MACROCYCLIC PEPTIDES AS DRUG CANDIDATES: RECENT

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MACROCYCLIC PEPTIDES AS DRUG CANDIDATES: RECENT PROGRESS AND REMAINING CHALLENGES Alexander A. Vinogradov, Yizhen Yi, and Hiroaki Suga J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b13178 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 15, 2019

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Journal of the American Chemical Society

MACROCYCLIC PEPTIDES AS DRUG CANDIDATES: RECENT PROGRESS AND REMAINING CHALLENGES Alexander A. Vinogradov, Yizhen Yin and Hiroaki Suga* Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ABSTRACT Peptides as a therapeutic modality attract much attention due to their synthetic accessibility, high degree of specific binding, and the ability to target protein surfaces traditionally considered “undruggable”. Unfortunately, at the same time, other pharmacological properties of a generic peptide, such as metabolic stability and cell permeability, are quite poor, which limits the success of de novo discovered biologically active peptides as drug candidates. Here, we review how macrocyclization as well as the incorporation of non-proteogenic amino acids and various conjugation strategies may be utilized to improve on these characteristics to create better drug candidates. We analyze recent progress and remaining challenges in improving individual pharmacological properties of bioactive peptides, and offer our opinion on interfacing these, often conflicting, considerations, to create balanced drug candidates as a potential way to make further progress in this area. INTRODUCTION Macrocyclic peptides possess a number of pharmacological characteristics distinct from other well-established therapeutic molecular classes, resulting in a versatile drug modality with a unique profile of advantages and limitations. Similar to small molecules, macrocyclic peptides are synthetically accessible, and thus they are amenable to lead optimization via traditional medicinal chemistry efforts. In this way, virtually any biophysical property of a peptide, for instance, its binding affinity and specificity, proteolytic stability, and/or solubility can be optimized and tailored to a specific application. On the other hand, various screening techniques routinely yield macrocyclic peptides capable of selective binding to relatively shallow protein surfaces often involved in clinically important protein-protein interactions (PPI), in a manner similar to antibody-based therapeutics and in contrast to small molecule approaches. Due to a unique combination of these advantages, macrocyclic peptides garnered a fair amount of attention from the scientific community in the recent years. The growing interest in the field is evidenced by the steadily increasing number of primary and secondary publications on the topic (Fig. 1), as well as by a number of recently established pharmaceutical companies focusing their drug development efforts primarily on macrocyclic peptides, which has led to several de novo discovered peptides entering clinical trials.1,2 Overall, in the past twenty or so years, much progress towards transforming macrocyclic peptides into a viable drug modality has been made. Continuous refinement of high-throughput screening strategies and rational design techniques led to the discovery of a number of biologically active peptides, and co-crystal structures with their targets revealed how these molecules interact with protein surfaces. Similarly, a lot of research has been done to identify factors responsible for cell permeability, proteolytic stability, and other properties critical in drug development. However, despite great progress in uncovering features helpful in improving these parameters, general strategies, especially for cell permeability and oral bioavailability, have not yet been fully realized. In this perspective, we summarize the current state of the field by analyzing the latest advances and remaining challenges associated with macrocyclic peptides as pharmaceutical candidates. We review the latest approaches to the discovery of functional peptides, and to the improvement of their cell uptake, metabolic stability, and other basic pharmacological properties. We analyze the interplay between these approaches and offer our opinion on achieving further progress in advancing macrocyclic peptides as a drug modality.

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DISCOVERY OF BIOACTIVE MACROCYCLIC PEPTIDES Macrocyclization and its benefits “Macrocyclic peptide” is an umbrella term for a remarkably diverse group of molecules, and as such, it may be difficult to put forth a meaningful definition. Nevertheless as the name suggests, macrocyclic peptides can be defined as predominantly peptidic structures bearing a ring spanning multiple amino acid residues. A variety of macrocyclization reactions has been developed over the years, and depending on the strategy, backbone cyclized, side-chain to side-chain cyclized, lariat peptides and other, more esoteric topologies can now be easily synthetically accessed (Fig. 2).3–5 At any rate, macrocyclization, the one feature that these structurally diverse molecules share, generally improves a number pharmacological properties of a peptide.6 Linear unconstrained oligopeptides with few exceptions are disordered in aqueous solvents: they can quickly sample a range of conformations, and thus exist in high entropy states. Macrocyclization imparts a degree of conformational restraint and functional group preorganization, making target engagement easier by minimizing entropic loss due to binding. In this way, macrocyclization contributes to stronger and more selective interaction with the target protein. It is a conceptually simple yet very effective strategy often used in nature. Many nonribosomally produced peptides (NRPs) and ribosomally produced post-translationally modified peptides (RiPPs) — two major classes of peptidic natural products — share macrocyclization as a common structural feature.7–10 The benefits of peptide macrocyclization and other strategies for conformational constraint were noticed in the seventies, in the studies on bioactivity of peptidic hormone analogues.11–13 Soon, macrocyclization became a common method for improving bioactivity of oligopeptides. Cyclization of RGD peptides to improve their binding affinity for integrin cell receptors demonstrates the success of this approach, as exemplified by the development of cilengitide, a backbone macrocyclic ligand acting as a potent (IC50 = 0.58 nM) and selective antagonist of αvβ3.14–16 With the development of the first high-throughput screening technologies in the early nineties, it became possible to analyze the utility of conformational constraint in a library format, and it quickly became apparent that constrained peptide libraries yield more bioactive hits.17 The modern technology for the discovery of functional peptides makes use of macrocyclization or similar strategies to “scaffold” amino acid residues into a conformation optimal for binding to target proteins.18–20 Several conceptually distinct discovery approaches can be delineated depending on the overall philosophy of the process and the type of utilized macrocyclization chemistry as discussed below. Stapling macrocyclization “Stapled” peptides are macrocyclic structures primarily designed to stabilize α-helical secondary structures through the cyclization performed on amino acid side chains displayed on the same helical face (the so-called i,i+4 or i,i+7 positions).21,22 Ring-closing methathesis on α,α-disubstituted olefin-bearing amino acid residues is a common reaction used for macrocyclization, but a variety of complementary approaches were also established and studied in detail.4 α-helices are often found on the interface of PPIs, and thus stapled α-helical peptide ligands disrupting such PPIs can be rationally designed. In this approach, the helical portion of one partner protein engaging in the interaction is identified and synthesized as a standalone peptide, which then can be stabilized into adopting an α-helical conformation via stapling macrocyclization, and further optimized using medicinal chemistry tools. Such ligand discovery strategy yielded a plethora of PPI inhibitors, including β-catenin binders,23 BCL-2 inhibitors,24,25 and HIV-1 inhibitors.26 For one such peptide, an inhibitor of MDMX and MDM2 interaction with p53, Aileron Therapeutics is conducting clinical trials to treat peripheral T cell lymphoma.27 De novo discovery of functional macrocyclic peptides For targets, which require their ligands to adopt a non-canonical structure, rational design approaches quickly become a lot more challenging and fall out of favor. In such cases, high-throughput screening of macrocyclic peptide libraries often becomes more fruitful. There are numerous approaches to the preparation and screening of such libraries, and these strategies differ in throughput, ability to incorporate arbitrary non-proteogenic — often “privileged” — amino acids, and in the overall topology of displayed peptides.


