Modulating Protein–Protein Interactions by Cyclic and Macrocyclic Peptides. Prominent Strategies and Examples

Cyclic and macrocyclic peptides constitute advanced molecules for modulating protein–protein interactions (PPIs). Although still peptide derivatives, they are metabolically more stable than linear counterparts, and should have a lower degree of flexibility, with more defined secondary structure conformations that can be adapted to imitate protein interfaces. In this review, we analyze recent progress on the main methods to access cyclic/macrocyclic peptide derivatives, with emphasis in a few selected examples designed to interfere within PPIs. These types of peptides can be from natural origin, or prepared by biochemical or synthetic methodologies, and their design could be aided by computational approaches. Some advances to facilitate the permeability of these quite big molecules by conjugation with cell penetrating peptides, and the incorporation of β-amino acid and peptoid structures to improve metabolic stability, are also commented. It is predicted that this field of research could have an important future mission, running in parallel to the discovery of new, relevant PPIs involved in pathological processes.     

Spectral properties and supramolecular inclusion complexes of β-cyclodextrin with flexible amphiphilic and rigid compounds

Spectral properties and inclusion complexes of β-cyclodextrin (β-CD) with nonionic amphiphiles and rigid 1-bromonaphthalene (BrN) was investigated in detail. Fluorescence and ¹H NMR measurements give new insights into inclusion of the hydrophobic moiety of amphiphiles into the cavity of β-CD. Their apparent stability constants were well correlated with the structure of the hydrophobic moiety of amphiphiles. The long and flexible hydrophobic moiety may occupy the cavity in the compressed manner. The phosphorescence quenching and the binding strength of BrN in ternary complexes indicate that the inclusion depth and the rigidity of BrN in the cavity of β-CD are predominant factors in determining its phosphorescence. Further inclusion of rigid BrN into the cavity drives the built-in phenyl group of amphiphiles to expose to bulk water phase to a greater extent. Comparative analyses of molecular sizes and models reveal that the flexible hydrocarbon chain of an amphiphile in supramolecular inclusion complexes was located inside the crowded cavity of β-CD due to the filling of rigid BrN into the cavity.     

Olefin ring-closing metathesis as a powerful tool in drug discovery and development – potent macrocyclic inhibitors of the hepatitis C virus NS3 protease

Peptidomimetic inhibitors of the hepatitis C NS3 protease often exhibit poor biopharmaceutical properties. Structure modification of a substrate-based tripeptide into a β-strand 15-membered ring scaffold provided a new class of peptidomimetics that are significantly superior as drug candidates to their acyclic precursors. Tripeptide dienes composed of three unnatural amino acid residues with numerous chiral centers were efficiently converted to macrocyclic peptides, in high diastereomeric purity, using ring-closing metathesis (RCM). The conformation of the acyclic diene and the protocol for the RCM reaction were investigated and optimized extensively in order to achieve an efficient synthesis of potential therapeutic agents for the treatment of hepatitis C infections. These studies provided the fist small molecule (BILN 2061) that was clinically validated for the treatment of hepatitis C infection in man and opened the door to a plethora of new pre-clinical pharmaceutical agents that can be made in multi kilogram quantities using RCM chemistry.     

Helical peptoid mimics of magainin-2 amide

A series of peptoid oligomers were designed as helical, cationic, and facially amphipathic mimics of the magainin-2 amide antibacterial peptide. We used circular dichroism spectroscopy to determine the conformation of these peptoids in aqueous buffer and in the presence of bacterial membrane-mimetic lipid vesicles, composed of a 7:3 mol ratio of POPE:POPG. We found that certain peptoids, which displayed characteristically helical CD in buffer and lipid vesicles, exhibit selective (nonhemolytic) and potent antibacterial activity against both Gram-positive and Gram-negative bacteria. In contrast, peptoids that exhibit weak CD, reminiscent of that of a peptide random coil, were ineffective antibiotics. In a manner similar to the natural magainin peptides, we find a correlation between peptoid lipophilicity and hemolytic propensity. We observe that a minimum length of approximately 12 peptoid residues may be required for antibacterial activity. We also see evidence that a helix length between 24 and 34 A may provide optimal antibacterial efficacy. These results provide the first example of a water-soluble, structured, bioactive peptoid.     

Natural product-like macrocyclic N-methyl-peptide inhibitors against a ubiquitin ligase uncovered from a ribosome-expressed de novo library

Naturally occurring peptides often possess macrocyclic and N-methylated backbone. These features grant them structural rigidity, high affinity to targets, proteolytic resistance, and occasionally membrane permeability. Because such peptides are produced by either nonribosomal peptide synthetases or enzymatic posttranslational modifications, it is yet a formidable challenge in degenerating sequence or length and preparing libraries for screening bioactive molecules. Here, we report a new means of synthesizing a de novo library of “natural product-like” macrocyclic N-methyl-peptides using translation machinery under the reprogrammed genetic code, which is coupled with an in vitro display technique, referred to as RaPID (random nonstandard peptides integrated discovery) system. This system allows for rapid selection of strong binders against an arbitrarily chosen therapeutic target. Here, we have demonstrated the selection of anti-E6AP macrocyclic N-methyl-peptides, one of which strongly inhibits polyubiqutination of proteins such as p53.     

