bond with the saccharide portion of the newly-formed glycosylated amino acid residue. A C-O-C linkage joins the amino acid and saccharide portions of the residue. For written convenience, such residues will be referred to hereinafter as a "monoglycosylated serine or threonine" .

Use of the word "aligned" herein means that when the two peptides are compared by their

sequences, those sequences are arranged so that there is the maximum identity of amino acid residues at each sequence position between them, Thus, if the native peptide hormone has a sequence of 35 residues and the synthetic peptide has 38 residues, the two sequences are first aligned to obtain the greatest sequence identity within the overlapping two lengths. Once the sequence alignment is considered, if the synthetic peptide hormone contains 35 residues that align with the native peptide hormone to provide at least an about 90% residue identity, the length and alignment criteria of the synthetic peptide hormone are fulfilled.

Each contemplated synthetic peptide hormone contains one monoglycosylated serine or threonine residue . In as much as there are no known naturally occurring monoglycosylated serine or threonine residues present in a naturally-occurring peptide hormone, such a glycosylated residue can constitute the required non-naturally-occurring (non-native) residue in a contemplated synthetic peptide hormone sequence . It is to be understood that a required non-native residue includes a monoglycosylated serine or threonine that is present at the same sequence position in the naturally-occurring form in a non- glycosylated form and/or a glycosylated serine or threonine that has replaced another residue at a given place in the sequence .

The native and synthetic peptide hormones also are preferably free of intrachain covalent linkages such as disulfides or amide bonds . Thus, a contemplated synthetic peptide hormone is preferably free of covalent cross-links such as disulfide or amide bonds. The alpha-helical secondary structure exhibited by the native and synthetic peptide hormones is amphipathic in that opposing polar and nonpolar faces of the helix are oriented along the long axis of the helix. The monoglycosylated serine or threonine residue is present in the sequence that constitutes the polar face of the amphipathic alpha- helical peptide. Preferably, that residue is situated near the center of the polar face of the amphipathic alpha-helix.

Several means are well known for predicting amphipathic helical structures in peptides and proteins. The reader's attention on this point is invited to Schiffer et al., Biophys J 7, 121-135 (1967); Jones et al., J Lipid Res, 33, 287-296

(1992); Phoenix et al., Curr Protein Pept Sci 3(2), 201-221 (April 2002); A. Drozdetskiy et al., Nucl. Acids Res 43 (W1) :W389-W394 (2015) ; K. Lin et al.

Bioinformatics 21,152-159 (2005) ; Buchan et al.; Nucl Acids Res 41 (W1): W340-W348 (2013) ; Gautier, et al, Bioinformatics 24(18) : 2101-2102 (2008); and Wadna et al., JOSS 3(31) : 1008 (2018) .

The presence of an amphipathic alpha- helical secondary structure in a peptide is typically determined by circular dichroism (CD) spectroscopy .

A CD spectroscopic determination is preferably carried out at a final peptide concentration of 50 mM in a 10 mM aqueous phosphate buffer at pH 7.4.

However, some native peptide hormones and their contemplated corresponding synthetic peptides do exhibit their secondary structure in aqueous media . As noted by Kezdy et al. , Proc Natl Acad Sci USA, 80:1137-1143 (1983), some cross-link-free peptides having sequence lengths of 10 to 50 residues that to not exhibit amphipathic alpha-helical structures in aqueous media do form amphipathic alpha-helical structures upon binding to cell surface proteins, lipids, phospholipids and the like. Kezdy et al. disclose methods to determine amphipathic alpha-helical secondary structures using egg lecithin vesicles and monolayer formation at an air-water interface . Separate X-ray crystallography analyses of the native peptide hormone and the synthetic peptide hormone bound to their receptor can also be useful . NMR techniques using as shown by NOSEY and chemical shift data can also be utilized to assay a GPCR-bound synthetic peptide hormone complex.

Still further, methanol, ethanol. acetonitrile, and trifluoroethanol alone or mixed with water can be used as solvents for circular dichroism determinations .

Exemplary GPCRs, their Native and Synthetic Agonists

The 178 residue proglucagon protein is a naturally occurring mammalian protein that is processed in vivo into shorter peptides (OniProtKB-

1275) . One of those peptides is known in the art as glucagon-like peptide-1 (GLP-1), and contains amiiio acid residues 92 through 128 of the proglucagon

Sequence . That peptide is relatively short-lived and is processed further into two peptides, both of which are also referred to in the art as gludagon-like peptide-1 (GLP-1) .

As further identifiers, a first of those peptides contains 31 residues of the proglucagon molecule and is referred to as GLP-1 (7-37). The second peptide contains 30 residues, is C-terminal amidated, and is further referred to as GLP-1 (7- 36) amide (UniProtKB-1275) . Any reference hereinafter to a GLP-1 peptide is directed to these latter two compounds, unless some other modifier is used to distinguish those peptides from the longer molecule. Both GLP-1 (7-37) and GLP-1 (7-36) amide bind to the GLP-1 receptor (GLP-1R) .

The present invention contemplates a synthetic GLP-1 derivative peptide whose sequence is shown below, as Formula 1 (SEQ ID NO: 1) , from left to right and in the direction from amino-terminus to carboxy-terminus in single letter amino acid code,

and R² are the same or different substituents that are hydrogen (H) or C₁-C₃ hydrocarbyl, with the proviso that only one of R¹ and R² is hydrogen, or R¹ and R² together with the depicted carbon to which they are bonded form a 5-7-membered ring;

R³ and are the same or different substituents that are hydrogen (H) or C₁-C₃ hydrocarbyl, or and

R⁴ together with the depicted carbon to which they are bonded form a 5-7-membered ring; R⁵, R⁶, R⁷ and R⁸ are the same or different and are H, C₁-C₁₈ hydrocarbyl, C₁-C₁₈ acyl, C₁-C₁₈ hydrocarbylsulfonyl, or either or both of R⁵ and R⁸ and R⁷ and R⁸ together with their depicted nitrogen atoms forms a 5-7-membered ring that optionally contains 1 or 2 additional hetero atoms that

independently are nitrogen, oxygen or sulfur, with the proviso that only one of R⁵ and R⁶ and R⁷ and R⁸ includes a hydrocarbyl, acyl or sulfonyl group having more than six carbon atoms; and

Sac is a glycosidically-linked C₅-C₆ saccharide, deoxysaccharide or 2- (N-acetylamino) C₅-C₆ saccharide or deoxysaccharide .

In one preferred embodiment, the saccharide of X₃ is O-G1cNAc. In another preference, R¹, R², R3 and are each methyl . As a further preference, R¹ and R² are both methyl and X2 is a valyl residue (V) . It is further preferred that X4 be G (Gly) , and that n be 0 (zero) so that the -(CH₂)_n group is absent.

It is also preferred that R⁷ and R⁸ each be hydrogen

(H) . It is further preferred that Q be hydrogen.

Exemplary preferred peptide sequences include G1, G2, G3 and G4, in which C₁= 2-amino- isobutyric acid (AIB) , and X₁ = X₂ = AIB; X₃ = -NH- CH [ (CH₂) -O- (G1cNAc) ] -C (O) -; and X₄ is G (Gly), are set out below along with their SEQ ID NOs:

(SEQ ID NO: 2) ;

(SEQ ID NO: 3) R^ and R⁴ are both methyl;

(SEQ ID NO: 4)

and

(SEQ ID NO: 5) R³, R², R³ and R⁴ are each methyl .

(Seq. ID. NO. : 6)

(SEQ ID NO: 7) .