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The oldest peptide screening platform, phage display,28 was successfully combined with a number of macrocyclization chemistries to generate libraries of macrocyclic peptides.29 Similar to the standard variant, DNA encoding randomized peptide sequences is ligated with the phage coat protein gene, and peptides are ribosomally synthesized following E. coli transduction. As a result, individual peptide sequences are displayed on the surface of a phage encoding these sequences. The library can then be cyclized with a reactive linker, which usually takes advantage of the unique reactivity of cysteine residues, and screened for biological activity. Diverse linkers for the preparation of macrocyclic peptide phage libraries have been developed. These chemistries include cysteine stapling with perfluoroaromatic compounds,30 cyclization with light-responsive linkers,31,32 thiother-forming tactics33 and disulfide bond formation,34 although the latter approach has somewhat fallen out of favor due to the inherent metabolic instability of the disulfide linkage.35 Similar ideas can be applied to generate bicyclic peptide libraries. In these approaches, a cysteine reactive trifunctional linker such as 1,3,5-tris(bromomethyl)benzene36 or 1,3,5-triacryloyl-1,3,5-triazinan37 is incubated with peptides containing three fixed cysteine residues to yield bicyclic libraries. Resulting constructs are even more conformationally restricted than their monocyclic counterparts, and it has been demonstrated that phage display screens on such libraries can yield potent and selective binders against various protein targets. Low nanomolar inhibitors of plasma kallikrein,36 FXIIa serine protease,38,39 and matrix metalloproteinase MMP-14 were discovered using this approach. The latter compound, BT1718, was further developed to increase its potency, target selectivity, and plasma stability, and it has recently entered clinical trials for treatment of solid tumors expressing MMP-14 as a bicyclic peptide-maytansinoid drug conjugate, performed by Bicycle therapeutics in collaboration with AstraZeneca.40 Another de novo discovery approach relying on in vivo production of macrocyclic peptides is split-intein circular ligation of peptides and proteins (SICLOPPS).41,42 In this method, a plasmid encoding a randomized extein sequence within a larger polypeptide containing two intein fragments is transformed into E. coli or other suitable host organism. Upon expression of the gene, intein splicing takes place, resulting in the formation of a head-to-tail cyclic extein peptide, which can be screened for biological activity with the use of a reverse two-hybrid system (RTHS). Intein-based macrocyclization can be further combined with genetic code reprogramming via the amber stop codon suppression to introduce non-proteogenic amino acids uniquely reactive towards cysteine side-chains, opening access to libraries of bicyclic peptides.43 A major advantage of SICLOPPS is the ability to perform functional (rather than affinity-based) selections owing to RTHS, and accordingly, this technique was used to discover a number of bioactive peptides — recently, head-to-tail cyclic hexapeptide inhibitors of BCL6 homodimerization44 and IDOL E3 ubiquitin ligase,45 with both ligands demonstrating low micromolar affinity for their targets. Discovery techniques based on variants of mRNA display have also rose to prominence. mRNA display can be used to screen vast libraries comprised of up to 1014 unique sequences — a feature that sets it apart from other approaches (for comparison, phage display and other techniques relying on in vivo production of libraries usually operate with 108-109 peptides).46 Similarly to aforementioned cyclization strategies, mRNA display can be utilized to produce peptides containing two fixed cysteine residues, which can be cyclized with a bifunctional linker such as dibromoxylene. This technique was applied to identify low nanomolar thrombin inhibitors,47 and Ra Pharmaceuticals adopted this strategy for their drug development efforts. Another mRNA display variant, termed random nonstandard peptide integrated discovery (RaPID) platform, utilizes flexible in vitro translation (FIT) to access macrocyclic peptides containing non-proteogenic amino acids.20 In FIT, E. coli translation machinery is combined with genetic code reprogramming achieved by the use of flexizymes, i.e. artificially evolved ribozymes capable of charging tRNA with a variety of non-proteogenic amino acids.48,49 This combination allows for the ribosomal incorporation of a wide variety of privileged nonproteogenic acids, which include α-hydroxyacids, N-methyl-, D-, β-amino acids, and amino acids bearing non-standard sidechains.50–52 The most common cyclization technique makes use of N-terminally installed chloroacetyl functionality, which under translation conditions spontaneously cyclizes with an internal cysteine to yield lariat peptides, although a number of other approaches to access mono- and bicyclic structures have been established.53 The combination of FIT with the incredible library diversity provided by mRNA display proved to be a powerful method for the discovery of bioactive macrocyclic peptides. RaPID screens were used to generate a number of high affinity and high selectivity binders. Examples include low nanomolar inhibitors of iPGM enzyme with good selectivity for C. elegans enzyme over other nematode species,54 and isoform-selective Akt kinase inhibitors among others.55



Bristol-Myers Squibb in collaboration with PeptiDream has recently initiated a molecular imaging study on one potent macrocyclic inhibitor of PD-1/PD-L1 interaction, which was originally identified using similar technology.56,57 Finally, synthetic chemistry approaches such as one-bead one-compound (OBOC) libraries can also be successfully utilized for the identification of peptides with designed biological activity.58,59 In principle, these methods allow for the greatest amount of freedom in terms of topology and cyclization design, while also allowing for the incorporation of arbitrary number of exotic amino acids. However, on-bead screens are generally laborious, which limits their throughput to ~106 compounds per screen. A number of other technical difficulties have somewhat limited the popularity of these approaches. Nevertheless, synthetic libraries are routinely used to discover bioactive macrocyclic peptides. Recently published examples include a two stage discovery of a potent head-to-tail cyclized cell permeable Ras inhibitor,60 and highly potent botulinum neurotoxin inhibitors cyclized with the use of alkyne/azide “click” reaction.61 Overall, a multitude of methods for the discovery of macrocyclic peptides with biological function of interest is available. Each of these techniques has unique advantages, best tailored to specific applications. The optimal discovery strategy should be informed by potential applications for macrocyclic peptides, specifics of the protein target, and available experimental expertise. That said, with so many general discovery platforms at our disposal, we venture to claim that finding macrocyclic peptides with biological activity of interest is no longer a bottleneck in the overall drug development process. Continuous development of techniques such as RaPID more or less transformed the challenge of discovering high affinity, high selectivity PPI inhibitors into a technical task. MAJOR CHALLENGES IN CREATING MACROCYCLIC PEPTIDE DRUG CANDIDATES Cellular uptake Advances in our understanding of factors governing cell penetrance of macrocyclic peptides have arguably not kept pace with discovery methods, and the challenge of developing a general strategy for creating peptides capable of accessing intracellular targets keeps tantalizing researchers. Although it is fair to say that a generic peptide sequence should not be expected to be readily uptaken by the cell, hundreds — if not thousands — of cell-penetrating peptides have been characterized,62 suggesting, at least in principle, the possibility of developing cell-permeable macrocyclic peptide drug candidates. Recent studies elucidated how various peptides penetrate biological membranes, and while many details remain unclear, several canonical entry mechanisms have been established (Fig. 3).63–65 The majority of peptides are uptaken either through pinocytosis/receptor-mediated endocytosis coupled to endosomal escape, or via facilitated diffusion through the cell membrane (will be reviewed in bioavailability section). Active transport of peptides by ABC-transporters and organic-anion transporters is also well documented. It should be noted, however, that these entry mechanisms are not mutually exclusive, and some research suggests that many cell permeable peptides can be uptaken via a combination of several pathways. For instance, one recent comprehensive study66 found that a number of hydrocarbon-stapled peptides showed up to 50% less accumulation in ATP-depleted cells, whereas accumulation of prototypical linear cationic Tat and poly-Arg8 peptides showed no such dependency. These results indicate that polycationic poly-Arg peptides permeate directly, in an energy independent manner, perhaps through passive diffusion facilitated by electrostatic interactions between arginine side chains and membrane phospholipids and sulfated proteoglycans,67,68 while stapled peptides are uptaken via a combination of energy dependent and independent pathways. At the same time, other stapled peptides, such as stapled BH3 helices, have been shown to enter the cytosol primarily through energy-dependent macropinocytosis followed by pinosomal escape.24 Regardless of the entry mechanism, several features are believed to be universally advantageous for cell permeability of a peptide. Stapling macrocyclization generally improves cellular penetrance, presumably by providing additional conformational stabilization. One study made use of live-cell microscopy, molecular dynamics simulations and analytical ultracentrifugation techniques to postulate that backbone rigidification and static presentation of guanidinium groups increases transduction of polycationic cellpenetrating peptides.69 In one key experiment, authors found that cyclic Arg10 internalized and localized to the cell nucleus five times faster than its linear analogue. Consistent with this hypothesis, numerous studies observed a correlation between cytosolic


