Random Non-standard Peptide Integrated Discovery Research Articles

De Novo Discovery of Pseudo‐Natural Prenylated Macrocyclic Peptide Ligands

Prenylation of peptides is widely observed in the secondary metabolites of diverse organisms, granting peptides unique chemical properties distinct from proteinogenic amino acids. Discovery of prenylated peptide agents has largely relied on isolation or genome mining of naturally occurring molecules. To devise a platform technology for de novo discovery of artificial prenylated peptides targeting a protein of choice, here we have integrated the thioether‐macrocyclic peptide (teMP) library construction/selection technology, so‐called RaPID (Random nonstandard Peptides Integrated Discovery) system, with a Trp‐C3‐prenyltransferase KgpF involved in the biosynthesis of a prenylated natural product. This unique enzyme exhibited remarkably broad substrate tolerance, capable of modifying various Trp‐containing teMPs to install a prenylated residue with tricyclic constrained structure. We constructed a vast library of prenylated teMPs and subjected it to in vitro selection against a phosphoglycerate mutase. This selection platform has led to the identification of a pseudo‐natural prenylated teMP inhibiting the target enzyme with an IC50 of 30 nM. Importantly, the prenylation was essential for the inhibitory activity, enhanced serum stability, and cellular uptake of the peptide, highlighting the benefits of peptide prenylation. This work showcases the de novo discovery platform for pseudo‐natural prenylated peptides, which is readily applicable to other drug targets.

De Novo Discovery of Pseudo-Natural Prenylated Macrocyclic Peptide Ligands.

Prenylation of peptides is widely observed in the secondary metabolites of diverse organisms, granting peptides unique chemical properties distinct from proteinogenic amino acids. Discovery of prenylated peptide agents has largely relied on isolation or genome mining of naturally occurring molecules. To devise a platform technology for de novo discovery of artificial prenylated peptides targeting a protein of choice, here we have integrated the thioether-macrocyclic peptide (teMP) library construction/selection technology, so-called RaPID (Random nonstandard Peptides Integrated Discovery) system, with a Trp-C3-prenyltransferase KgpF involved in the biosynthesis of a prenylated natural product. This unique enzyme exhibited remarkably broad substrate tolerance, capable of modifying various Trp-containing teMPs to install a prenylated residue with tricyclic constrained structure. We constructed a vast library of prenylated teMPs and subjected it to in vitro selection against a phosphoglycerate mutase. This selection platform has led to the identification of a pseudo-natural prenylated teMP inhibiting the target enzyme with an IC50 of 30 nM. Importantly, the prenylation was essential for the inhibitory activity, enhanced serum stability, and cellular uptake of the peptide, highlighting the benefits of peptide prenylation. This work showcases the de novo discovery platform for pseudo-natural prenylated peptides, which is readily applicable to other drug targets.

A RaPID Response to SARS‐CoV‐2

AbstractGenetically encoded peptide libraries are at the forefront of de novo drug discovery. The RaPID (Random Nonstandard Peptides Integrated Discovery) platform stands out due to the unique combination of flexible in vitro translation (FIT) and mRNA display. This enables the incorporation of non‐canonical amino acids, improving chemical diversity and allowing macrocyclisation of the peptide library. The resulting constrained peptides are valued for their strong binding affinity and stability, especially in the context of protein‐protein interactions. In response to SARS‐CoV‐2, the causative agent of the COVID‐19 pandemic, the RaPID system proved valuable in identifying high‐affinity ligands of viral proteins. Among many peptide ligands of SARS‐CoV‐2 spike and main protease (Mpro), several macrocycles stand out for their exceptional binding affinities. Structural data showcases distinct binding modes in complex with the receptor‐binding domain (RBD) of the spike glycoprotein or the catalytic active site of Mpro. However, translating these in vitro findings into clinical applications remains challenging, especially due to insufficient cell permeability.

Macrocyclic Peptides Closed by a Thioether-Bipyridyl Unit That Grants Cell Membrane Permeability.

