p-Chloropropynyl Phenylalanine, a Versatile Non-Canonical Amino Acid for Co-Translational Peptide Macrocyclization and Side Chain Diversification.

Macrocyclization has proven to be a beneficial strategy to improve upon some of the disadvantages of peptides as therapeutics. Nevertheless, many peptide cyclization strategies are not compatible with in vitro display technologies like mRNA display. Here we describe the novel amino acid p-chloropropynyl phenylalanine (pCPF). pCPF is a substrate for a mutant phenylalanyl-tRNA synthetase and its introduction into peptides via in vitro translation leads to spontaneous peptide macrocyclization in the presence of peptides containing cysteine. Macrocyclization occurs efficiently with a wide variety of ring sizes. Moreover, pCPF can be reacted with thiols after charging onto tRNA, enabling the testing of diverse ncAAs in translation. The versatility of pCPF should facilitate downstream studies of translation and enable the creation of novel macrocyclic peptide libraries.

Peptide Stapling with Boronate Esters- A Reversible Folding of (Artificial) Peptide Chain to α-Helix.

Stabilization of a peptide conformation via stapling strategy may be realized by the reversible or more often irreversible connection of side chains being in mutually appropriate geometry. An incorporation of phenylboronic acid and sugar residues (fructonic or galacturonic acid), attached to two lysine side chains via amide bonds and separated by 2, 3, or 6 other residues in the C-terminal fragment of RNase A introduces the intramolecular interaction stabilizing the α-helical organization. The boronate ester stapling is stabilized in mild basic conditions and may be switched off by acidification leading to unfolded organization of the peptide chain. We investigated the possibility of using switchable stapling by mass spectrometry, NMR and UV-CD spectroscopies, and DFT calculations.

Effect of macrocyclization and tetramethylrhodamine labeling on chemokine binding peptides.

Receptor-derived peptides have played an important role in elucidating chemokine-receptor interactions. For the inflammatory chemokine CXC-class chemokine ligand 8 (CXCL8), a site II-mimetic peptide has been derived from parts of extracellular loops 2 and 3 and adjacent transmembrane helices of its receptor CXC-class chemokine receptor 1 (Helmer et al., RSC Adv., 2015, 5, 25657). The peptide sequence with a C-terminal glutamine did not bind to CXCL8, whereas one with a C-terminal glutamate did but with low micromolar affinity. We sought to improve the affinity and protease stability of the latter peptide through cyclization while also cyclizing the former for control purposes. To identify a cyclization strategy that permits a receptor-like interaction, we conducted a molecular dynamics simulation of CXCL8 in complex with full-length CXC-class chemokine receptor 1. We introduced a linker to provide an appropriate spacing between the termini and used an on-resin side-chain-to-tail cyclization strategy. Upon chemokine binding, the fluorescence intensity of the tetramethylrhodamine (TAMRA)-labeled cyclic peptides increased whereas the fluorescence anisotropy decreased. Additional molecular dynamics simulations indicated that the fluorophore interacts with the peptide macrocycle so that chemokine binding leads to its displacement and observed changes in fluorescence. Macrocyclization of both 18-amino acid-long peptides led to the same low micromolar affinity for CXCL8. Likewise, both TAMRA-labeled linear peptides interacted with CXCL8 with similar affinities. Interestingly, the linear TAMRA-labeled peptides were more resistant to tryptic digestion than the unlabeled counterparts, whereas the cyclized peptides were not degraded at all. We conclude that the TAMRA fluorophore tends to interact with peptides altering their protease stability and behavior in fluorescence-based assays.

Helical Stabilization of Peptide Macrocycles by Stapled Architectures.

Over the past two decades, significant efforts have invested in the development of strategies for the stabilization of macrocyclic peptides with α-helix structure by stapling their architectures. These strategies can be divided into two categories: side chain to side chain cross-linking and N-terminal helix nucleation. These stable macrocyclic peptides have been applied in PPI inhibitors and self-assembly materials. Compared with unmodified short peptides, stable α-helix macrocyclic polypeptides have better biophysical properties including higher serum stability, cell permeability, and higher target affinity. This chapter will systematically introduce approaches for helical stabilization of peptide macrocycles, such as ring-closing metathesis (RCM), lactamisation, cycloadditions, reversible reactions, thioether formation as well as newly found sulfonium center formation and the common use of helical stabilized macrocyclic peptides.