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accumulation of stapled peptides and the degree of their α-helicity.68,70–72 Additionally, as most stapling reagents are rather hydrophobic, macrocyclization reaction increases peptide’s hydrophobicity on one helical face, and thus it often improves the overall amphipathicity of the structure, another feature commonly believed to be beneficial for cellular uptake. For instance, a family of small, amphipathic macrocyclic peptides composed primarily of arginine and aromatic side chain residues showed remarkable efficiencies of cytosolic entry and endosomal escape, apparently achieved via early endosome budding and the follow-up collapse of smaller vesicles.73 In another example, amphipathic arginine-rich peptides bicyclized with large perfluoroaromatic linkers were used to efficiently deliver oligonucleotide analogue cargo.74 In yet another study, authors postulated the importance of spatial separation between cationic and hydrophobic stretch, and, guided by this hypothesis, identified potent cell-penetrating inhibitors of β-catenin PPIs.75At the same time, research aimed at elucidating common biophysical determinants of cellular uptake for hydrocarbon-stapled peptides found70 that excessive positive charge combined with hydrophobicity may induce membrane disruption, as quantified by the extent of lactate dehydrogenase leakage from the cytosol. The same study also analyzed intracellular accumulation efficiency as a function of peptide’s HPLC retention time, and found that hydrophobicity was a major determinant of cellular penetrance, along with pI and α-helicity. Authors noted that the balance in all three parameters is important, as very hydrophobic or excessively positively charged peptides often led to membrane disruption and cytotoxicity. Receptor-mediated endocytosis is an alternative cell uptake mechanism with different structural requirements for the peptide.76,77 In this process, the peptide binds to a specific (in contrast to predominantly non-specific binding of polycationic and amphipathic peptides) cell surface receptor, triggering clathrin or caveolae-mediated internalization through endocytosis. Numerous peptides uptaken via this route are known, and in general, receptor-mediated endocytosis represents a powerful tool for cell entry.78 Natural receptor ligands and their mimics, such as engineered substance P, which specifically binds its cognate receptor, NK1R,79 or αvβ3/ αvβ5 integrin receptor binding cyclic RGD peptides are efficiently internalized,80 and can be used for cytosolic delivery of various cargo, including proteins, DNA, and even polystyrene microparticles.81 Nevertheless, the method is not without its share of limitations: not every ligand triggers an internalization event, and not every receptor undergoes clathrin-mediated endocytosis. In addition, engineering a secondary biological activity necessary for receptor binding onto a de novo discovered peptide may prove challenging. Transporter-mediated cell entry is another, admittedly significantly less explored mechanism of cellular uptake. A number of studies suggest that some peptide-like pharmaceuticals and oligopeptides are internalized with the assistance of proton-coupled oligopeptide transporters (SCL15).82 Many natural macrocyclic peptide toxins, such as α-amanitin,83 phalloidin,84 and microcystin-LR,85 as well as opioid peptides such as DPDPE, and deltorphin86 are uptaken by organic anion–transporting polypeptide (OATP) family proteins.87 Still, at present we lack complete knowledge of human transporter proteins and their substrate preferences required to generalize features of this entry mechanism. In theory, active transport may constitute a viable alternative to endocytosis-based cell uptake pathways. Clearly, macrocyclic peptides can be efficiently uptaken by the cell, at least in principle. It is also apparent that this incredibly versatile class of compounds can follow multiple avenues to enter the cell. Structural requirements naturally vary depending on a mechanism, and some of these requirements are better understood than others. But at any rate, the general strategy for transforming a bioactive peptide into a cell-permeable compound remains elusive. Oral bioavailability Similar to the problem of cell permeability, oral availability remains one of the biggest, largely unsolved, challenges for macrocyclic peptides.88 Orally bioavailable medications are easier to administer and to dose, which increases patient compliance [footnote: arguably, with the recent advances in injector technology, the barrier to intravenous injections has been significantly reduced, and thus, the patient compliance issue has become less critical], and makes bioavailability a desirable feature from the drug development standpoint. A number of orally bioavailable peptides, including a few clinically approved therapeutics, are well characterized,89 but in general peptides are not readily absorbed by the gastrointestinal (GI) tract into the blood stream.



