An expeditious route to sterically encumbered nonproteinogenic α-amino acid precursors using allylboronic acids

A diastereoselective allylation of N-tert-butane sulfinyl α-iminoesters using allylboronic acids is developed to obtain optically active non-proteinogenic α-amino acid precursors in good yields and diastereoselectivities. Gram-scale synthesis, broad tolerance of functional groups, excellent stereodivergence, post-synthetic modifications, and easy removal of the chiral auxiliary are some of the key highlights. The protocol is applicable to various amino acids and short peptides, resulting in the incorporation of these precursors at the N-terminal position.

A diastereoselective allylation of N-tert-butane sulfinyl α-iminoesters using allylboronic acids is developed to obtain optically active non-proteinogenic α-amino acid precursors in good yield and diastereoselectivities.     

Enabling chemical protein (semi)synthesis via reducible solubilizing tags (RSTs)

Chemical synthesis of proteins with poor solubility presents a challenging task. The existing solubilizing tag strategies are not suitable for the expressed protein segment. To address this issue, we report herein that solubilizing tags could be introduced at the side chain of the peptide and C-terminal peptide salicylaldehyde esters via a disulfide linker. Such reducible solubilizing tags (RSTs) are compatible with peptide salicylaldehyde ester-mediated Ser/Thr ligation and Cys/Pen ligation for purifying and ligating peptides with poor solubility. This strategy features operational simplicity and readily accessible materials. Both the protein 2B4 cytoplasmic tail and FCER1G protein have been successfully synthesized via this strategy. Of particular note, the RST strategy could be used for solubilizing the expressed protein segment for protein semi-synthesis of the HMGB1 protein.

The reducible solubilizing tag strategy served as a simple and powerful method for the chemical synthesis and semi-synthesis via Ser/Thr ligation and Cys/Pen ligation of extensive self-assembly peptides, membrane proteins with poor solubility.     

An economical approach for peptide synthesis via regioselective C–N bond cleavage of lactams

An economical, solvent-free, and metal-free method for peptide synthesis via C–N bond cleavage using lactams has been developed. The method not only eliminates the need for condensation agents and their auxiliaries, which are essential for conventional peptide synthesis, but also exhibits high atom economy. The reaction is versatile because it can tolerate side chains bearing a range of functional groups, affording up to >99% yields of the corresponding peptides without racemisation or polymerisation. Moreover, the developed strategy enables peptide segment coupling, providing access to a hexapeptide that occurs as a repeat sequence in spider silk proteins.

An economical, solvent-free, and metal-free method for peptide synthesis via C–N bond cleavage using lactams has been developed.     

Scalable synthesis and coupling of quaternary α-arylated amino acids: α-aryl substituents are tolerated in α-helical peptides

Quaternary amino acids are important tools for the modification and stabilisation of peptide secondary structures. Here we describe a practical and scalable synthesis applicable to quaternary alpha-arylated amino acids (Q4As), and the development of solid-phase synthesis conditions for their incorporation into peptides. Monomeric and dimeric α-helical peptides are synthesised with varying degrees of Q4A substitution and their structures examined using biophysical methods. Both enantiomers of the Q4As are tolerated in folded monomeric and oligomeric α-helical peptides, with the (R)-enantiomer slightly more so than the (S).

Both R and S enantiomers of Fmoc-protected amino acids bearing α-aryl substituents may be made on gram scale. Solid-phase synthesis leads to helical peptides unperturbed by the presence of these additional α-aryl groups.     

Amino acid bromides: their utilization for difficult couplings in solid-phase peptide synthesis

Use of N-protected-α-amino acid bromides for facile solid-phase synthesis of peptides (SPPS) containing extremely sterically hindered non-proteinogenic amino acids is presented. Amino acid bromides (Aaa-Br), generated in situ, were used for the synthesis of long chain homopeptides containing up to eight successive α-MeVal or Aib residues. SPPS of a heteropeptide containing a very bulky amino acid building block is also described. The choice of suitable N-protections is discussed.     

In vivo metal-catalyzed SeCT therapy by a proapoptotic peptide

Selective cell tagging (SeCT) therapy is a strategy for labeling a targeted cell with certain chemical moieties via a catalytic chemical transformation in order to elicit a therapeutic effect. Herein, we report a cancer therapy based on targeted cell surface tagging with proapoptotic peptides (Ac-GGKLFG-X; X = reactive group) that induce apoptosis when attached to the cell surface. Using either Au-catalyzed amidation or Ru-catalyzed alkylation, these proapoptotic peptides showed excellent therapeutic effects both in vitro and in vivo. In particular, co-treatment with proapoptotic peptide and the carrier–Ru complex significantly and synergistically inhibited tumor growth and prolonged survival rate of tumor-bearing mice after only a single injection. This is the first report of Ru catalyst application in vivo, and this approach could be used in SeCT for cancer therapy.

The combination of a proapoptotic peptide with covalent tagging and a carrier-Ru-complex inhibited tumor growth in mice after a single injection.     