Computationally designed peptide macrocycle inhibitors of New Delhi metallo-ß-lactamase 1.

The rise of antibiotic resistance calls for new therapeutics targeting resistance factors such as the New Delhi metallo-ß-lactamase 1 (NDM-1), a bacterial enzyme that degrades ß-lactam antibiotics. We present structure-guided computational methods for designing peptide macrocycles built from mixtures of l- and d-amino acids that are able to bind to and inhibit targets of therapeutic interest. Our methods explicitly consider the propensity of a peptide to favor a binding-competent conformation, which we found to predict rank order of experimentally observed IC50 values across seven designed NDM-1- inhibiting peptides. We were able to determine X-ray crystal structures of three of the designed inhibitors in complex with NDM-1, and in all three the conformation of the peptide is very close to the computationally designed model. In two of the three structures, the binding mode with NDM-1 is also very similar to the design model, while in the third, we observed an alternative binding mode likely arising from internal symmetry in the shape of the design combined with flexibility of the target. Although challenges remain in robustly predicting target backbone changes, binding mode, and the effects of mutations on binding affinity, our methods for designing ordered, binding-competent macrocycles should have broad applicability to a wide range of therapeutic targets.

Cooperative Stapling of Native Peptides at Lysine and Tyrosine or Arginine with Formaldehyde.

Stapling of peptides by intramolecular crosslinking of two neighboring amino acid side chains offers an important tool to modulate the structure and properties of peptides. In comparison to the stapling of artificially engineered peptide substrates, methods for stapling native peptides are more desirable for easier accessibility and genetic encodability. However, the existing strategy for selectivity control in the stapling of native peptides is relatively limited: the site of anchoring is often dominated by Cys, and the means for achieving the position selectivity among the same type of residues at different locations is lacking. We have developed a simple and powerful strategy for stapling native peptides at lysine residues with formaldehyde by the cooperation of nearby tyrosine or arginine residues. The stapling reactions can proceed with high efficiency and residue selectivity under mild conditions, and generate linchpins with distinct physiochemical properties. The new method for peptide stapling enables unique control of position-selectivity for substrates bearing multiple reaction sites by reactivity that can be readily built in the peptide sequence.

Strategies to Enhance Metabolic Stabilities.

Macrocyclic peptides are a unique class of molecules that display a relatively constrained peptidic backbone as compared to their linear counterparts leading to the defined 3-D orientation of the constituent amino acids (pharmacophore). Although they are attractive candidates for lead discovery owing to the unique conformational features, their peptidic backbone is susceptible to proteolytic cleavage in various biological fluids that compromise their efficacy. In this chapter we review the various classical and contemporary chemical and biological approaches that have been utilized to combat the metabolic instability of macrocyclic peptides. We note that any chemical modification that helps in providing either local or global conformational rigidity to these macrocyclic peptides aids in improving their metabolic stability typically by slowing the cleavage kinetics by the proteases.

Synthetic Cross-linking of Peptides: Molecular Linchpins for Peptide Cyclization.

Peptide-derived drugs constitute a significant fraction of therapeutic agents. In 2013, The global market of peptide therapeutics was ca. $19 billion; this value does not include revenue from insulin derivatives of $28 million. The combined sales of insulin and non-insulin peptide drugs is estimated to exceed $70 billion by 2019. A significant fraction of peptide-derived drugs is composed of an amino acid sequence and additional chemical functionalities that improve biological and pharmacological properties of the drug. In this review, we focus on synthetic cross-linkers that we refer to as "linchpins", which are commonly used to constrain the secondary structure of peptides and equip them with added benefits such as resistance to proteolytic degradation and conformational stability. The latter property leads to an increase in binding potency and increased bioavailability due to increased permeation through biological membranes. Some linchpins can even introduce properties not found in natural peptides such as light-responsiveness. Peptides cyclized by linchpins can be viewed as a sub-class of a larger family of peptide-derived drugs with desired pharmacological performance in vivo. To understand how chemical modifications by linchpins improve drug discovery, this review also briefly summarizes canonical examples of chemical modification used in modern peptide therapeutics.

Achieving Skeletal Diversity in Peptide Macrocycles through The Use of Heterocyclic Grafts.