Cyclic Peptides as Drugs for Intracellular Targets: The Next Frontier in Peptide Therapeutic Development

Developing macrocyclic peptides that can reach intracellular targets is a significant challenge. This review discusses the most recent strategies used to develop cell permeable cyclic peptides that maintain binding to their biological target inside the cell. Macrocyclic peptides are unique from small molecules because traditional calculated physical properties are unsuccessful for predicting cell membrane permeability. Peptide synthesis and experimental membrane permeability is the only strategy that effectively differentiates between cell permeable and cell impermeable molecules. Discussed are chemical strategies, including backbone N-methylation and stereochemical changes, which have produced molecular scaffolds with improved cell permeability. However, these improvements often come at the expense of biological activity as chemical modifications alter the peptide conformation, frequently impacting the compound's ability to bind to the target. Highlighted is the most promising approach, which involves side-chain alterations that improve cell permeability without impact binding events.     

Probing the structural requirements of peptoids that inhibit HDM2-p53 interactions

Many cellular processes are controlled by protein-protein interactions, and selective inhibition of these interactions could lead to the development of new therapies for several diseases. In the area of cancer, overexpression of the protein, human double minute 2 (HDM2), which binds to and inactivates the protein p53, has been linked to tumor aggressiveness and drug resistance. In general, inhibition of protein-protein interactions with synthetic molecules is challenging and currently remains a largely uncharted area for drug development. One strategy to create inhibitors of protein-protein interactions is to recreate the three-dimensional arrangement of side chains that are involved in the binding of one protein to another, using a nonnatural scaffold as the attachment point for the side chains. In this study, we used oligomeric peptoids as the scaffold to begin to develop a general strategy in which we could rationally design synthetic molecules that can be optimized for inhibition of protein-protein interactions. Structural information on the HDM2-p53 complex was used to design our first class of peptoid inhibitors, and we provide here, in detail, the strategy to modify peptoids with the appropriate side chains that are effective inhibitors of HDM2-p53 binding. While we initially tried to develop rigid, helical peptoids as HDM2 binders, the best inhibitors were surprisingly peptoids that lacked any helix-promoting groups. These results indicate that starting with rigid peptoid scaffolds may not always be optimal to develop new inhibitors.     

Role of rigidity on the activity of proteinase inhibitors and their peptide mimics

The Bowman-Birk inhibitors (BBIs) are a family of proteins that share a canonical loop structure whose presence in a conserved conformation is linked to their inhibitory activity. We study the conformational properties of the canonical loop using a graph theoretical approach as implemented in the floppy inclusions and rigid substructure topography (FIRST). We find that the canonical loop is an independent rigid cluster in the natural inhibitors. We have further used this technique to identify residues that play an important role in the structural rigidity of the protein by quantifying their contribution to the overall rigidity of the inhibitor. We find that the conserved elements among the natural and synthetic peptides are the ones that contribute the most to rigidity, even if they are located far from the active site, as rigidity effects are nonlinear and hence nonlocal. The results help to elucidate why certain mutations in the loop of the BBI produce peptides that fail to have the designed inhibitory activity.     

Drug Syntheses Beyond the Rule of 5

Drugs in the chemical space beyond the rule of 5 (bRo5) can modulate targets with difficult binding sites while retaining cell permeability and oral absorption. Reviewing the syntheses of bRo5 drugs approved since 1990 highlights synthetic chemistry's contribution to drug discovery in this space. Initially, bRo5 drugs were mainly natural products and semi-synthetic derivatives. Later, peptidomimetics and de novo designed compounds, that include up to seven chiral centres and macrocyclic rings became dominant. These drugs are prepared by total synthesis, sometimes by routes of more than 25 steps with stereocentres originating from the chiral pool, or being installed by chiral induction or enzymatic resolution. Interestingly, ring-closing metathesis proved to be the method of choice for macrocyclisation in hepatitis C virus protease inhibitors. We conclude that structural simplification, planning of synthetic routes regarding incorporation of stereocentres and macrocyclisation, as well as incorporation of structural knowledge and consideration of chameleonic properties in design, should facilitate drug discovery in bRo5 space.     