Enhancing proline-rich antimicrobial peptide action by homodimerization: influence of bifunctional linker

Antimicrobial peptides (AMPs) are host defense peptides, and unlike conventional antibiotics, they possess potent broad spectrum activities and, induce little or no antimicrobial resistance. They are attractive lead molecules for rational development to improve their therapeutic index. Our current studies examined dimerization of the de novo designed proline-rich AMP (PrAMP), Chex1-Arg20 hydrazide, via C-terminal thiol addition to a series of bifunctional benzene or phenyl tethers to determine the effect of orientation of the peptides and linker length on antimicrobial activity. Antibacterial assays confirmed that dimerization per se significantly enhances Chex1-Arg20 hydrazide action. Greatest advantage was conferred using perfluoroaromatic linkers (tetrafluorobenzene and octofluorobiphenyl) with the resulting dimeric peptides 6 and 7 exhibiting potent action against Gram-negative bacteria, especially the World Health Organization''s critical priority-listed multidrug-resistant (MDR)/extensively drug-resistant (XDR) Acinetobacter baumannii as well as preformed biofilms. Mode of action studies indicated these lead PrAMPs can interact with both outer and inner bacterial membranes to affect the membrane potential and stress response. Additionally, 6 and 7 possess potent immunomodulatory activity and neutralise inflammation via nitric oxide production in macrophages. Molecular dynamics simulations of adsorption and permeation mechanisms of the PrAMP on a mixed lipid membrane bilayer showed that a rigid, planar tethered dimer orientation, together with the presence of fluorine atoms that provide increased bacterial membrane interaction, is critical for enhanced dimer activity. These findings highlight the advantages of use of such bifunctional tethers to produce first-in-class, potent PrAMP dimers against MDR/XDR bacterial infections.

Homodimerization of a proline-rich antimicrobial peptide via bioconjugation to perfluoroaromatic linkers confers increased antimicrobial, antibiofilm and immunomodulatory activity. The dimers are promising new therapeutic leads against WHO priority multidrug resistant bacteria.     

Liposome fusion with orthogonal coiled coil peptides as fusogens: the efficacy of roleplaying peptides

Biological membrane fusion is a highly specific and coordinated process as a multitude of vesicular fusion events proceed simultaneously in a complex environment with minimal off-target delivery. In this study, we develop a liposomal fusion model system with specific recognition using lipidated derivatives of a set of four de novo designed heterodimeric coiled coil (CC) peptide pairs. Content mixing was only obtained between liposomes functionalized with complementary peptides, demonstrating both fusogenic activity of CC peptides and the specificity of this model system. The diverse peptide fusogens revealed important relationships between the fusogenic efficacy and the peptide characteristics. The fusion efficiency increased from 20% to 70% as affinity between complementary peptides decreased, (from K_F ≈ 10⁸ to 10⁴ M⁻¹), and fusion efficiency also increased due to more pronounced asymmetric role-playing of membrane interacting ‘K’ peptides and homodimer-forming ‘E’ peptides. Furthermore, a new and highly fusogenic CC pair (E₃/P1_K) was discovered, providing an orthogonal peptide triad with the fusogenic CC pairs P2_E/P2_K and P3_E/P3_K. This E₃/P1_k pair was revealed, via molecular dynamics simulations, to have a shifted heptad repeat that can accommodate mismatched asparagine residues. These results will have broad implications not only for the fundamental understanding of CC design and how asparagine residues can be accommodated within the hydrophobic core, but also for drug delivery systems by revealing the necessary interplay of efficient peptide fusogens and enabling the targeted delivery of different carrier vesicles at various peptide-functionalized locations.

We developed a liposomal fusion model system with specific recognition using a set of heterodimeric coiled coil peptide pairs. This study unravels important structure–fusogenic efficacy relationships of peptide fusogens.     

Catalyst-free arylation of sulfonamides via visible light-mediated deamination

A novel arylation of sulfonamides with boronic acids to afford numerous diaryl sulfones via a visible light-mediated N–S bond cleavage other than the typical transition-metal-catalyzed C(O)–N bond activation is described. This methodology, which represents the first catalyst-free protocol for the sulfonylation of boronic acids, is characterized by its simple reaction conditions, good functional group tolerance and high efficiency. Several successful examples for the late-stage functionalization of diverse sulfonamides indicate the high potential utility of this method in pharmaceutical science and organic synthesis.

The simple, catalyst-free sulfonylation of boronic acids with sulfonamides via a visible light-mediated N–S bond cleavage is described, affording diaryl sulfones with high efficiency. Late-stage functionalization of sulfonamide drugs was shown.     

Triazolinedione protein modification: from an overlooked off-target effect to a tryptophan-based bioconjugation strategy

Labelling of tyrosine residues in peptides and proteins has been reported to selectively occur via a ‘tyrosine-click’ reaction with triazolinedione reagents (TAD). However, we here demonstrate that TAD reagents are actually not selective for tyrosine and that tryptophan residues are in fact also labelled with these reagents. This off-target labelling remained under the radar as it is challenging to detect these physiologically stable but thermally labile modifications with the commonly used HCD and CID MS/MS techniques. We show that selectivity of tryptophan over tyrosine can be achieved by lowering the pH of the aqueous buffer to effect selective Trp-labelling. Given the low relative abundance of tryptophan compared to tyrosine in natural proteins, this results in a new site-selective bioconjugation method that does not rely on enzymes nor unnatural amino acids and is demonstrated for peptides and recombinant proteins.

A new strategy for selective tryptophan modification using triazolinedione (TAD) chemistry at pH 4 is shown on peptides and proteins. Additionally, off-target modification of tryptophan residues during the classical TAD-Y click reaction is uncovered.     