Another specific contemplated aspect of this invention is a synthetic parathyroid hormone (PTH) derivative peptide whose sequence is shown below, as Formula 2 and SEQ ID NO: 8, from left to right and in the direction from amino-terminus to carboxy-terminus :

-X₅ = F (Phe) or F-NR³ R⁴, with the proviso that only one of -C₁-, -C₂-, -X₃- and -X₄- is other than the stated amino acid residue; i.e. , His, Met, Glu and Lys; n = 0 or 1 and p = 1 or 2, with the proviso that when n = 1, p - 1, and when n = 0, p = 2;

R¹, R², R³ and R⁴ are the same or different and are H, C₁-C₁₈ hydrocarbyl, C₁-C₁₈ acyl

(hydrocaroby1) , C₁-C₁₈ hydrocarbylsulfonyl, or either

Or both of and and R³ and R⁴ together with their depicted nitrogen atoms forms a 5-7-membered ring that optionally contains 1 or 2 additional hetero atoms that independently are nitrogen, oxygen or sulfur, with the proviso that only one of R¹-, R², R^ and R⁴ includes a hydrocarbyl, acyl sulfonyl group contains more than six carbon atoms; and

Sac is a glycosidically-linked C₅-C₆ saccharide, deoxysaccharide or 2- (N-acetylamino) C₅-C₆

saccharide . Preferred saccharides are G1cNAc,

GalNAc, ribose, deoxyribose, mannose, glucose and galactose .

Four illustrative synthetic peptides were prepared based upon the above PTH sequence. Although about 3 or 4 sequential differences are permitted within the 90% identity criterion, each sequence contained only one such difference an O-G1cNAcylated serine. Those four peptides are set out below, from left to right and in the direction from amino- terminus to carboxy-terminus, along with their SEQ ID NOs.

(SEQ ID NO: 9}

(SEQ ID NO: 10)

(SEQ ID NO: 11)

(SEQ ID NO: 12)

Listed below are five illustrative native peptide hormones that provide illustrative bases for contemplated synthetic peptide hormones:

Secretin⁴⁰: 27 amino acid residues, C-terminal amidated, forms an alpha-helix at residues

5-13 and also residues 17-26. Binds to family B GPCRs, including those that mediate diabetes mellitus, bone disease, pain, inflammation, migraine, anxiety, depression, short bowel syndrome and even some neoplastic diseases:

(SEQ ID NO: 13);

Pancreatic polypeptide (PP)⁴¹: 36-amino acid residues whose C-terminus is amidated, forms an alpha-helix at residues 14-32. PP binds to the neuropeptide Y receptors that are class A GPCRs;

increases gastric emptying and gut motility.

Pancreatic polypeptide also relaxes the pyloric and ileocecocolic sphincters, the colon, and gallbladder. PP levels increase after ingestion of food and remain elevated from four to eight hours:

(SEQ ID NO: 14); b-Endorphin: 31-Amino acid residue peptide exhibits little alpha-helix content in water, but exhibit increasing amounts of alpha-helical

conformation with increasing concentrations of methanol and also in solutions of 3mM sodium

docecylsulfate (SDS) using CD spectroscopy⁴². There are two alpha-helical regions; one is at residues 4-11 and the other is at residues 21-29. Beta- endorphin binds to the opiate pain GPCRs mu, delta and kappa⁴³:

(SEQ ID NO: 15);

Human growth hormone-releasing fragment (hGRF) : This hormone is a 44-amino acid residue C-terminal amidated peptide that is reported to exhibit two nested alpha-helical regions residues 22- 29 when in low molar ratio aqueous SDS (e.g. 1.3:1- 3.3:1) and residues 19-32 in SDS high molar rations (e.g. 16:1 -72:1) as shown by NOSEY and chemical shift data⁴⁴. This peptide hormone binds to binding to the GPCR named growth hormone-releasing hormone receptor (GHRH-R) . Residues 1-29 are reported to be important for biological activity, and residues 1-29 are preferred⁴⁵:

(SEQ ID NO: 16);

Glucagon: 29 Amino acid residue peptide hormone that binds to the GPCR glucagon receptor.

The alpha-helical sequence is reported to be located at residues 19-27⁴⁶:

(SEQ ID NO: 17) .

Pharmaceutical Compositions and Methods

A contemplated synthetic peptide hormone was discussed and defined previously. Briefly, such a synthetic peptide hormone comprises a sequence of about 15 to about 50 amino acid residues that are at least about 90% sequentially identical to the same length aligned sequence of a naturally-occurring peptide hormone. A contemplated synthetic peptide hormone includes a raonoglycosylated serine or threonine residue in its sequence as a non-identical amino acid residue relative to the naturally- occurring (native) peptide hormone sequence; i.e., the naturally-occurring sequence does not include that glycosylated residue. The synthetic peptide hormone containing the monoglycosylated serine or threonine residue has a half-life in human serum at 37°C of about 25 to about 800 percent longer than the naturally-occurring peptide hormone.

In one embodiment, a composition contains a contemplated synthetic peptide hormone is present in an agonistic-effective amount dissolved or dispersed in a pharmaceutically acceptable diluent or medium.

Illustratively, a contemplated synthetic GLP-1 derivative peptide hormone of Formula 1 can be used in the manufacture of a medicament

(pharmaceutical composition) that is useful at for treating diabetes type-2. It is to be understood that a contemplated GLP-1 derivative peptide hormone exhibits activities in the standard assays

illustrated herein.

A contemplated GLP-1 synthetic derivative peptide hormone, medicament or pharmaceutical composition containing the same lowers the blood glucose level of a recipient mammal as in a subject in need thereof, as does the parent GLP-1 peptide. However, contrary to the parent GLP-1 peptide that is quite unstable to proteolysis in a mammalian blood stream, a contemplated GLP-1 derivative peptide is more stable in blood in vivo, and in plasma in vitro, while maintaining the parental potency, This maintained potency is also exhibited in in vitro cell-based assays as compared to parental GLP-1 itself.

Similarly, a contemplated PTH synthetic derivative peptide hormone, medicament or

pharmaceutical composition containing the same can treat osteoporosis of a recipient mammal as in a subject in need thereof, as does the parent PTH peptide.

When used in vitro and in vivo, a contemplated synthetic peptide hormone is typically present along with pharmaceutically acceptable salts, buffers and the like that collectively are referred to as pharmaceutically acceptable diluents as compared to those salts and buffers that can be present in a composition that is not intended for pharmaceutical use, as in an in vitro assay or during synthesis .

A contemplated synthetic peptide hormone can be provided for use by itself, or as a

pharmaceutically acceptable salt. A contemplated synthetic peptide hormone can contain basic and acidic side chains . As a result, one or both of cationic and anionic ions can be present as

counterions .

Exemplary salts useful for a contemplated synthetic peptide hormone include but are not limited to the following: cations—sodium, potassium,

mmagnesium, calcium, zinc, aluminum, choline, diethanol amine, ethylenediimine and procaine;

anions—sulfate, hydrochloride, hydro bromides, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate,

cyclopentanepropionate, dodecylsulfate,

ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy- ethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate,

palmoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, mesylate and undecanoate.

The reader is directed to Berge, J. Pharm. Sci. 1977 68(1):1-19 for lists of conmonly used pharmaceutically acceptable acids and bases that form pharmaceutically acceptable salts with pharmaceutical compounds .

In some cases, a salt can also be used as an aid in the isolation, purification or resolution of the compounds of this invention. In such uses, the acid used and the salt prepared need not be pharmaceutically acceptable .

As is seen from the data herein, a contemplated synthetic peptide hormone is active in in vitro assay studies at picomolar (pM) to

micromolar (mM) amounts. When used in an assay such as an in vitro assay, an illustrative contemplated GLP-1 synthetic peptide hormone or a PTH synthetic peptide hormone is present in the composition in an amount that is sufficient to provide a concentration of about 0.001 nanomolar (nM) to about 1000 nM, preferably about 0.1 nM to about 50 nM, to contact cells to be assayed. A composition is contemplated that contains an agonistic effective amount of a contemplated synthetic peptide hormone or a pharmaceutically acceptable salt thereof dissolved or dispersed in a physiologically (pharmaceutically) acceptable carrier. Such a composition can be administered to mammalian cells in vitro as in a cell culture to contact those cells, or the cells can be contacted in vivo as in a living, host mammal in need. As is seen from the results reported herein, illustrative effective amounts for laboratory mice were in the range of about 0.1 to about 10 mg/kg when provided by intraperitoneal administration.