Expedient synthesis of a novel asymmetric selectively deprotectable derivative of the ATAC scaffold

An efficient multigram scale synthesis of a new asymmetric triazacyclophane scaffold, the ATAC (Asymmetric-TAC) scaffold, bearing three selectively removable groups is described. This scaffold is slightly more rigid than our frequently used TAC (TriAzaCyclophane) scaffold. The synthesis of the required triamine is very high yielding without difficult steps or purifications and was also applied to a much improved synthesis of our original TAC scaffold. Especially the tedious first reaction step, that is, mono-oNBS-protection of a triamine could be omitted. The rigidity of the triazacyclophane ring in both TAC- and ATAC scaffolds has also been investigated using variable temperature ¹H NMR experiments.     

A versatile scaffold for site-specific modification of cyclic tetrapeptides

A novel scaffold that can be used to prepare conformationally homogeneous cyclic tetrapeptides equipped with a β-amino acid residue is disclosed. It is shown that regioselective structural modification can be accomplished using thiols and azide nucleophiles, commonly associated with rich downstream chemistry. The method should find application in efforts to constrain privileged tripeptide sequences in rigid molecular scaffolds.     

Peptide-Hypervalent Iodine Reagent Chimeras: Enabling Peptide Functionalization and Macrocyclization**

Herein, we report a novel strategy for the modification of peptides based on the introduction of highly reactive hypervalent iodine reagents—ethynylbenziodoxolones (EBXs)—onto peptides. These peptide-EBXs can be readily accessed, by both solution- and solid-phase peptide synthesis (SPPS). They can be used to couple the peptide to other peptides or a protein through reaction with Cys, leading to thioalkynes in organic solvents and hypervalent iodine adducts in water buffer. Furthermore, a photocatalytic decarboxylative coupling to the C-terminus of peptides was developed using an organic dye and was also successful in an intramolecular fashion, leading to macrocyclic peptides with unprecedented crosslinking. A rigid linear aryl alkyne linker was essential to achieve high affinity for Keap1 at the Nrf2 binding site with potential protein-protein interaction inhibition.     

Biosynthetic Strategies for Macrocyclic Peptides

Macrocyclic peptides are predominantly peptide structures bearing one or more rings and spanning multiple amino acid residues. Macrocyclization has become a common approach for improving the pharmacological properties and bioactivity of peptides. A variety of ribosomal-derived and non-ribosomal synthesized cyclization approaches have been established. The biosynthesis of backbone macrocyclic peptides using seven new emerging methodologies will be discussed with regard to the features and strengths of each platform rather than medicinal chemistry tools. The mRNA display variant, known as the random nonstandard peptide integrated discovery (RaPID) platform, utilizes flexible in vitro translation (FIT) to access macrocyclic peptides containing nonproteinogenic amino acids (NAAs). As a new discovery approach, the ribosomally synthesized and post-translationally modified peptides (RiPPs) method involves the combination of ribosomal synthesis and the phage screening platform together with macrocyclization chemistries to generate libraries of macrocyclic peptides. Meanwhile, the split-intein circular ligation of peptides and proteins (SICLOPPS) approach relies on the in vivo production of macrocyclic peptides. In vitro and in vivo peptide library screening is discussed as an advanced strategy for cyclic peptide selection. Specifically, biosynthetic bicyclic peptides are highlighted as versatile and attractive modalities. Bicyclic peptides represent another type of promising therapeutics that allow for building blocks with a heterotrimeric conjugate to address intractable challenges and enable multimer complexes via linkers. Additionally, we discuss the cell-free chemoenzymatic synthesis of macrocyclic peptides with a non-ribosomal catalase known as the non-ribosomal synthetase (NRPS) and chemo-enzymatic approach, with recombinant thioesterase (TE) domains. Novel insights into the use of peptide library tools, activity-based two-hybrid screening, structure diversification, inclusion of NAAs, combinatorial libraries, expanding the toolbox for macrocyclic peptides, bicyclic peptides, chemoenzymatic strategies, and future perspectives are presented. This review highlights the broad spectrum of strategy classes, novel platforms, structure diversity, chemical space, and functionalities of macrocyclic peptides enabled by emerging biosynthetic platforms to achieve bioactivity and for therapeutic purposes.     

Can a Proton be Encapsulated in Tetraamido/Diamino Quaternized Macrocycles in Aqueous Solution and Electric Field?

The proton‐binding behavior of solvated tetraamido/diamino quaternized macrocyclic compounds with rigid phenyl and flexible phenyl bridges in the absence or presence of an external electric field is investigated by molecular dynamics simulation. The proton can be held through H‐bonding interactions with the two carbonyl oxygen atoms in macrocycles containing rigid (phenyl) and flexible (propyl) bridges. The solute–solvent H‐bonding interactions cause the macrocyclic backbones to twist to different extents, depending on the different bridges. The macrocycle with the rigid phenyl linkages folds into a cuplike shape due to π–π interaction, while the propyl analogue still maintains the ellipsoidal ringlike shape with just a slight distortion. The potential energy required for proton transfer is larger in the phenyl‐containing macrocycle than in the compound with propyl units. When an external electric field with a strength of 2.5 V nm^?1 is exerted along the carbonyl oxygen atoms, a difference in proton encircling is exhibited for macrocycles with rigid and flexible bridges. In contrast to encapsulation of a proton in the propyl analogue, the intermolecular solute–solvent H‐bonding and intramolecular π–π stacking between the two rigid phenyl spacers leads to loss of the proton from the highly distorted cuplike macrocycle with phenyl bridges. The competition between intra‐ and intermolecular interactions governs the behavior of proton encircling in macrocycles.     