Construction of diverse peptide structural architectures via chemoselective peptide ligation

Herein, we report the development of a facile synthetic strategy for constructing diverse peptide structural architectures via chemoselective peptide ligation. The key advancement involved is to utilize the benzofuran moiety as the peptide salicylaldehyde ester surrogate, and Dap–Ser/Lys–Ser dipeptide as the hydroxyl amino functionality, which could be successfully introduced at the side chain of peptides enabling peptide ligation. With this method, the side chain-to-side chain cyclic peptide, branched/bridged peptides, tailed cyclic peptides and multi-cyclic peptides have been designed and successfully synthesized with native peptidic linkages at the ligation sites. This strategy has provided an alternative strategic opportunity for synthetic peptide development. It also serves as an inspiration for the structural design of PPI inhibitors with new modalities.

Methods of introducing peptide salicylaldehyde esters and hydroxyl amine functionality into the peptide side chain have been developed. Diverse peptide structural motifs were constructed via ligation with native amide linkages at the ligation sites.     

Conformational interplay in hybrid peptide–helical aromatic foldamer macrocycles

Macrocyclic peptides are an important class of bioactive substances. When inserting an aromatic foldamer segment in a macrocyclic peptide, the strong folding propensity of the former may influence the conformation and alter the properties of the latter. Such an insertion is relevant because some foldamer–peptide hybrids have recently been shown to be tolerated by the ribosome, prior to forming macrocycles, and can thus be produced using an in vitro translation system. We have investigated the interplay of peptide and foldamer conformations in such hybrid macrocycles. We show that foldamer helical folding always prevails and stands as a viable means to stretch, i.e. unfold, peptides in a solvent dependent manner. Conversely, the peptide systematically has a reciprocal influence and gives rise to strong foldamer helix handedness bias as well as foldamer helix stabilisation. The hybrid macrocycles also show resistance towards proteolytic degradation.

When peptides and helical aromatic foldamers are combined in a macrocycle, an interplay of their properties is observed, including helix handedness bias, helix stabilisation, peptide stretching and peptide resistance to proteolytic degradation.     

NHC and visible light-mediated photoredox co-catalyzed 1,4-sulfonylacylation of 1,3-enynes for tetrasubstituted allenyl ketones

The modulation of selectivity of highly reactive carbon radical cross-coupling for the construction of C–C bonds represents a challenging task in organic chemistry. N-Heterocyclic carbene (NHC) catalyzed radical transformations have opened a new avenue for acyl radical cross-coupling chemistry. With this method, highly selective cross-coupling of an acyl radical with an alkyl radical for efficient construction of C–C bonds was successfully realized. However, the cross-coupling reaction of acyl radicals with vinyl radicals has been much less investigated. We herein describe NHC and visible light-mediated photoredox co-catalyzed radical 1,4-sulfonylacylation of 1,3-enynes, providing structurally diversified valuable tetrasubstituted allenyl ketones. Mechanistic studies indicated that ketyl radicals are formed from aroyl fluorides via the oxidative quenching of the photocatalyst excited state, allenyl radicals are generated from chemo-specific sulfonyl radical addition to the 1,3-enynes, and finally, the key allenyl and ketyl radical cross-coupling provides tetrasubstituted allenyl ketones.

Unprecedented NHC and photocatalysis co-catalyzed radical 1,4-sulfonylacylation of 1,3-enynes has been realized, providing structurally diversified tetrasubstituted allenyl ketones via allenyl and ketyl radical cross-coupling.     

Solid-phase fluorescent BODIPY–peptide synthesis via in situ dipyrrin construction

Traditional fluorescent peptide chemical syntheses hinge on the use of limited fluorescent/dye-taggable unnatural amino acids and entail multiple costly purifications. Here we describe a facile and efficient protocol for in situ construction of dipyrrins on the N-terminus with 20 natural and five unnatural amino acids and the lysine''s side chain of selected peptides/peptide drugs through Fmoc-based solid-phase peptide synthesis. The new strategy enables the direct formation of boron–dipyrromethene (BODIPY)–peptide conjugates from simple aldehyde and pyrrole derivatives without pre-functionalization, and only requires a single-time chromatographic purification at the final stage. As a model study, synthesized EBNA1-targeting BODIPY1–Pep4 demonstrates intact selectivity in vitro, responsive fluorescence enhancement, and higher light cytotoxicity due to the photo-generation of cytotoxic singlet oxygen. This work offers a novel practical synthetic platform for fluorescent peptides for multifaceted biomedical applications.

Solid-phase fluorescent BODIPY–peptide synthesis via in situ dipyrrin construction offers an efficient fluorescent peptide synthetic platform for multifaceted biomedical applications.     

Spontaneous emergence of membrane-forming protoamphiphiles from a lipid–amino acid mixture under wet–dry cycles

Dynamic interplay between peptide synthesis and membrane assembly would have been crucial for the emergence of protocells on the prebiotic Earth. However, the effect of membrane-forming amphiphiles on peptide synthesis, under prebiotically plausible conditions, remains relatively unexplored. Here we discern the effect of a phospholipid on peptide synthesis using a non-activated amino acid, under wet–dry cycles. We report two competing processes simultaneously forming peptides and N-acyl amino acids (NAAs) in a single-pot reaction from a common set of reactants. NAA synthesis occurs via an ester–amide exchange, which is the first demonstration of this phenomenon in a lipid–amino acid system. Furthermore, NAAs self-assemble into vesicles at acidic pH, signifying their ability to form protocellular membranes under acidic geothermal conditions. Our work highlights the importance of exploring the co-evolutionary interactions between membrane assembly and peptide synthesis, having implications for the emergence of hitherto uncharacterized compounds of unknown prebiotic relevance.