Illustratively, those amounts along with the in vivo effective amounts for the GLP-l(7-37) derivative liraglutide (Victoza®) that contains an added palmitoyl side chain and one exchanged amino acid residue relative to native GLP-l(7-37) and the gila monster-derived 39 residue analog sold as

Extenden-4 provide a skilled worker a more than adequate basis to determine an effective amount of a GLP-1 synthetic peptide hormone or salt appropriate to the intended recipient and desired effect.

A contemplated composition is typically administered in vivo to a subject in need thereof a plurality of times within one month, such as weekly, and can be administered over a period of several months to several years. More usually, a

contemplated composition is administered a plurality of times over a course of treatment.

A contemplated pharmaceutical composition can be administered orally (perorally) or

parenterally, which is preferred, in a formulation containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. The term parenteral as used herein includes subcutaneous injections, intravenous (which is most preferred), intramuscular, intrasternal injection, or infusion techniques. Formulation of drugs is discussed in, for example. Hoover, John E.,

Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania; 1975 and Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980.

Solid dosage forms for oral administration can include capsules, tablets, pills, powders, and granules. The amount of a contemplated compound in a solid dosage form is as discussed previously, an amount sufficient to provide a concentration of about about 0.001 nanomolar (nM) to about 1000 nM,

preferably about 0.1 nM to about 50 nM in the serum or blood plasma. A solid dosage form can also be administered a plurality of times during a one week time period.

In such solid dosage forms, a compound of this invention is ordinarily combined with one or more adjuvants appropriate to the indicated route of administration. If administered per os, the

compounds can be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration. Such capsules or tablets can contain a controlled-release formulation as can be provided in a dispersion of active compound in hydroxypropylmethyl cellulose, In the case of capsules, tablets, and pills, the dosage forms can also comprise buffering agents such as sodium citrate, magnesium or calcium carbonate or

bicarbonate . Tablets and pills can additionally be prepared with enteric coatings.

A contemplated pharmaceutical composition is preferably adapted for parenteral administration. Thus, a pharmaceutical composition is preferably in liquid form when administered, and most preferably, the liquid is an aqueous liquid, although other liquids are contemplated as discussed below, and a presently most preferred composition is an injectable preparation.

Thus, injectable preparations, for example, sterile injectable aqueous or oleaginous solutions or suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable

preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water. Ringer's solution, and isotonic sodium chloride solution, phosphate-buffered saline.

Other liquid pharmaceutical compositions include, for example, solutions suitable for

parenteral administration. Sterile aqueous saline solutions of a contemplated synthetic peptide hormone or sterile solution of a contemplated synthetic peptide hormone in solvents comprising water, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration. In some aspects, a contemplated synthetic peptide hormone is provided as a dry powder that is to be dissolved in an appropriate liquid medium such as aqueous sodium chloride for injection prior to use.

In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides.

In addition, fatty acids such as oleic acid find use in the preparation of an injectable composition.

Dimethyl acetamide, surfactants including ionic and non-ionic detergents, polyethylene glycols can be used. Mixtures of solvents and wetting agents such as those discussed above are also useful .

Sterile solutions can be prepared by dissolving the active component in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the sterile compound in a previously sterilized solvent under sterile conditions.

A mammal in need of treatment (a subject) and to which a pharmaceutical composition containing a contemplated synthetic peptide hormone is

administered can be a primate such as a human, an ape such as a chimpanzee or gorilla, a monkey such as a cynomolgus monkey or a macaque, a laboratory animal such as a rat, mouse or rabbit, a companion animal such as a dog, cat, horse, or a food animal such as a cow or steer, sheep, lamb, pig, goat, llama or the like.

Where an in vitro assay is contemplated, a sample to be assayed such as cells and tissue can be used. These in vitro compositions typically contain the water, sodium or potassium chloride, and one or more buffer salts such as and acetate and phosphate salts, Hepes or the like, a metal ion chelator such as EDTA that are buffered to a desired pH value such as pH 4.0 -8.5, preferably about pH 7.2-7.4,

depending on the assay to be performed, as is well known.

Preferably, the pharmaceutical composition is in unit dosage form. In such form, the

composition is divided into unit doses containing appropriate quantities of the active compound. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparation, for example, in vials or ampules .

Results

During the course of studies on

O-G1cNAcylation of the Parkinson' s disease associated protein cr-synuclein, which is modified by O-G1cNAc in vivo, it was found that single modifications of this protein dramatically inhibited the proteolysis of a-synuclein by the protease calpain¹². Notably, this inhibition occurred despite the fact that the

O-G1cNAc modifications were very distant in primary sequence (10-15 amino acids) from certain cleavage sites.

Therefore, it was queried whether this property of "remote stabilization," by

O-G1cNAcylation could be repurposed for therapeutic peptides . Accordingly, we first synthesized four analogs of GLP-1 that contained a single O-G1cNAc at serine 18 or in combination with one or two

2-aminoisobutyric acid (AIB) residues at sequence positions 8 and/or 16, which have been shown to stabilize GLP-1 by the Geliman laboratory^{13, 14}. We then used a range of cell-based, in vitro, and in vivo assays to demonstrate that 0-G1cNAcylation does not compromise the agonism of GLP-1R by the peptide, but does indeed stabilize GLP-1 against cleavage by serum proteases, including DPP-4, resulting in improved glucose clearance in mice.

Surprisingly, we found that O-G1cNAc in combination with AIB substitutions not only further stabilized the peptide to cleavage by serum proteases [inhibited peptide cleavage by serum proteases] , but resulted in significantly increased biased agonism towards certain G-protein signaling pathways and reduced recruitment of b-arrestins compared the to AIB substitutions alone¹³'¹⁴, which could result in improved therapeutic properties . Finally, we used molecular modeling to show that a combination of O-G1cNAc in S18, and the AIB mutations in positions V16 and A8, can create new key interactions on the peptide-receptor interface that can facilitate the conformational changes leading to agonism bias.

Design and synthesis of O-G1cNAcylated variants of GLP-1

As mentioned above, biochemical and structural studies have demonstrated that the

N-terminal residues of GLP-1 are critical for making the appropriate interactions with GLP-1R that result in receptor activation. Additionally, the receptor makes additional interactions with the C-terminus of GLP-1 starting around residue 24.¹⁴ With these considerations in mind, we chose to place O-G1cNAc at serine 18 of GLP-1, as we hypothesized that this position would be on the polar face of the

amphipathic helix and solvent-exposed upon receptor binding. Accordingly, we prepared the suitably protected O-G1cNAcylated serine using a published procedure¹⁵ and prepared the peptides (G1-G4 ,

sometimes also referred to herein as C1-C4) using standard Fmoc-based solid phase peptide synthesis. Each peptide was purified.

Unmodified GLP-1 has significant «-helical character in solution. Therefore, we next used circular dichroism (CD) spectroscopy to examine the secondary structure of all of our peptides and found that neither O-G1cNAc nor the AIB substitutions had any noticeable consequences (Fig. 3) .

O-G1cNAcylation does not inhibit GLP-1R agonism alone but causes biased agonism in

combination with AIB. Endogenous binding of GLP-1 to the GLP-1R results in recruitment of the Ga_E

G-protein, subsequently stimulating the production of cyclic AMP (cAMP) from ATP and leading to glucose- stimulated insulin secretion¹⁶. To assess the ability of the O-G1cNAc-modified GLP-1 peptides G1-G4 to agonize human GLP-1R, cAMP accumulation was measured in Flpln CHO cells stably expressing human GLP-1R¹⁴.

As a positive control for full human GLP-1R activation, cells were treated with native GLP-1, which exhibited an EC₅₀= 4.1 pM (Fig. 1A and Table 1A) . O-G1cNAc peptide analogues G1-G4 showed very similar potencies (5.1-6.0 pM) that were not

significantly different from unmodified GLP-1.

Notably peptide G1 also resulted in a small but significant higher maximum efficacy compared to unmodified GLP-1.

