A broad-spectrum macrocyclic peptide inhibitor of the SARS-CoV-2 spike protein.

The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used messenger RNA (mRNA) display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor-binding domain, N-terminal domain, and S2 region, distal to the angiotensin-converting enzyme 2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target.

Exploration of macrocyclic peptide binders to the extracellular CRD domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1).

Elevated levels of receptor tyrosine kinase-like orphan receptor 1 (RORl) expression are observed in multiple hematological and solid tumors, but not in most of the healthy adult tissues, identifying ROR1 as an attractive target for tumor-specific therapy. Herein we will describe the discovery of macrocyclic peptides as binders of the extracellular Cysteine-Rich Domain (CRD) of human ROR1 via mRNA in vitro selection technology using the PDPS platform, followed by exploration of sidechain SAR of parent macrocycle peptides, fluorescently labeled analogs, and a Peptide Drug Conjugate (PDC). The parent macrocyclic peptides represented by Compound 1 and Compound 14 displayed nanomolar cell-based binding to ROR1 and relatively good internalization in 786-O and MDA-MB-231 tumor cell lines. However, these peptides were not observed to induce apoptosis in Mia PaCa-2 cells, a model pancreatic tumor cell line with a relatively low level of cell surface expression of ROR1.

Chemoenzymatic synthesis of macrocyclic peptides and polyketides via thioesterase-catalyzed macrocyclization.

Chemoenzymatic strategies that combine synthetic and enzymatic transformations offer efficient approaches to yield target molecules, which have been increasingly employed in the synthesis of bioactive natural products. In the biosynthesis of macrocyclic nonribosomal peptides, polyketides, and their hybrids, thioesterase (TE) domains play a significant role in late-stage macrocyclization. These domains can accept mimics of native substrates in vitro and exhibit potential for use in total synthesis. This review summarizes the recent advances of TE domains in the chemoenzymatic synthesis for these natural products that aim to address the common issues in classical synthetic approaches and increase synthetic efficiencies, which have the potential to facilitate further pharmaceutical research.

Bacterially Secretable Single-Chain Tandem Macrocyclic Peptides for High Affinity and Inhibitory Activity.

The inhibition of protein-protein interactions (PPIs) is an effective approach for therapy. Owing to their large binding surface areas to target proteins, macrocyclic peptides are suitable molecules for PPI inhibition. In this study, we developed single-chain tandem macrocyclic peptides (STaMPtides) that inhibits the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2). They were artificially designed to comprise two different VEGFR2-binding macrocyclic peptides linked in tandem by peptide linkers and secreted by Corynebacterium glutamicum. Most potent VEGFR2-inhibitory STaMPtides with length-optimized linkers exhibited >1000 times stronger inhibitory activity than their parental monomeric peptides, possibly due to the avidity effect of heterodimerization. Our approach of using STaMPtides for PPI inhibition may be used to inhibit other extracellular factors, such as growth factors and cytokines.

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.

Computational generation of an annotated gigalibrary of synthesizable, composite peptidic macrocycles.

Peptidomimetic macrocycles have the potential to regulate challenging therapeutic targets. Structures of this type having precise shapes and drug-like character are particularly coveted, but are relatively difficult to synthesize. Our laboratory has developed robust methods that integrate small-peptide units into designed scaffolds. These methods create macrocycles and embed condensed heterocycles to diversify outcomes and improve pharmacological properties. The hypothetical scope of the methodology is vast and far outpaces the capacity of our experimental format. We now describe a computational rendering of our methodology that creates an in silico three-dimensional library of composite peptidic macrocycles. Our open-source platform, CPMG (Composite Peptide Macrocycle Generator), has algorithmically generated a library of 2,020,794,198 macrocycles that can result from the multistep reaction sequences we have developed. Structures are generated based on predicted site reactivity and filtered on the basis of physical and three-dimensional properties to identify maximally diverse compounds for prioritization. For conformational analyses, we also introduce ConfBuster++, an RDKit port of the open-source software ConfBuster, which allows facile integration with CPMG and ready parallelization for better scalability. Our approach deeply probes ligand space accessible via our synthetic methodology and provides a resource for large-scale virtual screening.

