Semisynthesis of segmentally isotope-labeled and site-specifically palmitoylated CD44 cytoplasmic tail.

CD44, a ubiquitously expressed transmembrane receptor, plays a crucial role in cell growth, migration, and tumor progression. Dimerization of CD44 is a key event in signal transduction and has emerged as a potential target for anti-tumor therapies. Palmitoylation, a posttranslational modification, disrupts CD44 dimerization and promotes CD44 accumulation in ordered membrane domains. However, the effects of palmitoylation on the structure and dynamics of CD44 at atomic resolution remain poorly understood. Here, we present a semisynthetic approach combining solid-phase peptide synthesis, recombinant expression, and native chemical ligation to investigate the impact of palmitoylation on the cytoplasmic domain (residues 669-742) of CD44 (CD44ct) by NMR spectroscopy. A segmentally isotope-labeled and site-specifically palmitoylated CD44 variant enabled NMR studies, which revealed chemical shift perturbations and indicated local and long-range conformational changes induced by palmitoylation. The long-range effects suggest altered intramolecular interactions and potential modulation of membrane association patterns. Semisynthetic, palmitoylated CD44ct serves as the basis for studying CD44 clustering, conformational changes, and localization within lipid rafts, and could be used to investigate its role as a tumor suppressor and to explore its therapeutic potential.

Reflections on Navigating a Pandemic: Perspectives from the Chemical Biology Community.

The early-career researchers showcased in this ChemBioTalents special collection, and many others who have established their independent scientific careers over the last three years, have experienced a unique set of circumstances. The Covid-19 pandemic necessitated new forms of communication and interpersonal interactions: From online interviews and virtual networking to relocating and establishing labs during a pandemic, we faced many challenges, but also unexpected opportunities. In this perspective, we reflect on this unique and formative time through personal anecdotes and viewpoints, trying to capture diverse experiences from the Chemical Biology community and beyond. We have tried to get a broad and varied set of perspectives, however, the selection is biased towards researchers who were able to start their independent careers.1.

Synthesis and anti-parasitic activity of achiral N-benzylated phosphoramidic acid derivatives.

Synthetic pathways have been developed to access a series of N-benzylated phosphoramidic acid derivatives as novel, achiral analogues of the established Plasmodium falciparum 1-deoxy-d-xylulose-5-phosphate reductase (PfDXR) enzyme inhibitor, FR900098. Bioassays of the targeted compounds and their synthetic precursors have revealed minimal antimalarial activity but encouraging anti-trypanosomal activity - in one case with an IC50 value of 5.4 µM against Trypanosoma brucei, the parasite responsible for Nagana (African cattle sleeping sickness). The results of relevant in silico modelling and docking studies undertaken in the design and evaluation of these compounds are discussed.

Tumor-Targeting Immune System Engagers (ISErs) Activate Human Neutrophils after Binding to Cancer Cells.

Immune system engagers (ISErs) make up a new class of immunotherapeutics against cancer. They comprise two or more tumor-targeting peptides and an immune-stimulating effector peptide connected by inert polymer linkers. They are produced by solid phase peptide synthesis and share the specific targeting activities of antibodies (IgGs) but are much smaller in size and exploit a different immune-stimulating mechanism. Two ISErs (Y-9 and Y-59) that bind to the cancer cell markers integrin α3 and EphA2, respectively, are analyzed here with respect to their immune cell stimulation. We have previously shown that they activate formyl peptide receptors on myeloid immune cells and induce respiratory burst in neutrophils and myeloid chemotaxis in solution. It remained, however, unclear whether these molecules can stimulate immune cells while bound to tumor cells, an essential step in the hypothesized mode of action. Here, we demonstrate that ISEr Y-9 induced respiratory burst and caused a change in the shape of neutrophils when bound to the surface of protein A beads as a model of tumor cells. More importantly, tumor cell lines carrying receptor-bound Y-9 or Y-59 also activated neutrophils, evidenced by a significant change in shape. Interestingly, similar activation was induced by the supernatants of the cells incubated with ISEr, indicating that ISErs released from tumor cells, intact or degraded into fragments, significantly contributed to immune stimulation. These findings provide new evidence for the mode of action of ISErs, namely by targeting cancer cells and subsequently provoking an innate immune response against them.

Random coil shifts of posttranslationally modified amino acids.