Membrane permeability is an important factor that determines the virtue of peptides targeting intracellular molecules. By introducing a membrane penetration motif, some peptides exhibit better membrane permeabilities. Previous choices for such motifs have usually been polycationic sequences, but their protease vulnerabilities and modest endosome escapability remain challenging. Here, we report a strategy for macrocyclization of peptides closed by a hydrophobic bipyridyl (BPy) unit, which grants an improvement of their membrane permeability and proteolytic stability compared with the conventional polycationic peptides. We chemically prepared model macrocyclic peptides closed by a thioether-BPy unit and determined their cell membrane permeability, giving 200 nM CP50 (an indicative value of membrane permeability), which is 40-fold better than that of the ordinary thioether macrocycle consisting of the same sequence composition. To discover potent target binders consisting of the BPy unit, we reprogrammed the initiator with chloromethyl-BPy (ClMeBPy) for the peptide library synthesis with a downstream Cys residue(s) and executed RaPID (Random nonstandard Peptide Integrated Discovery) against the bromodomains of BRD4. One of the obtained sequences exhibited a single-digit nanomolar dissociation constant against BRD4 in vitro and showed approximately 2-fold and 10-fold better membrane permeability than positive controls, R9 and Tat peptides, respectively. Moreover, we observed an intracellular activity of the BPy macrocycle tagged with a proteasome target peptide motif (RRRG), resulting in modest but detectable degradation of BRD4. The present demonstration indicates that the combination of the RaPID system with an appropriate hydrophobic unit, such as BPy, would provide a potential approach for devising cell penetrating macrocycles targeting various intracellular proteins.

Combining Chemical Protein Synthesis and Random Nonstandard Peptides Integrated Discovery for Modulating Biological Processes.

ConspectusChemical manipulation of naturally occurring peptides offers a convenient route for generating analogs to screen against different therapeutic targets. However, the limited success of the conventional chemical libraries has urged chemical biologists to adopt alternative methods such as phage and mRNA displays and create libraries of a large number of variants for the screening and selection of novel peptides. Messenger RNA (mRNA) display provides great advantages in terms of the library size and the straightforward recovery of the selected polypeptide sequences. Importantly, the integration of the flexible in vitro translation (FIT) system with the mRNA display provides the basis of the random nonstandard peptides integrated discovery (RaPID) approach for the introduction of diverse nonstandard motifs, such as unnatural side chains and backbone modifications. This platform allows the discovery of functionalized peptides with tight binding against virtually any protein of interest (POI) and therefore shows great potential in the pharmaceutical industry. However, this method has been limited to targets generated by recombinant expression, excluding its applications to uniquely modified proteins, particularly those with post-translational modifications.Chemical protein synthesis allows a wide range of changes to the protein's chemical composition to be performed, including side chain and backbone modifications and access to post-translationally modified proteins, which are often inaccessible or difficult to achieve via recombinant expression methods. Notably, d-proteins can be prepared via chemical synthesis, which has been used in mirror image phase display for the discovery of nonproteolytic d-peptide binders.Combining chemical protein synthesis with the RaPID system allows the production of a library of trillions of cyclic peptides and subsequent selection for novel cyclic peptide binders targeting a uniquely modified protein to assist in studying its unexplored biology and possibly the discovery of new drug candidates.Interestingly, the small post-translational modifier protein ubiquitin (Ub), with its various polymeric forms, regulates directly or indirectly many biochemical processes, e.g., proteasomal degradation, DNA damage repair, cell cycle regulation, etc. In this Account, we discuss combining the RaPID approach against various synthetic Ub chains for selecting effective and specific macrocyclic peptide binders. This offers an advancement in modulating central Ub pathways and provides opportunities in drug discovery areas associated with Ub signaling. We highlight experimental approaches and conceptual adaptations required to design and modulate the activity of Lys48- and Lys63-linked Ub chains by macrocyclic peptides. We also present the applications of these approaches to shed light on related biological activities and ultimately their activity against cancer. Finally, we contemplate future developments still pending in this exciting multidisciplinary field.

A RaPID Macrocyclic Peptide That Inhibits the Formation of α-Synuclein Amyloid Fibrils.

There is considerable interest in drug discovery targeting the aggregation of α-synuclein (αSyn) since this molecular process is closely associated with Parkinson's disease. However, inhibiting αSyn aggregation remains a major challenge because of its highly dynamic nature which makes it difficult to form a stable binding complex with a drug molecule. Here, by exploiting Random non-standard Peptides Integrated Discovery (RaPID) system, we identified a macrocyclic peptide, BD1, that could interact with immobilized αSyn and inhibit the formation of fibrils. Furthermore, improving the solubility of BD1 suppresses the co-aggregation with αSyn fibrils while it kinetically inhibits more effectively without change in their morphology. We also revealed the molecular mechanism of kinetic inhibition, where peptides bind to fibril ends of αSyn, thereby preventing further growth of fibrils. These results suggest that our approach for generating non-standard macrocyclic peptides is a promising approach for developing potential therapeutics against neurodegeneration.