A Comprehensive Technology Platform for the Rapid Discovery of Peptide Inhibitors against SARS-CoV-2 Pseudovirus Infection.

We developed and validated a technology platform for designing and testing peptides inhibiting the infectivity of SARS-CoV-2 spike protein-based pseudoviruses. This platform integrates target evaluation, in silico inhibitor design, peptide synthesis, and efficacy screening. We generated a cyclic peptide library derived from the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor. The cell-free validation process by ELISA competition assays and Surface Plasmon Resonance (SPR) studies revealed that the cyclic peptide c9_05, but not its linear form, binds well to ACE2. Moreover, it effectively inhibited the transduction in HEK293, stably expressing the human ACE2 receptor of pseudovirus particles displaying the SARS-CoV-2 spike in the Wuhan or UK variants. However, the inhibitory efficacy of c9_05 was negligible against the Omicron variant, and it failed to impede the entry of pseudoviruses carrying the B.1.351 (South African) spike. These variants contain three or more mutations known to increase affinity to ACE2. This suggests further refinement is needed for potential SARS-CoV-2 inhibition. Our study hints at a promising approach to develop inhibitors targeting viral infectivity receptors, including SARS-CoV-2's. This platform also promises swift identification and evaluation of inhibitors for other emergent viruses.

Identification of key stabilizing interactions of amyloid-ß oligomers based on fragment molecular orbital calculations on macrocyclic ß-hairpin peptides.

Analyzing the electronic states and inter-/intra-molecular interactions of amyloid oligomers expand our understanding of the molecular basis of Alzheimer's disease and other amyloid diseases. In the current study, several high-resolution crystal structures of oligomeric assemblies of Aß-derived peptides have been studied by the ab initio fragment molecular orbital (FMO) method. The FMO method provides comprehensive details of the molecular interactions between the residues of the amyloid oligomers at the quantum mechanical level. Based on the calculations, two sequential aromatic residues (F19 and F20) and negatively charged E22 on the central region of Aß have been identified as key residues in oligomer stabilization and potential interesting pharmacophores for preventing oligomer formation.

Synthesis of L-cyclic tetrapeptides by backbone amide activation CyClick strategy.

Cyclic tetrapeptides exhibit high cellular permeability and a wide range of biological properties and thus have gained great interest in the field of medicinal chemistry. We synthesized highly strained 12-membered head to tail cyclic peptides with varying reactive amino acids, without oligomerization using the exclusively intramolecular CyClick chemistry. This occurs by a two-step process involving the low-energy formation of a 15 atom-containing cyclic imine, followed by a chemoselective ring contraction of the peptide backbone generating a highly strained 12 atom-containing cyclic tetrapeptide. This reaction exhibited high substrate scope and generated head to tail cyclic tetrapeptides with varying amino acids at the N-terminus, showing chemoselectivity without the need for side group protection.

Expanded Sequence Space of Radical S-Adenosylmethionine-Dependent Enzymes Involved in Post-translational Macrocyclization.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent one of the largest but primarily underexplored natural product families in bacteria. The genetically encoded nature of RiPPs simplifies the prediction and prioritization of their biosynthetic gene clusters (BGCs). We report a small peptide and enzyme co-occurrence analysis workflow (SPECO), which allowed us to identify 32 220 prospective rSAM-catalyzed RiPP BGCs from 161 954 bacterial genomes and prioritize 25 families with new biosynthetic architectures or precursor patterns. We characterized three new enzymes that respectively catalyze cysteine-glycine (BlaB), histidine-aliphatic side chain (ScaB), and tyrosine/histidine-arginine (VguB) cross-links. The cyclophane-forming enzyme ScaB exhibits broad substrate selectivity, allowing it to catalyze diverse triceptide formation. These results demonstrate the strength of the SPECO workflow in discovering new enzymes for peptide macrocyclization.

