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Transpeptidases are powerful tools for protein engineering but are largely restricted to acting at protein backbone termini. Alternative enzymatic approaches for internal protein labelling require bulky recognition motifs or non-proteinogenic reaction partners, potentially restricting which proteins can be modified or the types of modification that can be installed. Here we report a strategy for labelling lysine side chain ε-amines by repurposing an engineered asparaginyl ligase, which naturally catalyses peptide head-to-tail cyclization, for versatile isopeptide ligations that are compatible with peptidic substrates. We find that internal lysines with an adjacent leucine residue mimic the conventional N-terminal glycine–leucine substrate. This dipeptide motif enables efficient intra- or intermolecular ligation through internal lysine side chains, minimally leaving an asparagine C-terminally linked to the lysine side chain via an isopeptide bond. The versatility of this approach is demonstrated by the chemoenzymatic synthesis of peptides with non-native C terminus-to-side chain topology and the conjugation of chemically modified peptides to recombinant proteins.

Chemoenzymatic protein and peptide modification is a powerful means of generating defined, homogeneous conjugates for a range of applications. However, the use of transpeptidases is limited by the need to prepare synthetic peptide conjugates to be ligated, bulky recognition tags remaining in the product, and inefficient substrate turnover. Here, we report a peptide/protein labeling strategy that utilizes a promiscuous, engineered transpeptidase to irreversibly incorporate diverse, commercially available amines at a C-terminal asparagine. To demonstrate the utility of this approach, we prepare a protein–drug conjugate, generate a genetically inaccessible C-to-C protein fusion, and site specifically label both termini of a single protein in sequential steps.

Voltage-gated sodium (NaV) channels are pore-forming transmembrane proteins that play essential roles in excitable cells, and they are key targets for antiepileptic, antiarrhythmic, and analgesic drugs. We implemented a heterobivalent design strategy to modulate the potency, selectivity, and binding kinetics of NaVchannel ligands. We conjugated μ-conotoxin KIIIA, which occludes the pore of the NaVchannels, to an analogue of huwentoxin-IV, a spider-venom peptide that allosterically modulates channel gating. Bioorthogonal hydrazide and copper-assisted azide–alkyne cycloaddition conjugation chemistries were employed to generate heterobivalent ligands using polyethylene glycol linkers spanning 40–120 Å. The ligand with an 80 Å linker had the most pronounced bivalent effects, with a significantly slower dissociation rate and 4–24-fold higher potency compared to those of the monovalent peptides for the human NaV1.4 channel. This study highlights the power of heterobivalent ligand design and expands the repertoire of pharmacological probes for exploring the function of NaVchannels.

Structure–activity relationships for a series of small-molecule thiophenes resulted in potent and selective antagonism of human Complement C3a receptor. The compounds are about 100-fold more potent than the most reported antagonist SB290157. A new compound JR14a was among the most potent of the new antagonists in vitro, assessed by (a) inhibition of intracellular calcium release (IC5010 nM) induced in human monocyte-derived macrophages by 100 nM C3a, (b) inhibition of β-hexosaminidase secretion (IC508 nM) from human LAD2 mast cells degranulated by 100 nM C3a, and (c) selectivity for human C3aR over C5aR. JR14a was metabolically stable in rat plasma and in rat liver microsomes and efficacious in rats when given orally to suppress rat paw inflammation, macrophage and mast cell activation, and histopathology induced by intraplantar paw administration of a C3aR agonist. Potent C3aR antagonists are now available for interrogating C3a receptor activation and suppressing C3aR-mediated inflammation in mammalian physiology and disease.

Cyclotides are cysteine-rich plant-derived peptides composed of 28–37 amino acids with a head-to-tail cyclic backbone and a knotted arrangement of three conserved disulfide bonds. Their beneficial biophysical properties make them promising molecules for pharmaceutical and agricultural applications. The Violaceae plant family is the major cyclotide-producing family, and to date, every examined plant from this family has been found to contain cyclotides. The presence of cyclotides in Viola communiswas inferred by mass spectroscopy previously, but their sequences and properties had yet to be explored. In this study, the occurrence of cyclotides in this plant was investigated using proteomics and transcriptomics. Twenty cyclotides were identified at the peptide level, including two new members from the bracelet (Vcom1) and Möbius (Vcom2) subfamilies. Structural analysis of these newly identified peptides demonstrated a similar fold compared with cyclotides from the same respective subfamilies. Biological assays of Vcom1 and Vcom2 revealed them to be cytotoxic to Sf9 insect cell lines, with Vcom1 demonstrating higher potency than Vcom2. The results suggest that they could be further explored as insecticidal agents and confirm earlier general findings that bracelet cyclotides have more potent insecticidal activity than their Möbius relatives. Seven new cyclotide-like sequences were observed in the transcriptome of V. communis, highlighting the Violaceae as a rich source for new cyclotides with potential insecticidal activity. An analysis of sequences flanking the cyclotide domain in the various precursors from V. communisand other Violaceae plants revealed new insights into cyclotide processing and suggested the possibility of two alternative classes of N-terminal processing enzymes for cyclotide biosynthesis.

