Prolonged Activation of the GLP-1 Receptor via Covalent Capture.

The incretin gut hormone glucagon-like peptide-1 (GLP-1) has become a household name because of its ability to induce glucose-dependent insulin release with accompanying weight loss in patients. Indeed, derivatives of the peptide exert numerous pleiotropic actions that favorably affect other metabolic functions, and consequently, such compounds are being considered as treatments for a variety of ailments. The ability of native GLP-1 to function as a clinical drug is severely limited because of its short half-life in vivo. All of the beneficial effects of GLP-1 come from its agonism at the cognate receptor, GLP-1R. In our quest for long-lived activation of the receptor, we hypothesized that an agonist that had the ability to covalently cross-link with GLP-1R would prove useful. We here report the structure-guided design of peptide analogues containing an electrophilic warhead that could be covalently captured by a resident native nucleophile on the receptor. The compounds were evaluated using washout experiments, and resistance to such washing serves as an index of prolonged activation and covalent capture, which we use to tabulate longevity and robust long-lived GLP-1R agonism. The addition of SulF (cross-linkable warhead), an N-terminal trifluoroethyl group (for protease protection), and a C18 diacid lipid (protractor) all contributed to the increased wash resistance of GLP-1. The most effective compound based on the wash resistance metric, C2K26DAC18_K34SulF, has all three elements outlined and may serve as a blueprint and a proof-of-concept scaffold for the design of clinically useful molecules.

Molecular design of peptide therapeutics via N-terminal modification.

Glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, and glucagon are three naturally occurring peptide hormones that mediate glucoregulation. Several agonists representing appropriately modified native ligands have been developed to maximize metabolic benefits with reduced side-effects and many have entered the clinic as type 2 diabetes and obesity therapeutics. In this work, we describe strategies for improving the stability of the peptide ligands by making them refractory to dipeptidyl peptidase-4 catalyzed hydrolysis and inactivation. We describe a series of alkylations with variations in size, shape, charge, polarity, and stereochemistry that are able to engender full activity at the receptor(s) while simultaneously resisting enzyme-mediated degradation. Utilizing this strategy, we offer a novel method of modulating receptor activity and fine-tuning pharmacology without a change in peptide sequence.

Customizable Click Biochemistry Strategy for the Design and Preparation of Glucagon-like Peptide-1 Conjugates and Coagonists.

The development of oligomeric glucagon-like peptide-1 (GLP-1) and GLP-1-containing coagonists holds promise for enhancing the therapeutic potential of the GLP-1-based drugs for treating type 2 diabetes mellitus (T2DM). Here, we report a facile, efficient, and customizable strategy based on genetically encoded SpyCatcher-SpyTag chemistry and an inducible, cleavable self-aggregating tag (icSAT) scheme. icSAT-tagged SpyTag-fused GLP-1 and the dimeric or trimeric SpyCatcher scaffold were designed for dimeric or trimeric GLP-1, while icSAT-tagged SpyCatcher-fused GLP-1 and the icSAT-tagged SpyTag-fused GIP were designed for dual GLP-1/GIP (glucose-dependent insulinotropic polypeptide) receptor agonist. These SpyCatcher- and SpyTag-fused protein pairs were spontaneously ligated directly from the cell lysates. The subsequent icSAT scheme, coupled with a two-step standard column purification, resulted in target proteins with authentic N-termini, with yields ranging from 35 to 65 mg/L and purities exceeding 99%. In vitro assays revealed 3.0- to 4.1-fold increased activities for dimeric and trimeric GLP-1 compared to mono-GLP-1. The dual GLP-1/GIP receptor agonist exhibited balanced activity toward the GLP-1 receptor or the GIP receptor. All the proteins exhibited 1.8- to 3.0-fold prolonged half-lives in human serum compared to mono-GLP-1 or GIP. This study provides a generally applicable click biochemistry strategy for developing oligomeric or dual peptide/protein-based drug candidates.