Again, using the CHO-cell system, the ability of peptides G1-G4 to promote intracellular calcium mobilization was next measured, an established assay that reports on Gaq activation by

GLP-1R^14,17. In this assay, recruitment of Ga_q activates phospholipase Cb (PLCp) , catalyzing the formation of inositol-1, 4, 5-trisphosphate (IP₃) that can bind and open the IP₃-gated calcium channel, increasing calcium Concentration in the cytoplasm¹⁶.

We found GLP-1 to exhibit an EC₅₀= 58.5 nM (Fig. IB and Table 1A) in this assay, The O-G1cNAc peptide analogues G1 and G2 showed comparable potencies and maximum response relative to GLP-1. In contrast, O-G1cNAc peptide analogues G3 and G4 displayed reduced potencies relative to GLP-1 and their maximal response was reduced significantly, indicating that G3 and G4 could be biased Ga_s agonists .

In addition to these G-protein coupled pathways, GLP-1 has been shown to activate kinases through non-canonical pathways; therefore, we tested whether peptide analogues G1-G4 were assessed for their ability to promote ERK1/2 and ART

phosphorylation¹³'¹⁹. As expected, unmodified GLP-1 resulted in dose-dependent phosphorylation/activation of both ERK and ART (Fig. 1C, Fig. ID and Tables 1A & 1B) . Likewise, all of the O-G1cNAcylated analogs resulted in phosphorylation of both kinases that were essentially indistinguishable from the unmodified peptide (Fig. 2C, Fig. 2D and Tables 1A and 1B) .

Like most a G protein-coupled receptors (GPCRs) , GLP-1R activation can also result in recruitment of b-arrestin proteins that play a central role in receptor desensitization²⁰. An established bioluminescence resonance energy transfer (BRET) assay in FlpInCHO cells transiently expressing GLP-1R was used to evaluate this pathway²¹. In this assay, GLP-1 exhibited an EC₅₀= 34.8 nM for b-arrestin-l and an EC₅₀= 20.0 nM for

b-arrestin-2 (Fig. 1E, Fig. 1F and Table 1B) .

O-G1cNAc modified peptides G1-G3 displayed comparable EC₅₀ values arid curves to GLP-1 (Table 1B) .

In contrast, we observed both a shift in ECSQ and a substantial decline in the recruitment of the b-arrestins, relative to GLP-1, upon treatment with peptide G4 . These results further support peptide G4 as a biased agonist towards the Ga_s/cAMP signaling pathway. We previously showed that A1B substitution at both residues 8 and 16 can cause a modest bias in the same direction¹⁴. However, those magnitudes (about 15-25%) are considerably less than those observed for G4, indicating that the O-G1cNAc modification is playing a direct role in increasing bias.

Table 1A

Activity data for GLP and O-G1cNAcylated peptides G1-G4 in cAMP accumnlation, Ca²⁺ mobilization, ERK1/2 phosphorylation*

Table 1B

Activity date for GLP and O-GlcNAcylated peptides G1-G4 in AKT phosphorylation, and b-arrestin 1 or 2 recruitment*

*All studies were performed in FlpIn CHO cells either stably or transiently expressing human GLP-1R. PEC5Q and E_max values were calculated by logarithmic fitting in GraphPad. Values are the mean ± s.e.m. of at least four biological replicates .

Statistical significant difference from GLP-1 was calculated using either one way ANGVA followed by Tukey's post-hoc analysis (+) or students t-test (#) .

O-G1cNAc modification stabilizes GLP-1 from degradation and improves its function in vivo. To complement our ex vivo characterization of the GLP-1 analogs G1-G4, we next tested our original hypothesis that O-G1cNAcylation would improve the stability of GLP-1 in the presence of serum proteases .

Accordingly, unmodified GLP-1, Gl, or G4 were

incubated in triplicate in human serum for 24 hours .

After different lengths of time, aliquots were removed and analyzed by RP-HPLC to measure the remaining full-length peptide²². The resulting data were then fit to an exponential decay curve to calculate the peptide half-lives (Fig. 2A) .

Gratifyingly, the half-life of peptide Gl was twice that of unmodified GLP-1, confirming our O-G1cNAc hypothesis, and the incorporation of AIB units in G4 further increased this stability. Interestingly, we could identify the major cleavage product of GLP-1 and G1 by dipeptidyl peptidase-4 (DDP-4) by mass spectrometry and found that O-G1cNAcylation appears to inhibit this

proteolysis event despite the fact that this

modification is 10 amino acid residues away from the cleavage site in the primary sequence, Given this promising in vitro data, we next tested our peptides in vivo using a glucose clearance assay, or glucose tolerance test (GTT)²³.

More specifically, after fasting, mice (n = 5 per group) were injected intraperitoneally (IP) with 1 mg/kg of glucose and either vehicle, or vehicle plus GLP-1, or one of our O-G1cNAcylated analogs . After different lengths of time, tail blood-glucose levels were measured. In an acute GTT, all of the modified peptides (G1-G4) displayed significantly improved glucose disposal efficiency compared to unmodified GLP-1 (Fig. 2B) .

Notably, contemplated peptides G1-G4 lowered fasting blood glucose levels with a faster onset than GLP-1 as indicated by a greater correction of glucose levels at the 15 minute time point, These data fully support our cell-based assays that show near identical levels of cAMP accumulation after GLP- 1R activation by peptides G1-G4, and demonstrate that O-G1cNAc modification results in improved in vivo activity.

Structure-based modeling of the modified peptides to GLP-1R suggests the molecular

determinants of binding and signaling bias, In order to gain molecular insight into the effect of O-G1cNAc on GLP-1R binding by peptide Gl, and potentially more interestingly the biased agonism of peptide G4, we used molecular modeling based on a Cryo-EM structure of Unmodified GLP-1 bound to GLP-1R 9 as discussed hereinafter²⁴.

First, we generated a model of peptide Gl in complex with GLP-1R complex and found that the O-G1cNAc moiety introduced at 518 is accommodated in the shallow cleft located between the extracellular loops ECL2 and ECL3 of the 7 transmembrane (7TM) domain and the extracellular domain (ECD) of GLP-1R

(Fig. 4) . The energy-optimized conformation of the sugar makes hydrogen bond interactions with N300^ECL2 of the receptor and an internal hydrogen bond with D15 of GLP-1, which is further stabilized by a salt bridge with R376^ECL3, while maintaining the crucial salt bridge with the R380⁷·³⁴ residue, both of GLP-1R²⁵.

Additionally, introduction of the O-G1cNAc moiety leads to rearrangement of the Y19 and Q23 side chains in GLP-1, where Y19 forms anion-n interaction with receptor residue E139¹-³⁴ and an intramolecular hydrogen bond with Q23. This extended interaction network resulting from introduction of O-G1cNAc on SI8 is likely to contribute to increased rigidity in the GLP-R1-G1 complex .

Likewise, introduction of AIB in position V16 of G2 can impose additional conformational rigidity in the peptide . This model matches the experimental data, where both of these modifications of GLP-1 are well tolerated but do not have notable effects on signaling potency or bias.

The additional modification that confers signaling bias introduces AIB at position A8 at the very N-terminus of GLP-1, peptide G4. In this structural model, the additional methyl unit of AIB8 impinges on the receptor F367⁶·⁵⁶ phenyl ring, resulting in a dramatic shift of this side-chain.

This shift is likely to result in a dynamic change in the helix VI backbone itself, which is known to couple to downstream conformational changes,

facilitating G-protein signaling, while potentially compromising other signaling pathways (e.g.

b-arrestin) . Corroborating this hypothesis, our recent Cryo-EM structure with another biased agonist, Exendin-P5²⁶, shows a similar outward shift and rotation of residue F367⁶-⁵⁶ and the tip of helix VI (PDB ID 6B3J) , and Exendin-P5 has a bulky residue (V8) in the same potion as AIB8 in G4.