Despite their therapeutic potential, peptide macrocycles often suffer from drawbacks such as low membrane permeability, proteolytic instability, and conformational lability. As a result, there have been significant efforts to "depeptidize" amino acid-rich macrocycles through the incorporation of heterocyclic grafts into their backbones. In this concept article, we summarize selected recent methodologies that can be used to introduce heterocycles into cyclic peptides.

Tuning Sulfur Oxidation States on Thioether-Bridged Peptide Macrocycles for Modulation of Protein Interactions.

Thioethers, sulfoxides, and sulfonium ions, despite diverse physicochemical properties, all engage in noncovalent interactions with proteins. Thioether-containing macrocycles are also attracting attention as protein-protein interaction (PPI) inhibitors. Here, we used a model PPI between α-helical mixed lineage leukemia (MLL) protein and kinase-inducible domain interacting (KIX) domain to evaluate oxidation effects on sulfurcontaining macrocycle structure, stability, and protein affinity. Desolvation effects from various polarity states were evaluated computationally and experimentally at the side chain, amino acid, and peptide level. Sulfur-containing side chains spanned polarity ranges between all-hydrocarbon and lactam bridges for modulating solubility, cellular uptake, and affinity. Helical propensity studies showed that, although oxidized sulfur-containing side chains could be tolerated, conformational effects were sequence-dependent. In some cases, proteolytic stability, binding capacity with KIX, and increased helicity were obtained as first steps toward developing PPI inhibitors.

Ribosomal Synthesis of Thioether-Bridged Bicyclic Peptides.

Many biologically active peptides found in nature exhibit a bicyclic structure wherein a head-to-tail cyclic backbone is further constrained by an intramolecular linkage connecting two side chains of the peptide. Accordingly, methods to access macrocyclic peptides sharing this overall topology could be of significant value toward the discovery of new functional entities and bioactive compounds. With this goal in mind, we recently developed a strategy for enabling the biosynthesis of thioether-bridged bicyclic peptides in living bacterial cells. This method involves a split intein-catalyzed head-to-tail cyclization of a ribosomally produced precursor peptide, combined with inter-sidechain cross-linking through a genetically encoded cysteine-reactive amino acid. This approach can be applied to direct the formation of structurally diverse bicyclic peptides with high efficiency and selectivity in living Escherichia coli cells and provides a platform for the generation of combinatorial libraries of genetically encoded bicyclic peptides for screening purposes.

Discovery and optimization of peptide macrocycles.

INTRODUCTION: Macrocyclic peptides are generally more resistant to proteolysis and often have higher potency than linear peptides and so they are excellent leads in drug design. Their study is significant because they offer potential as a new generation of drugs that are potent and specific, and thus might have fewer side effects than traditional small molecule drugs. Areas covered: This article covers macrocyclic drug leads based on nature-derived cyclic peptides as well as synthetic cyclic peptides and close derivatives. The natural peptides include cyclotides, sunflower-derived peptides, theta-defensins and orbitides. Technologies to make engineered cyclic peptides covered here include cyclization via amino acid linkers, CLIPS, templates, and stapled peptides. Expert opinion: Macrocyclic peptides are promising drug leads and several are in clinical trials. The authors believe they offer key advantages over traditional small molecule drugs, as well as some advantages over protein-based 'biologics' such as antibodies or growth factors. These include the ability to penetrate cells and attack intracellular targets such as protein-protein interactions as well as to hit extracellular targets. Some macrocyclic peptides such as cyclotides offer the potential for production in plants, thus reducing manufacture costs and potentially increasing opportunities for their distribution to developing countries at low cost.

Neighbor-directed histidine N (τ)-alkylation: A route to imidazolium-containing phosphopeptide macrocycles.

Our recently discovered, selective, on-resin route to N(τ)-alkylated imidazolium-containing histidine residues affords new strategies for peptide mimetic design. In this, we demonstrate the use of this chemistry to prepare a series of macrocyclic phosphopeptides, in which imidazolium groups serve as ring-forming junctions. Interestingly, these cationic moieties subsequently serve to charge-mask the phosphoamino acid group that directed their formation. Neighbor-directed histidine N(τ)-alkylation opens the door to new families of phosphopeptidomimetics for use in a range of chemical biology contexts.