Potent De Novo Macrocyclic Peptides That Inhibit O-GlcNAc Transferase through an Allosteric Mechanism.

Glycosyltransferases are a superfamily of enzymes that are notoriously difficult to inhibit. Here we apply an mRNA display technology integrated with genetic code reprogramming, referred to as the RaPID (random non-standard peptides integrated discovery) system, to identify macrocyclic peptides with high binding affinities for O-GlcNAc transferase (OGT). These macrocycles inhibit OGT activity through an allosteric mechanism that is driven by their binding to the tetratricopeptide repeats of OGT. Saturation mutagenesis in a maturation screen using 39 amino acids, including 22 non-canonical residues, led to an improved unnatural macrocycle that is ≈40 times more potent than the parent compound (Ki app =1.5 nM). Subsequent derivatization delivered a biotinylated derivative that enabled one-step affinity purification of OGT from complex samples. The high potency and novel mechanism of action of these OGT ligands should enable new approaches to elucidate the specificity and regulation of OGT.

Potent De Novo Macrocyclic Peptides That Inhibit O‐GlcNAc Transferase through an Allosteric Mechanism

AbstractGlycosyltransferases are a superfamily of enzymes that are notoriously difficult to inhibit. Here we apply an mRNA display technology integrated with genetic code reprogramming, referred to as the RaPID (random non‐standard peptides integrated discovery) system, to identify macrocyclic peptides with high binding affinities for O‐GlcNAc transferase (OGT). These macrocycles inhibit OGT activity through an allosteric mechanism that is driven by their binding to the tetratricopeptide repeats of OGT. Saturation mutagenesis in a maturation screen using 39 amino acids, including 22 non‐canonical residues, led to an improved unnatural macrocycle that is ≈40 times more potent than the parent compound (Kiapp=1.5 nM). Subsequent derivatization delivered a biotinylated derivative that enabled one‐step affinity purification of OGT from complex samples. The high potency and novel mechanism of action of these OGT ligands should enable new approaches to elucidate the specificity and regulation of OGT.

Selective macrocyclic peptide modulators of Lys63-linked ubiquitin chains disrupt DNA damage repair

Developing an effective binder for a specific ubiquitin (Ub) chain is a promising approach for modulating various biological processes with potential applications in drug discovery. Here, we combine the Random Non-standard Peptides Integrated Discovery (RaPID) method and chemical protein synthesis to screen an extended library of macrocyclic peptides against synthetic Lys63-linked Di-Ub to discover a specific binder for this Ub chain. Furthermore, next-generation binders are generated by chemical modifications. We show that our potent cyclic peptide is cell-permeable, and inhibits DNA damage repair, leading to apoptotic cell death. Concordantly, a pulldown experiment with the biotinylated analog of our lead cyclic peptide supports our findings. Collectively, we establish a powerful strategy for selective inhibition of protein-protein interactions associated with Lys63-linked Di-Ub using cyclic peptides. This study offers an advancement in modulating central Ub pathways and provides opportunities in drug discovery areas associated with Ub signaling.

In Vitro Selection of Macrocyclic α/β3-Peptides against Human EGFR.

Here, we report ribosomal construction of thioether-macrocyclic α/β3-peptide libraries in which β-homoglycine, β-homoalanine, β-homophenylglycine, and β-homoglutamine are introduced by genetic code reprogramming. The libraries were applied to the RaPID (Random nonstandard Peptides Integrated Discovery) selection against human EGFR to obtain PPI (protein-protein interaction) inhibitors. The resulting peptides contained up to five β3-amino acid (β3AA) residues and exhibited outstanding binding affinity, PPI inhibitory activity, and proteolytic stability, which were attributed to the β3AAs included in the peptides. This showcase work has demonstrated that the use of such β3AAs enhances the drug-like properties of peptides, providing a unique platform for the discovery of de novo macrocycles against a protein of interest.

