Potent and stable polypeptide analogues via serine / threonine ligation.

Abstract

N 6 Disclosed are modified polypeptides comprising an amino acid sequence having a lysine or derivative thereof functionalized with a seryl or threonyl group, bioconjugates comprising the modified polypeptides, and methods for producing the bioconjugates.

Description

[Technical field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application No. 63 / 299,884, filed January 14, 2022, which is incorporated by reference in its entirety.

[0002] Array Listing Statement A computer readable form of the sequence listing is being filed together with this application by electronic filing and is hereby incorporated by reference in its entirety. The sequence listing is contained in a file having the file name "21-1624-WO.xml", created on December 23, 2022, and is 7kb in size. [Background technology]

[0003] Peptide therapeutics are rapidly being approved for clinical use due to their ability to bind to their targets with high affinity and specificity. Although many peptide drugs, such as teriparatide and angiotensin II, have been approved by the US Food and Drug Administration (FDA), they often suffer from poor pharmacokinetic profiles in vivo, which may result from proteolysis by endogenous enzymes. For example, GLP-1(7-37) has a half-life of only about 2 minutes due to degradation by dipeptidyl peptidase (DPP-4) cleavage at the N-terminal alanine 8. Current strategies aimed at addressing stability issues are inadequate, as they often compromise efficacy at the expense of stability. Summary of the Invention

[0004] In one aspect, the present disclosure provides a method for the preparation of a medicament for use in a pharmaceutical composition comprising: 6 The present invention provides a modified polypeptide comprising an amino acid sequence having a lysine or derivative thereof functionalized with a seryl or threonyl group at N 6 -L-seryl-functionalized lysine (e.g., [ka] N 6 -L-seryl-functionalized lysine derivatives (e.g. [ka] N 6 -L-threonyl-functionalized lysine (e.g., [ka] or N 6 -L-threonyl-functionalized lysine derivatives (e.g. [ka] Includes.

[0005] In certain embodiments, the polypeptide comprises an amino acid sequence that is at least 90%, or 92%, or 94%, or 95% or more identical to glucagon-like peptide 1 fragment 7-37 (GLP-1(7-37)), parathyroid hormone fragment 1-34 (PTH(1-34)), or peptide YY3-36 (PYY(3-36)). 6 In various embodiments, the peptide therapeutic is selected from the group consisting of the therapeutic peptides listed in Table 1, which have been modified to include a lysine or derivative thereof functionalized with a seryl or threonyl group at N 6 A functionalized lysine or derivative thereof is incorporated into an amino acid sequence in place of natural lysine or in place of a natural amino acid other than lysine, such as, but not limited to, serine.

[0006] In another embodiment, the present disclosure provides a bioconjugate comprising a modified polypeptide of any embodiment or combination of embodiments herein and a functional moiety, wherein the functional moiety is N 6-conjugated to the modified polypeptide at a seryl or threonyl group on a functionalized lysine or derivative thereof. In various embodiments, the functional moiety can include a polyethylene glycol molecule, a lipid molecule, a fluorescent molecule, a chemiluminescent molecule, a phosphorescent molecule, a radioisotope, an enzyme, an enzyme substrate, an affinity molecule, a ligand, an antigen, a hapten, an antibody, an antibody fragment, a peptide, a peptidomimetic, a protein, a chromogenic substrate, a contrast agent, an MRI contrast agent, a PET label, a phosphorescent label, or a combination thereof. In certain embodiments, the functional moiety includes: [ka] (for example, [ka] (for example, [ka] (wherein R=NH2, OH, or OCH3) (e.g., [ka]

[0007] The present disclosure also provides pharmaceutical compositions comprising one or more polypeptides and / or bioconjugates according to any embodiment or combination of embodiments herein and a pharma- ceutically acceptable carrier, solvent, adjuvant, and / or diluent.

[0008] In another aspect, the present disclosure provides a method for preparing a bioconjugate, the method comprising: 6 the method comprises contacting a modified polypeptide comprising an amino acid sequence having a lysine or derivative thereof functionalized with a seryl or threonyl group at N with a functionalized salicylaldehyde ester, the salicylaldehyde ester being 6 - reacts with a seryl or threonyl group on a functionalized lysine or derivative thereof to give a bioconjugate. In one embodiment, the functionalized salicylaldehyde ester has the formula: [ka] wherein R is a moiety that comprises a polyethylene glycol molecule, a lipid molecule, a fluorescent molecule, a chemiluminescent molecule, a phosphorescent molecule, a radioisotope, an enzyme, an enzyme substrate, an affinity molecule, a ligand, an antigen, a hapten, an antibody, an antibody fragment, a peptide, a peptidomimetic, a protein, a chromogenic substrate, a contrast agent, an MRI contrast agent, a PET label, a phosphorescent label, or a combination thereof; or a fluorescent molecule, biotin, a polyethylene glycol molecule, a lipid molecule, or a combination thereof.