Liposome Click Membrane Permeability Assay for Identifying Permeable Peptides.

PURPOSE: To develop a novel, target agnostic liposome click membrane permeability assay (LCMPA) using liposome encapsulating copper free click reagent dibenzo cyclooctyne biotin (DBCO-Biotin) to conjugate azido modified peptides that may effectively translocate from extravesicular space into the liposome lumen. METHOD: DBCO-Biotin liposomes were prepared with egg phosphatidylcholine and cholesterol by lipid film rehydration, freeze/thaw followed by extrusion. Size of DBCO-Biotin liposomes were characterized with dynamic light scattering. RESULTS: The permeable peptides representing energy independent mechanism of permeability showed higher biotinylation in LCMPA. Individual peptide permeability results from LCMPA correlated well with shifts in potency in cellular versus biochemical assays (i.e., cellular/ biochemical ratio) demonstrating quantitative correlation to intracellular barrier in intact cells. CONCLUSION: The study provides a novel membrane permeability assay that has potential to evaluate energy independent transport of diverse peptides.

Synthesis and structural characterization of a monocarboxylic inhibitor for GRB2 SH2 domain.

A monocarboxylic inhibitor was designed and synthesized to disrupt the protein-protein interaction (PPI) between GRB2 and phosphotyrosine-containing proteins. Biochemical characterizations show compound 7 binds with the Src homology 2 (SH2) domain of GRB2 and is more potent than EGFR1068 phosphopeptide 14-mer. X-ray crystallographic studies demonstrate compound 7 occupies the GRB2 binding site for phosphotyrosine-containing sequences and reveal key structural features for GRB2-inhibitor binding. This compound with a -1 formal charge offers a new direction for structural optimization to generate cell-permeable inhibitors for this key protein target of the aberrant Ras-MAPK signaling cascade.

Nonproteinogenic deep mutational scanning of linear and cyclic peptides.

High-resolution structure-activity analysis of polypeptides requires amino acid structures that are not present in the universal genetic code. Examination of peptide and protein interactions with this resolution has been limited by the need to individually synthesize and test peptides containing nonproteinogenic amino acids. We describe a method to scan entire peptide sequences with multiple nonproteinogenic amino acids and, in parallel, determine the thermodynamics of binding to a partner protein. By coupling genetic code reprogramming to deep mutational scanning, any number of amino acids can be exhaustively substituted into peptides, and single experiments can return all free energy changes of binding. We validate this approach by scanning two model protein-binding peptides with 21 diverse nonproteinogenic amino acids. Dense structure-activity maps were produced at the resolution of single aliphatic atom insertions and deletions. This permits rapid interrogation of interaction interfaces, as well as optimization of affinity, fine-tuning of physical properties, and systematic assessment of nonproteinogenic amino acids in binding and folding.

Structural Characterization of a Macrocyclic Peptide Modulator of the PD-1/PD-L1 Immune Checkpoint Axis.

The clinical success of PD-1/PD-L1 immune checkpoint targeting antibodies in cancer is followed by efforts to develop small molecule inhibitors with better penetration into solid tumors and more favorable pharmacokinetics. Here we report the crystal structure of a macrocyclic peptide inhibitor (peptide 104) in complex with PD-L1. Our structure shows no indication of an unusual bifurcated binding mode demonstrated earlier for another peptide of the same family (peptide 101). The binding mode relies on extensive hydrophobic interactions at the center of the binding surface and an electrostatic patch at the side. An interesting sulfur/π interaction supports the macrocycle-receptor binding. Overall, our results allow a better understanding of forces guiding macrocycle affinity for PD-L1, providing a rationale for future structure-based inhibitor design and rational optimization.

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.

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

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

Kinetic Selection in the Out-of-Equilibrium Autocatalytic Reaction Networks that Produce Macrocyclic Peptides.

