The coming of age of cyclic peptide drugs: an update on discovery technologies.

INTRODUCTION: Cyclic peptides are an established class of pharmaceuticals, with the ability to bind to a broader range of protein targets than traditional small molecules while also being capable of oral availability and cell penetration. Historically, cyclic peptide drugs have been discovered almost exclusively through natural product mining approaches; however, the last two decades have seen the development of display screening approaches capable of rapidly identifying de novo (i.e. not natural product derived) cyclic peptide ligands to targets of interest. AREAS COVERED: In this review, the authors describe the current clinical landscape for cyclic peptide pharmaceuticals. This article focuses on the discovery approaches that have led to the development of different classes of molecules and how the development of newer technologies, particularly phage and mRNA display, has broadened the clinical applicability of such molecules. EXPERT OPINION: The field of de novo cyclic peptide drug discovery is reaching maturity, with the first drugs identified through display screening approaches reaching the market in recent years. Many more are in clinical trials; however, significant technical challenges remain. Technological improvements will be required over the coming years to facilitate the identification of membrane permeable cyclic peptides capable of oral availability and targeting intracellular proteins.

Discovery of High Affinity Cyclic Peptide Ligands for Human ACE2 with SARS-CoV-2 Entry Inhibitory Activity.

The development of effective antiviral compounds is essential for mitigating the effects of the COVID-19 pandemic. Entry of SARS-CoV-2 virions into host cells is mediated by the interaction between the viral spike (S) protein and membrane-bound angiotensin-converting enzyme 2 (ACE2) on the surface of epithelial cells. Inhibition of this viral protein-host protein interaction is an attractive avenue for the development of antiviral molecules with numerous spike-binding molecules generated to date. Herein, we describe an alternative approach to inhibit the spike-ACE2 interaction by targeting the spike-binding interface of human ACE2 via mRNA display. Two consecutive display selections were performed to direct cyclic peptide ligand binding toward the spike binding interface of ACE2. Through this process, potent cyclic peptide binders of human ACE2 (with affinities in the picomolar to nanomolar range) were identified, two of which neutralized SARS-CoV-2 entry. This work demonstrates the potential of targeting ACE2 for the generation of anti-SARS-CoV-2 therapeutics as well as broad spectrum antivirals for the treatment of SARS-like betacoronavirus infection.

mRNA display reveals a class of high-affinity bromodomain-binding motifs that are not found in the human proteome.

Bromodomains (BDs) regulate gene expression by recognizing protein motifs containing acetyllysine. Although originally characterized as histone-binding proteins, it has since become clear that these domains interact with other acetylated proteins, perhaps most prominently transcription factors. The likely transient nature and low stoichiometry of such modifications, however, has made it challenging to fully define the interactome of any given BD. To begin to address this knowledge gap in an unbiased manner, we carried out mRNA display screens against a BD-the N-terminal BD of BRD3-using peptide libraries that contained either one or two acetyllysine residues. We discovered peptides with very strong consensus sequences and with affinities that are significantly higher than typical BD-peptide interactions. X-ray crystal structures also revealed modes of binding that have not been seen with natural ligands. Intriguingly, however, our sequences are not found in the human proteome, perhaps suggesting that strong binders to BDs might have been selected against during evolution.

Discovery and characterization of cyclic peptides selective for the C-terminal bromodomains of BET family proteins.

DNA-encoded cyclic peptide libraries can yield high-potency, high-specificity ligands against target proteins. We used such a library to seek ligands that could distinguish between paralogous bromodomains from the closely related bromodomain and extra-terminal domain family of epigenetic regulators. Several peptides isolated from a screen against the C-terminal bromodomain of BRD2, together with new peptides discovered in previous screens against the corresponding domain from BRD3 and BRD4, bound their targets with nanomolar and sub-nanomolar affinities. X-ray crystal structures of several of these bromodomain-peptide complexes reveal diverse structures and binding modes, which nevertheless display several conserved features. Some peptides demonstrate significant paralog-level specificity, although the physicochemical explanations for this specificity are often not clear. Our data demonstrate the power of cyclic peptides to discriminate between very similar proteins with high potency and hint that differences in conformational dynamics might modulate the affinity of these domains for particular ligands.

Two-Chain Mature Hepatocyte Growth Factor-Specific Positron Emission Tomography Imaging in Tumors Using 64Cu-Labeled HiP-8, a Nonstandard Macrocyclic Peptide Probe.