Serum-Stable and Selective Backbone-N-Methylated Cyclic Peptides That Inhibit Prokaryotic Glycolytic Mutases.

N-Methylated amino acids (N-MeAAs) are privileged residues of naturally occurring peptides critical to bioactivity. However, de novo discovery from ribosome display is limited by poor incorporation of N-methylated amino acids into the nascent peptide chain attributed to a poor EF-Tu affinity for the N-methyl-aminoacyl-tRNA. By reconfiguring the tRNA's T-stem region to compensate and tune the EF-Tu affinity, we conducted Random nonstandard Peptides Integrated Discovery (RaPID) display of a macrocyclic peptide (MCP) library containing six different N-MeAAs. We have here devised a "pool-and-split" enrichment strategy using the RaPID display and identified N-methylated MCPs against three species of prokaryotic metal-ion-dependent phosphoglycerate mutases. The enriched MCPs reached 57% N-methylation with up to three consecutively incorporated N-MeAAs, rivaling natural products. Potent nanomolar inhibitors ranging in ortholog selectivity, strongly mediated by N-methylation, were identified. Co-crystal structures reveal an architecturally related Ce-2 Ipglycermide active-site metal-ion-coordinating Cys lariat MCP, functionally dependent on two cis N-MeAAs with broadened iPGM species selectivity over the original nematode-selective MCPs. Furthermore, the isolation of a novel metal-ion-independent Staphylococcus aureus iPGM inhibitor utilizing a phosphoglycerate mimetic mechanism illustrates the diversity of possible chemotypes encoded by the N-MeAA MCP library.

The RaPID Platform for the Discovery of Pseudo-Natural Macrocyclic Peptides.

Although macrocyclic peptides bearing exotic building blocks have proven their utility as pharmaceuticals, the sources of macrocyclic peptide drugs have been largely limited to mimetics of native peptides or natural product peptides. However, the recent emergence of technologies for discovering de novo bioactive peptides has led to their reconceptualization as a promising therapeutic modality. For the construction and screening of libraries of such macrocyclic peptides, our group has devised a platform to conduct affinity-based selection of massive libraries (>1012 unique sequences) of in vitro expressed macrocyclic peptides, which is referred to as the random nonstandard peptides integrated discovery (RaPID) system. The RaPID system integrates genetic code reprogramming using the FIT (flexible in vitro translation) system, which is largely facilitated by flexizymes (flexible tRNA-aminoacylating ribozymes), with mRNA display technology.We have demonstrated that the RaPID system enables rapid discovery of various de novo pseudo-natural peptide ligands for protein targets of interest. Many examples discussed in this Account prove that thioether-closed macrocyclic peptides (teMPs) obtained by the RaPID system generally exhibit remarkably high affinity and specificity, thereby potently inhibiting or activating a specific function(s) of the target. Moreover, such teMPs are used for a wide range of biochemical applications, for example, as crystallization chaperones for intractable transmembrane proteins and for in vivo recognition of specific cell types. Furthermore, recent studies demonstrate that some teMPs exhibit pharmacological activities in animal models and that even intracellular proteins can be inhibited by teMPs, illustrating the potential of this class of peptides as drug leads.Besides the ring-closing thioether linkage in the teMPs, genetic code reprogramming by the FIT system allows for incorporation of a variety of other exotic building blocks. For instance, diverse nonproteinogenic amino acids, hydroxy acids (ester linkage), amino carbothioic acid (thioamide linkage), and abiotic foldamer units have been successfully incorporated into ribosomally synthesized peptides. Despite such enormous successes in the conventional FIT system, multiple or consecutive incorporation of highly exotic amino acids, such as d- and β-amino acids, is yet challenging, and particularly the synthesis of peptides bearing non-carbonyl backbone structures remains a demanding task. To upgrade the RaPID system to the next generation, we have engaged in intensive manipulation of the FIT system to expand the structural diversity of peptides accessible by our in vitro biosynthesis strategy. Semilogical engineering of tRNA body sequences led to a new suppressor tRNA (tRNAPro1E2) capable of effectively recruiting translation factors, particularly EF-Tu and EF-P. The use of tRNAPro1E2 in the FIT system allows for not only single but also consecutive and multiple elongation of exotic amino acids, such as d-, β-, and γ-amino acids as well as aminobenzoic acids. Moreover, the integration of the FIT system with various chemical or enzymatic posttranslational modifications enables us to expand the range of accessible backbone structures to non-carbonyl moieties prominent in natural products and peptidomimetics. In such systems, FIT-expressed peptides undergo multistep backbone conversions in a one-pot manner to yield designer peptides composed of modified backbones such as azolines, azoles, and ring-closing pyridines. Our current research endeavors focus on applying such in vitro biosynthesis systems for the discovery of bioactive de novo pseudo-natural products.