[0009] In another embodiment, the functionalized salicylaldehyde ester has the formula: [ka] (for example, [ka] (for example, [ka] (wherein R=NH2, OH, or OCH3) (e.g., [ka]

[0010] In various embodiments, the method further comprises the step of: 6 -seryl-lysine or its derivatives or N 6 In another embodiment, the modified polypeptide comprises an N-terminal serine or threonine, and the method further comprises protecting the N-terminal serine or threonine with a protecting group prior to contacting with the functionalized salicylaldehyde ester. [Brief description of the drawings]

[0011] [Figure 1]Chemical ligation at serine is shown. (Top) Previous efforts have primarily used serine / threonine ligation (STL) in protein chemical synthesis, semi-synthesis, or peptide cyclization. (Bottom) Cartoon of a method to generate site-specific modifications in-house using non-standard amino acids that contain the 1-amino-2-hydroxy functionality required for ligation. [Diagram 2] Design of GLP-1 peptide analogues. Primary sequences of GLP-1 (SEQ ID NO:1) and semaglutide (SEQ ID NO:4). Peptides G1 (SEQ ID NO:5) and G2 (SEQ ID NO:6), synthesized here via STL, contain a C18-PEG4 modification at position 26 together with an Aib residue at position 8 or only a C18-PEG4 modification at position 18. [Diagram 3] Lipidation does not affect cellular activity, stabilizes GLP-1 from proteolysis, and improves glucose clearance in vivo. (a) Lipid alone or together with Aib substitution does not affect the EC50 for cAMP production compared to unmodified GLP-1 (n=3). (a) Models of full-length (b) GLP-1R-semaglutide, (c) GLP-1R-G1, and (d) GLP-1R-G2 complexes. [Figure 4] Scheme 1. Site-specific modification of unprotected model peptides with STL. All reactions were carried out at a concentration of 10 mM in pyridine / acetic acid (1:1 v / v) followed by cleavage using TFA / H2O / i-Pr3SiH (94 / 5 / 1, v / v / v). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] All cited references are incorporated herein by reference in their entirety.

[0013] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. "And" is used herein synonymously with "or" unless expressly stated otherwise.

[0014] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), valine (Val; V), and alpha aminoisobutyric acid (AIB, B).

[0015] All embodiments of any aspect of the invention can be used in combination unless the context clearly indicates otherwise.

[0016] In a first aspect, the present disclosure provides a method for producing a cellular membrane comprising: 6 The present invention provides modified polypeptides comprising an amino acid sequence having a lysine or derivative thereof functionalized with a seryl or threonyl group at 25° C. As shown in the Examples below, the inventors have demonstrated that such modified polypeptides can be readily modified internally and site-specifically for a variety of applications in chemical biology, including the generation of potent and stable variants exemplified, but not limited to, by studies with semaglutide.

[0017] The polypeptide may include a functionalized lysine or a functionalized lysine derivative. 6 Any lysine derivative that can be functionalized with a seryl or threonyl group at may be used. In various embodiments, derivatives of lysine include, but are not limited to, the following: [ka]

[0018] For example, in certain embodiments, the modified polypeptide comprises: 6 -L-seryl-functionalized lysine (e.g., [ka] Or, N 6 -L-seryl-functionalized lysine derivatives (e.g. [ka] In certain embodiments, the modified polypeptide comprises: 6 -L-threonyl-functionalized lysine (e.g., [ka] or N 6 -L-threonyl-functionalized lysine derivatives (e.g. [ka] In some embodiments, N 6 The -functionalized lysine or derivative thereof includes a protecting group. For example, N 6 The -functionalized lysine may further include a Boc-protecting group or a t-butyl protecting group, or a combination thereof.

[0019] Preferred N 6 -functionalized lysine can be prepared as known in the art. For example, the preparation of certain functionalized lysine peptides, such as serine-lysine conjugates, can be found in CHP Cheung, J. Xu, CL Lee, Y. Zhang, R. Wei, D. Bierer, X. Hunag, and X. Li, Chem. Sci., 2021, 12, 7091, which is incorporated herein by reference in its entirety.

[0020] As will be appreciated by those skilled in the art, N 6 -Functionalized lysine or its derivative is incorporated into the amino acid sequence in place of natural lysine or in place of another natural amino acid. 6The N-functionalized lysine or derivative thereof can be incorporated in place of any amino acid in the unmodified polypeptide. 6 In another embodiment, the N-functionalized lysine or derivative thereof is incorporated in place of a lysine residue in the unmodified polypeptide. 6 -functionalized lysine or a derivative thereof is incorporated in place of a serine residue in the unmodified polypeptide. In one embodiment, the modified polypeptide comprises two or more N 6 -functionalized lysine or a derivative thereof.

[0021] The polypeptide may be any polypeptide that can benefit from functionalization to improve stability or other polypeptide properties. In various non-limiting embodiments, the polypeptide is 6 The therapeutic peptides may include modified versions of peptide therapeutics selected from the group consisting of the therapeutic peptides listed in Table 1, modified to include a lysine or derivative thereof functionalized with a seryl or threonyl group at 100 to 1500 . In some embodiments, the modified polypeptide is at least 90%, or 92%, or 94%, or 95%, or 96%, or 97%, or 98% or more identical to the amino acid sequence of the unmodified polypeptide. [Table 1] TIFF2025502171000020.tif133159

[0022] In one non-limiting embodiment, the modified polypeptide comprises an amino acid sequence that is at least 90%, or 92%, or 94%, or 95% or more identical to glucagon-like peptide 1 fragment 7-37 (GLP-1(7-37)) (SEQ ID NO:1). The amino acid sequence of GLP-1(7-37) is shown in FIG.

