Tirzepatide, GIP(1-42) and GIP(1-30) display unique signaling profiles at two common GIP receptor variants, E354 and Q354.

Type 2 diabetes (T2D) and obesity are prevalent metabolic disorders affecting millions of individuals worldwide. A new effective therapeutic drug called tirzepatide for the treatment of obesity and T2D is a dual agonist of the GIP receptor and GLP-1 receptor. Tirzepatide is clinically more effective than GLP-1 receptor agonists but the reasons why are not well understood. Tirzepatide reportedly stimulates the GIP receptor more potently than the GLP-1 receptor. However, tirzepatide signaling has not been thoroughly investigated at the E354 (wildtype) or Q354 (E354Q) GIP receptor variants. The E354Q variant is associated increased risk of T2D and lower body mass index. To better understand GIP receptor signaling we characterized the activity of endogenous agonists and tirzepatide at both GIP receptor variants. Using Cos7 cells we examined wildtype and E354Q GIP receptor signaling, analyzing cAMP and IP1 accumulation as well as AKT, ERK1/2 and CREB phosphorylation. GIP(1-42) and GIP(1-30)NH2 displayed equipotent effects on these pathways excluding CREB phosphorylation where GIP(1-30)NH2 was more potent than GIP(1-42) at the E354Q GIP receptor. Tirzepatide favored cAMP signaling at both variants. These findings indicate that tirzepatide is a biased agonist towards Gαs signaling and suggests it equally activates the wildtype and E354Q GIP receptor variants. We also observed differences between the pharmacology of the GIP receptor variants with endogenous peptides, which may help to explain differences in phenotype. These findings contribute to a comprehensive understanding of GIP receptor signaling, and will aid development of therapies combating T2D and obesity.

Leveraging the Mukaiyama oxidation-reduction condensation reaction for on-resin aryl thio-esterification for bio-conjugation.

A room-temperature Mukaiyama oxidation-reduction condensation inspired thioesterification methodology has been developed to afford aryl Cα-terminal peptide thioesters on-resin. The conditions herein feature mild reactions compatible with all Fmoc-SPPS protocols offering direct access to this critical arylthioester scaffold. This one-pot synthesis to aryl-thioester functionalised peptides facilitates peptide/protein synthesis by native chemical ligation.

The Antimicrobial Peptide Capitellacin: Chemical Synthesis of Analogues to Probe the Role of Disulphide Bridges and Their Replacement with Vinyl Sulphides.

Capitellacin (1) is a 20-residue antimicrobial ß-hairpin, produced by the marine polychaeta (segmented worms) Capitella teletai. Since its discovery in 2020, only very limited studies have been undertaken to understand capitellacin's structure-activity relationship (SAR). Using fast-flow Fmoc-SPPS, a focused library of capitellacin analogues was prepared to systematically study the influence of the two disulphide bridges on its structure and activity, and their replacement with a vinyl sulphide as a potential bioisostere. Upon studying the resulting peptides' antimicrobial activity and secondary structure, the most terminal disulphide emerged as the most critical element for maintaining both bioactivity and the secondary structure, properties which were demonstrated to be closely interlinked. The removal of the innermost disulphide bridge or disulphide replacement with a vinyl sulphide emerged as strategies with which to tune the activity spectrum, producing selectivity towards E. coli. Additionally, an enantiomeric d-capitellacin analogue revealed mechanistic insights, suggesting that chirality may be an inherent property of capitellacin's bacterial membrane target, or that a hitherto unknown secondary mechanism of action may exist. Additionally, we propose the Alloc protecting group as a more appropriate alternative to the common Dde group during fast-flow Fmoc-SPPS, in particular for short-chain diamino acids.

Chemical Modifications on the αvß6 Integrin Targeting A20FMDV2 Peptide: A Review.

Integrin proteins have received a significant increase in attention in recent scientific endeavors. The current trend uses the pre-established knowledge that the arginyl-glycyl-aspartic acid (RGD) structural motif present in the A20FMDV2 peptide is highly selective for the integrin class αvß6 which is overexpressed in many cancer types. This review will provide an extensive overview of the existing literature research to date to the best of our knowledge, highlighting significant improvements and drawbacks of structure-activity relationships (SAR) work undertaken, aiding future research to identify established SAR for an informed design of future A20FMDV2 mimetic inhibitors. Herein, the review aims to collate the existing structural chemical modifications present on A20FMDV2 in the literature to highlight key structural analogues that display more potent biological activity.