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.

De novo macrocyclic peptides dissect energy coupling of a heterodimeric ABC transporter by multimode allosteric inhibition.

ATP-binding cassette (ABC) transporters constitute the largest family of primary active transporters involved in a multitude of physiological processes and human diseases. Despite considerable efforts, it remains unclear how ABC transporters harness the chemical energy of ATP to drive substrate transport across cell membranes. Here, by random nonstandard peptide integrated discovery (RaPID), we leveraged combinatorial macrocyclic peptides that target a heterodimeric ABC transport complex and explore fundamental principles of the substrate translocation cycle. High-affinity peptidic macrocycles bind conformationally selective and display potent multimode inhibitory effects. The macrocycles block the transporter either before or after unidirectional substrate export along a single conformational switch induced by ATP binding. Our study reveals mechanistic principles of ATP binding, conformational switching, and energy transduction for substrate transport of ABC export systems. We highlight the potential of de novo macrocycles as effective inhibitors for membrane proteins implicated in multidrug resistance, providing avenues for the next generation of pharmaceuticals.

Lasso-grafting of macrocyclic peptide pharmacophores yields multi-functional proteins

Protein engineering has great potential for devising multifunctional recombinant proteins to serve as next-generation protein therapeutics, but it often requires drastic modifications of the parental protein scaffolds e.g., additional domains at the N/C-terminus or replacement of a domain by another. A discovery platform system, called RaPID (Random non-standard Peptides Integrated Discovery) system, has enabled rapid discovery of small de novo macrocyclic peptides that bind a target protein with high binding specificity and affinity. Capitalizing on the optimized binding properties of the RaPID-derived peptides, here we show that RaPID-derived pharmacophore sequences can be readily implanted into surface-exposed loops on recombinant proteins and maintain both the parental peptide binding function(s) and the host protein function. We refer to this protein engineering method as lasso-grafting and demonstrate that it can endow specific binding capacity toward various receptors into a diverse set of scaffolds that includes IgG, serum albumin, and even capsid proteins of adeno-associated virus, enabling us to rapidly formulate and produce bi-, tri-, and even tetra-specific binder molecules.

Structure–activity relationship of ipglycermide binding to phosphoglycerate mutases

Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex.

Development of cyclic peptides with potent in vivo osteogenic activity through RaPID-based affinity maturation

Osteoporosis is caused by a disequilibrium between bone resorption and bone formation. Therapeutics for osteoporosis can be divided into antiresorptives that suppress bone resorption and anabolics which increase bone formation. Currently, the only anabolic treatment options are parathyroid hormone mimetics or an anti-sclerostin monoclonal antibody. With the current global increases in demographics at risk for osteoporosis, development of therapeutics that elicit anabolic activity through alternative mechanisms is imperative. Blockade of the PlexinB1 and Semaphorin4D interaction on osteoblasts has been shown to be a promising mechanism to increase bone formation. Here we report the discovery of cyclic peptides by a novel RaPID (Random nonstandard Peptides Integrated Discovery) system-based affinity maturation methodology that generated the peptide PB1m6A9 which binds with high affinity to both human and mouse PlexinB1. The chemically dimerized peptide, PB1d6A9, showed potent inhibition of PlexinB1 signaling in mouse primary osteoblast cultures, resulting in significant enhancement of bone formation even compared to non-Semaphorin4D-treated controls. This high anabolic activity was also observed in vivo when the lipidated PB1d6A9 (PB1d6A9-Pal) was intravenously administered once weekly to ovariectomized mice, leading to complete rescue of bone loss. The potent osteogenic properties of this peptide shows great promise as an addition to the current anabolic treatment options for bone diseases such as osteoporosis.