We report the chemical synthesis of scorpion toxin Cn2, a potent and highly selective activator of the human voltage-gated sodium channel NaV1.6. In an attempt to decouple channel activation from channel binding, we also synthesized the first analogue of this toxin, Cn2[E15R]. This mutation caused uncoupling of the toxin’s excitatory and depressant activities, effectively resulting in a NaV1.6 inhibitor. In agreement with the in vitro observations, Cn2[E15R] is antinociceptive in mouse models of NaV1.6-mediated pain.

Protein ligases of defined substrate specificity are versatile tools for protein engineering. Upon completion of the reaction, the products of currently reported protein ligases contain the amino acid sequence that is recognized by that same ligase, resulting in repeated cycles of ligation and hydrolysis as competing reactions. Thus, previous efforts to sequentially label proteins at distinct positions required ligases of orthogonal specificity. A recombinant Oldenlandia affinisasparaginyl endopeptidase, OaAEP1, is promiscuous for incoming nucleophiles. This promiscuity enabled us to define a nucleophile composed of natural amino acids that is ligated efficiently to the substrate yet yields a product that is poorly recognized by OaAEP1. Proteins modified with an efficient recognition module could be readily modified to yield a defined product bearing a cleavage-resistant motif, whereas proteins containing this inferior recognition motif remained essentially unmodified. We demonstrate the versatility of the N- or C-terminal protein modifications obtainable with this approach and modify the N- and C-termini of a single substrate protein in a sequential, site-specific manner in excellent yield.

Antifibrinolytic drugs provide important pharmacological interventions to reduce morbidity and mortality from excessive bleeding during surgery and after trauma. Current drugs used for inhibiting the dissolution of fibrin, the main structural component of blood clots, are associated with adverse events due to lack of potency, high doses, and nonselective inhibition mechanisms. These drawbacks warrant the development of a new generation of highly potent and selective fibrinolysis inhibitors. Here, we use the 14-amino acid backbone-cyclic sunflower trypsin inhibitor-1 scaffold to design a highly potent (Ki= 0.05 nM) inhibitor of the primary serine protease in fibrinolysis, plasmin. This compound displays a million-fold selectivity over other serine proteases in blood, inhibits fibrinolysis in plasma more effectively than the gold-standard therapeutic inhibitor aprotinin, and is a promising candidate for development of highly specific fibrinolysis inhibitors with reduced side effects.

Many naturally occurring peptides have poor proteolytic stability, which limits their therapeutic applications. Cyclotides are plant-derived cyclic peptides that resist proteolysis due to their highly constrained structure, comprising a head-to-tail cyclic backbone and three disulfide bonds that form a cystine-knotted core. This structure makes them useful as scaffolds onto which peptide sequences (epitopes) can be grafted. In this study, VHH7, an alpaca-derived nanobody that targets murine class II MHC molecules, was used for the targeted delivery of cyclotides to antigen-presenting cells (APCs). The cyclotides MCoTI-I, and MCoTI-I with a HA-tag (YPYDVPDYA) grafted into loop 6 (MCoTI-HA), were tested for immunogenic properties. To produce the requisite VHH7-peptide conjugates, a site-specific sortase A-catalyzed reaction in combination with a copper-free strain-promoted cycloaddition reaction was used. MCoTI-I alone did not display any obvious antibody response, thus showing the capacity of cyclotides as immunologically silent scaffolds. By contrast, MCoTI-I conjugated to VHH7 elicited antibodies against cyclic or linear MCoTI-I, thus suggesting a simple and robust approach for targeting cyclotides to APCs, and potentially to other cell types. A similar antibody response was observed when MCoTI-HA was conjugated to VHH7, but there was no reactivity toward a linear HA-tag itself, suggesting differences in conformational constraint between cyclotide-presented and linear epitopes. Studies of commercially available HA antibodies applied to MCoTI-HA confirmed that the conformation of peptide immunogens affects their reactivity. Thus, the production of antibodies that recognize constrained epitopes may benefit from engraftment onto scaffolds such as cyclotides. More broadly, this study validates that a prototypic cyclotide, a member of a peptide family that has proven to be useful as drug design scaffolds in many other studies, can efficiently reach a specific target in vivo.