Engineering of a Biologically Active Glycosylated Glucagon-Like Peptide-1 Analogue.

Glucagon-like peptide receptor (GLP-1R) agonists (e.g., semaglutide, liraglutide, etc.) are efficient treatment options for people with type 2 diabetes and obesity. The manufacturing method to produce semaglutide, a blockbuster GLP-1 drug on the market, involves multistep synthesis. The large peptide has a hydrophobic fatty acid side chain that makes it sparingly soluble, and its handling, purification, and large-scale production difficult. The growing demand for semaglutide that the manufacturer is not capable of addressing immediately triggered a worldwide shortage. Thus, we have developed a potential alternative analogue to semaglutide by replacing the hydrophobic fatty acid with a hydrophilic human complex-type biantennary oligosaccharide. Our novel glycoGLP-1 analogue was isolated in an ∼10-fold higher yield compared with semaglutide. Importantly, our glycoGLP-1 analogue possessed a similar GLP-1R activation potency to semaglutide and was biologically active in vivo in reducing glucose levels to a similar degree as semaglutide.

An Update on Dipeptidyl Peptidase-IV Inhibiting Peptides.

Diabetes is a chronic metabolic disorder. According to the International Diabetes Federation, about 537 million people are living with diabetes. The two types of diabetes are type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM), among which the population affected by T2DM is relatively higher. A major reason for T2DM is that insulin stimulation is hampered due to the inactivation of incretin hormones. Dipeptidyl peptidase-IV (DPP-IV) is a serine protease that is directly involved in the inactivation of incretin hormones, e.g., glucagon-like peptide-1 (GLP-1). Therefore, the inhibition of DPP-IV can be a promising method for managing T2DM, in addition to other enzyme inhibition strategies, such as inhibition of α-amylase and α -glucosidase. Currently, about 12 different gliptin drugs are available in the market that inhibit DPP-IV in a dose-dependent manner. Instead of gliptins, 'peptides' can also be employed as an alternative and promising way to inhibit DPP-IV. Peptide inhibitors of DPP-IV have been identified from various plants and animals. Chemically synthesized peptides have also been experimented for inhibiting DPP-IV. Most peptides have been analysed by biochemical assays, whereas some in vitro assays have also been reported. Molecular docking analysis has been applied to comprehend the mechanism of inhibition. In this review, certain aspects of natural as well as synthetic peptides are described that have been proven to inhibit DPP-IV.

Differential Responses of the GLP-1 and GLP-2 Receptors to N-Terminal Modification of a Dual Agonist.

Class B1 G protein-coupled receptors (GPCRs), collectively, respond to a diverse repertoire of extracellular polypeptide agonists and transmit the encoded messages to cytosolic partners. To fulfill these tasks, these highly mobile receptors must interconvert among conformational states in response to agonists. We recently showed that conformational mobility in polypeptide agonists themselves plays a role in activation of one class B1 GPCR, the receptor for glucagon-like peptide-1 (GLP-1). Exchange between helical and nonhelical conformations near the N-termini of agonists bound to the GLP-1R was revealed to be critical for receptor activation. Here, we ask whether agonist conformational mobility plays a role in the activation of a related receptor, the GLP-2R. Using variants of the hormone GLP-2 and the designed clinical agonist glepaglutide (GLE), we find that the GLP-2R is quite tolerant of variations in α-helical propensity near the agonist N-terminus, which contrasts with signaling at the GLP-1R. A fully α-helical conformation of the bound agonist may be sufficient for GLP-2R signal transduction. GLE is a GLP-2R/GLP-1R dual agonist, and the GLE system therefore enables direct comparison of the responses of these two GPCRs to a single set of agonist variants. This comparison supports the conclusion that the GLP-1R and GLP-2R differ in their response to variations in helical propensity near the agonist N-terminus. The data offer a basis for development of new hormone analogues with distinctive and potentially useful activity profiles; for example, one of the GLE analogues is a potent agonist of the GLP-2R but also a potent antagonist of the GLP-1R, a novel form of polypharmacology.