Synthesis of lipoamino acids via ester–amide exchange under prebiotically plausible wet-dry cycling conditions that results in vesicles at acidic pH.     

Small peptide diversification through photoredox-catalyzed oxidative C-terminal modification

A photoredox-catalyzed oxidative decarboxylative coupling of small peptides is reported, giving access to a variety of N,O-acetals. They were used as intermediates for the addition of phenols and indoles, leading to novel peptide scaffolds and bioconjugates. Amino acids with nucleophilic side chains, such as serine, threonine, tyrosine and tryptophan, could also be used as partners to access tri- and tetrapeptide derivatives with non-natural cross-linking.

A photoredox approach for the generation of N-acyliminiums derived from peptides enabling diversification via Friedel–Crafts reactions.     

A novel tyrosine hyperoxidation enables selective peptide cleavage

A novel tyrosine hyperoxidation enabling selective peptide cleavage is reported. The scission of the N-terminal amide bond of tyrosine was achieved with Dess–Martin periodinane under mild conditions, generating a C-terminal peptide fragment bearing the unprecedented hyperoxidized tyrosine motif, 4,5,6,7-tetraoxo-1H-indole-2-carboxamide, along with an intact N-terminal peptide fragment. This reaction proceeds with high site-selectivity for tyrosine and exhibits broad substrate scope for various peptides, including those containing post-translational modifications. More importantly, this oxidative cleavage was successfully applied to enable sequencing of three naturally occurring cyclic peptides, including one depsipeptide and one lipopeptide. The linearized peptides generated from the cleavage reaction significantly simplify cyclic peptide sequencing by MS/MS, thus providing a robust tool to facilitate rapid sequence determination of diverse cyclic peptides containing tyrosine. Furthermore, the highly electrophilic nature of the hyperoxidized tyrosine unit disclosed in this work renders it an important electrophilic target for the selective bioconjugation or synthetic manipulation of peptides containing this unit.

A Tyr-selective peptide cleavage was reported using Dess–Martin periodinane. The cleavage generates an unprecedented hyperoxidized tyrosine motif in the C-terminal fragment and showed excellent site-specificity and broad scope for various peptides.     

An integrated platform approach enables discovery of potent,selective and ligand-competitive cyclic peptides targeting the GIP receptor

In any drug discovery effort, the identification of hits for further optimisation is of crucial importance. For peptide therapeutics, display technologies such as mRNA display have emerged as powerful methodologies to identify these desired de novo hit ligands against targets of interest. The diverse peptide libraries are genetically encoded in these technologies, allowing for next-generation sequencing to be used to efficiently identify the binding ligands. Despite the vast datasets that can be generated, current downstream methodologies, however, are limited by low throughput validation processes, including hit prioritisation, peptide synthesis, biochemical and biophysical assays. In this work we report a highly efficient strategy that combines bioinformatic analysis with state-of-the-art high throughput peptide synthesis to identify nanomolar cyclic peptide (CP) ligands of the human glucose-dependent insulinotropic peptide receptor (hGIP-R). Furthermore, our workflow is able to discriminate between functional and remote binding non-functional ligands. Efficient structure–activity relationship analysis (SAR) combined with advanced in silico structural studies allow deduction of a thorough and holistic binding model which informs further chemical optimisation, including efficient half-life extension. We report the identification and design of the first de novo, GIP-competitive, incretin receptor family-selective CPs, which exhibit an in vivo half-life up to 10.7 h in rats. The workflow should be generally applicable to any selection target, improving and accelerating hit identification, validation, characterisation, and prioritisation for therapeutic development.

mRNA display generates vast datasets of protein binders. Bioinformatic clustering of the sequences combined with high throughput synthesis and analysis methods allow efficient prioritisation of hits for in vivo experiments.