This analysis suggests that the distinct interaction of N-terminal residues of the biased ligand with helix VI, and particular the F367^6.56 side chain, might serve as a major determinant of

G-protein bias in GLP-1R. As noted above, AIB modifications without the O-G1cNAcylation induce much less bias¹³·¹⁴. We propose that this may be due to flexibility in the ligand, which can absorb the introduced strain of the AIB8 - F367⁶·⁵⁶ interaction.

In contrast, when all 3 changes are combined in the G4 ligand, the increased overall rigidity of ligand-GLP-1 interactions by O-G1cNAc on SI8 and AIB16 results in very pronounced bias effect from the AIB8 mutation. In other words, whereas AIB8 creates a lever for inducing the conformational change in helix VI, the other two modifications in the peptide enhance the foothold for this lever and make it more rigid.

Thus, all three mutations contribute to the dramatic changes in signaling profile and introduce ligand bias. Although the model effectively

describes our cellular data, direct structural information may be needed to further validate the mechanism of biased mode of action of the G4 ligand.

This model is validated by our experimental data, where the O-G1cNAcylation of Seri8 is

accommodated by GLP-1R and does not inhibit potency of our GLP-1 peptide analogs in the cell-based signaling assays. Moreover, mutation of key residues within this pocket alter the potency and/or maximum response of GLP-1, G1 and G4 peptides when assessed in both cAMP and pERKl/2, albeit there are

distinctions in the extent of these effects when comparing between cAMP and pERKl/2 and between ligands, as shown in Tables 3A and 3B, respectively, below.

Table 3A¹

¹ Notes follow Table 3B.

Table 3B¹

¹ All studies were performed in Flpln CHO cells either stably or transiently expressing wildtype or mutant human GLP-1R. pEC50,

Emax, and error (±s.e.m. ) were calculated by nonlinear fitting (three parameter logistic fit for all cAMP data, and four parameter fit for pERK) in GraphPad . Values are from at least four replicates . P values were calculated for each ligand using one-way ANOVA followed by Dunnett's post-test with wildtype as the control. Statistically significant differences (P<0.05) are denoted by *.

For example, L142A reduces potency for all ligands in cAMF but has more limited effect in pERKl/2 whereas R376AECL3 had a greater effect of pERKl/2 potency relative to cAMF. Ala mutation of

N300ECL2 and R380ECL3 heavily impaired cAMP and pERKl/2 signaling mediated by all three peptides, but G4 was more sensitive to these mutations in cAMP.

For pERKl/2, potency was impacted for all three peptides, but maximal response was only reduced for G1 and G4 . These results are in excellent agreement with the additional interactions predicted by the modelling between the sugar of the O-GlcNAc peptides and receptor residues N300, R376, and R38G.

Design and synthesis of O-G1cNAcylated variants of PTH

In the case of PTH, the O-G1cNAcylation sites on the crystal structure of PTH (15-34) bound to the extracellular domain of PTHR1²¹. More

specifically, the individual O-G1cNAc moieties were mapped along the a-helical face of PTH that points away from the extracellular domain of PTHR1, yielding peptides P1-P4. As with GLP-1, Fmoc-based solid phase peptide synthesis was employed, the peptides were purified by RP-HPLC, and characterized them by ESI-MS.

PTH also tends to form an a-helix in aqueous solution, CD spectroscopy was used to examine any effects of O-G1cNAcylation (Fig. 6) . Similar to

GLP-1, modification on peptides PI and P2 had little- to-no effect on the structure of PTH. However, peptides P3 and P4 were more unstructured in solution with P4 having the largest degree of disorder.

O-GlcNAcylation analogs of PTH maintain canonical cAMP signaling

Much like GLP-1R, activation of PTHR1 can result in the recruitment of Gets that promotes cAMP production and associated downstream signaling events in bone and the kidneys to regulate blood calcium levels . To test the ability of our PTH analogs (n = 4) to stimulate cAMP production, we used a HEK-293 cell line, GP-2.3, which stably overexpresses the human PTHR1 and a cAMP sensor Table 2, as well as a human osteoblastic cell line (SGS-72) that

endogenously expresses PTHR1 Table 2, below. Table 2

¹ All studies were performed in the indicated cell lines that either overexpress (GP-2.3) or endogenously produce

(SGS-72) PTHR1. PEC₅₀ , E_max, and error (±s.e.m. ) were c_{alculated by nonlinear fitting (thr}ee parameters logistic fit) in GraphPad. Values are from at least four

biological replicates. P values were calculated using one-way ANOVA followed by a Dunnett' s post-test .

Statistically significant differences are denoted by *.

Overall, there was limited impact of the O-G1cNAcylation modification on PTH-mediated cAMP responses, with only small reductions in potency observed, in a peptide- and cell-specific manner. However, similar to what we observed with GLP-1 modifications, the most robust effect was an

enhancement of Emax, particularly in the case of the GP-2.3 model .

O-GlcMAc modification inhibits PTH serum degradation while maintaining its in vivo function

To determine if the O-G1cNAc modified PTH analogs could also enhance serum stability, we selected one of the analogs, P2 for further

characterization. P2 was selected as it displayed the best potency compared to unmodified PTH in the endogenous PTHR1 expression-model (SGS-72, Table 2) .

Thus, either P2 or PTH was incubated with human serum for 48 hours (n = 3 for each peptide) ex vivo. As with the GLP-1 peptides, aliquots of the reaction mixture were removed after different lengths of time and analyzed by RP-HPLC and peptide half- lives were calculated. Strikingly, we found that P2 was approximately twice as stable as unmodified PTH, an increase in stability very similar to that of G1 compared to GLP-1.

We next tested these two peptides in vivo measuring blood Ca²⁺ levels in mice, which increase upon PTH administration through release of calcium from bone. Specifically, either PTH or P2 was injected subcutaneously into the tail vein at a dose of 20 nmol/kg. We then analyzed the ionizable Ca²⁺ levels from blood that was withdrawn immediately before injection and at various time points after injection.

As expected, we observed an increase in blood Ca^2÷ levels upon administration of PTH.

However, despite the increased serum stability of P2, we did not observe an additional increase in blood

Ca²⁺ over PTH. Nevertheless, these data indicate that O-G1cNAcylation can function as a general stabilizer of peptides in serum.

Structure-based modeling of the modified peptides to PTHR1 suggests the molecular determinants of binding and signaling bias, Analogous to the work described above for the effect of O-G1cNAc on GLP-1R binding by peptide Gl, we used molecular modeling based on the high resolution crystal structure of ePTH, a peptide agonist with 79% sequence homology to PTH, in complex with the full length PTH1R (2.5 A) .⁴⁸

First, we generated a model of P2 in complex with PTH1R and we found that the O-G1cNAc moiety introduced at Metis can be accommodated in the proximity of the stalk region spanning between the 7TM domain and the ECD (170-179 residues) . The optimized conformation of the O-G1cNAc moiety is accommodated in the receptor model even without conformational changes in the protein or peptide backbone . Moreover, there is a crystallographic water molecule located in the proximity of residue Met18, which is coordinated by Glu22 and Agr25 of ePTH, and might also form an additional hydrogen bond to the O-G1cNAc moiety.

Taken together, the model suggests that any observed increase in the maximal signaling from P2 can be explained by the shape complementarity and polar contacts with the PTH1R residues N176 and El80 in the stalk region as well as overall increased rigidity of the complex (Figs. 4C and 4D) .

DISCUSSION

Inspired by our studies showing that

O-G1cNAcylation of the much larger a-synuclein (140 amiino acid residues) inhibits its proteolytic degradation¹², we report here that this property is portable to the much shorter peptide hormones GLP-1, PTH and others having up to about 50 amino acid residues . More specifically, we first found that modification of GLP-1 at S18 (peptide G1) had no detrimental effects on the ability of GLP-1 to engage its receptor in living cells and initiate the physiologically-relevant signaling pathways (Fig. 1A- 1F) .

However, this monosaccharide does indeed slow the degradation of GLP-1 in human serum in vitro by 2-fold compared to the unmodified peptide (Fig.