To count as bioavailable, a peptide must meet a number of criteria. First, it needs to be metabolically and chemically stable, such that it can survive low pH environment of the stomach, treatment with various digestive proteases found at virtually every step of the process, and first pass clearance. Second, the peptide must cross a layer of enterocytes in the small intestine to reach the blood stream. This absorption can happen in two major ways: paracellular, through tight junctions between the cells forming the GI tract, and transcellular, which involves double crossing through enterocyte’s cell membrane. Although some examples of paracellular transport have been described, for the most part peptides are thought to be absorbed via transcytosis. This process is conceptually analogous to the issue of cell penetrance, as it requires two membrane crossing events to occur, but for the same reason it arguably represents an even more formidable challenge. Transcytosis via active transport (as discussed above) has long been documented, but by and large recent research focused on utilizing passive diffusion as a means to achieve oral bioavailability for macrocyclic peptides.90,91 Regular peptides should not be expected to rapidly diffuse through cellular membranes at physiologically relevant concentrations: most de novo discovered bioactive peptides violate Lipinski’s rules (also rule of five; RO5), which is a set of empirically determined criteria to estimate whether a molecule will diffuse through biological membranes at an appreciable rate.92 In most cases, to provide for strong and selective binding to its target, a peptide needs to display a number of amino acid side-chains, and thus it must reach a certain size. For instance, macrocyclic peptides with nanomolar binding affinities discovered with RaPID platform, as well as bicyclic peptide obtained from phage display screens, average around 1800 Da in molecular weight (see below), and thus they far exceed the empirical 500 Da molecular weight cutoff imposed by the RO5. Similarly, macrocyclic peptides have too many hydrogen bond donors (HBD) and acceptors (HBA), as well as large polar surface areas to passively diffuse through biological membranes.93 However, there are remarkable exceptions to these rules of thumb: most notably, cyclosporine A (CSA, Fig. 4). CSA is a backbonecyclic NRP used in clinic as an orally bioavailable immunosuppressant. This peptide is able to passively diffuse through biological membranes, albeit slower than small molecule drugs: CSA’s diffusion coefficient is 2.5·10−7 cm/s vs. 10−5 to 10−6 cm/s for “regular” small molecule therapeutics.94 Because of its unique properties CSA and its analogues have served as a model system for studying factors responsible for passive diffusion of peptides. Several important factors have been identified. CSA is fairly conformationally flexible, as it can adopt at least 6 different conformations in polar solvents such as methanol.95,96 The conformational switch hypothesis posits that the intermolecular hydrogen bonding configuration observed in polar aqueous solvents needs to rearrange to a network of intramolecular, shielded hydrogen bonds, minimizing the energy requirement for the phase transition.97,98 This hypothesis found some experimental evidence, as it has been shown that CSA folds into a compact structure featuring four intramolecular hydrogen bonds in non-polar carbon tetrachloride,99 a conformation, which may facilitate its entry into a hydrophobic phase. Additionally, CSA features seven N-methylated amino acids which also have been shown to be important for its facile diffusion through biological membranes, as in addition to decreasing the number of HBDs N-methylated amides point to solvent in carbon tetrachloride, shielding polar functionalities.99 Modified peptide backbones lacking amide protons, which act as HBDs, is a feature also found in other bioavailable peptidic natural products, and as a consequence, this strategy has received a fair amount of attention.100–102 For example, N-methylation has been found instrumental in achieving high oral bioavailability of N-methylated β-strand decapeptides in rat models,103 and it was helpful for many other model peptides.104–106 At the same time, it is clear that N-methylation is not a panacea: one study discovered that Nmethylation may decrease oral bioavailability of a peptide, using sanguinamide A analogues as a model system.107 In another report, authors prepared increasingly N-methylated analogues of octreotide, expecting to see increase in cellular uptake with increasing lipophilicity of the peptides, but observed no such trend.108,109 Similarly, replacing a peptide bond with an ester (such peptides are often called depsipeptides) removes an HBD, often leading to increased oral bioavailability at the expense of metabolic stability, as esters are often quickly degraded in biological milieu. Depsipeptide linkages are found in some bioavailable natural products: for instance, anthelmintic PF1022A cyclic octapeptide features four ester bonds,110 and antibiotic cyclic hexapeptide beauvericin has three esters and three N-methyl amides,111 effectively leaving it with no HBDs. Other approaches towards reducing the number of HBDs include the use of N-alkylated peptoids and peptoid-peptide hybrids, as described in a recent study where authors incorporated cycloalanine residues to create a cell-permeable


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variant of HIV Rev protein-binding peptide,112 and the use of oxa- and thiazole moieties, which are heavily featured in both RiPPs and NRPs (Fig. 4). It should be noted however, that since passive diffusion of peptides disfavors excessive HBDs and HBAs, many orally available peptides display predominantly hydrophobic amino acid side chains (Leu, Ile, Val, Trp, Phe, etc), which often leads to extremely low solubility of these compounds: for example, CSA is practically insoluble in water, and has to be administered as an emulsion. Accessing conformations outside of the established Ramachandran space may also facilitate passive diffusion. Accordingly, D-amino acid replacements,113 as well the use of α,α-disubstituted amino acids, prominently used in hydrocarbon-stapled peptides, has been suggested as a means to improve bioavailability.114 In fact, macrocyclization itself is a key feature shared by most bioavailable peptides, including many natural product peptides, and it is believed that, analogously to the benefits for binding, a degree of conformational constraint and functional group preorganization is a pre-requisite for passive diffusion across biological membranes. Overall, the latest progress in understanding the drivers of passive diffusion of peptides has uncovered a number of structural requirements. It became possible to de novo design an orally available macrocyclic peptide, but despite these advances achieving oral availability for bioactive drug candidate peptides remains a challenging task, due to the fact that such compounds often bear numerous functionalities heavily disfavoring passive diffusion. Perhaps, fresh insights will be required to make further progress in this area. Metabolic stability Peptides are natural substrates of ubiquitous endo- and exopeptidases functioning in every tissue and every cell of the body: the human genome encodes for more than 550 protease genes.115 As a consequence, linear oligopeptides composed of proteogenic amino acids are rapidly degraded in vivo, and their elimination half-lives (t1/2) rarely exceed several minutes. Nevertheless, structural features conferring resistance to proteolysis are fairly well-understood, and thus fine-tuning proteolytic stability of a macrocyclic peptide is arguably easier than modulating its other pharmacological properties. Introducing any non-proteogenic elements into the structure of a peptide will render it more resistant to proteolysis. 116 A recurring theme of this review — effectiveness of macrocyclization in improving a range of pharmacological properties of peptides — is also relevant here. Macrocyclic peptides demonstrate higher resistance towards proteases in vitro and in vivo as compared to their linear counterparts. As an example, macrocyclic C-terminal hexapeptide of substance P had half-life in liver slices ~30 min, compared to 4 min demonstrated by its linear analogue.117 In another work, less than 5% of backbone cyclic analogues of melanocyte-stimulating hormone degraded when treated with brush border membrane vesicles for 90 min, whereas the concentration of the linear analogue decreased by 40% after 40 min of the identical treatment.118 Bicyclization introduces additional strain to the peptide structure, and may act as a tool to further increase proteolytic stability of a drug candidate. Accordingly, the aforementioned BT1718 is retained in plasma for more than 20 hours.119 The use of non-standard amino acids will also normally increase proteolytic stability of macrocyclic peptides. Seven N-methylated residues collectively render CSA resistant to proteolysis, and lead to six hour plasma half-life.120 Another clinically approved peptide drug, degarelix, is an acyclic structure, which utilizes a number of D-amino acids, exotic non-natural side chains as well as acylated N-terminus (Fig. 5) to gain extraordinary metabolic stability: the drug is retained in plasma for months, with elimination half-life reaching up to 70 days.121 Introduction of α,α-disubstituted amino acids also stabilizes the peptide against proteolytic degradation, and accordingly, a number of bioactive protease-resistant stapled peptides have been reported.114,122 Since proteolysis ultimately revolves around peptide bond cleavage, replacing the amide with its non-cleavable or more stable bioisosteres will make a peptide more metabolically stable, while minimally perturbing the overall structure.123 Thioamides, structures characterized by a single atom O→S replacement, are especially interesting in this regard. For instance, a GLP-1 analogue bearing a single thioamide in a critical position (Fig. 6) showed 750-fold higher stability towards degradation by dipeptidyl peptidase 4 compared to wild type GLP-1, while retaining full AMP activation ability.124 Such stabilization is noteworthy considering that the thioamide analogue is still an unconstrained linear peptide consisting largely of proteogenic amino acids.