Abstract 683: Identification of a macrocyclic peptide that blocks the CD47-SIRPα interaction and its antitumor effect

Abstract Much recent attention has focused on the modification of immune responses as a basis for new cancer treatments. Immune checkpoint inhibitors, such as monoclonal antibodies (mAbs) to CTLA-4, PD-1, and PD-L1, have provided clinical benefit in patients with a variety of types of cancer, and a growing number of agents targeting immune checkpoints are in preclinical and clinical development. SIRPα is a transmembrane protein that is abundant in myeloid cells, such as macrophages and dendritic cells. SIRPα interacts with another transmembrane protein CD47 through their ectodomains, and they constitute the cell-cell communication system (the CD47-SIRPα system). We previously demonstrated that the interaction of SIRPα on macrophages with CD47 on tumor cells prevented the phagocytosis of tumor cells by macrophages. In contrast, blockage by anti-SIRPα antibodies of the CD47-SIRPα interaction enhanced the phagocytic activity of macrophages against antibody-opsonized tumor cells, suggesting that the CD47-SIRPα axis acts as an immune checkpoint pathway involved in tumor escape from immunosurveillance. Here, by the use of random nonstandard peptide integrated discovery (RaPID) system, we have identified novel macrocyclic peptides that bind to the extracellular domain of SIRPα with high affinity. These SIRPα-binding macrocyclic peptides inhibited the CD47-SIRPα interaction. Moreover, one of the SIRPα-binding macrocycles promoted the phagocytosis of antibody-opsonized tumor cells by macrophages in vitro and enhanced the antitumor effect of mAbs against tumor antigens in xenograft or syngeneic mouse models of cancer. Our results thus suggest that macrocyclic peptides against SIRPα hold promise as a novel tool for cancer immunotherapy. Citation Format: Daisuke Hazama, Yoji Murata, Takeshi Okamoto, Daisuke Tanaka, Mariko Sakamoto, Yuka Kakuchi, Yasuyuki Saito, Takenori Kotani, Yoshihiro Nishimura, Takashi Matozaki. Identification of a macrocyclic peptide that blocks the CD47-SIRPα interaction and its antitumor effect [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 683.

Revolutionary therapeutic peptides

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. Macrocyclic peptides are accessible by not only chemical synthesis but also ribosomal synthesis. Particularly, recent inventions of the genetic code reprogramming integrated with an in vitro display format, referred to as RaPID (Random non-standard Peptides Integrated Discovery) system, have enabled us to screen mass libraries (>1 trillion members) of non-standard peptides containing multiple non-proteinogenic amino acids, giving unique properties of peptides distinct from conventional peptides, e.g. greater proteolytic stability, higher affinity (low nM to sub nM dissociation constants similar to antibodies), and superior pharmacokinetics. The field is rapidly growing evidenced by increasing interests from industrial sectors, including small start-ups as well as mega-pharmas, toward drug development efforts on macrocyclic peptides, which has led to several de novo discovered peptides entering clinical trials. This lecture discusses the aforementioned screening technology, the RaPID system, and several showcases of therapeutic potentials of macrocyclic peptides.

Generation of non-standard macrocyclic peptides specifically binding TSC-22 homologous gene-1

Transforming growth factor-β 1 (TGFβ1)-stimulated clone 22 (TSC22) family includes proteins containing a leucine zipper domain and a TSC-box that are highly conserved during evolution. Currently, limited data are available on the function of this protein family, especially of TSC-22 homologous gene-1 (THG-1)/TSC22 domain family member 4 (TSC22D4). Similar to other family members, THG-1 functions depending on its interaction with the partner proteins and it is suggested to mediate a broad range of biological processes. THG-1-specific binding molecules will be instrumental for elucidating its functions. Therefore, the Random non-standard Peptide Integrated Discovery (RaPID) system was modified using commercially available materials and used for selecting macrocyclic peptides (MCPs) that bind to THG-1. Several MCPs were identified to bind THG-1. Fluorescein- and biotin-tagged MCPs were synthesized and employed as THG-1 detection probes. Notably, a fluorescein-tagged MCP specifically detected THG-1-expressing cells. Biotin-tagged MCPs can be successfully used for Enzyme-Linked Protein Sorbent Assay (ELISA) like assay of THG-1 protein and affinity-precipitation of purified THG-1 and endogenous THG-1 in esophageal squamous cell carcinoma cell lysates. The modified RaPID system rapidly and successfully identified THG-1-binding MCPs in vitro and the synthesized THG-1 binding MCPs are useful alternatives acting for antibodies.