In-silico study of the interactions between acylated glucagon like-peptide-1 analogues and the native receptor.

We have performed a series of multiple molecular dynamics (MD) simulations of glucagon-like peptide-1 (GLP-1) and acylated GLP-1 analogues in complex with the endogenous receptor (GLP-1R) to obtain a molecular understanding of how fatty acid (FA) chain structure, acylation position on the peptide, and presence of a linker affect the binding. MD simulations were analysed to extract heatmaps of receptor-peptide interaction patterns and to determine the free energy of binding using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approach. The extracted free energies from MM-PBSA calculations are in qualitative agreement with experimentally determined potencies. Furthermore, the interaction patterns seen in the receptor-GLP-1 complex simulations resemble previously reported binding interactions validating the simulations. Analysing the receptor-GLP-1 analogue complex simulations, we found that the major differences between the systems stem from FA interactions and positioning of acylation in the peptide. Hydrophobic interactions between the FA chain and a hydrophobic patch on the extracellular domain contribute significantly to the binding affinity. Acylation on Lys26 resulted in noticeably more interactions between the FA chain and the extracellular domain hydrophobic patch than found for acylation on Lys34 and Lys38, respectively. The presence of a charged linker between the peptide and FA chain can potentially stabilise the complex by forming hydrogen bonds to arginine residues in the linker region between the extracellular domain and the transmembrane domain. A molecular understanding of the fatty acid structure and its effect on binding provides important insights into designing acylated agonists for GLP-1R.Communicated by Ramaswamy H. Sarma.

Molecular simulations of zwitterlation-induced conformation and dynamics variation of glucagon-like peptide-1 and insulin.

Zwitterionic materials have shown their ability to improve the circulation time and stability of proteins. Zwitterionic peptides present unique potential because genetic technology can fuse them to any wild-type protein. One critical question is the effect of the fusing zwitterionic peptides on the conformation and dynamics of the original protein domain. To shed light on this question, we investigate the conformation and dynamics of six artificial proteins composed of two small therapeutic polypeptide and protein (glucan-like peptide-1 and insulin) and a zwitterionic (glutamic acid-lysine)10 peptide in an explicit solvent using molecular dynamics simulations. The zwitterionic peptide is fused to the N- and C-terminal of the glucan-like peptide-1 and the chain A and B of the insulin. We analyze the conformation and dynamics variation of the polypeptide and protein domain using root mean square deviation, root mean square fluctuation, solvent accessible surface area, and secondary structure distributions. The simulation results show that the zwitterlation induces substantial changes in the conformation of the glucan-like peptide-1 and a moderate change in the conformation of the insulin, while the two polypeptide and protein remain folded. The glucan-like peptide-1 presents a full α-helix conformation when zwitterlated at the C-terminal. The zwitterionic location also plays a role in the conformational change. These zwitterlation-induced conformation variations indicate a comprehensive relationship between zwitterlation and protein stability and activity.

Mutational Landscape of the Proglucagon-Derived Peptides.

Strong efforts have been placed on understanding the physiological roles and therapeutic potential of the proglucagon peptide hormones including glucagon, GLP-1 and GLP-2. However, little is known about the extent and magnitude of variability in the amino acid composition of the proglucagon precursor and its mature peptides. Here, we identified 184 unique missense variants in the human proglucagon gene GCG obtained from exome and whole-genome sequencing of more than 450,000 individuals across diverse sub-populations. This provides an unprecedented source of population-wide genetic variation data on missense mutations and insights into the evolutionary constraint spectrum of proglucagon-derived peptides. We show that the stereotypical peptides glucagon, GLP-1 and GLP-2 display fewer evolutionary alterations and are more likely to be functionally affected by genetic variation compared to the rest of the gene products. Elucidating the spectrum of genetic variations and estimating the impact of how a peptide variant may influence human physiology and pathophysiology through changes in ligand binding and/or receptor signalling, are vital and serve as the first important step in understanding variability in glucose homeostasis, amino acid metabolism, intestinal epithelial growth, bone strength, appetite regulation, and other key physiological parameters controlled by these hormones.