The search for proteolytically stable sequences at the discovery stage represents another, conceptually different strategy towards the problem of metabolic stability. In this approach, a counter-selection against proteases of choice can be added alongside primary target selections, which in theory should yield proteolytically stable biologically active peptides. In one such work,125 authors performed mRNA display selection against signaling protein Gαi1 on a pre-selected macrocyclic peptide library treated with chymotrypsin, and obtained fully functional ligands, which had 100-fold increased human serum stability compared to a macrocyclic peptide discovered in the absence of protease pre-treatment (t1/2 ~ 28 h vs. 0.33 h). Interestingly, with the exception of the linker used for cyclization, this impressive stabilization was achieved for a fully proteogenic sequence without the use of non-standard amino acids. This research highlights the fact that a variety of strategies for increasing proteolytic stability of a peptide are available. It should be noted, however, that the exact pathways of in vivo metabolism are not always investigated in great detail. For fully proteogenic peptides proteolysis is the most common degradation route, but other pathways should not be discounted, especially for macrocyclic structures bearing multiple non-standard amino acids. For instance, one study on the metabolic stability of CXCR7 modulators with exotic side-chains revealed that the macrocyclic peptide degraded in NADPH-supplied rat liver microsomes much faster (t1/2 ~ 45 min) than in microsomes lacking NADPH (t1/2 > 2 h), suggesting that cytochrome P450 enzymes play a role in oxidative degradation of the peptide.126 Renal clearance Metabolic stability is not the only concern pertaining to the challenge of plasma retention. Exogenous peptides can be rapidly filtered in the kidney even if they are chemically intact. The glomeruli in the kidney is a porous structure able to efficiently filter out of the plasma molecules with a mass below approximately 5-10 kDa.127 Most macrocyclic peptides fall in this category, and since peptides are also poorly reabsorbed through the renal tubule, they undergo rapid renal clearance. In contrast to the challenges discussed above, typical amino acid modifications (N-methyl-, D-, β-, and α,α-disubstituted amino acids) are not effective in fighting fast renal clearance, and thus different strategies are needed. Increasing the effective hydrodynamic radius of a peptide such that it is no longer filtered out is the primary approach for extending its plasma retention time. Peptide modification which enhances its albumin binding is a classic technique that follows this logic. Human serum albumin (HSA) is a 66 kDa plasma protein with long plasma retention time (t1/2 ~ 20 d), which binds and transports lipophilic molecules. Although sufficiently hydrophobic peptides may display satisfactory plasma protein binding, in general the conjugation of a fatty acid or a similar lipophilic moiety to a peptide to enhance its HSA binding is required to extend its plasma half-life.128 Two clinically approved peptide-based medications, levemir,129 a long-acting insulin analogue, and liraglutide,130 a GLP-1 analogue, both take advantage of fatty acid conjugation (C14 for levemir, and C16 for liraglutide) to extend their plasma circulation times. In the case of liraglutide, the plasma half-life improved by a factor of 15 compared to an unmodified GLP-1 analogue (Fig. 6). Another conceptually similar strategy is the tethering of a bioactive peptide to an HSA-binding peptide. For instance, bicyclic inhibitors of urokinase-type plasminogen activator (uPA) conjugated to albumin-binding peptide demonstrated up to 50-fold longer plasma retention times (t1/2 ~ 1 d), and improved proteolytic stability compared to the bicyclic ligand alone.131 Other methods include covalent conjugation to proteins with long serum half-lives,132,133 and to biocompatible polymers such as PEG,134 polysialic acid135 and hydroxyethyl starch.136 Overall, it may be said that fast renal clearance of macrocyclic peptides is an issue which needs to be taken into account, although a number of general strategies to overcome this challenge have been developed. Nevertheless, these strategies are not without limitations, as any conjugation to excessively lipophilic moieties runs the risk of compromising solubility, biological activity, and in some cases immunogenicity of the resulting construct. An overview of the literature on the topic137 reveals that in most cases it is possible to prolong peptide plasma residence to several hours, and up to a few days. But still, reaching serum retention comparable to antibody therapeutics may prove challenging. Immunogenicity


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In general, oligopeptides are believed to be poor immunogens.138 However, worth noting is the fact that at least two late stage clinical trials for linear peptides in the last decade were discontinued due to immunogenicity issues. First was taspoglutide, a long acting GLP-1 analogue used for diabetes type II treatment, which occasionally led to serious hypersensitivity in patients, presumably through the formation of anti-drug antibodies, which were detectable in about 50% of the patients.139 Similarly, peginesatide was a PEGylated erythropoietin analogue (2.5 kDa peptide chain; functional as a dimer) pursued by Affimax and Takeda Pharmaceuticals. The drug was withdrawn from the market after a small fraction of patients started developing severe hypersensitivity reactions including lifethreatening anaphylaxis.140 These examples demonstrate that medium size (3-5 kDa) peptides may lead to serious immunogenicity issues, even if the peptide is based on the human sequence, as in the case of taspoglutide (Fig. 6). For smaller macrocyclic peptides not enough research has been published to warrant generalizations. Even less is known about the effect that non-standard amino acids exert on the overall immunogenicity of a peptide. D-amino acids are perhaps best studied in this regard, but even in this case some controversy remains.141 Early studies showed no immunogenicity of D-amino acid copolymers in a variety of animal models,142 but later research revealed that dosage is of critical importance, as immune paralysis occurs at higher doses.143 In a number of separate studies, chemically prepared D-proteins, rubredoxin144 and VEGFA antagonist,145 elicited no immune response in mice at any dosage, possibly because folded, non-digestible D-polypeptides are not presentable onto MHC. However, shorter epitope-like D-amino acid sequences do appear to be able to elicit robust immune response. A recent study describes a peptide comprised entirely of D-amino acids, which acted as a full agonist of CD8+ T cells, and was able to protect naive mice by subcutaneous vaccination by conferring protection from subsequent lethal influenza challenge.146 In short, further research shedding light on the factors driving immunogenicity or non-immunogenicity of macrocyclic peptides is critical in advancing our understanding of in vivo fates of these biomolecules. PERSPECTIVE It is fair to say that macrocyclic peptides as a potential drug modality are experiencing a surge of interest. We attribute this phenomenon primarily to the identification of a plethora of structurally intriguing NRP and RiPP natural products, propelled by the revolution in sequencing technology, and to the development and further refinement of efficient de novo discovery methods. These advances led to the current state of affairs, where discovery of a lead compound with designed biological activity no longer represents a major bottleneck in the overall early drug discovery process. Optimization of other pharmacological properties, i.e. membrane permeability, oral bioavailability, and metabolic stability as discussed above, may prove significantly more challenging. Fine tuning any one of these parameters may be practical, but the true challenge lies in interfacing every biophysical property with each other, creating a balanced drug candidate. For instance, oral bioavailability through passive diffusion similar to CSA models requires peptides with high lipophilicity and minimal intramolecularly unsatisfied HBD and HBA. At the same time, polar and charged functionalities displayed by a bioactive peptide often play a critical role in securing high degree of specificity and binding affinity to its target protein. Similarly, effective cell penetration often requires polycationic amphipathic structures, which are rarely — if ever — obtained from naive selections with display technology such as mRNA or phage display. Both of these popular techniques tend to yield non-canonically folded macrocyclic peptides with a fine balance between positive, polar and hydrophobic side chains orderly situated to optimally fit their complementary protein surface. As such a balance is at odds with other pharmacological properties, it is unfortunate that these otherwise powerful de novo discovery platforms require painstaking lead optimization to create viable drug candidates. To provide a basis for these speculations, we performed a meta-analysis of macrocyclic peptide ligands developed with the use of RaPID, one of the most prolific discovery platforms to date. Our analysis included published selections against 16 therapeutically relevant targets, ranging in their structure and function from soluble cytosolic enzymes to large transmembrane receptors. Naturally, depending on their tertiary structure and a number of other considerations, these targets should be expected to exhibit different binding preferences, which may limit the usefulness of such an analysis. Yet at the same time, we found that most analyzed peptides share a number of features, and thus we attempt to make certain generalizations about an “average bioactive macrocyclic peptide”.