Most eukaryotic proteins are modified during and/or after translation, regulating their structure, function and localisation. The role of posttranslational modifications (PTMs) in both normal cellular processes and in diseases is already well recognised and methods for detection of PTMs and generation of specifically modified proteins have developed rapidly over the last decade. However, structural consequences of PTMs and their specific effects on protein dynamics and function are not well understood. Furthermore, while random coil NMR chemical shifts of the 20 standard amino acids are available and widely used for residue assignment, dihedral angle predictions and identification of structural elements or propensity, they are not available for most posttranslationally modified amino acids. Here, we synthesised a set of random coil peptides containing common naturally occurring PTMs and determined their random coil NMR chemical shifts under standardised conditions. We highlight unique NMR signatures of posttranslationally modified residues and their effects on neighbouring residues. This comprehensive dataset complements established random coil shift datasets of the 20 standard amino acids and will facilitate identification and assignment of posttranslationally modified residues. The random coil shifts will also aid in determination of secondary structure elements and prediction of structural parameters of proteins and peptides containing PTMs.

Native chemical ligation in protein synthesis and semi-synthesis.

Native chemical ligation (NCL) provides a highly efficient and robust means to chemoselectively link unprotected peptide and protein segments to generate proteins. The ability to incorporate non-proteinogenic amino acids (e.g.d-amino acids or fluorescent labels) and post-translational modifications into proteins by stitching together peptide fragments has driven extremely important developments in peptide and protein science over the past 20 years. Extensions of the original NCL concept (including the development of thiol- and selenol-derived amino acids and desulfurisation and deselenisation methods), improved access to peptide thioesters, and the use of the methodology in combination with recombinantly expressed polypeptide fragments (termed Expressed Protein Ligation, EPL) have helped to further expand the utility of the methodology. Over the past five years, there has been a dramatic increase in the number of proteins that have been accessed by total chemical synthesis and semi-synthesis, including a large range of modified proteins; new records have also been set with regards to the size of proteins that can now be accessed via ligation chemistry. Together these efforts have not only contributed to a better understanding of protein structure and function, but have also driven innovations in protein science. In this tutorial review, we aim to provide the reader with the latest developments in NCL- and EPL-based ligation technologies as well as illustrated examples of using these methods, together with synthetic logic, to access proteins and modified proteins for biological study.

Synthetic Cancer-Targeting Innate Immune Stimulators Give Insights into Avidity Effects.

Multispecific and multivalent antibodies are seen as promising cancer therapeutics, and numerous antibody fragments and derivatives have been developed to exploit avidity effects that result in increased selectivity. Most of these multispecific and multivalent antibody strategies make use of recombinant expression of antigen-binding modules. In contrast, chemical synthesis and chemoselective ligations can be used to generate a variety of molecules with different numbers and combinations of binding moieties in a modular and homogeneous fashion. In this study we synthesized a series of targeted immune system engagers (ISErs) by using solid-phase peptide synthesis and chemoselective ligations. To explore avidity effects, we constructed molecules bearing different numbers and combinations of two "binder" peptides that target ephrin A2 and integrin α3 receptors and an "effector" peptide that binds to formyl peptide receptors and stimulates an immune response. We investigated various strategies for generating multivalent and multispecific targeted innate immune stimulators and studied their activities in terms of binding to cancer cells and stimulation of immune cells. This study gives insights into the influence that multivalency and receptor density have on avidity effects and is useful for the design of potential anticancer therapeutics.

Multifunctional αvß6 Integrin-Specific Peptide-Pt(IV) Conjugates for Cancer Cell Targeting.

Increasing the specificity of cancer therapy, and thereby decreasing damage to normal cells, requires targeting to cancer-cell specific features. The αvß6 integrin is a receptor involved in cell adhesion and is frequently up-regulated in cancer cells compared to normal cells. We have selected a peptide ligand reported to bind specifically to the ß6 integrin and have synthesized a suite of multispecific molecules to explore the potential for targeting of cancer cells. A combination of solid-phase peptide synthesis and chemoselective ligations was used to synthesize multifunctional molecules composed of integrin-targeting peptides, cytotoxic platinum(IV) prodrugs, and fluorescent or affinity probes joined with flexible linkers. The modular synthesis approach facilitates the construction of peptide-drug conjugates with various valencies and properties in a convergent manner. The binding and specificity of the multifunctional peptide conjugates were investigated using a cell line transfected with the ß6 integrin and fluorescence microscopy. This versatile and highly controlled approach to synthesizing labeled peptide-drug conjugates has the potential to target potent cytotoxic drugs specifically to cancer cells, reducing the doses required for effective treatment.