Role of Proglucagon Peptides in Osmoregulation.

Glucagon-like peptide-1 (GLP-1), a product of partial proteolysis of proglucagon, is involved not only in regulation of carbohydrates, but also in water-salt metabolism. The study examined the role of proglucagon derivatives GLP-1, GLP-2, and oxyntomodulin in rat osmoregulation. Of them, only blood plasma GLP-1 increased in response to water load (20 ml/kg). Administration of glucose (1.5 g/kg) elevated GLP-1 and oxyntomodulin but did not change the level of GLP-2. GLP-1 accelerated excretion of excess water during hyperhydration, whereas GLP-2 decreased this parameter. No physiological effects of oxyntomodulin in the kidneys were revealed. Probably, the blood levels of proglucagon derivatives are independently regulated for each peptide. In contrast to GLP-2 and oxyntomodulin, GLP-1 is involved in osmoregulation.

Molecular simulation of zwitterionic polypeptides on protecting glucagon-like peptide-1 (GLP-1).

Owing to their anti-fouling properties, zwitterionic polypeptides demonstrate great advantage on protecting protein drugs. When conjugated to glucagon-like peptide-1 (GLP-1), a drug for type-II diabetes, zwitterionic polypeptides confer better pharmacokinetics than uncharged counterparts. However, its microscopic mechanism is still unclear due to the complicated conformational space. To address this challenge, this work explored the interaction modes of GLP-1 with the unconnected repeat units, instead of the full-length polypeptides. The three repeat units are two zwitterionic pentapeptides VPKEG and VPREG, and one uncharged control VPGAG. Our molecular simulations revealed that the helical conformation of GLP-1 was stabilized by adding 40 polypeptides. Both VPGAG and VPREG formed dense packing shells around GLP-1, but the driving forces were hydrophobic and electrostatic interactions, respectively. In contrast, the packing shell composed of VPKEG was most loose, while could still stabilize GLP-1. The moderate electrostatic interactions endowed VPKEG an anti-fouling property, thereby avoiding non-specific interaction with other amino acids. The strong electrostatic interactions exerted by arginine promoted atomic contacts between VPREG and other residues, making it as "hydrophobic" as VPGAG. In summary, the combination of hydrophobic and moderate electrostatic interactions in VPKEG brings about a subtle balance between stabilizing GLP-1 and avoiding non-specific interaction.

Bariatric surgery induces a new gastric mucosa phenotype with increased functional glucagon-like peptide-1 expressing cells.

Glucagon-Like Peptide-1 (GLP-1) undergoes rapid inactivation by dipeptidyl peptidase-4 (DPP4) suggesting that target receptors may be activated by locally produced GLP-1. Here we describe GLP-1 positive cells in the rat and human stomach and found these cells co-expressing ghrelin or somatostatin and able to secrete active GLP-1 in the rats. In lean rats, a gastric load of glucose induces a rapid and parallel rise in GLP-1 levels in both the gastric and the portal veins. This rise in portal GLP-1 levels was abrogated in HFD obese rats but restored after vertical sleeve gastrectomy (VSG) surgery. Finally, obese rats and individuals operated on Roux-en-Y gastric bypass and SG display a new gastric mucosa phenotype with hyperplasia of the mucus neck cells concomitant with increased density of GLP-1 positive cells. This report brings to light the contribution of gastric GLP-1 expressing cells that undergo plasticity changes after bariatric surgeries, to circulating GLP-1 levels.

Directed evolution of conformation-specific antibodies for sensitive detection of polypeptide aggregates in therapeutic drug formulations.