The interaction between the cell–cell adhesion proteins CD2 and CD58 plays a crucial role in lymphocyte recruitment to inflammatory sites, and inhibitors of this interaction have potential as immunomodulatory drugs in autoimmune diseases. Peptides from the CD2 adhesion domain were designed to inhibit CD2:CD58 interactions. To improve the stability of the peptides, β-sheet epitopes from the CD2 region implicated in CD58 recognition were grafted into the cyclic peptide frameworks of sunflower trypsin inhibitor and rhesus theta defensin. The designed multicyclic peptides were evaluated for their ability to modulate cell–cell interactions in three different cell adhesion assays, with one candidate, SFTI-a, showing potent activity in the nanomolar range (IC50: 51 nM). This peptide also suppresses the immune responses in T cells obtained from mice that exhibit the autoimmune disease rheumatoid arthritis. SFTI-a was resistant to thermal denaturation, as judged by circular dichroism spectroscopy and mass spectrometry, and had a half-life of ∼24 h in human serum. Binding of this peptide to CD58 was predicted by molecular docking studies and experimentally confirmed by surface plasmon resonance experiments. Our results suggest that cyclic peptides from natural sources are promising scaffolds for modulating protein–protein interactions that are typically difficult to target with small-molecule compounds.

ABSTRACTThe plant defensin NaD1 is a potent antifungal molecule that also targets tumor cells with a high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity against fungi and no activity against tumor cells. All plant defensins have a common tertiary structure known as a cysteine-stabilized α-β motif which consists of an α helix and a triple-stranded β-sheet stabilized by four disulfide bonds. The chimeras were produced by replacing loops 1 to 7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and is essential for the potent cytotoxic effect of NaD1 on tumor cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P2binding and the tumor cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules, even though they bound poorly to PI(4,5)P2, suggesting that additional mechanisms operate against fungal cells. Unexpectedly, the loop 1B swap chimera was 10 times more active than NaD1 against filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to that of the natural variants.

Scorpion α-toxins are invaluable pharmacological tools for studying voltage-gated sodium channels, but few structure–function studies have been undertaken due to their challenging synthesis. To address this deficiency, we report a chemical engineering strategy based upon native chemical ligation. The chemical synthesis of α-toxin OD1 was achieved by chemical ligation of three unprotected peptide segments. A high resolution X-ray structure (1.8 Å) of synthetic OD1 showed the typical βαββ α-toxin fold and revealed important conformational differences in the pharmacophore region when compared with other α-toxin structures. Pharmacological analysis of synthetic OD1 revealed potent α-toxin activity (inhibition of fast inactivation) at Nav1.7, as well as Nav1.4 and Nav1.6. In addition, OD1 also produced potent β-toxin activity at Nav1.4 and Nav1.6 (shift of channel activation in the hyperpolarizing direction), indicating that OD1 might interact at more than one site with Nav1.4 and Nav1.6. Investigation of nine OD1 mutants revealed that three residues in the reverse turn contributed significantly to selectivity, with the triple OD1 mutant (D9K, D10P, K11H) being 40-fold more selective for Nav1.7 over Nav1.6, while OD1 K11V was 5-fold more selective for Nav1.6 than Nav1.7. This switch in selectivity highlights the importance of the reverse turn for engineering α-toxins with altered selectivity at Navsubtypes.

Going pro: The first facile Pro–Cys ligation using a preformed prolyl selenoester is reported (see scheme; P=peptide). In a comparative study of peptide selenoesters in native chemical ligation peptide α‐selenoesters are shown to be superior acyl donors and result in rate enhancements of at least two orders of magnitude when compared to the well‐established peptide α‐thioesters. This method permits rapid chemical ligation even at previously intractable sites, such as Pro‐Cys.

Pro‐blemlos: Die Pro‐Cys‐Ligation mit einem zuvor gebildeten Prolylselenoester wird vorgestellt (siehe Schema; P=Peptid). In einer vergleichenden Studie erwiesen sich Peptid‐α‐Selenoester als die besten Acyldonoren; sie steigerten die Reaktionsgeschwindigkeit gegenüber den etablierten Peptid‐α‐Thioestern um zwei Größenordnungen. Damit gelingt die Ligation auch an Stellen wie Pro‐Cys, für die bisher keine Methode verfügbar war.