In recent years, peptides have emerged as powerful therapeutic agents¹ and the most prominent examples can match antibodies in selectivity and potency. This development has been greatly aided by breakthroughs in half life extension technologies by formulation or chemical modification. A key advantage of engineered peptides is their ease of production, and while antibodies are generally considered to be too large for efficient oral uptake in therapeutically relevant amounts, the small size of peptides allows the potential for oral dosing with significant progress made in recent years.^2,3 Despite these advantages, most drug development projects heavily rely on antibodies, most likely due to the ease of de novo antibody discovery by evolutionarily optimised methods. Modern in vivo or in vitro display technologies, such as phage display and mRNA display, allow for the identification of potent and specific peptide ligands through iterative screening of peptide pools containing trillions of randomised sequences.^4,5 These technologies have the potential to yield a plethora of de novo peptides which could be engineered into powerful therapeutics. However, these techniques are surprisingly scarce in clinical pipelines (compared to antibodies or small molecules) despite being known for decades and most peptides in late-stage discovery are derivatives of naturally occurring molecules.¹ Challenges in hit prioritisation and time-consuming validation could at least partly be responsible for this observation. While classical approaches would rely on Sanger sequencing of the most abundant clones (yielding very few hits), the advent of next generation sequencing (NGS) has revolutionized the information content available from de novo peptide screens.⁴ In the majority of studies, however, this information is unused and only a handful of the most abundant sequences are followed up by chemical synthesis for testing of biological activity.⁵ We rationalized that powerful bioinformatic data mining methods would best utilise the vast information contained in peptide display datasets. To prove this, we combined chemical synthesis and high throughput binding analysis on data from mRNA display. We show that these methods identify more diverse series of hits with different modes of binding as starting points for peptide drug discovery efforts, especially when a functional binder to a specific binding pocket is desired (Scheme 1).Open in a separate windowScheme 1Workflow of peptide hit identification, prioritisation, and validation.We chose the type 2 GPCR glucose-dependent insulinotropic peptide receptor (GIP-R) as a challenging and clinically relevant test case.⁶ Its natural ligand GIP and the close relative glucagon-like peptide 1 (GLP-1) are incretins, i.e., hormones which are secreted after oral nutrient uptake and which augment glucose-dependent insulin release from pancreatic beta cells.⁷ GLP-1 receptor agonists have been reported to not only have effects in metabolic disease through increasing insulin release, but also promote satiety by delaying gastric emptying, and are being investigated for their protective effects in obesity,^8,9 heart disease,¹⁰ and diabetic kidney disease.¹¹ Conversely, the biology of GIP is less clearly understood, and both GIP-R agonists¹² as well as antagonists^13,14 are under investigation as potential therapeutics in the fields of type 2 diabetes and obesity. Previous approaches to target the GIP-R thus far have made use of analogues of GIP itself,^15,16 or GIP R specific antibodies^14,17–20 and peptide display work was based on dual GIP-GLP-1 analogue libraries.^14,19,20 As such, the GIP-R represented an ideal target for our mRNA based workflow as we strived to identify potent, ligand competitive, and subfamily selective de novo binders, which could provide valuable tools to understand GIP-R biology and serve as starting points for drug discovery efforts.We employed mRNA display to identify cyclic peptide ligands towards the biotinylated extracellular domain (ECD) of the human GIP receptor (residues 22–138 of hGIP-R), which has been shown to retain binding to GIP.²¹ Initially, we established conditions for efficient cyclisation by disulphide bond formation for representative library peptides (Fig. S3^†), before conducting iterative rounds of mRNA display^5,22 using a nucleotide library encoding two conserved cysteines for macrocyclization flanking a region of 4 to 12 random amino acid sequence. Negative selections were performed against biotin-loaded streptavidin-functionalised magnetic beads, followed by positive selection against bead-immobilised hGIP-R ECD. Sufficient library enrichment was obtained for hGIP-R after five rounds of selections (percentage recovery relative to input > 0.1%) (Fig. S4a^†). NGS of enriched output cDNA was carried out and peptides were clustered by similarity for each selection round (R1–R5). All sequences exceeding six reads in R5 (the top 3160 sequences) were compared against each other using pairwise local alignments in order to derive a complete distance matrix. Single-linkage hierarchical clustering was then performed on the distance matrix, choosing a threshold for cutting the relative dendrogram into clusters based on a statistical optimality criterion.²³ For this dataset, the sequence similarity threshold for assigning a sequence to a cluster was set at 0.38, and the fewest number of members that a cluster was allowed to contain was 20. This generated 13 clusters (assigned letters A through M), where the largest cluster, A, contained 1880 sequences, while the two least populated clusters L and M contained 21 sequences each, and the unclustered sequences were assigned to a “noise” cluster (termed cluster 0), encompassing 263 sequences (Fig. 1).Open in a separate windowFig. 1High throughput pairwise clustering analysis of 3160 Round 5 output sequences selected against hGIP-R ECD. Lighter colour indicates closer similarity between individual sequences (ESI Data 1^†).In the next step, we selected peptides from each cluster (A-M, 0) based on sequence diversity and abundance in R5 for parallel solid-phase peptide synthesis (SPPS) in a 96 well format. The N-terminus was capped with an acetyl group (Ac) to mimic the fMet in mRNA-display and a C-terminal FLAG tag was added to each peptide to ensure peptide solubility in buffer by serving as a source of negative charge, analogous to C-terminal mRNA that is present during the selection screens.²⁴ Peptides were assigned identifiers with cluster ID and their rank order based on their abundance in R5 (e.g. B_5). Following synthesis and cleavage, the peptides were macrocyclized via disulphide bond formation in 20% DMSO in buffer.²⁵ Peptide identity, purity, and complete macrocyclization was confirmed by UPLC-MS and peptide concentrations were determined by UPLC-CAD (ESI Data 3^†). For the initial high throughput characterisation, peptides were used without further purification (average purity 50–70%). To establish the binding properties, we performed high-throughput single-concentration biolayer interferometry (BLI) measurements, using biotinylated hGIP-R ECD immobilised on streptavidin-functionalised biosensors. Select peptides with favourable binding potencies were resynthesized, purified and their K_d determined by multiple concentration BLI measurements (Fig. 2). The results were generally in good agreement with the single concentration data.Open in a separate windowFig. 