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.

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.

Posttranslational modifications of α-conotoxins: sulfotyrosine and C-terminal amidation stabilise structures and increase acetylcholine receptor binding.

Conotoxins are peptides found in the venoms of marine cone snails. They are typically highly structured and stable and have potent activities at nicotinic acetylcholine receptors, which make them valuable research tools and promising lead molecules for drug development. Many conotoxins are also highly modified with posttranslational modifications such as proline hydroxylation, glutamic acid gamma-carboxylation, tyrosine sulfation and C-terminal amidation, amongst others. The role of these posttranslational modifications is poorly understood, and it is unclear whether the modifications interact directly with the binding site, alter conotoxin structure, or both. Here we synthesised a set of twelve conotoxin variants bearing posttranslational modifications in the form of native sulfotyrosine and C-terminal amidation and show that these two modifications in combination increase their activity at nicotinic acetylcholine receptors and binding to soluble acetylcholine binding proteins, respectively. We then rationalise how these functional differences between variants might arise from stabilization of the three-dimensional structures and interactions with the binding sites, using high-resolution nuclear magnetic resonance data. This study demonstrates that posttranslational modifications can modulate interactions between a ligand and receptor by a combination of structural and binding alterations. A deeper mechanistic understanding of the role of posttranslational modifications in structure-activity relationships is essential for understanding receptor biology and could help to guide structure-based drug design.

Segmental and site-specific isotope labelling strategies for structural analysis of posttranslationally modified proteins.

Posttranslational modifications can alter protein structures, functions and locations, and are important cellular regulatory and signalling mechanisms. Spectroscopic techniques such as nuclear magnetic resonance, infrared and Raman spectroscopy, as well as small-angle scattering, can provide insights into the structural and dynamic effects of protein posttranslational modifications and their impact on interactions with binding partners. However, heterogeneity of modified proteins from natural sources and spectral complexity often hinder analyses, especially for large proteins and macromolecular assemblies. Selective labelling of proteins with stable isotopes can greatly simplify spectra, as one can focus on labelled residues or segments of interest. Employing chemical biology tools for modifying and isotopically labelling proteins with atomic precision provides access to unique protein samples for structural biology and spectroscopy. Here, we review site-specific and segmental isotope labelling methods that are employed in combination with chemical and enzymatic tools to access posttranslationally modified proteins. We discuss illustrative examples in which these methods have been used to facilitate spectroscopic studies of posttranslationally modified proteins, providing new insights into biology.

Site-specific modification and segmental isotope labelling of HMGN1 reveals long-range conformational perturbations caused by posttranslational modifications.

Interactions between histones, which package DNA in eukaryotes, and nuclear proteins such as the high mobility group nucleosome-binding protein HMGN1 are important for regulating access to DNA. HMGN1 is a highly charged and intrinsically disordered protein (IDP) that is modified at several sites by posttranslational modifications (PTMs) - acetylation, phosphorylation and ADP-ribosylation. These PTMs are thought to affect cellular localisation of HMGN1 and its ability to bind nucleosomes; however, little is known about how these PTMs regulate the structure and function of HMGN1 at a molecular level. Here, we combine the chemical biology tools of protein semi-synthesis and site-specific modification to generate a series of unique HMGN1 variants bearing precise PTMs at their N- or C-termini with segmental isotope labelling for NMR spectroscopy. With access to these precisely-defined variants, we show that PTMs in both the N- and C-termini cause changes in the chemical shifts and conformational populations in regions distant from the PTM sites; up to 50-60 residues upstream of the PTM site. The PTMs investigated had only minor effects on binding of HMGN1 to nucleosome core particles, suggesting that they have other regulatory roles. This study demonstrates the power of combining protein semi-synthesis for introduction of site-specific PTMs with segmental isotope labelling for structural biology, allowing us to understand the role of PTMs with atomic precision, from both structural and functional perspectives.

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.

Deciphering protein post-translational modifications using chemical biology tools.

Proteins carry out a wide variety of catalytic, regulatory, signalling and structural functions in living systems. Following their assembly on ribosomes and throughout their lifetimes, most eukaryotic proteins are modified by post-translational modifications; small functional groups and complex biomolecules are conjugated to amino acid side chains or termini, and the protein backbone is cleaved, spliced or cyclized, to name just a few examples. These modifications modulate protein activity, structure, location and interactions, and, thereby, control many core biological processes. Aberrant post-translational modifications are markers of cellular stress or malfunction and are implicated in several diseases. Therefore, gaining an understanding of which proteins are modified, at which sites and the resulting biological consequences is an important but complex challenge requiring interdisciplinary approaches. One of the key challenges is accessing precisely modified proteins to assign functional consequences to specific modifications. Chemical biologists have developed a versatile set of tools for accessing specifically modified proteins by applying robust chemistries to biological molecules and developing strategies for synthesizing and ligating proteins. This Review provides an overview of these tools, with selected recent examples of how they have been applied to decipher the roles of a variety of protein post-translational modifications. Relative advantages and disadvantages of each of the techniques are discussed, highlighting examples where they are used in combination and have the potential to address new frontiers in understanding complex biological processes.