[0023] In another embodiment, the polypeptide comprises an amino acid sequence that is at least 90%, or 92%, or 94%, or 95% or more identical to parathyroid hormone fragment 1-34 (PTH(1-34)) (SEQ ID NO:2). SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF(SEQ ID NO:2)

[0024] In further embodiments, the polypeptide comprises an amino acid sequence that is at least 85%, or 90%, or 92%, or 95% or more identical to peptide YY3-36 (PYY(3-36)) (SEQ ID NO:3). IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY(SEQ ID NO:3)

[0025] In another aspect, the present disclosure provides a modified polypeptide of the present disclosure described herein, and 6 -Provides a bioconjugate comprising a functional moiety conjugated to a modified polypeptide at a seryl or threonyl group on a functionalized lysine or derivative thereof. The modified polypeptides of the present disclosure can accept any functional moiety deemed appropriate for the intended use. In a non-limiting embodiment, the functional moiety can comprise a polyethylene glycol molecule, a lipid molecule, a fluorescent molecule, a chemiluminescent molecule, a phosphorescent molecule, a radioisotope, an enzyme, an enzyme substrate, an affinity molecule, a ligand, an antigen, a hapten, an antibody, an antibody fragment, a peptide, a peptidomimetic, a protein, a chromogenic substrate, a contrast agent, an MRI contrast agent, a PET label, a phosphorescent label, or a combination thereof. In one particular embodiment, the functional moiety can comprise a fluorescent molecule, biotin, a polyethylene glycol molecule, a lipid molecule, or a combination thereof. In other embodiments, the functional moiety can comprise a moiety selected from the group consisting of: [ka] (for example, [ka] (for example, [ka] (wherein R=NH2, OH, or OCH3) (e.g., [ka]

[0026] In various of these embodiments, the functional moiety increases the in vivo stability of the bioconjugate relative to the in vivo stability of the native (unmodified) polypeptide. As shown in the Examples below, and non-limitingly exemplified by studies with semaglutide, the bioconjugates of the disclosure are potent and stable analogs of unmodified polypeptides, and have improved stability relative to the unmodified polypeptide.

[0027] The present disclosure also provides pharmaceutical compositions comprising one or more polypeptides and / or bioconjugates according to the present disclosure described herein, wherein the modified polypeptide comprises a therapeutic protein or peptide, and a pharma- ceutically acceptable carrier, solvent, adjuvant, and / or diluent.The pharmaceutical compositions of the present disclosure can be used, for example, in a method for treating a subject in need of treatment for a disorder that the polypeptide is designed to treat.The pharmaceutical compositions can comprise, in addition to the polypeptide or bioconjugate of the present disclosure, (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative, and / or (g) a buffer.

[0028] In some embodiments, the buffer in the pharmaceutical composition is Tris buffer, histidine buffer, phosphate buffer, citrate buffer or acetate buffer.The pharmaceutical composition may also contain a lyoprotectant, such as sucrose, sorbitol or trehalose.In certain embodiments, the pharmaceutical composition contains a preservative, such as benzalkonium chloride, benzethonium, chlorhexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof.In other embodiments, the pharmaceutical composition contains a bulking agent, such as glycine. In yet other embodiments, the pharmaceutical composition comprises a surfactant, such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleate, or a combination thereof. The pharmaceutical composition may also comprise a tonicity adjuster, such as a compound that makes the formulation substantially isotonic or isosmotic with human blood. Exemplary tonicity adjusters include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the pharmaceutical composition further comprises a stabilizer, e.g., a molecule that, when combined with the protein of interest, substantially prevents or reduces chemical and / or physical instability of the protein of interest in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0029] The polypeptides and / or bioconjugates may be the only active agents in a pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for the intended use.

[0030] In another aspect, the present disclosure provides methods for preparing the bioconjugates of the present disclosure described herein. Such methods include 6 the method comprises contacting a modified polypeptide comprising an amino acid sequence having a lysine or derivative thereof functionalized with a seryl or threonyl group at N with a functionalized salicylaldehyde ester, the salicylaldehyde ester being 6 -functionalized lysine or its derivative to yield a bioconjugate. In embodiments where the modified polypeptide contains a protecting group, the method of preparing the bioconjugate may further include treating the modified polypeptide to remove the protecting group.

[0031] Any suitable functionalized salicylaldehyde ester can be used in the method. In one embodiment, the functionalized salicylaldehyde ester has the formula: [ka] wherein R is a moiety comprising a polyethylene glycol molecule, a lipid molecule, a fluorescent molecule, a chemiluminescent molecule, a phosphorescent molecule, a radioisotope, an enzyme, an enzyme substrate, an affinity molecule, a ligand, an antigen, a hapten, an antibody, an antibody fragment, a peptide, a peptidomimetic, a protein, a chromogenic substrate, an imaging agent, an MRI contrast agent, a PET label, a phosphorescent label, or a combination thereof.

[0032] In one embodiment, R is a fluorescent molecule, biotin, a polyethylene glycol molecule, a lipid molecule, or a combination thereof. In another embodiment, the functionalized salicylaldehyde ester has a formula selected from the group consisting of: [ka] (for example, [ka] (for example, [ka] (wherein R=NH2, OH, or OCH3) (e.g., [ka]

[0033] Many peptide-based therapeutics are unstable due to protease-catalyzed degradation. In further embodiments, the R moiety increases the in vivo stability of the bioconjugate compared to the in vivo stability of the modified polypeptide. The modified polypeptide may be according to any embodiment or combination of embodiments described herein. In vivo stability can be determined by incubating the peptide in human serum and monitoring the peptide by chromatography, for example, reverse-phase HPLC. In certain embodiments, the bioconjugate has a half-life in human serum of at least 12 hours, for example, at least 16 hours, or at least 24 hours, or at least 30 hours.

[0034] The modified polypeptide can be contacted with the functionalized salicylaldehyde ester in any suitable solution.In one embodiment, the modified polypeptide is contacted with the functionalized salicylaldehyde ester in a solution containing a nitrogen base and an organic acid, for example, a pyridine / acetic acid solution.The pyridine / acetic acid can be in any suitable ratio in the solution, for example, in the range of 0.2:1 to 5:1 v / v pyridine:acetic acid, for example, but not limited to, a 1:1 v / v ratio.