In this analysis, for each of the 16 targets, we analyzed up to three best ligands (as judged by KD, IC50 or other relevant quantitative activity measure) for each protein, which resulted in a set of 46 peptides (Supplementary Table 1). An average ligand was 15.07 amino acid residues long (Fig. 7B), and most peptides clustered around this value (s.d. = 1.69). No ligands were longer than 20 amino acids or shorter than 13 residues, which is worth noting, because in most cases a library peptide length was not fixed at a single value, as most libraries contained a mixture of sequences ranging from 6 residues up to 14 or 17. This observation lends some support to the idea that a ligand needs to be of a certain size to properly fold and create a binding surface complementary to its target. Seven crystal structures of these macrocyclic peptides in complex with their targets are available (PDB: 4L3O, 3VVR, 5B4W, 5KGL, 5KEZ, 5LY1, 5O45). A cursory glance at the binding interfaces reveals that both hydrophobic and hydrophilic residues engage in specific target interactions while a number other amino acids (commonly proline, glycine, and the cysteine utilized for macrocyclization) help scaffolding these residues in place for optimal binding. Analysis of amino acid composition of these peptides revealed that most (43/46; 93%) ligands bear at least one charged amino acid, with an average of 2.57±1.54 (Fig. 7B). Thus, an average ligand consisted of 17±10% charged amino acids. Most peptides demonstrated a fine balance between hydrophobic and hydrophilic residues: an average ligand had 45±13% hydrophobic residues, and no peptide was more than 67% hydrophobic. Remarkably, these results were in close agreement with an analogous analysis performed for bicyclic peptides discovered from phage display screens, even though library designs, compositions and even the topology of discovered peptides were quite different. Bicyclic ligands were on average 15.33±1.87 residues long, and were composed on average of 20±10% charged and 50±9% hydrophobic amino acids. At the same time, bicyclic peptides were generally lighter (average molecular weight 1804±237) and less lipophilic (average cLogP -9.8±2.5) than RaPID peptides (1924±185 Da and -5.5±3.3 cLogP), but at any rate, every analyzed ligand resided far from the prototypical CSA on the cLogP vs. molecular weight plot (Fig. 7A). Therefore, the aforementioned speculations appear to be supported by this analysis: de novo discovered functional peptides mismatch conventionally developed archetypes for passively diffusing lipophilic or actively internalized amphipathic structures. We envision several potential solutions to this dilemma. A recent work from our laboratory describes a successful RaPID screen using a heavily genetically reprogrammed library (11 non-standard amino acids) depleted of charged and hydrophilic amino acids.147 The selection still yielded potent (KD ~ 40 nM) and selective interleukin-6 receptor (αIL6R) binders, demonstrating for the first time the utility of such “charge-depleted” libraries in generating natural product-like lipophilic compounds (Fig. 8). Additionally, this work demonstrated that in a RaPID format, about half (11/23) of the encoded amino acids can be successfully reprogrammed to privileged non-standard analogues, pointing towards the possibility of exploring heavily reprogrammed libraries with a skewed monomer composition to introduce structural bias and to promote the discovery of ligands with certain biophysical characteristics. This approach can be further complemented by saturated mutagenesis with both proteogenic and various non-proteogenic amino acids achieved via the combination of RaPID selection and deep sequencing.148 Such a technique allows for the facile optimization of ligand structures to generate macrocyclic peptides with improved cLogP values. Another potential way to generate macrocyclic ligands with an improved pharmacological profile is multilayered selections, that is, screens for multiple activities simultaneously. The aforementioned work125 on the mRNA display discovery of protease resistant Gαi1 inhibitors paves the way in performing such experiments. At the same time, it should be admitted that screens for more elaborate activities (cell permeability is an example) may prove disproportionately harder to interface with regular binding selections. Finally, we anticipate that active transport mechanisms can be leveraged to solve the issues of low cell permeability and bioavailability at least in some cases. A number of natural product macrocyclic peptides are known to be actively transported across cell membranes, and with improved understanding of mechanisms underlying active transport, these pathways may be mimicked to deliver therapeutic peptides to the cytosol. Alternatively, various conjugation strategies may be utilized to the same effect. A recent proof-of-concept work on the transcytosis of GM1-conjugated peptides has demonstrated a feasibility of such approaches.149 To summarize, the field of macrocyclic peptides is progressing by leaps and bounds. Recent research elucidated many ways in which pharmacological properties of macrocyclic peptides can be fine-tuned and improved, and with the advent of efficient methods to generate biologically active ligands, de novo designed macrocyclic peptides are poised to become a viable drug modality. Interfacing


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a number of different, often conflicting considerations for improving individual pharmacological properties, and the establishment of an integrated discovery platform able to achieve such interfacing represent, perhaps, the single biggest challenge remaining in the field. Also important will be generalizing the understanding of factors behind cellular uptake and bioavailability, as well as other critical in vivo characteristics, such as immunogenicity and metabolic fates of macrocyclic peptides, especially those which contain a number of exotic amino acids and are generally proteolytically stable. We believe that the remaining hurdles will be overcome in the not so distant future. SUPPORTING INFORMATION Table S1 listing macrocyclic peptide ligands discovered from RaPID and phage display with bicyclic peptide screens. AUTHOR INFORMATION Corresponding Author *[email protected] ORCID Alexander Vinogradov: 0000-0002-8899-0533 Hiroaki Suga: 0000-0002-5298-9186 Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS We are thankful to our current and past co-workers at the Suga laboratory for creating an intellectually stimulating environment that helped in the preparation of this manuscript. A.V. would also like to thank Dr. Zachary Gates (MIT) for insightful discussions throughout the course of this work. The body of the works discussed in this article from the Suga laboratory was supported by many Japanese funding agencies, recently by JST CREST (JPMJCR12L2), AMED (am0301001h0004 and am0101090j0001), and JSPS Kakenhi-S (26220204). REFERENCES (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13)