Synthesis, Structure-Activity Relationship Study, Bioactivity, and Nephrotoxicity Evaluation of the Proposed Structure of the Cyclic Lipodepsipeptide Brevicidine B.

The brevicidines represent a novel class of nonribosomal antimicrobial peptides that possess remarkable potency and selectivity toward highly problematic and resistant Gram-negative pathogenic bacteria. A recently discovered member of the brevicidine family, coined brevicidine B (2), comprises a single amino acid substitution (from d-Tyr2 to d-Phe2) in the amino acid sequence of the linear moiety of brevicidine (1) and was reported to exhibit broader antimicrobial activity against both Gram-negative (MIC = 2-4 µgmL-1) and Gram-positive (MIC = 2-8 µgmL-1) pathogens. Encouraged by this, we herein report the first total synthesis of the proposed structure of brevicidine B (2), building on our previously reported synthetic strategy to access brevicidine (1). In agreement with the original isolation paper, pleasingly, synthetic 2 demonstrated antimicrobial activity toward Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae (MIC = 4-8 µgmL-1). Interestingly, however, synthetic 2 was inactive toward all of the tested Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus strains. Substitution of d-Phe2 with its enantiomer, and other hydrophobic residues, yields analogues that were either inactive or only exhibited activity toward Gram-negative strains. The striking difference in the biological activity of our synthetic 2 compared to the reported natural compound warrants the re-evaluation of the original natural product for purity or possible differences in relative configuration. Finally, the evaluation of synthetic 1 and 2 in a human kidney organoid model of nephrotoxicity revealed substantial toxicity of both compounds, although 1 was less toxic than 2 and polymyxin B. These results indicate that modification to position 2 may afford a strategy to mitigate the nephrotoxicity of brevicidine.

Poly-γ-glutamylation of biomolecules.

Poly-γ-glutamate tails are a distinctive feature of archaeal, bacterial, and eukaryotic cofactors, including the folates and F420. Despite decades of research, key mechanistic questions remain as to how enzymes successively add glutamates to poly-γ-glutamate chains while maintaining cofactor specificity. Here, we show how poly-γ-glutamylation of folate and F420 by folylpolyglutamate synthases and γ-glutamyl ligases, non-homologous enzymes, occurs via processive addition of L-glutamate onto growing γ-glutamyl chain termini. We further reveal structural snapshots of the archaeal γ-glutamyl ligase (CofE) in action, crucially including a bulged-chain product that shows how the cofactor is retained while successive glutamates are added to the chain terminus. This bulging substrate model of processive poly-γ-glutamylation by terminal extension is arguably ubiquitous in such biopolymerisation reactions, including addition to folates, and demonstrates convergent evolution in diverse species from archaea to humans.

Polymyxin B and ethylenediaminetetraacetic acid act synergistically against Pseudomonas aeruginosa and Staphylococcus aureus.

Polymyxin B and ethylenediaminetetraacetic acid are antimicrobials possessing antibiofilm activity. They act by displacement and chelation, respectively, of divalent cations in bacterial membranes and may therefore act synergistically when applied in combination. If so, this combination of agents may be useful for the treatment of diseases like cystic fibrosis (CF), in which biofilms are present on the respiratory epithelium. We used checkerboard assays to investigate the synergy between these agents using reference strains Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 6538 in planktonic form. We then determined the efficacy of each agent against biofilms of both species grown on 96-pin lids and proceeded to combination testing against the P. aeruginosa reference strain and 10 clinical isolates from patients with CF. Synergism was observed for planktonic forms of both species and for biofilms of P. aeruginosa. The susceptibility of biofilms of P. aeruginosa clinical isolates to these agents was variable compared to the laboratory reference strain. This combination of agents may be useful in the management of biofilm-associated conditions, particularly those amenable to topical therapies. These results provide a basis upon which the antimicrobial and antibiofilm efficacy of preparations containing these agents may be enhanced.IMPORTANCEBacteria living in biofilms produce a protective matrix which makes them difficult to kill. Patients with severe respiratory disease often have biofilms. Polymyxin B is an antibiotic commonly used in topical medications, such as eye drops and nasal sprays. Ethylenediaminetetraacetic acid (EDTA) is used widely as a preservative in medication but also has antimicrobial properties. It has been hypothesized that Polymyxin B and EDTA could have a synergistic relationship: when used in combination their antimicrobial effect is enhanced. Here, we evaluated the levels at which Polymyxin B and EDTA work together to kill common pathogens Pseudomonas aeruginosa and Staphylococcus aureus. We found that Polymyxin B and EDTA were synergistic. This synergy may be useful in the management of planktonic infection with P. aeruginosa and S. aureus, or biofilm infection with P. aeruginosa. This synergy may be beneficial in the treatment of respiratory biofilms, in which P. aeruginosa biofilms are common.