2A) . Although this level of stabilization is at first glance modest, O-G1cNAcylation significantly improves glucose disposal in vivo (Fig. 2B) , a property that we attribute to its increased

circulation time. Remarkably, the increased

stability of GLP-1 extends to the major cleavage of the peptide at residue 8 by the protease DDP4 that is 10 amino acid residues away from the site of O-G1cNAc modification, mirroring our a-synuclein results.

We believe that this "remote stabilization" property of O-G1cNAc will make it significantly more flexible in its application than other strategies that target the specific site of proteolysis, For example, the N-terminus of GLP-1 is essential for signaling, and previous strategies that directly stabilize the amide bond at residue 8⁷'²⁷ or use larger hydrophilic groups 28 can result in reduced signaling potency.

This flexibility of remote stabilization by O-G1cNAc renders it applicable to other peptide hormones or even proteins, as the modification would likely be placed in a solvent-exposed position distant from the proteolytically-labile residues that are also key for receptor binding, This is being currently explored.

In addition to this single modification of GLP-1 by O-G1cNAc, we also explored the potential for additional effects when glycosylation was combined with backbone modifications. Serendipitously, two AIB substitutions at residues 8 and 16 of 61,

producing G4, resulted in fairly large biased agonism towards cAMP accumulation and away from Ca²⁺

mobilization and arrestin recruitment (Figs. 1B, 1E and 1F) . This result demonstrates that O-G1cNAe cap be combined with other strategies, including other natural and unnatural amino acids, to further improve the clinical properties of peptides, Furthermore, because O-G1cNAc is a native modification of oither intracellular proteins it has a low potential to elicit immune responses.

Given these interesting biological results and potential clinical applications, we next used molecular modeling to examine how O-G1cNAcylation in G1 affected binding to the GLP-1 receptor and found that this modification was mostly solvent exposed, as hypothesized. In this model, the N-terminus of 61 can adopt a confirmation similar to the unmodified peptide consistent with our cell-based results that show very little changes in signaling between GLP-1 and Cl (Figs. 1A-1F) .

We also wanted to explore the basis of 64' s signaling profile in our cell assays and again used molecular modeling . Strikingly, we found that an AIB substitution at residue 8 in GLP-1 clashed with

F367^6·56 of the receptor in a manner similar to that of our previous Cyro-EM structure of GLP-1R in complex with the biased agonist Exendin-P5²⁶, which is consistent with the cellular signaling profile of 64

(Figs . 1A-1F) . We hypothesize that the positioning of O-G1cNAc in a cleft of GLP-1R, and potentially the specific interactions around the 2-N-acetate,

rigidify GLP-1 with respect to the receptor and allow it to "push harder" against helix VI than either G1 or the AIB substitutions alone .

EXPERIMENTAL PROCEDURES

Materials

Dulbecco' s Modified Eagles Medium (DMEM) , Fluo-4 acetoxymethyl ester and fetal bovine serum (FBS) were purchased from Life Technologies

(Carlsbad, CA) . Polyethylenimine (PEI) was purchased from Polysciences (Warrington, PA) . AlphaScreen® reagents, LANCE® cAMP reagents and 384-well

ProxiPlates^TM were purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA) . SureFire® extracellular signal-regulated kinases 1 and 2

(ERK1/2) and protein kinase B (Akt) reagents were obtained from TGR Biosciences (Adelaide, SA,

Australia) . Coelenterazine H was purchased from Nanolight Techology (Pinetop, AZ) . GLP-1 (7-37) was purchased from American Peptide (Sunnyvale, CA) .

All solvents and reagents were purchased from commercial sources (Sigma-Aldrich, Fluka, EMD, Novagen, etc. ) and used without any further

purification. All aqueous solutions were prepared using ultrapure laboratory grade water (deionized, filtered, and sterilized) obtained from an in-house ELGA® water purification system and filter sterilized with 0.45 mm syringe filters (VWR) before use.

Reverse-phase high-performance liquid chromatography (RP-HPLC) was performed using an Agilent Technologies 1200 Series HPLC instrument with a diode array detector. Unless otherwise stated, the following HPLC buffers were used: buffer A, 0.1% trifluoroacetic acid (TFA) in H₂O; buffer B, 0.1% TFA and 90% acetonitrile (ACN) in H2O. Mass spectra were acquired on an API 3000 LC/MS-MS system (Applied Biosystems/MDS SCIEX) .

Peptide Syntheses

All peptides were synthesized using standard Fmoc solid-phase chemistry on Rink amide

ChemMatrix® (PCAS BioMatrix, 0.45 ramol/g) resin using HBTU (5 equiv, Novabiochem) and DIEA (10 equiv,

Sigma) in DMF for 1 hour. For activated glycosylated amino acids, 2 equivalents of monomer in 3 mL of DMF was coupled overnight (about 18 hours) . When peptides were completed, acetyl groups were

deprotected with hydrazine monohydrate [80% (v/v) in MeOH] twice for 30 minutes with mixing. All peptides were then cleaved using (95:2.5:2.5 TFA/H₂O/

triisopropylsilane) for 3.5 hours at room

temperature, precipitated out of cold ether, and purified by reverse-phase HPLC using preparative chromatography. All peptides were characterized by mass analysis using ESI-MS, and the sequence purity was assessed by analytical HPLC.

Circular Dichroism Spectroscopy

Circular dichroism spectra were recorded on a Jasco J-815 CD spectrometer. Peptides were freshly diluted to a final concentration of 50 mM in 10 mM phosphate buffer (pH 7.4) prior to sample

measurement. Spectra were recorded from 250 to 190 nm with a 0.1 nm data pitch, a 50 nm min^-1 scanning speed, a 4 second data integration time, a 1 nm bandwidth, and a 1 ran path length with 3

accumulations, at 25°C. All data were background- subtracted from a sample containing only phosphate buffer.

Cell Culture Chinese Hamster Ovary (CHO) Flpln cells stably transfected with an N-terminally double cmyc labeled human GLP-1R (CHO Flpln huGLP-1R) were generated using Gateway technology (Invitrogen) as previously described²⁹, These cells were used for all studies, with the exception of b-arrestin recruitment assays, for which CHO Flpln parental cells were transiently cotransfected with C-terminally RlucS labeled huGLP-lR and C-terminally YFP labeled b-arrestin-l or p-arrestin-2. For transient

transfections, cells were seeded at 3 x 10⁶ cells per 10 cm dish or prepared in a 10 mL FBS-enriched DMEM suspension at a density of 3 x 10^€ cells, and

transfected using a 1:6 ratio of DNArPEI, prepared in 150 mM NaC1 for 15 minutes room temperature (RT) and then added to cells. Cells transfected in suspension were then immediately seeded at a density of 30,000 cells/well into 96-well white culture plates, In all cases, total DNA transfected did not exceed 5 pg, with receptorib-arrestin transfected at a ratio of

1:4. All cells were maintained in DMEM supplemented with 5% FBS in a humidified environment at 37°C in 5% C(½.

GLP-1 cAMP Accumulation Assay

CHO F1pln huGLP-1R cells were seeded at a density of 30,000 cells/well into 96-well culture plates and incubated overnight at 37°C in 5% CO2, and peptide-mediated cAMP generation was carried out using the LANCE® cAMP protocol (PerkinElmer) as described previously¹⁴. All values were converted to concentration of cAMP using a cAMP standard curve performed in parallel, and data subsequently

normalized to the response elicited by 10 mM

forskolin. Intracellular Ca²⁺ Mobilization Assay

CHO Flpln huGLP-lR cells were seeded at a density of 30,000 cells/well into 96-well culture plates and incubated overnight (about 18 hours) at 37°C in 5% CO₂, and peptide-mediated intracellular Ca²⁺ mobilization was determined as described

previously¹⁷. The peak value following peptide addition was used to create concentration-response curves . Data were normalized to the maximal response elicited by 100 mM ATP.