[0035] The modified polypeptide may be contacted with the functionalized salicylaldehyde ester at any suitable temperature range. In one embodiment, the modified polypeptide is contacted with the functionalized salicylaldehyde ester at a temperature ranging from about 18° C. to 27° C. for a period of time sufficient to form an N,O-benzylidene acetal intermediate. In a further embodiment, the N,O-benzylidene acetal intermediate is reacted under acidic conditions for a period of time sufficient to form the bioconjugate. Any suitable acidic conditions may be used. In one embodiment, the acidic conditions include the use of trifluoroacetic acid.

[0036] In a further embodiment, the method comprises: 6 -seryl-lysine or its derivatives or N 6 It further comprises incorporating -threonyl-lysine or a derivative thereof into the synthesis of the modified polypeptide.

[0037] In another embodiment, when the modified polypeptide comprises an N-terminal serine or threonine, the method may further comprise protecting the N-terminal serine or threonine with a protecting group prior to contacting with the functionalized salicylaldehyde ester. Any suitable protecting group may be used, including, but not limited to, allylserine or N-terminal acetylation.

[0038] During any of the processes for preparation of the subject compounds, polypeptides, or bioconjugates, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules concerned. This is consistent with standard studies, such as JFW McOmie, “Protective Groups in Organic Chemistry,” Plenum Press, London and New York 1973, TW Greene and PGM Wuts, “Protective Groups in Organic Synthesis,” 3rd edition, Wiley, New York 1999, “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York. 1981, “Methoden der organischen Chemie,” Houben-Weyl, 4.sup.th edition, Vol. 15 / l, Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide, Proteine,” Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and / or Jochen Lehmann, “Chemie der This can be achieved by conventional protecting groups as described in Kohlenhydrate: Monosaccharide and Derivate,” Georg Thieme Verlag, Stuttgart 1974. The protecting groups can be removed at a convenient subsequent stage using methods known in the art.

[0039] Working Example Peptide and protein bioconjugation allows the site-specific introduction of various functional groups for different applications, e.g., proteomics and high-resolution imaging. One such method is Ser / Thr ligation (STL), a chemoselective reaction occurring between a C-terminal salicylaldehyde ester and an N-terminal fragment containing a serine or threonine residue that undergoes reversible imine formation via aldehyde capture. After the acyl shift, a stable N,O-benzylidene acetal intermediate is cleaved by acid, liberating the native serine / threonine bond at the ligation site. For example, the N,O-benzylidene acetal intermediate only forms in the presence of a 1-amino-2-hydroxy functional group, present for N-terminal serine or threonine. Here we use non-standard amino acids containing a 1-amino-2-hydroxy functional group to internally and site-specifically modify peptides for a variety of applications in chemical biology, e.g., the generation of potent and stable variants of GLP-1(7-37) (Figure 1).

[0040] To assess the scope and generality of this approach, we first synthesized biotin, cyanine-3, palmitic acid analogs, and monodisperse polyethylene glycol salicylaldehyde esters in one step from commercially available starting materials. All probes were then site-specifically incorporated onto a model peptide containing a 1-amino-2-hydroxy nonstandard amino acid (Scheme 1; Figure 4). After bioconjugation, monitored by high-performance liquid chromatography, all products were characterized by electrospray ionization mass spectrometry.

[0041] Next, we investigated how this bioconjugation strategy could be utilized to enhance the stability of peptide therapeutics. The most common strategies to extend the half-life of peptide and protein therapeutics are PEGylation and lipidation. Two GLP-1(7-37) drugs, semaglutide and liraglutide, are lipidated and are currently used to control blood glucose for the treatment of type 2 diabetes. Both PEGylation and lipidation provide protection from protease-catalyzed degradation. In addition, lipidation promotes binding to circulating human albumin, which results in the release of the drug at a slow, constant rate.

[0042] With this in mind, we used STL to synthesize two analogues of GLP-1 similar to semaglutide, containing hybrid PEG and fatty acid side chains (Figure 2). The first peptide (G1) was modified at lysine 26, the same position as semaglutide. As this site is further away from the DPP-4 cleavage site at alanine 8, we also included 2-aminoisobutyric acid (AIB) instead of alanine 8 (similar to semaglutide) to provide additional stability. The main difference between semaglutide and G1 is that, besides minor side chain modifications, G1 maintains the native lysine 34, so that conjugation is site-specific with STL. The second peptide (G2) was modified at serine 18. In this particular case, we choose to omit Aib at alanine 8, as the lipid is closer to the N-terminus and may provide better shielding against proteolysis. The characterization data for the two analogues are summarized in Table 2. [Table 2]

[0043] Many biochemical and structural studies have shown that an extended amphipathic α-helix in GLP-1 is involved in high affinity binding interactions with the extracellular domain of the GLP receptor. To assess how these modifications disrupt secondary structure, we used circular dichroism (CD) spectroscopy to observe changes to GLP-1. In contrast to GLP-1 (which exhibits a characteristic helical fold), both G1 and G2 also exhibit helical structure, however less than native GLP-1. This data is consistent with the lipid modifications found on semaglutide, and it is believed that this loss of structure is induced by lipid modifications.