Factor XIIa (FXIIa) is a promising target for developing new drugs that prevent thrombosis without causing bleeding complications. A native cyclotide (MCoTI-II) is gaining interest for engineering FXIIa-targeted anticoagulants as this peptide inhibits FXIIa but not other coagulation proteases. Here, we engineered the native biosynthetic cyclization loop of MCoTI-II (loop 6) to generate improved FXIIa inhibitors. Decreasing the loop length led to gains in potency up to 7.7-fold, with the most potent variant having five residues in loop 6 (Ki= 25 nM). We subsequently examined sequence changes within loop 6 and an adjacent loop, with substitutions at P4 and P2′ producing a potent FXIIa inhibitor (Ki= 2 nM) that displayed more than 700-fold selectivity, was stable in human serum, and blocked the intrinsic coagulation pathway in human plasma. These findings demonstrate that engineering the biosynthetic cyclization loop can generate improved cyclotide variants, expanding their potential for drug discovery.

In this work, cysteine staples were used as a late-stage functionalization strategy to diversify peptides and build conjugates targeting the melanocortin G-protein-coupled receptors [melanocortin receptor-1 (MC1R) and MC3R–MC5R]. Monocyclic and bicyclic agonists based on sunflower trypsin inhibitor-1 were used to generate a selection of stapled peptides that were evaluated for binding (pKi) and functional activation (pEC50) of the melanocortin receptor subtypes. Stapled peptides generally had improved activity, with aromatic stapled peptides yielding selective MC1R agonists, including a xylene-stapled peptide (2) with an EC50of 1.9 nM for MC1R and >150-fold selectivity for MC3R and MC4R. Selected stapled peptides were further functionalized with linkers and payloads, generating a series of conjugated peptides with potent MC1R activity, including one pyridazine-functionalized peptide (21) with picomolar activity at MC1R (Ki58 pM; EC50< 9 pM). This work demonstrates that staples can be used as modular synthetic tools to tune potency and selectivity in peptide-based drug design.

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Prenylated proteins with non‐native functionalities are generally very difficult to obtain by recombinant or enzymatic means. The semisynthesis of preparative amounts of prenylated Rab guanosine triphosphatases (GTPases) from recombinant proteins and synthetic prenylated peptides depends largely on the availability of functionalised prenylated peptides corresponding to the proteins' native structure or modifications thereof. Here, we describe and compare solution‐phase and solid‐phase strategies for the generation of peptides corresponding to the prenylated C terminus of Rab7 GTPase. The solid‐phase with utilisation of a hydrazide linker emerges as the more favourable approach. It allows a fast and practical synthesis of pure peptides and gives a high degree of flexibility in their modification. To facilitate the analysis of semisynthetic proteins, the synthesised peptides were equipped with a fluorescent group. Using the described approach, we introduced fluorophores at several different positions of the Rab7 C terminus. The position of the incorporated fluorescent groups in the peptides did not influence the protein‐ligation reaction, as the generated peptides could be ligated onto thioester‐tagged Rab7. However, it was found that the positioning of the fluorescent group had an influence on the functionality of the Rab7 proteins; analysis of the interaction of the semisynthetic Rab7 proteins with REP (Rab escort protein) and GDI (guanosine diphosphate dissociation inhibitor) molecules revealed that modification of the peptide side chains or of the C‐terminal isoprenoid did not significantly interfere with complex formation. However, functionalisation of the C terminus was found to have an adverse effect on complex formation and stability, possibly reflecting low structural flexibility of the Rab GDI/REP molecules in the vicinity of the lipid‐binding site.

We have designed a new class of highly potent bivalent melanocortin receptor ligands based on the nature-derived bicyclic peptide sunflower trypsin inhibitor 1 (SFTI-1). Incorporation of melanotropin pharmacophores in each of the two turn regions of SFTI-1 resulted in substantial gains in agonist activity particularly at human melanocortin receptors 1 and 3 (hMC1R/hMC3R) compared to monovalent analogues. In in vitrobinding and functional assays, the most potent molecule, compound 6, displayed low picomolar agonist activity at hMC1R (pEC50> 10.3; EC50< 50 pM; pKi: 10.16 ± 0.04; Ki: 69 ± 5 pM) and is at least 30-fold more selective for this receptor than for hMC3R, hMC4R, or hMC5R. The results are discussed in the context of structural homology models of hMCRs in complex with the developed bivalent ligands.

For Abstract see ChemInform Abstract in Full Text.