2(a and b) Single concentration hGIP-R ECD binding K_d values and dissociation rates for the most abundant peptide clusters, and corresponding abundances/reads of peptide sequences in the final round of selection against GIP-R ECD. All peptides contain an N-terminal Ac group and C-terminal FLAG tag and are cyclised as disulphides. (c) Radiolabelled [¹²⁵I]-GIP displacement from hGIPR#5/BHK Creluc 2P cells by cyclic peptides at a single concentration of 1000 nM CPs (x axis), and 100 nM CPs (y axis) (CPM = counts per minute). All peptides were tested crude without prior purification (ESI Data 2^†).We identified several peptides (>50% of our panel) with nanomolar binding affinities toward the hGIP-R from multiple sequence clusters, particularly from the two most abundant clusters A and B (Fig. 2a and b). However, potent binders were also identified in less abundant sequence families. In particular, peptide M_46 was the second most potent binder amongst the resynthesized peptide panel, while only accumulating 0.2% of the reads compared to the most abundant peptide A_0. This highlights the benefit of following up on peptide hits independent of their ranking based on sequencing reads. Encouragingly, weak-to-no binding was observed from peptide members of cluster 0 (Fig. S8^†), corresponding to bioinformatic ‘noise’ in the NGS data, which likely consists of singleton sequences and possible sequencing errors. The FLAG tag itself did not bind to hGIP-R ECD. For members of clusters containing an internal cysteine, e.g. peptide C_24, we generated single cysteine-to-methionine mutants for each site (Met2, Met8, and Met13) and deduced the disulphide pattern from the binding data (i.e. a bond between Cys2 or Cys8 for C_24, see Fig. S8^†). In the spirit of the high throughput nature of the workflow, we did not follow up on peptides which failed during parallel synthesis, as the goal was the identification of one hit sequence. As a consequence, we suggest that the pursuit of peptides with complex folds or unpaired cysteines is only indicated if no functional hits can be identified otherwise.The non-biased selection process means that high affinity CPs can be generated against any sites on hGIP-R ECD. We hypothesized that each cluster represents peptides which bind at a specific target binding site, with the possibility of different clusters having overlapping or distinct binding sites on the receptor. In this study, we were interested in identifying inhibitory peptide families which are able to either compete or disrupt the natural ligand, whether through direct competition or allosteric binding. To investigate this, we selected representative CPs from each cluster and performed competitive displacement assays with ¹²⁵I-GIP in BHK CreLuc 2P cells stably expressing hGIP-R. Notably, this assay shows (competitive) binding to the full length GIP-R, whereas our selection and initial screen had been performed against the ECD. As shown in Fig. 2c, only peptides from clusters B and M were found to displace ¹²⁵I-GIP from hGIP-R (as measured at peptide concentrations of 100 nM and 1000 nM). Interestingly, cluster B contains a LWPF motif at the C-terminus, while cluster M has a related LPWF motif at the N-terminus, indicating a potentially conserved binding site. None of the members of other clusters were found to displace ¹²⁵I-GIP, even those which were determined to have nanomolar binding affinities to the hGIP R ECD by BLI, including the most abundant peptide family of the selection (cluster A), which covered over 50% of the whole dataset. This highlights the value of the clustering approach, as enrichment of the sequences during mRNA display is driven only by binding affinities of encoded CPs to hGIP-R ECD, but our subsequent clustering analyses served to reveal different functionalities of these CPs. Thus, the high-throughput prioritisation approach is likely to have a higher chance of identifying functional ligands, rather than those prioritised based on observed amounts of NGS reads.Encouraged by these results, we focussed our subsequent work on cluster B, namely the structurally most diverse members B_3, B_5, and B_68. B_68 had < 20% purity in the 96 well synthesis and did not reveal any binding in the initial high throughput binding assay but was included in the follow up. Further analysis of the sequence list showed another structurally diverse member of cluster B, B_1275, which we hypothesized would also exhibit potent binding and GIP competition, despite the low levels of sequencing reads. These four sequences were scaled up by traditional SPPS and further investigated in multi-concentration BLI and competitive binding assays. All purified peptides showed nanomolar binding and competition of the ¹²⁵I-GIP from BHK cells stably expressing hGIP-R (Fig. 4). Furthermore, none of these ligands were found to displace radiolabelled GLP-1 from GLP-1R, nor glucagon from GCG-R (Fig. S11^†). The parent CP sequences also do not bear any significant sequence homology with native GIP, nor any known interactors of hGIP-R, suggesting that these are the first reported examples of de novo incretin receptor selective and competitive peptides for hGIP-R.Open in a separate windowFig. 4(a) BLI binding K_d values and radiolabelled [¹²⁵I]-GIP displacement IC₅₀ data for purified peptides. Multiple concentrations of CPs were used to determine these values. (*) uncertain values due to limited solubility of CP. (b) Stability of selected peptides upon incubation with human plasma at 37 °C. (c) In vivo plasma exposure levels of selected peptides upon i.v. dosing to rats. All data are presented as mean ± SEM of three independent experiments.To optimise the biophysical and physicochemical properties of the CPs, and to identify a suitable attachment point for half-life extending moieties, such as albumin binders, we sought to establish a detailed structure–activity relationship (SAR) on cluster B. We chose the most and least abundant of our cluster B lead peptides (B_3 and B_1275) as starting points for full amino acid mutation scans.^24,26 We focussed on natural amino acids only to keep the option for (semi-)recombinant expression of the compounds, should large amounts be needed for further development stages. Single mutation variant peptides were synthesised in a 96 well format for each amino acid in the variable region contained between the two conserved cysteine residues C2 and C13. Methionine and cysteine were not included in the mutational scan due to potential oxidation, and asparagine was not included due to the risk of deamidation and isomerisation to iso-aspartate. Additionally, scrambled peptides were included in the panel. The binding affinities of these mutant peptides to hGIP-R ECD were then determined by single concentration BLI experiments.As shown in Fig. 3a and b, the consensus sequence of cluster B (positions 9–12, LWPF) was found to be the most intolerant to mutation, confirming this region to be critical for binding. Proline at position 11 was found to be completely intolerant to replacement by any other amino acid, suggesting that this residue is critical for maintaining conformationally restricted binding interface of CPs. While residues 9–12 are hydrophobic in both parent peptides, it is unlikely that the interaction of the peptides to hGIP-R ECD is unspecific, as none of the scrambled peptides (e.g. LWPF) were found to bind, including the transposed mutants of B_3 and B_1275, with interchanged L9 and W10. The data shows some general trends for both B_3 and B_1275. In general, positions 3 and 6 (both F in the parent peptides) favour aromatic residues, position 8 favours aliphatic residues, while residues 4, 5, and 7 are fairly tolerant to mutation. The single amino acid scan did not reveal any substitutions that led to remarkable improvements in binding, suggesting that these peptides may either already be optimised for binding through several rounds of mRNA-display selection, or that further binding improvements would only be achieved by synergistic action of multiple substituted residues. The latter could ideally be investigated by mRNA display based affinity maturation experiments in follow up studies.