Harnessing the power of a photoinitiated thiol-ene "click" reaction for the efficient synthesis of S-lipidated collagen model peptide amphiphiles.

A photoinitiated thiol-ene "click" reaction was used to synthesize S-lipidated collagen model peptide amphiphiles. Use of 2-iminothiolane provided an epimerization-free thiol handle required for thiol-ene based incorporation of lipid moieties onto collagen-based peptide sequences. This approach not only led to improvements in the triple helical characteristics of the resulting collagen model peptides but also increased the aqueous solubility of the peptide amphiphiles. As a result, this methodology holds significant potential for the design and advancement of functional peptide amphiphiles, offering enhanced capabilities across a wide range of applications.

NMR Shows Why a Small Chemical Change Almost Abolishes the Antimicrobial Activity of Glycocin F.

Glycocin F (GccF), a ribosomally synthesized, post-translationally modified peptide secreted by Lactobacillus plantarum KW30, rapidly inhibits the growth of susceptible bacteria at nanomolar concentrations. Previous studies have highlighted structural features important for its activity and have shown the absolute requirement for the Ser18 O-linked GlcNAc on the eight-residue loop linking the two short helices of the (C-X6-C)2 structure. Here, we show that an ostensibly very small chemical modification to Ser18, the substitution of the Cα proton with a methyl group, reduces the antimicrobial activity of GccF 1000-fold (IC50 1.5 µM cf. 1.5 nM). A comparison of the GccFα-methylSer18 NMR structure (PDB 8DFZ) with that of the native protein (PDB 2KUY) showed a marked difference in the orientation and mobility of the loop, as well as a markedly different positioning of the GlcNAc, suggesting that loop conformation, dynamics, and glycan presentation play an important role in the interaction of GccF with as yet unknown but essential physiological target molecules.

On-Resin Conjugation of the Ruthenium Anticancer Agent Plecstatin-1 to Peptide Vectors.

Ruthenium piano-stool complexes have been explored for their anticancer activity and some promising compounds have been reported. Herein, we conjugated a derivative of plecstatin-1 to peptides in order to increase their cancer cell targeting ability. For this purpose, plecstatin-1 was modified at the arene ligand to introduce a functional amine handle (3), which resulted in a compound that showed similar activity in an in vitro anticancer activity assay. The cell-penetrating peptide TAT48-60, tumor-targeting neurotensin8-13, and plectin-targeting peptide were functionalized with succinyl or ß-Ala-succinyl linkers under standard solid-phase peptide synthesis (SPPS) conditions to spatially separate the peptide backbones from the bioactive metal complexes. These modifications allowed for conjugating precursor 3 to the peptides on resin yielding the desired metal-peptide conjugates (MPCs), as confirmed by high-performance liquid chromatography (HPLC), NMR spectroscopy, and mass spectrometry (MS). The MPCs were studied for their behavior in aqueous solution and under acidic conditions and resembled that of the parent compound plecstatin-1. In in vitro anticancer activity studies in a small panel of cancer cell lines, the TAT-based MPCs showed the highest activity, while the other MPCs were virtually inactive. However, the MPCs were significantly less active than the small molecules plecstatin-1 and 3, which can be explained by the reduced cell uptake as determined by inductively coupled plasma MS (ICP-MS). Although the MPCs did not display potent anticancer activities, the developed conjugation strategy can be extended toward other metal complexes, which may be able to utilize the targeting properties of peptides.

Factors influencing on-resin depsipeptide bond formation: case studies on daptomycin- and brevicidine-derived sequences.