A comparative study of synthetic and semisynthetic approaches for ligating the epidermal growth factor to a bivalent scaffold.

A prominent target of monoclonal antibodies as targeted therapies for cancer is the epidermal growth factor receptor, which is overexpressed on the surface of various cancer cell types. Its natural binder, the epidermal growth factor (EGF), is a 53 amino acid polypeptide. Anticancer synthetic targeted immune system engagers (ISErs) comprising two 'binder' peptides, which are attached to a scaffold conveying immune stimulating 'effector' properties, via monodisperse polyethylene glycol chains. So far, preparation of ISErs has been limited to the use of small peptides (8-20 amino acids) as binding functionalities, and they have been entirely synthesized by solid phase peptide synthesis. Here, we describe a synthetic and a semisynthetic approach for the preparation of an ISEr bearing two murine EGF molecules as binding entities (ISEr-EGF2 ). EGF was either synthesized in segments by solid phase peptide synthesis or expressed recombinantly and ligated to the scaffold by native chemical ligation. We report the successful generation of synthetic and semisynthetic ISEr-EGF2 as well as several challenges encountered during the synthesis and ligations. We demonstrate the application of native chemical ligation for the design of larger ISEr constructs, facilitating new objectives for the coupling of small binder peptides and larger proteins to multivalent ISEr scaffolds. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Mirror Images of Antimicrobial Peptides Provide Reflections on Their Functions and Amyloidogenic Properties.

Enantiomeric forms of BTD-2, PG-1, and PM-1 were synthesized to delineate the structure and function of these ß-sheet antimicrobial peptides. Activity and lipid-binding assays confirm that these peptides act via a receptor-independent mechanism involving membrane interaction. The racemic crystal structure of BTD-2 solved at 1.45 Å revealed a novel oligomeric form of ß-sheet antimicrobial peptides within the unit cell: an antiparallel trimer, which we suggest might be related to its membrane-active form. The BTD-2 oligomer extends into a larger supramolecular state that spans the crystal lattice, featuring a steric-zipper motif that is common in structures of amyloid-forming peptides. The supramolecular structure of BTD-2 thus represents a new mode of fibril-like assembly not previously observed for antimicrobial peptides, providing structural evidence linking antimicrobial and amyloid peptides.

Approaches to the stabilization of bioactive epitopes by grafting and peptide cyclization.

Peptides are attracting increasing interest from the pharmaceutical industry because of their specificity and ability to address novel targets, including protein-protein interactions. However, typically they require stabilization for therapeutic applications owing to their susceptibility to degradation by proteases. Advances in the ability to chemically synthesize peptides and the development of new side-chain and backbone ligation strategies provide new tools to stabilize bioactive peptide epitopes. Two such epitopes are LyP1, a nine residue peptide that localizes to tumor cells and has potential as an anticancer therapeutic, and RGDS, a tetrapeptide shown to bind to survivin and induce apoptosis. Here we applied a variety of strategies for the stabilization of LyP1 and RGDS, including side-chain cyclization using "click" chemistry and "grafting" the epitopes into two naturally occurring cyclic peptide scaffolds, i.e., θ-defensins and cyclotides. NMR data showed that the three-disulfide θ-defensin and cyclotide scaffolds accommodated the LyP1 and RGDS epitopes but that scaffolds with fewer disulfide bonds were structurally compromised by inclusion of the LyP1 epitope. LyP1, LyP1-, and RGDS-grafted peptides that were largely unstructured also had reduced resistance to degradation in human serum, showing that grafting into a stable cyclic scaffold is an effective strategy for increasing the stability of a bioactive peptide epitope. Overall, the study demonstrates several methods for stabilizing peptide epitopes using side-chain or backbone cyclization and illustrates their potential in peptide drug design.

Efficient enzymatic cyclization of an inhibitory cystine knot-containing peptide.