[0044] Endogenous binding of GLP-1 to the GLP-1R results in intracellular rearrangements that allow recruitment of G-proteins, which then stimulate the production of cyclic AMP (cAMP) from ATP, leading to glucose-stimulated insulin secretion. 19 To assess the ability of lipid-modified GLP-1 analogues G1 and G2 to activate the human GLP-1R, cAMP accumulation was measured in CHO-K1 cells overexpressing the human GLP-1R. Cells were first treated with native GLP-1 and semaglutide as reference agonists, which had ECs of 3.3 ± 0.6 nM and 0.60 ± 0.2 nM (mean ± sem, n = 3), respectively. 50 In comparison, both G1 and G2 performed better than the unmodified peptide and were roughly equivalent to semaglutide, with EC values ​​of 0.97 ± 0.2 nM and 0.73 ± 0.2 nM, respectively. 50 (mean ± sem, n = 3). This data suggests that lipid modifications on either Lys26 or Ser18 do not significantly perturb endogenous function. To complement the in vitro pharmacological profiling of lipid-modified GLP-1 analogues G1 and G2, the stability of the compounds relative to native GLP-1 and semaglutide in human serum was compared using reversed-phase high-performance liquid chromatography (RP-HPLC) (data not shown). As expected, native GLP-1 showed a relatively short half-life in this assay, with t 1 / 2= 3.5 h, because the N-terminal Ala8 residue is readily cleaved. In contrast, semaglutide shows little sign of degradation up to 48 h, because its stability is significantly enhanced by Aib addition and lipid modification at Ala8. These half-lives are consistent with previous reports. Relative to native GLP-1, G1 has a significantly improved stability profile, t 1 / 2 = approximately 40 hours. Although G1 contains an Aib substituted with Ala8 to prevent cleavage by DPP4, the data suggest that other proteases present in human serum may degrade G1 at other sites. Finally, G2 proved to be highly stable, with a >14-fold increase in stability over native GLP-1 and very comparable to semaglutide.

[0045] Given the encouraging activation and stability data, the peptides were tested in vivo using a standard glucose tolerance test (GTT). Both G1 and G2 showed improved glucose disposal efficiency compared to unmodified GLP-1 (data not shown), consistent with their in vitro data. These data highlight the ability of lipidation to significantly increase stability without compromising potency, thus resulting in improved in vivo activity in mouse models of glucose disposal.

[0046] To gain molecular insight into how G1 and G2 interact with GLP-1R, we performed computational modeling of the corresponding ligand-receptor complexes as described in the experimental methods. The GLP-1R peptide binding model is based on the recently published Cryo-EM structure of GLP-1R complexed with unmodified GLP-1 peptide. The generated models of semaglutide, G1, and G2 in their complexes with GLP-1R suggest that lipidation at either serine 18 or lysine 26 is solvent exposed and likely would not interfere with critical contacts involved in binding or activation (Fig. 3b-d). In addition, the lipid modification of G1 is closer to the N-terminus of the peptide, suggesting that there is probably an exposed degradation site between the C-terminal Aib residue and the lipid modification.

[0047] In conclusion, we introduce a robust site-specific bioconjugation strategy that relies on "serine ligation". A large number of salicyaldehyde ester probes can be easily synthesized in one step from commercially available carboxylic acids, generating peptides with stable linkages for a variety of applications in chemical biology and medicine. Unlike the chemoselective chemistry currently used to introduce lipid functional groups on semaglutide, which involves mutation of any other native lysine residues in the native sequence, our site-specific strategy does not require this. N-terminal serine or threonine residues in the peptide may compete for modification; however, this can be circumvented by using simple protecting group strategies, e.g., allylic serine or N-terminal acetylation. We applied this technique to generate potent and stable GLP-1 analogs attached with hybrid PEG and fatty acid side chains similar to the widely used diabetes drug semaglutide. Both compounds were equipotent to semaglutide in their ability to activate the GLP-1R, showed significantly improved stability profiles in human serum versus native GLP-1, and outperformed GLP-1 in vivo.

[0048] In certain embodiments, the STL bioconjugation strategy can be used to generate potent and stable analogs of other GPCRs, such as PTH(1-34). In addition, in certain embodiments, STL bioconjugation is combined with amber stop codon technology to expand the generation of modified peptides of the present disclosure by eliminating solid-phase peptide synthesis. This approach demonstrates the feasibility of generating peptides for a variety of applications, with an emphasis on therapeutic peptides.

[0049] Abbreviation GPCR, G protein-coupled receptor; DPP-4, dipeptidyl peptidase; STL, Ser / Thr ligation; Aib, 2-aminoisobutyric acid; GTT, glucose tolerance test.

[0050] Materials: Biotin was purchased from Sigma Aldrich. Cyanine3 acid was purchased from lumiprobe. C18-PEG4-COOH and t-boc-N-amide-sPEG8-acid were purchased from creative PEGworks and Quanta Biodesign, respectively. Semaglutide acetate was purchased from Bachem. The synthesis of Lys-Ser dipeptide was carried out as previously reported by WuXi AppTec (CHP Cheung, J. Xu, CL Lee, Y. Zhang, R. Wei, D. Bierer, X. Hunag, and X. Li, Chem. Sci., 2021, 12, 7091). All other reagents were obtained from commercial sources and used without further purification. All aqueous solutions were prepared using ultra-high purity laboratory grade water (deionized, filtered, and sterile) obtained from an in-house ELGA water purification system. Reverse-phase high performance liquid chromatography (RP-HPLC) was performed using an Agilent Technologies 1260 series HPLC instrument equipped with a diode array detector. For analytical analysis, a C18 reverse-phase HPLC column was used (Higgins). Samples were eluted with a 5-95% acetonitrile / water gradient (0.1% TFA) in 45 min at a flow rate of 1 mL / min and monitored at 214 nm. For purification, a semi-preparative C18 reverse-phase HPLC column was used (Higgins). Samples were eluted with a 5-65% or 25-95% acetonitrile / water gradient (0.1% TFA) in 35 min at a flow rate of 5.0 mL / min and monitored at 220 nm. Mass spectra were acquired on an Agilent LC-TOF or Thermo ESI direct inject mass spec.