GLP-1 ERK1/2 and Akt Phosphorylation Assays CHO Flpln huGLP-lR cells were seeded at a density of 30,000 cells/well into 96-well culture plates and incubated overnight (about 18 hours) at 37°C in 5% CO2, and peptide-mediated ERK1/2 and Akt phosphorylation was determined using the AlphaScreen® SureFire® protocol (PerkinEInter) as described previously²⁹. Initial phosphorylation studies were performed over a 1 hour time course to determine the peak phosphorylation time point. Cells were

stimulated with peptide at the time required to cause a maximal response (pERKl/2, 6 minutes; pAkt, 5 minutes) . Data were normalized to the response of 10% FBS at 6 minutes (pERKl/2) or 4 minutes (pAkt) (peak FBS response) .

GLP-1 b-Arrestin Recruitment Assay Twenty-four hours following transient transfection, CHO Flpln cells expressing the huGLP-lR and b-arrestin-l or b-arrestin-2 were seeded from 10 cm dishes into 96-well white culture plates at a density of 30,000 cells/well, and incubated for a further 24 hours at 37°C in 5% CO2 prior to assay.

CHO Flpln cells transiently expressing the huGLP-lR and b-arrestin-l or b-arrestin-2 using the suspension method were incubated for 48 hours at 37°C in 5% CO₂ prior to assay.

Peptide-mediated recruitment of b-arrestin- 1 or b-arrestin-2 was conducted using BRET, as previously described²¹. Initial recruitment studies were performed over a 15 ininute time course to determine the peak BRET signal. Subsequent studies were performed at the time required to cause a maximal BRET response (b-arrestin-l, 3 minutes;

b-arrestin-2, 2.5 minutes). Data were corrected for vehicle-treated cells (Hank's buffered salt

solution) .

Data Analysis

All data were analyzed using Prism 7.0 (GraphPad Software Inc. , San Diego, CA) . For all analyses, the data were unweighted and each y value (mean of replicates for each individual study) was considered an individual point. Concentration response signaling data were analyzed with a three- parameter logistic equation as described previously²³:

where "Bottom" represents the y value in the absence of ligand (s), "Top" represents maximal stimulation in the presence of ligand(s), "[A]" is the molar

concentration of agonist and "EC₅₀" is the molar concentration of ligand required to generate a response halfway between Top and Bottom. pERK1/2 concentration response signaling data were also analyzed with a biphasic dose response model:

where the parameters are as listed above and LogEC_50(I) and LogEC_50(II) are the midpoint potencies for the phases 1 and 2, respectively, "nHr" and "nH_II" are their respective Hill slopes, and "Frac" is the more potent fraction of the curve.

Glucose Tolerance Test

Male C57BL/6J mice (Jackson Laboratory) were tested between 10 and 12 weeks of age. Mice were group-housed on a 12:12 light-dark cycle in a temperature-controlled environment with free access to standard chow diet and water. Mice were divided into groups (n= 5) and fasted 18 hours (overnight) . Mice were injected intraperitoneally with compound 20 minutes prior to intraperitoneal injection of glucose bolus at lg per kilogram body weight. Blood glucose level was monitored at the indicated time points using an AphaTRAK2 glucose meter Zoetis Services LLC (Parsippany, New Jersey) . All mouse studies were approved by and performed according to the guidelines of the Institutional Animal Care and Use Committee of the Scripps Research Institute.

Human Serum Stability Assay

Stock solutions of the peptides (500 mM in PBS, pH 7.4) and reconstituted normal human serum (Thermo Scientific) were pre-equilibrated to 37°C for 15 minutes in separate tubes. For each replicate, peptides and serum were combined to a final

concentration of 50 mM and 50% (v/v) respectively, final volume of 150 mL. The reaction tubes were then incubated at 37ºC with 300 rpm shaking in an

Eppendorf® ThermoMixer®.

At each time point, 20 pL of the reaction was taken from each replicate and diluted with four- fold excess of cold acetone. The serum proteins were allowed to precipitate fop 15 minutes at: -20°C and were removed by centrifugation at 14,000 g, 4°C for 10 minutes. The supernatant was transferred to a new tube, diluted with the same volume of water, and were stored at -20°C until HPLC analysis.

Chromatographic separations were performed on a Cl8 reversed phase analytical column (Higgins Analytical, 150x4.6 mm, 5 mM pore size) using a 30- 5G%B linear gradient over 30 minutes, Solvent A consisted of 0.1% (v/v) TFA in water, and solvent B contained 90% acetonitrile, 0.1% TFA in water, Peak areas corresponding to the intact peptide (as confirmed by MALDI-TOF) were obtained by manual integration using Agilent OpenLab CDS ChemStation.

PTH cAMP Accumulation Assay

Agonist activity at the human PTHR1 was assessed using HEK-293-derived cell lines that stably express the Glosensor cAMP reporter (Promega Corp.) along with wild-type human PTHR1 (GP-2.3 cells), The cells were cultured in 96-well plates and treated 24- 48 hours after reaching confluency. Cells were pre- incubated with CO2 independent media (Life Sciences) containing d-luciferin (0.5 mM) in 96-well plates at room temperature until a stable baseline level of luminescence was established (30 minutes) . Then, varying concentrations of test ligands were added, and the time course of luminescence response was recorded using a Perkin Elmer plate reader following peptide addition. The maximal luminescence response (observed about 10-30 minutes after ligand addition) was expressed as per-cent of the maximum response

observed for used for generating dose response curves. Reported EC₅₀ values are the average of 4 independent studies, each in duplicate, Data were fitted to a sigmoidal dose-response model with variable slope.

In Vivo Calcemic Response

Mice (GDI, female, age 11 weeks) were treated in accordance with the ethical guidelines adopted by Massachusetts General Hospital. Mice (n= 5 per compound) were injected subcutaneously with vehicle (10 mM citric acid/150 mM NaCl/0.05% Tween®- 80, pH 5.0) containing PTH(1-34) or modified analog each at a dose of 20 nmol/kg body weight. Tail vein blood was withdrawn just prior to injection (t = Q) and at times thereafter and immediately measured for Ca²⁺ concentration using a Siemens RapidLab 348 Ca²VpH analyzer.

Molecular Modeling

The GLP-1R-G1 and GLP-1R-G4 models were created based on Cryo-EM structure of GLP-lR-GLP-1 complex (PDB ID: 5VAI)²⁴. The N-terminal 1-28 residues of the GLP-1R are not resolved in the structural template, and likely highly flexible and were not modeled. The residue R380 side chain, unresolved in the structure was modeled and

additional minimization of the model at the tip of helix VII, where R380 is located was performed to restore the known key interaction between D15 of GLP-1 and R380 of GLP-1R²⁵.

The G1 and G4 peptides were prepared by adding O-G1cNAc modification, and two 2-amino- isobutyric acid (AIB) residues to the GLPl, followed by extensive energy-based conformational optimization of the GLP-1R-G1 and GLP-1R-G4 complexes in ICM3.8- Pro molecular modeling software (Molsoft LLC, San

Diego) . Optimization runs were performed for all peptide side chains and receptor side chains located 5 A from the peptide, The GLP-1R-G4 model was compared to the Cryo-EM structure of GLP-1R complex with a bias agonist Exendin-P5 (PDB ID 6B3J)²⁶.

The PTH1R-P2 model was generated based on high resolution structure of PTHlR-ePTH complex (PDB:

6FJ3)²⁸. The P2 peptide was generated by mutating the stabilizing point mutations in PTH and mutating residue 18 into serine and adding O-G1cNAc

modification, respectively, The PTH1R-P2 complex was submitted to energy based conformational optimization including O-G1cNAc modification and side chains located 5 A from the peptide. Optimization was performed in ICM3.8-Pro molecular modeling software

(Molsoft LLC, San Diego) .

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. Each of the patents, patent applications and articles cited herein is

incorporated by reference.

The foregoing description and the examples are intended as illustrative and are not to be taken as limiting. Still other variations within the spirit and scope of this invention are possible and will readily present themselves to those skilled in the art.