Biologics such as peptides and proteins possess a number of attractive attributes that make them particularly valuable as therapeutics, including their high activity, high specificity, and low toxicity. However, one of the key challenges associated with this class of drugs is their propensity to aggregate. Given the safety and immunogenicity concerns related to polypeptide aggregates, it is particularly important to sensitively detect aggregates in therapeutic drug formulations as part of the quality control process. Here, we report the development of conformation-specific antibodies that recognize polypeptide aggregates composed of a GLP-1 receptor agonist (liraglutide) and their integration into a sensitive immunoassay for detecting liraglutide amyloid fibrils. We sorted single-chain antibody libraries against liraglutide fibrils using yeast surface display and magnetic-activated cell sorting, and identified several antibodies with high conformational specificity. Interestingly, these antibodies cross-react with amyloid fibrils formed by several other polypeptides, revealing that they recognize molecular features common to different types of fibrils. Moreover, we find that our immunoassay using these antibodies is >50-fold more sensitive than the conventional method for detecting liraglutide aggregation (Thioflavin T fluorescence). We expect that our systematic approach for generating a sensitive, aggregate-specific immunoassay can be readily extended to other biologics to improve the quality and safety of formulated drug products.

Differential GLP-1R Binding and Activation by Peptide and Non-peptide Agonists.

Peptide drugs targeting class B1 G-protein-coupled receptors (GPCRs) can treat multiple diseases; however, there remains substantial interest in the development of orally delivered non-peptide drugs. Here, we reveal unexpected overlap between signaling and regulation of the glucagon-like peptide-1 (GLP-1) receptor by the non-peptide agonist PF 06882961 and GLP-1 that was not observed for another compound, CHU-128. Compounds from these patent series, including PF 06882961, are currently in clinical trials for treatment of type 2 diabetes. High-resolution cryoelectron microscopy (cryo-EM) structures reveal that the binding sites for PF 06882961 and GLP-1 substantially overlap, whereas CHU-128 adopts a unique binding mode with a more open receptor conformation at the extracellular face. Structural differences involving extensive water-mediated hydrogen bond networks could be correlated to functional data to understand how PF 06882961, but not CHU-128, can closely mimic the pharmacological properties of GLP-1. These findings will facilitate rational structure-based discovery of non-peptide agonists targeting class B GPCRs.

Sulfono-γ-AApeptides as Helical Mimetics: Crystal Structures and Applications.

Foldamers have defined and predictable structures, improved resistance to proteolytic degradation, enhanced chemical diversity, and are versatile in their mimicry of biological molecules, making them promising candidates in biomedical and material applications. However, as natural macromolecules exhibit endless folding structures and functions, the exploration of the applications of foldamers remains crucial. As such, it is imperative to continue to discover unnatural foldameric architectures with new frameworks and molecular scaffolds. To this end, we recently developed a new class of peptidomimetics termed ″γ-AApeptides", oligomers of γ-substituted-N-acylated-N-aminoethyl amino acids, which are inspired by the chiral peptide nucleic acid backbone. To date γ-AApeptides have been shown to be resistant to proteolytic degradation and possess limitless potential to introduce chemically diverse functional groups, demonstrating promise in biomedical and material sciences. However, the structures of γ-AApeptides were initially unknown, rendering their rational design for the mimicry of a protein helical domain impossible in the beginning, which limited their potential development. To our delight, in the past few years, we have obtained a series of crystal structures of helical sulfono-γ-AApeptides, a subclass of γ-AApeptides. The single-crystal X-ray crystallography indicates that sulfono-γ-AApeptides fold into unprecedented and well-defined helices with unique helical parameters. On the basis of the well-established size, shape, and folding conformation, the design of sulfono-γ-AApeptide-based foldamers opens a new avenue for the development of alternative unnatural peptidomimetics for their potential applications in chemistry, biology, medicine, materials science, and so on.In this Account, we will outline our journey on sulfono-γ-AApeptides and their application as helical mimetics. We will first briefly introduce the design and synthetic strategy of sulfono-γ-AApeptides and then describe the crystal structures of helical sulfono-γ-AApeptides, including left-handed homogeneous sulfono-γ-AApeptides, right-handed 1:1 α/sulfono-γ-AA peptide hybrids, and right-handed 2:1 α/sulfono-γ-AA peptide hybrids. After that, we will illustrate the potential of helical sulfono-γ-AApeptides for biological applications such as the disruption of medicinally relevant protein-protein interactions (PPIs) of BCL9-ß-catenin and p53-MDM2/MDMX as well as the mimicry of glucagon-like peptide 1 (GLP-1). In addition, we also exemplify their potential application in material science. We expect that this Account will shed light on the structure-based design and function of helical sulfono-γ-AApeptides, which can provide a new and alternative way to explore and generate novel foldamers with distinctive structural and functional properties.