[0051] Peptide synthesis: All peptides were synthesized using standard Fmoc solid phase chemistry on either 2-Chlorotrityl ProTide (CEM, 0.45 mmol / g) or Rink amide ChemMatrix (PCAS BioMatrix, 0.45 mmol / g) resins using a Liberty Blue peptide synthesizer from CEM. Coupling was performed using DIC (5 eq., Novabiochem) and Oxyma (10 eq., Sigma) in DMF, followed by Fmoc deprotection with 20% piperidine. All peptides were then cleaved with (95:2.5:2.5 TFA / H2O / triisopropylsilane) for 3.5 h at room temperature, precipitated from cold ether, and purified by reversed-phase HPLC and using preparative chromatography. All peptides were characterized by mass spectrometry using ESI-MS and sequence purity was assessed by analytical HPLC.

[0052] Circular dichroism spectroscopy: Circular dichroism spectra were recorded on a Jasco J-1500 CD spectrometer. Peptides were freshly diluted to a final concentration of 50 μM in 10 mM phosphate buffer (pH 7.4) before sample measurement. Spectra were recorded at 25° C. from 250 to 190 nm with 0.1 nm data pitch, 50 nm min-1 scan speed, 4 s data integration time, 1 nm bandwidth, and 1 mm path length, using 3 accumulations. All data were background subtracted from samples containing only phosphate buffer.

[0053] GLP-1 cAMP accumulation assay: GLP-1 receptor activation was measured in CHO-K1 cells overexpressing human GLP1R using the cAMP Hunter eXpress assay kit (Eurofins DiscoverX Corporation, #95-0062E2CP2M). All reagents were from the assay kit unless otherwise stated. All were performed according to the manufacturer's instructions. All data was analyzed using Prism 8.3.0 (GraphPad Software Inc., San Diego, CA).

[0054] Salicylaldehyde ester synthesis and ligation reaction conditions: Synthesis of salicylaldehyde esters: Biotin, Cyanine3, C18-PEG4-COOH, and t-boc-N-amido-sPEG8-acid were added to salicylaldehyde (1.1 eq.), DIC (1.2 eq), and DMAP (0.1 eq) in dry DCM (2-4 mL). The reaction(s) were allowed to proceed overnight to afford the salicylaldehyde esters, which were purified by semi-preparative HPLC.

[0055] General procedure for ligation to model peptides: Salicylaldehyde ester (1 equiv.) and peptide fragment (1.0 equiv.) were dissolved in pyridine / acetic acid (1:1 v / v) to a final concentration of approximately 0.025-0.05 M. The reaction(s) were stirred at room temperature and monitored using LC / MS. After completion of the reaction, the solvent was removed by lyophilization and the intermediate product was treated with TFA / H2O / i-Pr3SiH (94 / 5 / 1, v / v / v) for 10 min (2 h for boc-bearing PEG) to yield the product containing a native amide bond at the ligation site.

[0056] General procedure for ligation to GLP-1(7-37): GLP-1(7-37) (10 mg) was dissolved in pyridine / acetic acid (1:1 v / v) to a final concentration of approximately 10 mM, and the corresponding salicylaldehyde ester (1 equiv.) was added. The reaction was stirred at room temperature and monitored using HPLC. After completion of the reaction, the solvent was removed by lyophilization and the intermediate was treated with TFA / H2O / i-Pr3SiH (94 / 5 / 1, v / v / v) for 15 min and for 2 h for Boc-protected PEG to give the product containing a native amide bond at the ligation site.