Two-chain hepatocyte growth factor (tcHGF), the mature form of HGF, is associated with malignancy and anticancer drug resistance; therefore, its quantification is an important indicator for cancer diagnosis. In tumors, activated tcHGF hardly discharges into the systemic circulation, indicating that tcHGF is an excellent target for molecular imaging using positron emission tomography (PET). We recently discovered HGF-inhibitory peptide-8 (HiP-8) that binds specifically to human tcHGF with nanomolar affinity. The purpose of this study was to investigate the usefulness of HiP-8-based PET probes in human HGF knock-in humanized mice. 64Cu-labeled HiP-8 molecules were synthesized using a cross-bridged cyclam chelator, CB-TE1K1P. Radio-high-performance liquid chromatography-based metabolic stability analyses showed that more than 90% of the probes existed in intact form in blood at least for 15 min. In PET studies, significantly selective visualization of hHGF-overexpressing tumors versus hHGF-negative tumors was observed in double-tumor-bearing mice. The accumulation of labeled HiP-8 into the hHGF-overexpressing tumors was significantly reduced by competitive inhibition. In addition, the radioactivity and distribution of phosphorylated MET/HGF receptor were colocalized in tissues. These results demonstrate that the 64Cu-labeled HiP-8 probes are suitable for tcHGF imaging in vivo, and secretory proteins like tcHGF can be a target for PET imaging.

Antiviral cyclic peptides targeting the main protease of SARS-CoV-2.

Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of the Random nonstandard Peptide Integrated Discovery (RaPID) mRNA display on a chemically cross-linked SARS-CoV-2 Mpro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of Mpro complexed with a selenoether analogue of the highest-affinity peptide revealed key binding interactions, including glutamine and leucine residues in sites S1 and S2, respectively, and a binding epitope straddling both protein chains in the physiological dimer. Several of these Mpro peptide inhibitors possessed antiviral activity against SARS-CoV-2 in vitro with EC50 values in the low micromolar range. These cyclic peptides serve as a foundation for the development of much needed antivirals that specifically target SARS-CoV-2.

De novo macrocyclic peptides for inhibiting, stabilizing, and probing the function of the retromer endosomal trafficking complex.

The retromer complex (Vps35-Vps26-Vps29) is essential for endosomal membrane trafficking and signaling. Mutation of the retromer subunit Vps35 causes late-onset Parkinson's disease, while viral and bacterial pathogens can hijack the complex during cellular infection. To modulate and probe its function, we have created a novel series of macrocyclic peptides that bind retromer with high affinity and specificity. Crystal structures show that most of the cyclic peptides bind to Vps29 via a Pro-Leu­containing sequence, structurally mimicking known interactors such as TBC1D5 and blocking their interaction with retromer in vitro and in cells. By contrast, macrocyclic peptide RT-L4 binds retromer at the Vps35-Vps26 interface and is a more effective molecular chaperone than reported small molecules, suggesting a new therapeutic avenue for targeting retromer. Last, tagged peptides can be used to probe the cellular localization of retromer and its functional interactions in cells, providing novel tools for studying retromer function.

An Ultrapotent and Selective Cyclic Peptide Inhibitor of Human ß-Factor XIIa in a Cyclotide Scaffold.

Cyclotides are plant-derived peptides with complex structures shaped by their head-to-tail cyclic backbone and cystine knot core. These structural features underpin the native bioactivities of cyclotides, as well as their beneficial properties as pharmaceutical leads, including high proteolytic stability and cell permeability. However, their inherent structural complexity presents a challenge for cyclotide engineering, particularly for accessing libraries of sufficient chemical diversity to design potent and selective cyclotide variants. Here, we report a strategy using mRNA display enabling us to select potent cyclotide-based FXIIa inhibitors from a library comprising more than 1012 members based on the cyclotide scaffold of Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II). The most potent and selective inhibitor, cMCoFx1, has a pM inhibitory constant toward FXIIa with greater than three orders of magnitude selectivity over related serine proteases, realizing specific inhibition of the intrinsic coagulation pathway. The cocrystal structure of cMCoFx1 and FXIIa revealed interactions at several positions across the contact interface that conveyed high affinity binding, highlighting that such cyclotides are attractive cystine knot scaffolds for therapeutic development.

Discovery of Cyclic Peptide Ligands to the SARS-CoV-2 Spike Protein Using mRNA Display.