^24,26 To gain insight into the binding mode of CPs to hGIP-R on the atomistic level we employed a two-step modelling protocol that first generates multiple conformations of the complex and then selects the final conformation based on stability (for details, see ESI^†). As our peptides were demonstrated to be ¹²⁵I-GIP-competitive, we directly folded them inside the GIP binding site of hGIP-R using Rosetta (Fig. 3c and d; results for B_1275 shown). Briefly, each of the four crucial residues according to the SAR data (Leu9, Trp10, Pro11 or Phe12) was placed in the GIP binding site and the rest of the peptide was grown around it. The obtained conformations (24 000 in total) were further clustered and the representative poses of the four most populated clusters were submitted to molecular dynamics (MD) simulations (600 ns for each pose) to assess their stability. The model with the lowest L_RMSD (Root Mean Square Deviation of the ligand backbone during MD trajectory) was selected for further characterization (Fig. 3c). In this model, the C-terminal half of the peptide faces the GIP binding site forming most of the interactions with hGIP-R. This is in line with the obtained SAR data that the C-terminal region is in general less tolerant to mutagenesis compared to the N-terminus. Furthermore, the Trp10 residue overlaps with the position of Phe22 in GIP which is known to be crucial for its binding to the receptor (Fig. 3d). Finally, overall conformation of the peptide resembles that of a hairpin with the turn located on Phe6 and His7, which are the only two positions (together with the peptide termini, see below) tolerating mutagenesis to a proline.Open in a separate windowFig. 3Heat maps showing K_d (nM) values for binding of single-residue mutant peptides derived from B3 (a) and B1275 (b) to biotinylated hGIP-R ECD as determined by single-concentration BLI. The columns indicate the amino acid changes compared to the parent sequence (displayed at the top of the table). All peptides featured a C-terminal Cys and were tested in crude form. Peptides that were not tested or those where synthesis failed are marked as grey boxes. None of the scrambled mutant peptides showed any binding. (c) Atomistic model of the cyclic peptide B_1275 and GIP receptor complex suggested by molecular modelling. The LWPF sequence of the peptide is shown in red, while the rest of the molecule is coloured by the atom name (carbon in yellow, nitrogen in blue, oxygen in red, and sulphur in orange). The receptor is show in grey; the binding site residues (L35, W39, M67 and Y87) are highlighted in green. (d) Overlay of the crystal structure of GIP complexed with hGIP receptor (PDB 2QKH) and atomistic model of cyclic peptide B_1275 in the binding site of the GIP receptor. Note that the positions of W10 of the cyclic peptide and F22 of GIP overlap.Next, we wanted to use the accrued binding and SAR information to design peptides for in vivo studies. For these, peptides need to fulfil two crucial requirements. Firstly, they need to be stable in plasma and secondly, they need to be protected from renal clearance. The latter is crucial to the development of any peptide therapeutic as even protease resistant peptides usually exhibit plasma half lives in the range of a few minutes. Attachment of a fatty acid-based albumin binder (termed protractor) is one of the most employed strategies to extend peptide half-life and we opted for a 2xOEG-gGlu-C18 diacid albumin binder for our peptide hits.^3,27–29 The binding model suggested that the addition of an albumin binding moiety is tolerated at the N- or C-terminus, as these are solvent exposed. Further evidence that replacement of the Met in position 1 with Ala is possible for both B_3 and B_1275 without loss of affinity and the ease of synthesis prompted us to design four peptides with a protractor attached to the N-terminus (B_3.1, B_5.1, B_68.1 and B_1275.1). Furthermore, we synthesized linear versions of the parent peptides in order to investigate the importance of macrocyclization on binding and stability (B_3.2, B_5.2, B_68.2 and B_1275.2). Additionally, we synthesized peptides without FLAG tag to prove that binding indeed is mediated by the macrocycle and not the tag (B_1275.3). During the purification of the latter, we realized that solubility was low (as expected from the rather hydrophobic sequence), complicating purification and analysis. Thus, we utilized our SAR understanding to design variants with improved biophysical properties by reducing the hydrophobicity and increasing the charge at positions where mutation was tolerated (especially positions 1, 4 and 7). Furthermore, we replaced the Met residues to avoid oxidation of the sulphur atom. We focussed on B_1275 as we anticipated from the sequence that this peptide is most hydrophilic, as also indicated by the shortest retention time in HPLC chromatography (Table S1^†). All resulting peptides containing a FLAG tag (B_1275.7 through B_1275.11) were soluble at the relevant conditions, however, only B_1275.5 showed high solubility without the tag, as measured by nephelometry at different PEG concentrations (Fig. S14^†). Pleasingly, addition of an albumin binder (B_1275.6) did not negatively affect solubility. Analysis of the peptides by multi-concentration BLI and radio-GIP displacement showed that indeed N-terminal modification was allowed for most peptides (Fig. 4). None of the linear variants (B_3.2, B_5.2, B_68.2, B_1275.2) showed binding, suggesting that macrocyclization is crucial for the interaction. Most mutant peptides, however, retained binding, including the triple mutant B_1275.9 (M1A, T4R, H7E; K_d at 31 nM) and quadruple mutants B_1275.7 (M1A, F3W, H5E, H7E, K_d at 101 nM) and B_1275.8 (M1S, T4R, H5A, H7Q, K_d at 184 nM), showing the power of having detailed SAR information available for multi-factorial optimisation of hits. Interestingly, in contrast to the binding data obtained from F11P mutants from high throughput mutagenesis studies (Fig. 3), B_1275.10 with a F11P substitution did not show any binding, which exemplifies the limits of high throughput SAR analysis using crude peptides, as the terminal P possibly interferes with cyclisation and might lead to multi- or polymers which interfere with the BLI assay.Finally, we tested a panel of the optimised peptides for stability in human plasma and determined in vivo pharmacokinetic parameters in rats. Most cyclic peptides (B_3.1, B_1275.3, B_1275.4, B_1275.5 and B_1275.6) showed no decrease in plasma stability over the course of 5 h, including in the presence of a FLAG tag or of a protractor (Fig. 4b). While parent B_1275 had a t_1/2 of 3.5 h, the two linear peptides tested (B_3.2 and B_1275.2) exhibited low levels of degradation (t_1/2ca. 2 h, see Table S2 in ESI^† for exact values), and the biologically active linear peptides of both GIP and GLP-1 were more rapidly degraded (t_1/2ca. 35–45 min), which highlights the benefits of cyclic peptides in terms of druggability. Having established the plasma stability, we turned our attention to the in vivo half-life. We chose two protracted parent peptides (B_3.1 and B_1275.1) and the optimised B_1275 variants with FLAG tag (B_1275.4), without FLAG tag (B_1275.5) and with protractor but without FLAG tag (B_1275.6). The two non-protracted peptides exhibit very short half-lives (B_1275.4 = 3.5 min, B_1275.5 = 1.8 min), whereas the peptides carrying an albumin binder show significant plasma exposure over a long period of time (t_1/2, B_3.1 = 9.8 h,B_1275.1 = 10.7 h B_1275.6 = 4.2 h), with half-lives being increased by more than 100 fold (Fig. 4d) (as a comparison, the GLP-1 analogue semaglutide, which is dosed once-weekly in humans has a t_1/2 in rats of 7 h.)²⁸     