[0057] References (1)Muttenthaller, M.; King, GF; Adams, DJ; Alewood, PF Trends in Peptide Discovery. Nat. Rev. Drug Discov. 2021, 20, 309-325. (2)Trujillo, J. M.; Nuffer, W.; Ellis, S. L. GLP-1 Receptor Agonists: a Review of Head-to-Head Clinical Studies. Ther. Adv. Endocrinol. Metab. 2015, 6 (1), 19-28。 (3)Al-Merani, A. S.; Brooks, P. D.; Chapman, J. B.; Munday, A. K. The Half-Lives of Angiotensin II, Angiotensin II-Amide, Angiotensin III, Sar1-Ala8- Angiotensin II and Renin in the Circulatory System of the Rat. J. Physiol. 1978, 278, 471-490。 (4)Lovshin, J. A.; Drucker, D. J. Incretin-Based Therapies for Type 2 Diabetes Mellitus. Nat. Rev. Endocrinol. 2009, 5 (5), 262-269。 (5)Green, B. D.; Mooney, M. H.; Gault, V. A.; Irwin, N.; Bailey, C. J.; Harriott, P.; Greer, B.; O’Harte, F. P. M.; Flatt, P. R. N-Terminal His(7)-Modification of Glucagon-Like Peptide-1(7-36) Amide Generates Dipeptidyl Peptidase IV-Stable Analogues with Potent Antihyperglycaemic Activity. J. Endocrinol. 2004, 180 (3), 379-388。 (6)Chen, X.; Mietlicki-Baase, E. G.; Barrett, T. M.; McGrath, L. E.; Koch-Laskowski, K.; Ferrie, J. J.; Hayes, M. R.; Petersson, E. J. Thioamide Substitution Selectively Modulates Proteolysis and Receptor Activity of Therapeutic Peptide Hormones. J. Am. Chem. Soc. 2017, 139 (46), 16688-16695。 (7)Lin, S., Yang, X., Jia, S., Weeks, A. M., Hornsby, M., Lee, P. S., Nichiporuk, R. V., Iavarone, A. T., Wells, J. A., Toste, F. D., and Chang, C. J. Redox-Based Reagents for Chemoselective Methionine Bioconjugation. Science. 2017, 355, 597-602。 (8)Gupta, A.; Rivera-Molina, F.; Xi, Z.; Toomre, D.; Schepartz, A. Endosome Motility Defects Revealed at Super-Resolution in Live Cells Using HIDE Probes. Nat. Chem. Biol. 2020, 16, 408-414。 (9)Zhang, Y.; Xu, C.; Lam, H. Y.; Lee, C. L.; Li, X. Protein Chemical Synthesis by Serine and Threonine Ligation. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (37), 6657-6662。 (10)Levine, P. M.; Craven, T. W.; Bonneau, R.; Kirshenbaum, K. emi-Synthesis of Peptoid-Protein Hybrids by Chemical Ligation at Serine. Org. Lett. 2014, 16 (2), 512-515。 (11)Knudsen, L. B., Nielsen, P. F.; Huusfeldt, P. O.; Johansen, N. L.; Madsen, K.; Pedersen, F. Z.; Thoegersen, H.; Wilken, M.; Agersoe, H. Potent Derivatives of Glucagon-Like Peptide-1 with Pharmacokinetic Properties Suitable for Once Daily Administration. J. Med. Chem. 2000, 43 (9), 1664-1669。 (12)Jensen, L.; Helleberg, H.; Roffel, A.; Van Lier J. J.; Bjoernsdottir, I.; Pedersen, P. J.; Rowe, E.; Derving Karsbol, J.; Pedersen, M. L.; Absorption, Metabolism and Excretion of the GLP-1 Analogue Semaglutide in Humans and Nonclinical Species. Eur. J. Pharm. Sci. 2017, 104, 31-41。 (13)Menacho-Melgar, R.; Decker, J. S.; Hennigan, J. N.; Lynch, M. D.; A Review of Lipidation in the Development of Advanced Protein and Peptide Therapeutics. J. Control. Release. 2019, 10 (295), 1-12。 (14)Spector, A. A. Fatty Acid Binding to Plasma Albumin. J. Lipi. Res. 1975, 16 (3), 165-179。 (15)Zhang, Y.; Sun, B.; Feng, D.; Hu, H.; Chu, M.; Qu, Q.; Tarrasch, J. T.; Li, S.; Sun Kobilka, T.; Kobilka, B. K.; Skiniotis, G. Cryo-EM Structure of the Activated GLP-1 Receptor in Complex with a G Protein. Nature. 2017, 546 (7657), 248-253。 (16)Levine, P. M.; Balana, A. T.; Sturchler, E.; Koole, C.; Noda, H.; Zarzycka, B.; Daley, E. J.; Truong, T. T.; Katritch, V.; Gardella, T. J.; Wootten, D.; Sexton, P. M.; McDonald, P.; Pratt, M. R. O-GlcNAc Engineering of GPCR Peptide-Agonists Improves Their Stability and In Vivo Activity. J. Am. Chem. Soc. 2019, 141, 14210-14219。 (17)Adelhorst, K.; Hedegaard, B. B.; Knudsen, L. B., Kirk, O. Structure Activity Studies of Glucagon-like Peptide-1. J. Biol. Chem. 1994, 269 (9), 6275-6278。 (18)Venanzi, M.; Savioli, M.; Cimino, R.; Gatto, E.; Palleschi, A.; Ripani, G.; Cicero, D.; Placidi, E.; Orvieto, F.; Bianchi, E. A Spectroscopic and Molecular Dynamics Study on the Aggregation Process of a Long-Lasting Lipidated Therapeutic Peptide: The Case of Semaglutide. Soft Matter. 2020, 16, 10122-10131。 (19)Zhang, R.; Xie, X. Tools for GPCR Drug Discovery. Acta Pharmacol. Sin. 2012, 33 (3), 372-384。 (20)Fraher, L. J.; Klein, K.; Marier, R.; Freeman, D.; Hendy, G. N.; Goltzman, D.; Hodsman, A. B. Comparison of the Pharmacokinetics of Parenteral Parathyroid Hormone-(1-34) [PTH-(1-34)] and PTH-Related Peptide-(1-34) in Healthy Young Humans. J. Clin. Endocrinol. Metab. 1995, 80 (1), 60-64。

Claims

1. N ６ A modified polypeptide comprising an amino acid sequence having a lysine or a derivative thereof functionalized with a seryl or threonyl group at 25.degree.

2. The N ６ -L-seryl-functionalized lysine (e.g. 【Chemistry 1】 Or, the N ６ -L-seryl-functionalized lysine derivatives (e.g. 【Chemistry 2】 The modified polypeptide of claim 1 , comprising:

3. The N ６ -L-threonyl-functionalized lysine (e.g. 【Chemistry 3】 Or, the N ６ -L-threonyl-functionalized lysine derivatives (e.g. 【Chemistry 4】 The modified polypeptide of claim 1 , comprising:

4. 4. The modified polypeptide of any one of claims 1 to 3, wherein the polypeptide comprises an amino acid sequence that is at least 90%, or 92%, or 94%, or 95% or more identical to Glucagon-like peptide 1 fragment 7-37 (GLP-1(7-37)).