Depsipeptides are an important class of bioactive natural products, where a growing number of genome-mined structures that display anti-microbial activity are macrocyclic depsipeptides. Chemically, peptide ester (depsipeptide) bond formation often displays low yields, and thereby hampers efforts to access these structures for structure-activity studies. Herein, we present a systematic study of the variables that influence depsipeptide bond formation on-resin, using simplified sequences derived from antibiotic peptides, daptomycin and brevicidine, prepared via Fmoc-based solid-phase synthesis. Our study highlights reaction solvent as the key determinant, where switching the solvent from DMF to DCM in almost all cases increased the amount of depsipeptide product. Limiting the number of amino-acids N-terminal to the reactive alcohol was also noted to significantly improve the acylation efficiency. The impact of different N-terminal and side-chain protecting groups, as well as stereochemistry, was also investigated. Additives to the reaction, such as inclusion of surfactants for esterification of long hydrophobic sequences, did not improve conversion. 6-ClHOBt, often added to improve acylation efficiency, notably decreased the amount of depsipeptide observed. Lastly, no significant difference between polystyrene and Tentagel® (PEG-decorated) resins were found for these sequences.

Flow-Based Fmoc-SPPS Preparation and SAR Study of Cathelicidin-PY Reveals Selective Antimicrobial Activity.

Antimicrobial peptides (AMPs) hold promise as novel therapeutics in the fight against multi-drug-resistant pathogens. Cathelicidin-PY (NH2-RKCNFLCKLKEKLRTVITSHIDKVLRPQG-COOH) is a 29-residue disulfide-cyclised antimicrobial peptide secreted as an innate host defence mechanism by the frog Paa yunnanensis (PY) and reported to possess broad-spectrum antibacterial and antifungal properties, exhibiting low cytotoxic and low hemolytic activity. Herein, we detail the total synthesis of cathelicidin-PY using an entirely on-resin synthesis, including assembly of the linear sequence by rapid flow Fmoc-SPPS and iodine-mediated disulfide bridge formation. By optimising a synthetic strategy to prepare cathelicidin-PY, this strategy was subsequently adapted to prepare a bicyclic head-to-tail cyclised derivative of cathelicidin-PY. The structure-activity relationship (SAR) of cathelicidin-PY with respect to the N-terminally positioned disulfide was further probed by preparing an alanine-substituted linear analogue and a series of lactam-bridged peptidomimetics implementing side chain to side chain cyclisation. The analogues were investigated for antimicrobial activity, secondary structure by circular dichroism (CD), and stability in human serum. Surprisingly, the disulfide bridge emerged as non-essential to antimicrobial activity and secondary structure but was amenable to synthetic modification. Furthermore, the synthetic AMP and multiple analogues demonstrated selective activity towards Gram-negative pathogen E. coli in physiologically relevant concentrations of divalent cations.

The Anti-Tubercular Aminolipopeptide Trichoderin A Displays Selective Toxicity against Human Pancreatic Ductal Adenocarcinoma Cells Cultured under Glucose Starvation.

Pancreatic ductal adenocarcinoma remains a highly debilitating condition with no effective disease-modifying interventions. In our search for natural products with promising anticancer activity, we identified the aminolipopeptide trichoderin A as a potential candidate. While it was initially isolated as an antitubercular peptide, we provide evidence that it is also selectively toxic against BxPC-3 and PANC-1 human pancreatic ductal adenocarcinoma cells cultured under glucose deprivation. This has critical implications for the pancreatic ductal adenocarcinoma, which is characterized by nutrient deprivation due to its hypovascularized network. We have also successfully simplified the trichoderin A peptide backbone, allowing greater accessibility to the peptide for further biological testing. In addition, we also conducted a preliminary investigation into the role of peptide lipidation at the N-terminus. This showed that analogues with longer fatty acyl chains exhibited superior cytotoxicity than those with shorter acyl chains. Further structural optimization of trichoderin A is anticipated to improve its biological activity, whilst ongoing mechanistic studies to elucidate its intracellular mechanism of action are conducted in parallel.

Novel Fluorescently Labeled PACAP and VIP Highlight Differences between Peptide Internalization and Receptor Pharmacology.