Disulfide-rich peptides isolated from cone snails are of great interest as drug leads due to their high specificity and potency toward therapeutically relevant ion channels and receptors. They commonly contain the inhibitor cystine knot (ICK) motif comprising three disulfide bonds forming a knotted core. Here we report the successful enzymatic backbone cyclization of an ICK-containing peptide κ-PVIIA, a 27-amino acid conopeptide from Conus purpurascens, using a mutated version of the bacterial transpeptidase, sortase A. Although a slight loss of activity was observed compared to native κ-PVIIA, cyclic κ-PVIIA is a functional peptide that inhibits the Shaker voltage-gated potassium (Kv) channel. Molecular modeling suggests that the decrease in potency may be related to the loss of crucial, but previously unidentified electrostatic interactions between the N-terminus of the peptide and the Shaker channel. This hypothesis was confirmed by testing an N-terminally acetylated κ-PVIIA, which shows a similar decrease in activity. We also investigated the conformational dynamics and hydrogen bond network of cyc-PVIIA, both of which are important factors to be considered for successful cyclization of peptides. We found that cyc-PVIIA has the same conformational dynamics, but different hydrogen bond network compared to those of κ-PVIIA. The ability to efficiently cyclize ICK peptides using sortase A will enable future protein engineering for this class of peptides and may help in the development of novel therapeutic molecules. Biotechnol. Bioeng. 2016;113: 2202-2212. © 2016 Wiley Periodicals, Inc.

Arginine side-chain modification that occurs during copper-catalysed azide-alkyne click reactions resembles an advanced glycation end product.

Dehydroascorbate is a by-product of copper-catalysed azide-alkyne click (CuAAC) reactions and also forms advanced glycation end products (AGEs) in tissues undergoing oxidative stress. Here we isolate and characterize an arginine-dehydroascorbate adduct formed during CuAAC reactions, investigate strategies for preventing its formation, and propose its biological relevance as an AGE.

Transforming conotoxins into cyclotides: Backbone cyclization of P-superfamily conotoxins.

Peptide backbone cyclization is a widely used approach to improve the activity and stability of small peptides but until recently it had not been applied to peptides with multiple disulfide bonds. Conotoxins are disulfide-rich conopeptides derived from the venoms of cone snails that have applications in drug design and development. However, because of their peptidic nature, they can suffer from poor bioavailability and poor stability in vivo. In this study two P-superfamily conotoxins, gm9a and bru9a, were backbone cyclized by joining the N- and C-termini with short peptide linkers using intramolecular native chemical ligation chemistry. The cyclized derivatives had conformations similar to the native peptides showing that backbone cyclization can be applied to three disulfide-bonded peptides with cystine knot motifs. Cyclic gm9a was more potent at high voltage-activated (HVA) calcium channels than its acyclic counterpart, highlighting the value of this approach in developing active and stable conotoxins containing cyclic cystine knot motifs.

The cyclic cystine ladder in θ-defensins is important for structure and stability, but not antibacterial activity.

θ-Defensins are ribosomally synthesized cyclic peptides found in the leukocytes of some primate species and have promising applications as antimicrobial agents and scaffolds for peptide drugs. The cyclic cystine ladder motif, comprising a cyclic peptide backbone and three parallel disulfide bonds, is characteristic of θ-defensins. In this study, we explore the role of the cyclic peptide backbone and cystine ladder in the structure, stability, and activity of θ-defensins. θ-Defensin analogues with different numbers and combinations of disulfide bonds were synthesized and characterized in terms of their NMR solution structures, serum and thermal stabilities, and their antibacterial and membrane-binding activities. Whereas the structures and stabilities of the peptides were primarily dependent on the number and position of the disulfide bonds, their antibacterial and membrane-binding properties were dependent on the cyclic backbone. The results provide insights into the mechanism of action of θ-defensins and illustrate the potential of θ-defensin analogues as scaffolds for peptide drug design.

The cyclic cystine ladder of theta-defensins as a stable, bifunctional scaffold: A proof-of-concept study using the integrin-binding RGD motif.