Fit to be tied: conformation-directed macrocyclization of peptoid foldamers

Covalent macrocyclic constraints can be readily installed on N-substituted glycine "peptoid" oligomer substrates. Cu(I)-catalyzed [3+2] cycloaddition reactions were conducted on solid support to ligate peptoid side chain azide and alkyne functionalities. Intramolecular macrocycle formation is facilitated by preorganizing the reactive groups across one turn of the helical secondary structure. These results confirm that conformational ordering can be exploited to assist the macrocyclization of folded oligomers.     

Heterodivalent Linked Macrocyclic β-Sheets with Enhanced Activity against Aβ Aggregation: Two Sites Are Better Than One

This paper reports a series of heterodivalent linked macrocyclic β-sheets 6 that are not only far more active against amyloid-β (Aβ) aggregation than their monovalent components 1a and 1b but also are dramatically more active than their homodivalent counterparts 4 and 5. The macrocyclic β-sheet components 1a and 1b comprise pentapeptides derived from the N- and C-terminal regions of Aβ and molecular template and turn units that enforce a β-sheet structure and block aggregation. Thioflavin T fluorescence assays show that heterodivalent linked macrocyclic β-sheets 6 delay Aβ(1-40) aggregation 6-8-fold at equimolar concentrations and substantially delay aggregation at substoichiometric concentrations, while homodivalent linked macrocyclic β-sheets 4 and 5 and monovalent macrocyclic β-sheets 1a and 1b only exhibit more modest effects at equimolar or greater concentrations. A model to explain these observations is proposed, in which the inhibitors bind to and stabilize the early β-structured Aβ oligomers and thus delay aggregation. In this model, heterodivalent linked macrocyclic β-sheets 6 bind to the β-structured oligomers more strongly, because N-terminal-derived component 1a can bind to the N-terminal-based core of the β-structured oligomers, while the C-terminal-derived component 1b can achieve additional interactions with the C-terminal region of Aβ. The enhanced activity of the heterodivalent compounds suggests that polyvalent inhibitors that can target multiple regions of amyloidogenic peptides and proteins are better than those that only target a single region.     

Macrocyclic scaffold for the collagen triple helix

[structure: see text] Three strands of natural collagen are linked by covalent bonds prior to their folding into a triple helix. We report on a synthetic collagen in which the strands are pendent on a rigid macrocyclic scaffold of C(3) symmetry. The scaffold confers substantial conformational stability upon the collagen triple helix and makes its folding independent of concentration, both desirable attributes for exploring and exploiting synthetic collagens.     

Cyclic peptoids

Foldamers are an intriguing family of biomimetic oligomers that exhibit a propensity to adopt stable secondary structures. N-Substituted glycine oligomers, or "peptoids", are a prototypical example of these foldamer systems and are known to form a helix resembling that of polyproline type I. Ongoing studies seek to improve the stability of peptoid folding and to discover new secondary structure motifs. Here, we report that peptoids undergo highly efficient head-to-tail macrocyclization reactions. A diverse array of peptoid sequences from pentamers to 20mers were converted to macrocyclic products within 5 min at room temperature. The introduction of the covalent constraint enhances conformational ordering, allowing for the crystallization of a cyclic peptoid hexamer and octamer. We present the first X-ray crystallographic structures of peptoid hetero-oligomers, revealing that peptoid macrocycles can form a reverse-turn conformation.     

Polycationic calix[8]arenes able to recognize and neutralize heparin

A mutual induced fit mechanism is responsible for the exceptional complexation performances exhibited by calix[8]arene polycations towards heparin. The recognition process was studied in comparison with two other heparin antagonists: protamine and polylysine. The arrangement of multiple functional groups on the flexible macrocyclic scaffold of calix[8]arene, with respect to the conformationally rigid protamine and low ordered polylysine, allowed a mutual adaptability between calixarene polycations and heparin, significantly enhancing the recognition performances. Fluorescence, NMR titration, and activated partial thromboplastin time (aPTT) experiments confirmed that these calixarene derivatives have a very high specificity and affinity towards heparin neutralization as in aqueous solution as in blood. Analogous results were obtained with low molecular weight heparin (LMWH) whose effect protamine is unable to completely reverse.