The related peptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) have diverse biological functions in peripheral tissues and the central nervous system. Therefore, these peptides and their three receptors represent potential drug targets for several conditions, including neurological and pain-related disorders. However, very little is known about how these peptides regulate their receptors through processes such as internalization. Therefore, we developed tools to study receptor regulation through the synthesis of fluorescently labeled analogues of PACAP-38, PACAP-27, and VIP using copper-mediated 1,3-dipolar cycloaddition of the Cy5 fluorophore. The functionality of Cy5-labeled peptides at their receptors was confirmed in cAMP accumulation assays. Internalization of the Cy5-labeled peptides was then examined and quantified at two distinct PAC1 receptor splice variants, VPAC1 and VPAC2 receptors in transfected cells. All labeled peptides were functional, exhibiting comparable cAMP pharmacology to their unlabeled counterparts and underwent internalization in a time-dependent manner. Temporal differences in the internalization profiles were observed between Cy5-labeled peptides at the PAC1n, PAC1s, VPAC1, and VPAC2 receptors. Interestingly, the pattern of Cy5-labeled peptide activity differed for cAMP accumulation and internalization, indicating that these peptides differentially stimulate cAMP accumulation and internalization and therefore display biased agonism. This novel insight into PACAP-responsive receptor signaling and internalization may provide a unique avenue for future therapeutic development. The fluorescently labeled PACAP and VIP peptides described herein, which we validated as tools to study receptor internalization, will have utility across a broad range of applications and provide greater insight into this receptor family.

Characterization of Antibodies against Receptor Activity-Modifying Protein 1 (RAMP1): A Cautionary Tale.

Calcitonin gene-related peptide (CGRP) is a key component of migraine pathophysiology, yielding effective migraine therapeutics. CGRP receptors contain a core accessory protein subunit: receptor activity-modifying protein 1 (RAMP1). Understanding of RAMP1 expression is incomplete, partly due to the challenges in identifying specific and validated antibody tools. We profiled antibodies for immunodetection of RAMP1 using Western blotting, immunocytochemistry and immunohistochemistry, including using RAMP1 knockout mouse tissue. Most antibodies could detect RAMP1 in Western blotting and immunocytochemistry using transfected cells. Two antibodies (844, ab256575) could detect a RAMP1-like band in Western blots of rodent brain but not RAMP1 knockout mice. However, cross-reactivity with other proteins was evident for all antibodies. This cross-reactivity prevented clear conclusions about RAMP1 anatomical localization, as each antibody detected a distinct pattern of immunoreactivity in rodent brain. We cannot confidently attribute immunoreactivity produced by RAMP1 antibodies (including 844) to the presence of RAMP1 protein in immunohistochemical applications in brain tissue. RAMP1 expression in brain and other tissues therefore needs to be revisited using RAMP1 antibodies that have been comprehensively validated using multiple strategies to establish multiple lines of convincing evidence. As RAMP1 is important for other GPCR/ligand pairings, our results have broader significance beyond the CGRP field.

PAC1, VPAC1, and VPAC2 Receptor Expression in Rat and Human Trigeminal Ganglia: Characterization of PACAP-Responsive Receptor Antibodies.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide expressed in the trigeminal ganglia (TG). The TG conducts nociceptive signals in the head and may play roles in migraine. PACAP infusion provokes headaches in healthy individuals and migraine-like attacks in patients; however, it is not clear whether targeting this system could be therapeutically efficacious. To effectively target the PACAP system, an understanding of PACAP receptor distribution is required. Therefore, this study aimed to characterize commercially available antibodies and use these to detect PACAP-responsive receptors in the TG. Antibodies were initially validated in receptor transfected cell models and then used to explore receptor expression in rat and human TG. Antibodies were identified that could detect PACAP-responsive receptors, including the first antibody to differentiate between the PAC1n and PAC1s receptor splice variants. PAC1, VPAC1, and VPAC2 receptor-like immunoreactivity were observed in subpopulations of both neuronal and glial-like cells in the TG. In this study, PAC1, VPAC1, and VPAC2 receptors were detected in the TG, suggesting they are all potential targets to treat migraine. These antibodies may be useful tools to help elucidate PACAP-responsive receptor expression in tissues. However, most antibodies exhibited limitations, requiring the use of multiple methodologies and the careful inclusion of controls.

Synthesis, Antibacterial Activity, and Nephrotoxicity of Polymyxin B Analogues Modified at Leu-7, d-Phe-6, and the N-Terminus Enabled by S-Lipidation.