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Claims

CLAIMS:

1. A synthetic peptide hormone comprising a sequence of about 15 to about 50 amino acid residues that are at least about 90 percent identical to the same length and aligned sequence of a

naturally-occurring peptide hormone; each of the synthetic and naturally-occurring peptide sequences exhibiting an amphipathic alpha-helical secondary structure; said synthetic peptide hormone including a monoglycosylated serine or threonine residue in its sequence as a non-identical residue of the synthetic peptide hormone sequence that is present in the sequence of polar face of the helical structure; said synthetic peptide hormone containing said

monoglycosylated serine or threonine residue has a half-life in human serum at 37°C of about 25 to about 800 percent longer than the naturally-occurring peptide hormone.

2. The synthetic peptide hormone according to claim 1, wherein each of said synthetic and naturally-occurring peptide hormone peptides is free of a covalent intrachain linkage.

3. The synthetic peptide hormone according to claim 1, wherein each of said synthetic and naturally-occurring peptide hormone is an agonists of a G protein-coupled receptor (GPCR) .

4. The synthetic peptide hormone according to claim 1 that comprises a sequence length of about 20 to about 45 residues.

5. The synthetic peptide hormone according to claim 1, wherein said alpha-helical secondary structure is determined by circular dichroism (CD) spectroscopy.

6. The synthetic peptide hormone according to claim 1, wherein said naturally-occurring peptide hormone has the sequence of the glucagon-like peptide-1 GLP-1 (7-37) (SEQ ID NO: 6) or GLP-1 (7- 3) amide .

7. The synthetic peptide hormone according to claim 6, wherein said GLP-1 has the amino acid residue sequence of GLP-1 (7-37) or GLP-1 (7-36) amide whose amino acid residue sequence, from left to right and from amino-terminus to carboxy-terminus,

corresponds to Formula 1 (SEQ ID NO: 1) :

R¹ and R² are the same or different substituents that are hydrogen (H) or C₁-C₃ hydrocarbyl, with the proviso that only one of R¹ and R² is hydrogen, or R¹ and R² together with the depicted carbon to which they are bonded form a 5-7-membered ring; R³ and R⁴ are the same or different substituents that are hydrogen (H) or C₁-C₃ hydrocarbyl, or R³ and

R⁴ together with the depicted carbon to which they are bonded form a 5-7-membered ring;

R⁵, R⁶, R⁷ and R⁸ are the same or different and are H, C₁-C₁₈ hydrocarbyl, C₁-C₁₈ acyl, C₁-C₁₈ hydrocarbylsulfonyl, or either or both of R⁵ and R⁶ and R⁷ and R⁸ together with their depicted nitrogen atoms forms a 5-7-membered ring that optionally contains 1 or 2 additional hetero atoms that

independently are nitrogen, oxygen or sulfur, with the proviso that only one of R⁵ and R⁶ and and R⁸ includes a hydrocarbyl, acyl or sulfonyl group that contains more than six carbon atoms; and

Sac is a glycosidically-linked C₅-C₆ saccharide, deoxysaccharide or 2- (N-acetylamino) C₅-C₆ saccharide or deoxysaccharide.

8. The synthetic peptide hormone according to claim 7, wherein the saccharide (Sac) of X₃ is

O-G1cNAc, X₄ is a glycyl residue (G) , and n and m are both zero (SEQ ID NO: 2) .

9. The synthetic peptide hormone according to claim 8, wherein R¹, R², R³ and R⁴ are each methyl (SEQ ID NO: 5) .

10. The synthetic peptide hormone according to claim 8, wherein the R¹and R² of C₁ are both methyl, and X₂ is a valyl residue (V) (SEQ ID NO: 4) .

11. The synthetic peptide hormone

according to claim 8, wherein X₁ is an alanyl residue

(A) and R³ and R⁴ are both methyl (SEQ ID NO: 3) .

12. The synthetic peptide hormone

according to claim 1, wherein said naturally- occurring peptide hormone has the sequence of the parathyroid hormone (PTH) peptide whose sequence is shown below, as Formula 2 and SEQ ID NO: 7, from left to right and in the direction from amino-terminus to carboxy-terminus :

(SEQ ID NO: 7)

wherein

-X₅ = F (Phe) or F-NR³R⁴, with the proviso that only one of -X^-, -X₂-, -X3- and -X4- is other than the stated amino acid residue; i.e., His, Met, Glu and Lys;

n = 0 or 1 and p = 1 or 2, with the proviso that when n = 1, p = 1, and when n = 0, p = 2;

R¹, R², R³ and R⁴ are the same or different and are H, C₁-C₁₈ hydrocarbyl, C₁-C₁₈ acyl

(hydrocaroby 1) , C₁-C₁₈ hydrocarbylsulfonyl, or either or both of R¹ and R² and R³ and R⁴ together with their depicted nitrogen atoms forms a 5-7-membered ring that optionally contains 1 or 2 additional hetero atoms that independently are nitrogen, oxygen or sulfur, with the proviso that only one of R¹, R², R³ and R⁴ includes a hydrpcarbyl, acyl or sulfonyl group that contains more than six carbon atoms; and Sac is a glycosidically-linked C₅-C₆ saccharide, deoxysaccharide or 2- (N-acetylamino) C₅-C₆

saccharide .

13. The synthetic peptide hormone according to claim 6, wherein said glycosidically- linked C₅-C₆ saccharide, deoxysaccharide or 2- (N-acetylamino) C₅-C₆ saccharide is selected from the group consisting of G1cNAc, GalNAc, ribose,

deoxyribose, mannose, glucose and galactose.

14. The synthetic peptide hormone according to claim 13, wherein said glycosidically- linked 2- (N-acetylamino) C₅-C₆ saccharide is G1cNAc.

15. The synthetic peptide hormone according to claim 12 that is one Or more of:

(SEQ ID NO: 8);

(SEQ ID NO: 9) ;

(SEQ ID NO: 10) ; and

(SEQ ID NO: 11) .

16. A conqposition containing an agonistic effective amount of a synthetic peptide hormone according to claim 1 dissolved or dispersed in a pharmaceutically acceptable carrier.

17. The pharmaceutical composition according to claim 16, wherein said synthetic peptide hormone is a GLP-1 derivative peptide of Formula 1 (SEQ ID NO: 1) that is present in an IR- and/or GLP- 1R- agonist effective amount dissolved or dispersed in a pharmaceutically acceptable carrier.

18. The pharmaceutical composition according claim 17, wherein said GLP-1 derivative peptide of Formula 1 is a peptide of SEQ ID NO: 2.

19. The pharmaceutical composition according claim 17, wherein said GLP-1 derivative peptide of Formula 1 is a peptide of SEQ ID NO: 5.

20. The pharmaceutical composition according to claim 16, wherein said synthetic peptide hormone is a PTH derivative peptide of Formula 2 (SEQ ID NO: 8) dissolved or dispersed in a

pharmaceutically acceptable carrier.

21. The pharmaceutical composition according claim 20, wherein said PTH derivative peptide of Formula 2 is a peptide of SEQ ID NO: 10.

22. A method of treating a mammalian G protein-coupled receptor-mediated [GPCR-mediated] condition of a mammal that comprises administering an agonistic effective amount of a synthetic peptide hormone according to claim 1 dissolved or dispersed in a pharmaceutically acceptable carrier to that mammal having that GPCR-mediated condition.

23. The method of treating according to claim 22, wherein said administration is repeated as needed to maintain said mammal.

24. The method of treating according to claim 22, wherein said GPCR-mediated condition is type-2 diabetes that comprises administering said pharmaceutical composition contains an IR- or GLP- 1R- agonist effective amount of a peptide of Formula 1 (SEQ ID NO: 1) .

25. The method of treating according to claim 24, wherein said administration is repeated as needed to maintain said mammal.

26. The method of treating according to claim 22, wherein said synthetic peptide hormone is a peptide of SEQ ID NO: 8.

27. The method of treating according to claim 26, wherein said administration is repeated as needed to maintain said mammal.