Peptides have the specificity and size required to target the protein-protein interactions involved in many diseases. Some cyclic peptides have been utilised as scaffolds for peptide drugs because of their stability; however, other cyclic peptide scaffolds remain to be explored. θ-Defensins are cyclic peptides from mammals; they are characterised by a cyclic cystine ladder motif and have low haemolytic and cytotoxic activity. Here we demonstrate the potential of the cyclic cystine ladder as a scaffold for peptide drug design by introducing the integrin-binding Arg-Gly-Asp (RGD) motif into the θ-defensin RTD-1. The most active analogue had an IC50 of 18 nM for the αv ß3 integrin as well as high serum stability, thus demonstrating that a desired bioactivity can be imparted to the cyclic cystine ladder. This study highlights how θ-defensins can provide a stable and conformationally restrained scaffold for bioactive epitopes in a ß-strand or turn conformation. Furthermore, the symmetry of the cyclic cystine ladder presents the opportunity to design peptides with dual bioactive epitopes to increase activity and specificity.

The chemistry and biology of theta defensins.

Cyclic peptides are found in a diverse range of organisms and are characterized by their stability and role in defense. Why is only one class of cyclic peptides found in mammals? Possibly we have not looked hard enough for them, or the technologies needed to identify them are not fully developed. We also do not yet understand their intriguing biosynthesis from two separate gene products. Addressing these challenges will require the application of chemical tools and insights from other classes of cyclic peptides. Herein, we highlight recent developments in the characterization of theta defensins and describe the important role that chemistry has played in delineating their modes of action. Furthermore, we emphasize the potential of theta defensins as antimicrobial agents and scaffolds for peptide drug design.

Tyrosine Sulfation Modulates the Binding Affinity of Chemokine-Targeting Nanobodies.

Chemokines are an important family of small proteins integral to leukocyte recruitment during inflammation. Dysregulation of the chemokine-chemokine receptor axis is implicated in many diseases, and both chemokines and their cognate receptors have been the targets of therapeutic development. Analysis of the antigen-binding regions of chemokine-binding nanobodies revealed a sequence motif suggestive of tyrosine sulfation. Given the well-established importance of post-translational tyrosine sulfation of receptors for chemokine affinity, it was hypothesized that the sulfation of these nanobodies may contribute to chemokine binding and selectivity. Four nanobodies (16C1, 9F1, 11B1, and 11F2) were expressed using amber codon suppression to incorporate tyrosine sulfation. The sulfated variant of 16C1 demonstrated significantly improved chemokine binding compared to the non-sulfated counterpart, while the other nanobodies displayed equipotent or reduced affinity upon sulfation. The ability of tyrosine sulfation to modulate chemokine binding, both positively and negatively, could be leveraged for chemokine-targeted sulfo-nanobody therapeutics in the future.

Exploring DOXP-reductoisomerase binding limits using phosphonated N-aryl and N-heteroarylcarboxamides as DXR inhibitors.

DOXP-reductoisomerase (DXR) is a validated target for the development of antimalarial drugs to address the increase in resistant strains of Plasmodium falciparum. Series of aryl- and heteroarylcarbamoylphosphonic acids, their diethyl esters and disodium salts have been prepared as analogues of the potent DXR inhibitor fosmidomycin. The effects of the carboxamide N-substituents and the length of the methylene linker have been explored using in silico docking studies, saturation transfer difference NMR spectroscopy and enzyme inhibition assays using both EcDXR and PfDXR. These studies indicate an optimal linker length of two methylene units and have confirmed the importance of an additional binding pocket in the PfDXR active site. Insights into the constraints of the PfDXR binding site provide additional scope for the rational design of DXR inhibitors with increased ligand-receptor interactions.

Structural characterization of the cyclic cystine ladder motif of θ-defensins.

The θ-defensins are, to date, the only known ribosomally synthesized cyclic peptides in mammals, and they have promising antimicrobial bioactivities. The characteristic structural motif of the θ-defensins is the cyclic cystine ladder, comprising a cyclic peptide backbone and three parallel disulfide bonds. In contrast to the cyclic cystine knot, which characterizes the plant cyclotides, the cyclic cystine ladder has not been as well described as a structural motif. Here we report the solution structures and nuclear magnetic resonance relaxation properties in aqueous solution of three representative θ-defensins from different species. Our data suggest that the θ-defensins are more rigid and structurally defined than previously thought. In addition, all three θ-defensins were found to self-associate in aqueous solution in a concentration-dependent and reversible manner, a property that might have a role in their mechanism of action. The structural definition of the θ-defensins and the cyclic cystine ladder will help to guide exploitation of these molecules as structural frameworks for the design of peptide drugs.