The COVID-19 pandemic, caused by SARS-CoV-2, has led to substantial morbidity, mortality, and disruption globally. Cellular entry of SARS-CoV-2 is mediated by the viral spike protein, and affinity ligands to this surface protein have the potential for applications as antivirals and diagnostic reagents. Here, we describe the affinity selection of cyclic peptide ligands to the SARS-CoV-2 spike protein receptor binding domain (RBD) from three distinct libraries (in excess of a trillion molecules each) by mRNA display. We identified six high affinity molecules with dissociation constants (K D) in the nanomolar range (15-550 nM) to the RBD. The highest affinity ligand could be used as an affinity reagent to detect the spike protein in solution by ELISA, and the cocrystal structure of this molecule bound to the RBD demonstrated that it binds to a cryptic binding site, displacing a ß-strand near the C-terminus. Our findings provide key mechanistic insight into the binding of peptide ligands to the SARS-CoV-2 spike RBD, and the ligands discovered in this work may find future use as reagents for diagnostic applications.

Potent Cyclic Peptide Inhibitors of FXIIa Discovered by mRNA Display with Genetic Code Reprogramming.

The contact system comprises a series of serine proteases that mediate procoagulant and proinflammatory activities via the intrinsic pathway of coagulation and the kallikrein-kinin system, respectively. Inhibition of Factor XIIa (FXIIa), an initiator of the contact system, has been demonstrated to lead to thrombo-protection and anti-inflammatory effects in animal models and serves as a potentially safer target for the development of antithrombotics. Herein, we describe the use of the Randomised Nonstandard Peptide Integrated Discovery (RaPID) mRNA display technology to identify a series of potent and selective cyclic peptide inhibitors of FXIIa. Cyclic peptides were evaluated in vitro, and three lead compounds exhibited significant prolongation of aPTT, a reduction in thrombin generation, and an inhibition of bradykinin formation. We also describe our efforts to identify the critical residues for binding FXIIa through alanine scanning, analogue generation, and via in silico methods to predict the binding mode of our lead cyclic peptide inhibitors.

Cyclic peptides can engage a single binding pocket through highly divergent modes.

Cyclic peptide library screening technologies show immense promise for identifying drug leads and chemical probes for challenging targets. However, the structural and functional diversity encoded within such libraries is largely undefined. We have systematically profiled the affinity, selectivity, and structural features of library-derived cyclic peptides selected to recognize three closely related targets: the acetyllysine-binding bromodomain proteins BRD2, -3, and -4. We report affinities as low as 100 pM and specificities of up to 106-fold. Crystal structures of 13 peptide-bromodomain complexes reveal remarkable diversity in both structure and binding mode, including both α-helical and ß-sheet structures as well as bivalent binding modes. The peptides can also exhibit a high degree of structural preorganization. Our data demonstrate the enormous potential within these libraries to provide diverse binding modes against a single target, which underpins their capacity to yield highly potent and selective ligands.

Discovery of Potent Cyclic Sulfopeptide Chemokine Inhibitors via Reprogrammed Genetic Code mRNA Display.

Targeting chemokine signaling is an attractive avenue for the treatment of inflammatory disorders. Tyrosine sulfation is an important post-translational modification (PTM) that enhances chemokine-receptor binding and is also utilized by a number of pathogenic organisms to improve the binding affinity of immune-suppressive chemokine binding proteins (CKBPs). Here we report the display selection of tyrosine-sulfated cyclic peptides using a reprogrammed genetic code to discover high-affinity ligands for the chemokine CCL11 (eotaxin-1). The selected cyclic sulfopeptides possess high affinity for the target chemokine (as well as one or more of the related family members CCL2, CCL7 and CCL24) and inhibit CCL11 activation of CC chemokine receptor 3 (CCR3). This work demonstrates the utility of exploiting native PTMs as binding motifs for the generation of new leads for medicinal chemistry.

Macrocyclic peptides that inhibit Wnt signalling via interaction with Wnt3a.

Here we report de novo macrocyclic peptide binders to Wnt3a, a member of the Wnt protein family. By means of the Random non-standard Peptides Integrated Discovery (RaPID) system, we have performed in vitro selection against the complex of mouse Wnt3a (mWnt3a) with human afamin (hAFM) to discover macrocyclic peptides that bind mWnt3a with K D values as tight as 110 nM. One of these peptides, WAp-D04 (Wnt-AFM-peptide-D04), was able to inhibit the receptor-mediated signaling process, which was demonstrated in a Wnt3a-dependent reporter cell-line. Based on this initial hit, we applied a block-mutagenesis scanning display to identify a mutant inhibitor, WAp-D04-W10P, with 5-fold greater potency in a reporter assay. This work represents the first instance of molecules capable of inhibiting Wnt signaling through direct interaction with a Wnt protein, a molecular class for which targeting has been challenging due its highly hydrophobic nature.

The Road Ahead for the Development of Macrocyclic Peptide Ligands.