Recent Advances in Peptide-Based Approaches for Cancer Treatment.

BACKGROUND: Peptide-based pharmaceuticals have recently experienced a renaissance due to their ability to fill the gap between the two main classes of available drugs, small molecules and biologics. Peptides combine the high potency and selectivity typical of large proteins with some of the characteristic advantages of small molecules such as synthetic accessibility, stability and the potential of oral bioavailability. METHODS: In the present manuscript we review the recent literature on selected peptide-based approaches for cancer treatment, emphasizing recent advances, advantages and challenges of each strategy. RESULTS: One of the applications in which peptide-based approaches have grown rapidly is cancer therapy, with a focus on new and established targets. We describe, with selected examples, some of the novel peptide-based methods for cancer treatment that have been developed in the last few years, ranging from naturally-occurring and modified peptides to peptidedrug conjugates, peptide nanomaterials and peptide-based vaccines. CONCLUSION: This review brings out the emerging role of peptide-based strategies in oncology research, critically analyzing the advantages and limitations of these approaches and the potential for their development as effective anti-cancer therapies.

Protein Chemistry Looking Ahead: 8th Chemical Protein Synthesis Meeting 16-19 June 2019, Berlin, Germany.

The 8th Chemical Protein Synthesis meeting took place in Berlin in June 2019, covering broad topics in protein chemistry, ranging from synthetic methodology to applications in medicine and biomaterials. The meeting was also the culmination of the Priority Program SPP1623 on "Chemoselective Reactions for the Synthesis and Application of Functional Proteins" funded by the German Science Foundation (DFG) from 2012 to 2018. We present highlights from presentations at the forefront of the field, grouped into broad themes that illustrate how the field of protein chemistry is looking ahead to new discoveries and applications.

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.

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.

Multifunctional Scaffolds for Assembling Cancer-Targeting Immune Stimulators Using Chemoselective Ligations.

Chemoselective ligations allow chemical biologists to functionalise proteins and peptides for biomedical applications and to probe biological processes. Coupled with solid phase peptide synthesis, chemoselective ligations enable not only the design of homogeneous proteins and peptides with desired natural and unnatural modifications in site-specific locations but also the design of new peptide and protein topologies. Although several well-established ligations are available, each method has its own advantages and disadvantages and they are seldom used in combination. Here we have applied copper-catalyzed azide-alkyne "click," oxime, maleimide, and native chemical ligations to develop a modular synthesis of branched peptide and polymer constructs that act as cancer-targeting immune system engagers (ISErs) and functionalised them for detection in biological systems. We also note some potential advantages and pitfalls of these chemoselective ligations to consider when designing orthogonal ligation strategies. The modular synthesis and functionalization of ISErs facilitates optimisation of their activity and mechanism of action as potential cancer immunotherapies.

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.

Advancing the Frontiers of Chemical Protein Synthesis-The 7th CPS Meeting, Haifa, Israel.

The 7th Chemical Protein Synthesis Meeting took place in September 2017 in Haifa, Israel, bringing together 100 scientists from 11 countries. The cutting-edge scientific program included new synthetic strategies and ligation auxiliaries, novel insights into protein signaling and post-translational modifications, and a range of promising therapeutic applications.

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.

Synthetic integrin-binding immune stimulators target cancer cells and prevent tumor formation.

Immuno-oncology approaches mainly utilize monoclonal antibodies or protein-based scaffolds that bind with high affinity to cancer cells and can generate an immune response. Peptides can also bind with high affinity to cancer cells and are intermediate in size between antibodies and small molecules. They are also synthetically accessible and therefore easily modified to optimize their stability, binding affinity and selectivity. Here we describe the design of immune system engagers (ISErs), a novel class of synthetic peptide-based compounds that bind specifically to cancer cells and stimulate the immune system. A prototype, Y9, targets integrin α3, which is overexpressed on several cancer cells, and activates the immune system via a formyl methionine-containing effector peptide. Injection of Y9 leads to immune cell infiltration into tissue and prevents tumor formation in a guinea pig model. The anti-tumor activity and synthetic accessibility of Y9 illustrate that ISErs could be applied to a wide variety of targets and diseases.

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.

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.

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.