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Uppalapati, M.; Lee, D. J.; Mandal, K.; Li, H.; Miranda, L. P.; Lowitz, J.; Kenney, J.; Adams, J. J.; Ault-Riché, D.; Kent, S. B. H.; et al. A Potent d Protein Antagonist of VEGF-A Is Nonimmunogenic, Metabolically Stable, and Longer-Circulating in Vivo. ACS Chem. Biol. 2016, 11, 1058. Miles, J. J.; Tan, M. P.; Dolton, G.; Edwards, E. S. J.; Galloway, S. A. E.; Laugel, B.; Clement, M.; Makinde, J.; Ladell, K.; Matthews, K. K.; et al. Peptide Mimic for Influenza Vaccination Using Nonnatural Combinatorial Chemistry. J. Clin. Invest. 2018, 128, 1569. Passioura, T.; Liu, W.; Dunkelmann, D.; Higuchi, T.; Suga, H. Display Selection of Exotic Macrocyclic Peptides Expressed under a Radically Reprogrammed 23 Amino Acid Genetic Code. J. Am. Chem. Soc. 2018, 140, 11551. Rogers, J. M.; Passioura, T.; Suga, H. Nonproteinogenic Deep Mutational Scanning of Linear and Cyclic Peptides. Proc. Natl. Acad. Sci. 2018, 115, 10959. Garcia-Castillo, M. D.; Chinnapen, D. J. F.; Te Welscher, Y. M.; Gonzalez, R. J.; Softic, S.; Pacheco, M.; Mrsny, R. J.; Ronald Kahn, C.; von Andrian, U. H.; Lau, J.; et al. Mucosal Absorption of Therapeutic Peptides by Harnessing the Endogenous Sorting of Glycosphingolipids. Elife 2018, 7, e34469. Tillett, D.; Dittmann, E.; Erhard, M.; von Döhren, H.; Börner, T.; Neilan, B. A. Structural Organization of Microcystin Biosynthesis in Microcystis Aeruginosa PCC7806: An Integrated Peptide-Polyketide Synthetase System. Chem. Biol. 2000, 7, 753. Yamagishi, Y.; Shoji, I.; Miyagawa, S.; Kawakami, T.; Katoh, T.; Goto, Y.; Suga, H. Natural Product-like Macrocyclic N-Methyl-Peptide Inhibitors against a Ubiquitin Ligase Uncovered from a Ribosome-Expressed de Novo Library. Chem. Biol. 2011, 18, 1562. Rentero Rebollo, I.; McCallin, S.; Bertoldo, D.; Entenza, J. M.; Moreillon, P.; Heinis, C. Development of Potent and Selective S. Aureus Sortase A Inhibitors Based on Peptide Macrocycles. ACS Med. Chem. Lett. 2016, 7, 606. Rhodes, C. A.; Dougherty, P. G.; Cooper, J. K.; Qian, Z.; Lindert, S.; Wang, Q. E.; Pei, D. Cell-Permeable Bicyclic Peptidyl Inhibitors against NEMO-IκB Kinase Interaction Directly from a Combinatorial Library. J. Am. Chem. Soc. 2018, 140, 12102. Taori, K.; Paul, V. J.; Luesch, H. Structure and Activity of Largazole, a Potent Antiproliferative Agent from the Floridian Marine Cyanobacterium Symploca Sp. J. Am. Chem. Soc. 2008, 130, 1806. Mascio, C. T. M.; Mortin, L. I.; Howland, K. T.; Van Praagh, A. D. G.; Zhang, S.; Arya, A.; Chuong, C. L.; Kang, C.; Li, T.; Silverman, J. A. In Vitro and in Vivo Characterization of CB-183,315, a Novel Lipopeptide Antibiotic for Treatment of Clostridium Difficile. Antimicrob. Agents Chemother. 2012, 56, 5023. Schwochert, J.; Turner, R.; Thang, M.; Berkeley, R. F.; Ponkey, A. R.; Rodriguez, K. M.; Leung, S. S. F.; Khunte, B.; Goetz, G.; Limberakis, C.; et al. Peptide to Peptoid Substitutions Increase Cell Permeability in Cyclic Hexapeptides. Org. Lett. 2015, 17, 2928. Dong, J. Z.; Shen, Y.; Zhang, J.; Tsomaia, N.; Mierke, D. F.; Taylor, J. E. Discovery and Characterization of Taspoglutide, a Novel Analogue of Human Glucagon-like Peptide-1, Engineered for Sustained Therapeutic Activity in Type 2 Diabetes. Diabetes, Obes. Metab. 2011, 13, 19. Gutniak, M. K.; Linde, B.; Holst, J. J.; Efendic, S. Subcutaneous Injection of the Incretin Hormone Glucagon-Like Peptide 1 Abolishes Postprandial Glycemia in NIDDM. Diabetes Care 1994, 17, 1039. Jensen, L.; Helleberg, H.; Roffel, A.; van Lier, J. J.; Bjørnsdottir, I.; Pedersen, P. J.; Rowe, E.; Derving Karsbøl, J.; Pedersen, M. L. Absorption, Metabolism and Excretion of the GLP-1 Analogue Semaglutide in Humans and Nonclinical Species. Eur. J. Pharm. Sci. 2017, 104, 31.


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Figure 1. Publications and citations on the topic of macrocyclic peptides for the 1990 to 2017 time period. Data was obtained from Web of Science for the query “macrocyclic peptide”. Both publication and citation numbers trend upwards to indicate a continuous increase of interest in the field.



Figure 2. The diversity of macrocyclization approaches. Five functional peptides of different topology illustrate common macrocyclization strategies. Microcystin-LR utilizes head-to-tail cyclization, a motif often observed in NRP natural products;150 E6AP Ub ligase inhibitor, a lariat peptide discovered from a RaPID screen,151 demonstrates a thioether linkage created via an SN2 chemistry on a cysteine side-chain with an N-terminal chloroacetyl moiety; bicyclic Sortase A inhibitor discovered from a phage display screen152 is prepared via a cyclization of three cysteine residues with 1,3,5-tris(bromomethyl)benzene linker; BH3 α-helical peptide24 makes use of olefin methathesis to establish a side-chain-to-side-chain macrocycle; NEMO inhibitor discovered from a synthetic combinatorial library153 cyclizes three primary amines with 1,3,5-benzenetricarboxylic acid to yield a unique bicyclic architecture. Functional groups involved in macrocyclization are highlighted in purple; amino acids with non-proteogenic side-chains — red; D-, β- and α,α-disubstituted amino acids — green; N-methylated amino acids — blue. For BH3 peptide, the linear portion of the peptide is abbreviated using the standard one letter amino acid code.


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Figure 3. An overview of canonical cell uptake mechanisms. Left to right: polycationic peptides such such poly-Arg and TAT are thought to cross the cell membrane via facilitated diffusion enabled by an electrostatic interaction with heparan sulfate displayed on cell surface receptors; RGD peptides serve as a model to illustrate internalization via receptor-mediated endocytosis, in this case exemplified by the interaction of RGD with integrin receptors, which leads to the formation of clathrin-coated pits followed by endosome formation; numerous stapled and amphipathic macrocyclic peptides cross the cell membrane via pinocytosis followed by pinosomal escape; OATP family proteins are involved in active transport of oligopeptides and some cyclic natural products, such as α-amanitin; cyclosporine A represents lipophilic macrocyclic peptides which permeate the cell membrane in a passive way, akin to small molecules.



Figure 4. Common structural features found in bioavailable macrocyclic peptides, as exemplified by cyclosporine A, largazole,154 surotomycin,155 and a model cell-permeable hexapeptide/peptoid hybrid.156 Amino acids with nonproteogenic side-chains are highlighted in red; D-amino acids (green); N-methylated amino acids, depsipeptide linkages and heterocyclic backbones (blue).