5. 4. The modified polypeptide of any one of claims 1 to 3, wherein the polypeptide comprises an amino acid sequence that is at least 90%, or 92%, or 94%, or 95% or more identical to parathyroid hormone fragment 1-34 (PTH(1-34)), or wherein the polypeptide comprises an amino acid sequence that is at least 85%, or 90%, or 92%, or 95% or more identical to peptide YY3-36 (PYY(3-36)).

6. The polypeptide is N ６ 4. The modified polypeptide of any one of claims 1 to 3, comprising a peptide therapeutic selected from the group consisting of therapeutic peptides listed in Table 1, modified to include a lysine or derivative thereof functionalized with a seryl or threonyl group at

7. The N ６ The modified polypeptide according to any one of claims 1 to 6, wherein a functionalized lysine or a derivative thereof is incorporated into the amino acid sequence in place of a natural lysine.

8. The N ６ The modified polypeptide according to any one of claims 1 to 6, wherein a functionalized lysine or a derivative thereof is incorporated into the amino acid sequence in place of a naturally occurring amino acid other than lysine.

9. The modified polypeptide of claim 8, wherein the natural amino acid is serine.

10. A bioconjugate comprising a modified polypeptide according to any one of claims 1 to 9 and a functional moiety, said functional moiety being attached to said modified polypeptide by said N ６ - Bioconjugates that are conjugated at seryl or threonyl groups on functionalized lysine or its derivatives.

11. 11. The bioconjugate of claim 10, wherein the functional moiety comprises a polyethylene glycol molecule, a lipid molecule, a fluorescent molecule, a chemiluminescent molecule, a phosphorescent molecule, a radioisotope, an enzyme, an enzyme substrate, an affinity molecule, a ligand, an antigen, a hapten, an antibody, an antibody fragment, a peptide, a peptidomimetic, a protein, a chromogenic substrate, a contrast agent, an MRI contrast agent, a PET label, a phosphorescent label, or a combination thereof.

12. The bioconjugate of claim 10 , wherein the functional moiety comprises a fluorescent molecule, biotin, a polyethylene glycol molecule, a lipid molecule, or a combination thereof.

13. The bioconjugate of claim 10, wherein the functional moiety comprises: 【Chemistry 5】 (for example, 【Chemistry 6】 (for example, 【Chemistry 7】 (In the formula, R=NH ２ , OH, or OCH ３ ) (e.g., 【Chemistry 8】

14. The bioconjugate of any one of claims 10-13, wherein the functional moiety increases the in vivo stability of the bioconjugate compared to the in vivo stability of the native polypeptide.

15. A pharmaceutical composition comprising one or more polypeptides and / or bioconjugates according to any one of claims 1 to 14, and a pharma- ceutically acceptable carrier, solvent, adjuvant and / or diluent.

16. 1. A method for preparing a bioconjugate, comprising: N ６ contacting a modified polypeptide comprising an amino acid sequence having a lysine or derivative thereof functionalized with a seryl or threonyl group with a functionalized salicylaldehyde ester; Including, The salicylaldehyde ester is ６ - reacting with said seryl or threonyl group on a functionalized lysine or derivative thereof to obtain said bioconjugate.

17. The functionalized salicylaldehyde ester has the formula: 【Chemistry 9】 17. The method of claim 16, wherein R is a moiety that comprises a polyethylene glycol molecule, a lipid molecule, a fluorescent molecule, a chemiluminescent molecule, a phosphorescent molecule, a radioisotope, an enzyme, an enzyme substrate, an affinity molecule, a ligand, an antigen, a hapten, an antibody, an antibody fragment, a peptide, a peptidomimetic, a protein, a chromogenic substrate, a contrast agent, an MRI contrast agent, a PET label, a phosphorescent label, or a combination thereof; or a fluorescent molecule, biotin, a polyethylene glycol molecule, a lipid molecule, or a combination thereof.

18. 17. The method of claim 16, wherein the functionalized salicylaldehyde ester has the formula: 【Chemistry 10】 (for example, 【Chemistry 11】 (for example, 【Chemistry 12】 (In the formula, R=NH ２ , OH, or OCH ３ ) (e.g., 【Chemistry 13】

19. The method of claim 16 , wherein the R moiety increases the in vivo stability of the bioconjugate compared to the in vivo stability of the modified polypeptide.

20. The method according to any one of claims 16 to 19, wherein the modified polypeptide is as defined in any one of claims 2 to 9.

21. The method of any of claims 16 to 20, wherein the modified polypeptide is contacted with the functionalized salicylaldehyde ester in a pyridine / acetic acid solution; or the modified polypeptide is contacted with the functionalized salicylaldehyde ester in a pyridine / acetic acid solution in a 1:1 v / v ratio.

22. 22. The method of any of claims 16-21, wherein the modified polypeptide is contacted with the functionalized salicylaldehyde ester at a temperature in the range of about 18°C ​​to 27°C for a period of time sufficient to form an N,O-benzylidene acetal intermediate.

23. 23. The method of claim 22, wherein the N,O-benzylidene acetal intermediate is reacted under acidic conditions for a period of time sufficient to allow the bioconjugate to form.

24. 24. The method of claim 23, wherein trifluoroacetic acid is used.

25. N ６ -seryl-lysine or its derivatives or N ６ The method of any one of claims 16 to 24, further comprising introducing -threonyl-lysine or a derivative thereof into the synthesis of the modified polypeptide.

26. 25. The method of any of claims 16-24, wherein the modified polypeptide comprises an N-terminal serine or threonine, and the method further comprises protecting the N-terminal serine or threonine with a protecting group prior to contacting with the functionalized salicylaldehyde ester.

27. 27. The method of claim 26, wherein the protecting group is selected from allyl serine and N-terminal acetylation.

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