An evolution-inspired strategy to design disulfide-rich peptides tolerant to extensive sequence manipulation

Natural disulfide-rich peptides (DRPs) are valuable scaffolds for the development of new bioactive molecules and therapeutics. However, there are only a limited number of topologically distinct DRP folds in nature, and most of them suffer from the problem of in vitro oxidative folding. Thus, strategies to design DRPs with new constrained topologies beyond the scope of natural folds are desired. Herein we report a general evolution-inspired strategy to design new DRPs with diverse disulfide frameworks, which relies on the incorporation of two cysteine residues and a random peptide sequence into a precursor disulfide-stabilized fold. These peptides can spontaneously fold in redox buffers to the expected tricyclic topologies with high yields. Moreover, we demonstrated that these DRPs can be used as templates for the construction of phage-displayed peptide libraries, enabling the discovery of new DRP ligands from fully randomized sequences. This study thus paves the way for the development of new DRP ligands and therapeutics with structures not derived from natural DRPs.

A general method was developed to design multicyclic peptides with diverse disulfide frameworks amenable to random peptide library design, enabling the development of new disulfide-rich peptide ligands and therapeutics with structures not derived from natural peptides.     

Late-stage modification of peptides and proteins at cysteine with diaryliodonium salts

The modification of peptides and proteins has emerged as a powerful means to efficiently prepare high value bioconjugates for a range of applications in chemical biology and for the development of next-generation therapeutics. Herein, we report a novel method for the chemoselective late-stage modification of peptides and proteins at cysteine in aqueous buffer with suitably functionalised diaryliodonium salts, furnishing stable thioether-linked synthetic conjugates. The power of this new platform is showcased through the late-stage modification of the affibody zEGFR and the histone protein H2A.

New operationally simple platform for the chemoselective arylation of cysteine in peptides and proteins to access a variety of high value bioconjugates.