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Figure 5. Chemical structure of degarelix, an FDA approved medication used in the treatment of prostate cancer. The peptide demonstrates an extraordinary long plasma circulation time (t1/2 ~ 40-70 days) owing to many nonproteogenic elements in its structure.121 Amino acids with non-proteogenic side-chains are highlighted in red; D-amino acids (green).



Figure 6. GLP-1 and its analogues as a model for extending plasma retention of a peptide. GLP-1, an endogenous linear peptide hormone involved in glucose metabolism, is proteolytically unstable and thus is unsuitable as a medication on its own. Its modified analogues serve as an illustration of various strategies for increasing proteolytic stability and protein binding, both of which are critical in achieving long plasma circulation times. A) Consensus structure shared between GLP-1 and its analogues. Amino acid sequence is given in the standard one letter code. B) Chemical structures of GLP-1 peptides and their respective t1/2 values. The values for t1/2 were taken for GLP-1 from refs. 124,157,158; for GLP-1 F7A8S from ref. 124; for liraglutide from ref. 130; for taspoglutide from ref. 157; for semaglutide from ref. 159.


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Figure 7. Analysis of macrocyclic peptide ligands obtained from RaPID and phage display screens. A) Calculated lipophilicity (cLogP; calculated with OSIRIS Property Explorer) as a function of molecular weight for the ligands discovered with RaPID (blue), or with phage display on bicyclic peptides (green). All ligands are bigger and less lipophilic than CSA (red). IL6R-2 ligand from Fig. 8 is shown in purple for comparison. B) Analysis of amino acid sequence length and crude composition by amino acid type: ligands discovered with the use of two different techniques show remarkable similarities.



Figure 8. Chemical structure of interleukin-6 receptor ligand (IL6R-2) obtained from a RaPID screen of a “charge depleted” library.147 This ligand is smaller and significantly more lipophilic than most compounds identified from RaPID screens. Amino acids with non-proteogenic side-chains are highlighted in red; N-methylated amino acids (blue); functional groups involved in macrocyclization (purple).


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TOC figure.



Figure 1. Publications and citations on the topic of macrocyclic peptides for the 1990 to 2017 time period. Data was obtained from Web of Science for the query “macrocyclic peptide”. Both publication and citation numbers trend upwards to indicate a continuous increase of interest in the field. 222x105mm (300 x 300 DPI)


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Figure 2. The diversity of macrocyclization approaches. Five functional peptides of different topology illustrate common macrocyclization strategies. Microcystin-LR utilizes head-to-tail cyclization, a motif often observed in NRP natural products;150 E6AP Ub ligase inhibitor, a lariat peptide discovered from a RaPID screen,151 demonstrates a thioether linkage created via an SN2 chemistry on a cysteine side-chain with an N-terminal chloroacetyl moiety; bicyclic Sortase A inhibitor discovered from a phage display screen152 is prepared via a cyclization of three cysteine residues with 1,3,5-tris(bromomethyl)benzene linker; BH3 αhelical peptide24 makes use of olefin methathesis to establish a side-chain-to-side-chain macrocycle; NEMO inhibitor discovered from a synthetic combinatorial library153 cyclizes three primary amines with 1,3,5 benzenetricarboxylic acid to yield a unique bicyclic architecture. Functional groups involved in macrocyclization are highlighted in purple; amino acids with non-proteogenic side-chains — red; D-, β- and α,α-disubstituted amino acids — green; N-methylated amino acids — blue. For BH3 peptide, the linear portion of the peptide is abbreviated using the standard one letter amino acid code. 445x355mm (300 x 300 DPI)



Figure 3. An overview of canonical cell uptake mechanisms. Left to right: polycationic peptides such poly-Arg and TAT are thought to cross the cell membrane via facilitated diffusion enabled by an electrostatic interaction with heparan sulfate displayed on cell surface receptors; RGD peptides serve as a model to illustrate internalization via receptor-mediated endocytosis, in this case exemplified by the interaction of RGD with integrin receptors, which leads to the formation of clathrin-coated pits followed by endosome formation; numerous stapled and amphipathic macrocyclic peptides cross the cell membrane via pinocytosis followed by pinosomal escape; OATP family proteins are involved in active transport of oligopeptides and some cyclic natural products, such as α-amanitin; cyclosporine A represents lipophilic macrocyclic peptides which permeate the cell membrane in a passive way, akin to small molecules. 444x196mm (300 x 300 DPI)


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Figure 4. Common structural features found in bioavailable macrocyclic peptides, as exemplified by cyclosporine A, largazole,154 surotomycin,155 and a model cell-permeable hexapeptide/peptoid hybrid.156 Amino acids with non-proteogenic side-chains are highlighted in red; D-amino acids (green); N-methylated amino acids, depsipeptide linkages and heterocyclic backbones (blue). 222x434mm (300 x 300 DPI)



Figure 5. Chemical structure of degarelix, an FDA approved medication used in the treatment of prostate cancer. The peptide demonstrates an extraordinary long plasma circulation time (t1/2 ~ 40-70 days) owing to many non-proteogenic elements in its structure.121 Amino acids with non-proteogenic side-chains are highlighted in red; D-amino acids (green). 222x103mm (300 x 300 DPI)


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Figure 6. GLP-1 and its analogues as a model for extending plasma retention of a peptide. GLP 1, an endogenous linear peptide hormone involved in glucose metabolism, is proteolytically unstable and thus is unsuitable as a medication on its own. Its modified analogues serve as an illustration of various strategies for increasing proteolytic stability and protein binding, both of which are critical in achieving long plasma circulation times. A) Consensus structure shared between GLP-1 and its analogues. Amino acid sequence is given in the standard one letter code. B) Chemical structures of GLP-1 peptides and their respective t1/2 values. The values for t1/2 were taken for GLP-1 from refs. 124,157,158; for GLP-1 F7A8S from ref. 124; for liraglutide from ref. 130; for taspoglutide from ref. 157; for semaglutide from ref. 159. 444x326mm (300 x 300 DPI)



Figure 7. Analysis of macrocyclic peptide ligands obtained from RaPID and phage display screens. A) Calculated lipophilicity (cLogP; calculated with OSIRIS Property Explorer) as a function of molecular weight for the ligands discovered with RaPID (blue), or with phage display on bicyclic peptides (green). All ligands are bigger and less lipophilic than CSA (red). IL6R-2 ligand from Fig. 8 is shown in purple for comparison. B) Analysis of amino acid sequence length and crude composition by amino acid type: ligands discovered with the use of two different techniques show remarkable similarities. 222x255mm (300 x 300 DPI)


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Figure 8. Chemical structure of interleukin-6 receptor ligand (IL6R-2) obtained from a RaPID screen of a “charge depleted” library.147 This ligand is smaller and significantly more lipophilic than most compounds identified from RaPID screens. Amino acids with non-proteogenic side-chains are highlighted in red; Nmethylated amino acids (blue); functional groups involved in macrocyclization (purple). 222x132mm (300 x 300 DPI)



Graphic abstract 82x44mm (300 x 300 DPI)


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MACROCYCLIC PEPTIDES AS DRUG CANDIDATES: RECENT

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