(EX-4)2-Fc, an effective long-acting GLP-1 receptor agonist, reduces obesity-related inflammation by inhibiting leptin expression.

Obesity has been recognized as a low-grade, chronic inflammatory disease that leads to an increase in obesity-associated disorders, including type 2 diabetes (T2D), fatty liver diseases and cancer. Glucagon-like peptide-1 (GLP-1) is an effective drug for T2D, and it not only has glucose-regulating effects but also has anti-inflammatory effects in obesity. In our previous study, we designed a novel GLP-1 analogue, (EX-4)2-Fc, which has been shown to reduce body weight and improve glucose tolerance in vivo. In this study, we observed that (EX-4)2-Fc also has anti-inflammatory functions in adipose tissue. After the treatment of diet-induced obesity (DIO) mice with (EX-4)2-Fc, we found that the inflammatory response in adipose tissue was significantly attenuated. (Ex-4)2-Fc can reduce obesity-associated proinflammatory cytokine levels and macrophage numbers in DIO mice. In addition, (EX-4)2-Fc treatment resulted in proinflammatory M1-type macrophages beginning to transform into anti-inflammatory M2-type macrophages. The inflammatory mitogen-activated protein kinase (MAPK) signalling pathway and nuclear factor kappa B (NF-κB) were altered in adipose tissue after (EX-4)2-Fc treatment. Leptin has been proven to be closely related to immunity, and we demonstrated that the effect of (EX-4)2-Fc on adipocyte inflammation was related to leptin. The data suggested that (EX-4)2-Fc could modulate the inflammatory response by inhibiting the expression of leptin in adipose tissue.

Rational design and biological evaluation of gemfibrozil modified Xenopus GLP-1 derivatives as long-acting hypoglycemic agents.

Novel methods for peptides structural modification and bioactivity optimization are highly needed in peptide-based drug discovery. Herein, we explored the use gemfibrozil (GFZ) as an albumin binder to enhance the stability and improve the bioactivity of peptides. Short-acting Xenopus glucagon-like peptide-1 (xGLP-1) analogues with anti-diabetic activity were selected as the starting point. Mono-GFZ conjugation, peptide sequence hybridization, and dimeric-GFZ derivatization were successively used to generate novel GFZ-xGLP-1 conjugates, biologically screened by various in vitro and in vivo models. Dimeric-GFZ modified conjugate 3b was finally identified as a promising anti-diabetic candidate with high albumin binding affinity, enhanced in vivo stability in SD rats, and long-acting hypoglycemic activity in db/db mice. Moreover, GFZ endowed 3b with strong lipid-regulating ability in DIO and db/db mice. In a twelve-week study, chronic administration of 3b in db/db mice resulted in sustained glycemic control, to a greater extent than liraglutide and semaglutide. In addition, 3b showed comparable therapeutic efficacies to liraglutide and semaglutide on HbA1c and pancreas islets protection. Our studies reveal 3b as a potential candidate for the treatment of metabolic diseases and indicate dimeric-GFZ modification as a novel method for peptide optimization.