With the post-antibiotic era rapidly approaching, many have turned their attention to developing new treatments, often by structural modification of existing antibiotics. Polymyxins, a family of lipopeptide antibiotics that are used as a last line of defense in the clinic, have recently developed resistance and exhibit significant nephrotoxicity issues. Using thiol-ene chemistry, the facile preparation of six unique S-lipidated building blocks was demonstrated and used to generate lipopeptide mimetics upon incorporation into solid-phase peptide synthesis (SPPS). We then designed and synthesized 38 polymyxin analogues, incorporating these unique building blocks at the N-terminus, or to replace hydrophobic residues at positions 6 and 7 of the native lipopeptides. Several polymyxin analogues bearing one or more S-linked lipids were found to be equipotent to polymyxin, showed minimal kidney nephrotoxicity, and demonstrated activity against several World Health Organisation (WHO) priority pathogens. The S-lipidation strategy has demonstrated potential as a novel approach to prepare innovative new lipopeptide antibiotics.

Synthesis and Biological Evaluation of Termini-Modified and Cyclic Variants of the Connexin43 Inhibitor Peptide5.

Peptide5 is a 12-amino acid mimetic peptide that corresponds to a region of the extracellular loop 2 (EL2) of connexin43. Peptide5 regulates both cellular communication with the cytoplasm (hemichannels) and cell-to-cell communication (gap junctions), and both processes are implicated in neurological pathologies. To address the poor in vivo stability of native peptide5 and to improve its activity, twenty-five novel peptide5 mimetics were designed and synthesized. All the analogues underwent biological evaluation as a hemichannel blocker and as a gap junction disruptor, and several were assessed for stability in human serum. From this study, it was established that several acylations on the N-terminus were tolerated in the hemichannel assay. However, the replacement of the L-Lys with an N-methylated L-Lys to give H-VDCFLSRPTE-N-MeKT-OH showed good hemichannel and gap junction activity and was more stable in human serum. The cyclic peptide variants generally were not tolerated in either the hemichannel and gap junction assay although several possessed outstanding stability in human serum.

Synthetic Studies towards the Calcium-Dependent Lipopeptide Antibiotic Cadaside B.

In the current global crisis of antimicrobial resistance, antimicrobial peptides represent a promising source of alternative antibiotics. Recently discovered cadaside B, a novel calcium-dependent antibiotic, exhibits potent antimicrobial activity towards Gram-positive pathogens including multi-drug resistant strains. These properties, coupled with a novel structure, non-cytotoxicity, and low likelihood of developing resistance render cadaside B an important synthetic target. Herein, a synthetic strategy towards cadaside B is reported with the key steps involving on-resin depsipeptide bond formation and solution-phase macrolactamization. Good agreement of the synthetic cadaside B MS/MS fragmentation pattern was observed with the natural product, but a different 1 H NMR spectrum and absence of antimicrobial activity suggest an undetected epimerization event took place during the synthesis. Herein the findings of our synthetic journey and suggestions for future directions are presented.

Synthesis and Systematic Study on the Effect of Different PEG Units on Stability of PEGylated, Integrin-αvß6-Specific A20FMDV2 Analogues in Rat Serum and Human Plasma.

A20FMDV2 is a 20-mer peptide that exhibits high selectivity and affinity for the tumour-related αvß6 integrin that can compete with extracellular ligands for the crucial RGD binding site, playing a role as a promising αvß6-specific inhibitor for anti-cancer therapies. Unfortunately, the clinical value of A20FMDV2 is limited by its poor half-life in blood caused by rapid renal excretion and its reported high susceptibility to serum proteases. The incorporation of poly (ethylene glycol) chains, coined PEGylation, is a well-established approach to improve the pharmacokinetic properties of drug molecules. Here, we report a systematic study on the incorporation of a varying number of ethylene glycol units (1-20) into the A20FMDV2 peptide to establish the effects of PEGylation size on the peptide stability in both rat serum and human plasma. In addition, the effect of acetyl and propionyl PEGylation handles on peptide stability is also described. Selected peptide analogues were assessed for integrin-αvß6-targeted binding, showing good specificity and activity in vitro. Stability studies in rat serum established that all of the PEGylated peptides displayed good stability, and an A20FMDV2 peptide containing twenty ethylene glycol units (PEG20) was the most stable. Surprisingly, the stability testing in human plasma identified shorter PEGs (PEG2 and PEG5) as more resistant to degradation than longer PEGs, a trend which was also observed with affinity binding to integrin αvß6.