Macrocyclic peptides make up an emerging class of candidate therapeutics and chemical probes, with properties that make them potentially applicable to a wide range of targets that are intractable using current pharmacological agents. Additionally, a number of biochemical screening strategies have been developed, particularly over the past decade, that allow for the massively parallel screening of cyclic peptide libraries of up to 1 trillion compounds or more, leading to the isolation of molecules with exceptional target affinity, selectivity, and bioactivity. Clinical development of compounds derived from such screens is already underway, but the nature of these molecules means that such development is likely to follow pathways different from those of traditional small molecule drugs or well-established biologics such as monoclonal antibodies. In addition, recent work has shown that the biochemical techniques used to identify macrocyclic peptides can also be used to rapidly characterize and optimize them. These findings are likely to facilitate the development of these compounds as chemical probes and as therapeutics for areas of unmet medical need.

Macrocyclic peptide-based inhibition and imaging of hepatocyte growth factor.

Activation of hepatocyte growth factor (HGF) by proteolytic processing is triggered in cancer microenvironments, and subsequent signaling through the MET receptor is involved in cancer progression. However, the structure of HGF remains elusive, and few small/medium-sized molecules can modulate HGF. Here, we identified HiP-8, a macrocyclic peptide consisting of 12 amino acids, which selectively recognizes active HGF. Biochemical analysis and real-time single-molecule imaging by high-speed atomic force microscopy demonstrated that HiP-8 restricted the dynamic domains of HGF into static closed conformations, resulting in allosteric inhibition. Positron emission tomography using HiP-8 as a radiotracer enabled noninvasive visualization and simultaneous inhibition of HGF-MET activation status in tumors in a mouse model. Our results illustrate the conformational change in proteolytic activation of HGF and its detection and inhibition by a macrocyclic peptide, which may be useful for diagnosis and treatment of cancers.

De Novo Discovery of Nonstandard Macrocyclic Peptides as Noncompetitive Inhibitors of the Zika Virus NS2B-NS3 Protease.

The Zika virus presents a major public health concern due to severe fetal neurological disorders associated with infections in pregnant women. In addition to vaccine development, the discovery of selective antiviral drugs is essential to combat future epidemic Zika virus outbreaks. The Zika virus NS2B-NS3 protease, which performs replication-critical cleavages of the viral polyprotein, is a promising drug target. We report the first macrocyclic peptide-based inhibitors of the NS2B-NS3 protease, discovered de novo through in vitro display screening of a genetically reprogrammed library including noncanonical residues. Six compounds were selected, resynthesized, and isolated, all of which displayed affinities in the low nanomolar concentration range. Five compounds showed significant protease inhibition. Two of these were validated as hits with submicromolar inhibition constants and selectivity toward Zika over the related proteases from dengue and West Nile viruses. The compounds were characterized as noncompetitive inhibitors, suggesting allosteric inhibition.

Ribosomal Synthesis of Backbone-Cyclic Peptides Compatible with In Vitro Display.

Backbone-cyclic peptides are an attractive class for therapeutic development. However, in vitro display technologies coupled with ribosomal synthesis are intrinsically inapplicable to such "phenotypes" because of loss of the C-terminal peptide region linking to "genotype". Here, we report a methodology enabling the display of backbone-cyclic peptides. To achieve this, genetic code reprogramming was utilized to implement a rearrangement strategy involving the ribosomal incorporation of a designer initiator containing a thiazolidine-protected cysteine and 2-chloroacetoamide (ClAc) side chain, followed by an α-thio acid and cysteine at downstream positions. Upon expression of the linear peptide, spontaneous thioester rearrangement occurs between the α-thioester and the thiol group of the cysteine, liberating the α-thio group and resulting in cross-linking to the upstream ClAc side-chain group. Then selective deprotection of the thiazolidine-protected cysteine immediately promotes intramolecular native chemical ligation, as demonstrated for various sequences and ring sizes. In this approach, the backbone-cyclic peptides retain their C-terminal peptide regions via the side-chain thioether covalent linkage, making them compatible with in vitro display.

Structural Features and Binding Modes of Thioether-Cyclized Peptide Ligands.

Macrocyclic peptides are an emerging class of bioactive compounds for therapeutic use. In part, this is because they are capable of high potency and excellent target affinity and selectivity. Over the last decade, several biochemical techniques have been developed for the identification of bioactive macrocyclic peptides, allowing for the rapid isolation of high affinity ligands to a target of interest. A common feature of these techniques is a general reliance on thioether formation to effect macrocyclization. Increasingly, the compounds identified using these approaches have been subjected to x-ray crystallographic analysis bound to their respective targets, providing detailed structural information about their conformation and mechanism of target binding. The present review provides an overview of the target bound thioether-closed macrocyclic peptide structures that have been obtained to date.