Silica-Coated Nanoparticles with a Core of Zinc, l-Arginine, and a Peptide Designed for Oral Delivery.

Nanoparticle constructs for oral peptide delivery at a minimum must protect and present the peptide at the small intestinal epithelium in order to achieve oral bioavailability. In a reproducible, scalable, surfactant-free process, a core was formed with insulin in ratios with two established excipients and stabilizers, zinc chloride and l-arginine. Cross-linking was achieved with silica, which formed an outer shell. The process was reproducible across several batches, and physicochemical characterization of a single batch was confirmed in two independent laboratories. The silica-coated nanoparticles (SiNPs) entrapped insulin with high entrapment efficiency, preserved its structure, and released it at a pH value present in the small intestine. The SiNP delivered insulin to the circulation and reduced plasma glucose in a rat jejunal instillation model. The delivery mechanism required residual l-arginine in the particle to act as a permeation enhancer for SiNP-released insulin in the jejunum. The synthetic process was varied in terms of ratios of zinc chloride and l-arginine in the core to entrap the glucagon-like peptide 1 analogue, exenatide, and bovine serum albumin. SiNP-delivered exenatide was also bioactive in mice to some extent following oral gavage. The process is the basis for a platform for oral peptide and protein delivery.

Inhibiting the fibrillation of a GLP-1-like peptide.

Aggregation, including the formation of fibrils, poses significant challenges for the development of therapeutic peptides. To prepare stable peptide formulations, some understanding of the mechanisms underpinning the fibrillation process is required. A thioflavin T fluorescence assay was first used to determine the fibrillation profile of a GLP-1-like peptide (G48) at conditions being considered to formulate the peptide. G48 concentrations ranged from 0 to 600 µM and three pH values (pH 3.7, 7.4 and 8.5) were evaluated. Kinetic data demonstrate that G48 displays a pH-dependent aggregation profile. At pH 3.7, which is below the isoelectric point of G48 (pI ~ 5), kinetics representative of amorphous aggregates forming via a nucleation-independent mechanism were seen. At pH 7.4 and 8.5 (pH > pI) typical nucleation-dependent aggregation kinetics were observed. The weight concentration of ß-sheet rich aggregates (FLmax) correlated inversely with net charge, so lower FLmax values were observed at pH 3.7 and 8.5 than at pH 7.4. Incorporation of a non-ionic surfactant (polysorbate 80) into the peptide solution suppressed the fibrillation of G48 at all pH values and maintained the native peptide conformation, whereas a phenolic co-formulant (ferulic acid) had minimal effects on fibril growth. Peptide fibrillation, which can occur within a range of formulation concentrations and pH values, can hence be inhibited by the judicious use of excipients.

Prolonged half-life of small-sized therapeutic protein using serum albumin-specific protein binder.

Many small-sized proteins and peptides, such as cytokines and hormones, are clinically used for the treatment of a variety of diseases. However, their short half-life in blood owing to fast renal clearance usually results in a low therapeutic efficacy and frequent dosing. Here we present the development of a human serum albumin (HSA)-specific protein binder with a binding affinity of 4.3nM through a phage display selection and modular evolution approach to extend the blood half-life of a small-sized therapeutic protein. As a proof-of-concept, the protein binder composed of LRR (Leucine-rich repeat) modules was genetically fused to the N-terminus of Glucagon-like Peptide-1 (GLP-1). The fused GLP-1 was shown to have a significantly improved pharmacokinetic property: The terminal half-life of the fused GLP-1 increased to approximately 10h, and the area under the curve was 5-times higher than that of the control. The utility and potential of our approach was demonstrated by the efficient control of the blood glucose level in type-2 diabetes mouse models using the HSA-specific protein binder-fused GLP-1 over a prolonged time period. The present approach can be effectively used in enhancing the efficacy of small-sized therapeutic proteins and peptides through an enhanced blood circulation time.