Autocatalytic reaction networks are instrumental for validating scenarios for the emergence of life on Earth and for synthesizing life de novo. Here, we demonstrate that dimeric thioesters of tripeptides with the general structure (Cys-Xxx-Gly-SEt)2 form strongly interconnected autocatalytic reaction networks that predominantly generate macrocyclic peptides up to 69 amino acids long. Some macrocycles of 6-12 amino acids were isolated from the product pool and were characterized by NMR spectroscopy and single-crystal X-ray analysis. We studied the autocatalytic formation of macrocycles in a flow reactor in the presence of acrylamide, whose conjugate addition to thiols served as a model "removal" reaction. These results indicate that even not template-assisted autocatalytic production combined with competing removal of molecular species in an open compartment could be a feasible route for selecting functional molecules during the pre-Darwinian stages of molecular evolution.

Dissecting the Binding Interactions of Teixobactin with the Bacterial Cell-Wall Precursor Lipid†II.

The prevalence of life-threatening, drug-resistant microbial infections has challenged researchers to consider alternatives to currently available antibiotics. Teixobactin is a recently discovered "resistance-proof" antimicrobial peptide that targets the bacterial cell wall precursor lipid II. In doing so, teixobactin exhibits potent antimicrobial activity against a wide range of Gram-positive organisms. Herein we demonstrate that teixobactin and several structural analogues are capable of binding lipid II from both Gram-positive and Gram-negative bacteria. Furthermore, we show that when combined with known outer membrane-disrupting peptides, teixobactin is active against Gram-negative organisms.

Affinity Maturation of Macrocyclic Peptide Modulators of Lys48-Linked Diubiquitin by a Twofold Strategy.

Messenger RNA display of peptides containing non-proteinogenic amino acids, referred to as RaPID system, has become one of the leading methods to express libraries consisting of more than trillion-members of macrocyclic peptides, which allows for discovering de novo bioactive ligands. Ideal macrocyclic peptides should have dissociation constants (KD ) as low as single-digit values in the nanomolar range towards a specific target of interest. Here, a twofold strategy to discover optimized macrocyclic peptides within this affinity regime is described. First, benzyl thioether cyclized peptide libraries were explored to identify tight binding hits. To obtain more insights into critical sequence information, sequence alignment was applied to guide rational mutagenesis for the improvement of their binding affinity. Using this twofold strategy, benzyl thioether macrocyclic peptide binders against Lys48-linked ubiquitin dimer (K48-Ub2) were successfully obtained that display KD values in the range 0.3-1.2 nm, which indicate binding two orders of magnitude stronger than those of macrocyclic peptides recently reported. Most importantly, this macrocyclic peptide also showed an improved cellular inhibition of the K48-Ub2 recognition by deubiquitinating enzymes and the 26S proteasome, resulting in the promotion of apoptosis in cancer cells.

Hit evaluation of an α-helical peptide: Ala-scan, truncation and sidechain-to-sidechain macrocyclization of an RNA polymerase Inhibitor.

RNA polymerase (RNAP) remains a relatively underexplored target with only rifampicin and fidaxomicin in clinical use. Hence, the concurrent rise in bacterial resistance rate urges the search for novel RNAP inhibitors with a novel mode of action. In this work, we investigated the impact of several systematic modifications including sidechain-to-sidechain macrocylization in the α-helical content and biological activity of a previously identified inhibitory sigma factor fragment. Ala-scan results, peptide truncation from both the N- and C-terminus and modifications inspired by other RNAP inhibitors revealed novel structure activity relationships but did not yield a superior sequence. Additionally, four insertion points for non-natural amino acids bearing side chains required for macrocylization were explored. Linear precursors showed improved stabilization of the α-helical content compared to the original sequence as demonstrated by circular dichroism (CD) spectroscopy. However, this increase in α-helicity did not translate into improved biological activity. Instead, complete abolishment of RNAP inhibitory activity occurred. We hypothesize three possible reasons for such a discrepancy and offer the basis for further optimization efforts for this peptidic RNAP inhibitor.