Synthesis of DNA-Encoded Bicyclic Peptides via Cysteine-Promoted Cyclization and Amide Condensation Reaction.

Macrocyclic peptides are promising scaffolds for drug discovery due to their structural rigidity and high target specificity. Here, we report a strategy for in vitro ribosomal translation of thioisoindole‐bridged bicyclic peptides. Central to this approach is a newly developed flexizyme substrate, Ac‐Ala(NtBA) Sc ‐CME, which features a semicarbazone‐masked 2‐nicotinoyl benzaldehyde sidechain. We show that this amino acid can be efficiently charged onto tRNA with flexizyme and incorporated into ribosomal peptides using a customized flexible in vitro translation (FIT) system. The semicarbazone group can be post‐translationally removed under mild conditions, triggering spontaneous intramolecular cyclization to cysteine and lysine sidechains in the same substrate to yield thioisoindole‐bridged bicyclic (TiB) peptides. This strategy was leveraged to synthesize structurally diverse bicyclic peptides with varying sequences and ring sizes. The method maintains the integrity of mRNA and is therefore compatible with mRNA display, which opens the possibility of constructing topologically defined bicyclic peptide libraries for therapeutic peptide discovery.

Bicyclic peptide ligands are promising molecules for the development of new therapeutics. They combine advantages from large protein therapeutics and small molecule drugs. Large combinatorial libraries of bicyclic peptides can be generated and screened by phage display using a recently developed strategy. Potent and selective bicyclic peptide inhibitors against several therapeutic targets have already been developed and their therapeutic potential is currently being evaluated in animal models. One aim of my thesis was the exploration of ring size diversity in bicyclic peptides. I generated a set of libraries of the format Cys-(Xaa)m-Cys-(Xaa)n-Cys, where 'm' and 'n' = 3, 4, 5 or 6, and performed affinity selections against the serine protease urokinase-type plasminogen activator. Bicyclic peptide inhibitors from virtually all ring size combinations were isolated, suggesting that many peptide formats can be accommodated in the active site of this enzyme. Moreover, they showed a large variety of consensus sequences and several of the identified consensus sequences were exclusively found in bicyclic peptides having specific ring size combinations. Having available multiple leads isolated from such bicyclic peptide libraries with variable ring sizes could be a great asset for the generation of high affinity binders. Additionally, other targets may have preferences for specific peptide constraints and the availability of these libraries increases the chances to isolate high affinity binders to any desired target. A second goal of my thesis was to apply high throughput sequencing technologies to phage display selections of bicyclic peptides, in order to identify a larger number of specific target-binding sequences and motifs. I developed a procedure to efficiently compare the sequences of large numbers of phage-selected peptides to identify target-binding peptide motifs based on abundance and sequence similarity. Applying this approach to phage isolated in selections against five different protein targets, I was able to identify rare target-binding peptide motifs and could more precisely define groups and sub-groups of consensus sequences. This information is valuable to choose peptide leads for drug development and facilitates the identification of epitopes. Moreover, binding motifs could be identified after a single round of phage panning. The final aim of my thesis was to discover bicyclic peptides that could be used as new antibiotics. Towards this end, I combined the newly generated variable ring size libraries and high-throughput sequencing procedures. I focused on the development of inhibitors of Staphylococcus aureus sortase A, an antivirulence target for which no potent and specific inhibitors have been reported. For the isolation of bicyclic peptide inhibitors to this target, the ring size diversity of the libraries turned out to be key. Inhibitors all shared the same motif in a loop of 5 residues. Further characterization of their effects on S. aureus showed that they could inhibit sortase-mediated incorporation of external substrates on the staphylococcal cell wall. However, they were not sufficiently potent to compete with the native substrates of the enzyme. More potent inhibitors are needed to effectively inhibit sortase A on S. aureus cells, and the bicyclic peptide inhibitors isolated constitute promising leads for the development of future antisortase therapeutics.

Macrocyclic peptides are promising scaffolds for drug discovery due to their structural rigidity and high target specificity. Here, we report a strategy for in vitro ribosomal translation of thioisoindole-bridged bicyclic peptides. Central to this approach is a newly developed flexizyme substrate, Ac-Ala(NtBA)Sc-CME, which features a semicarbazone-masked 2-nicotinoyl benzaldehyde sidechain. We show that this amino acid can be efficiently charged onto tRNA with flexizyme and incorporated into ribosomal peptides using a customized flexible in vitro translation (FIT) system. The semicarbazone group can be post-translationally removed under mild conditions, triggering spontaneous intramolecular cyclization to cysteine and lysine sidechains in the same substrate to yield thioisoindole-bridged bicyclic (TiB) peptides. This strategy was leveraged to synthesize structurally diverse bicyclic peptides with varying sequences and ring sizes. The method maintains the integrity of mRNA and is therefore compatible with mRNA display, which opens the possibility of constructing topologically defined bicyclic peptide libraries for therapeutic peptide discovery.

Protein–protein interactions (PPIs) are challenging targets for small molecules because their binding sites usually do not contain well-defined pockets for small molecules to bind. Biologic drugs (e.g., monoclonal antibodies) are effective against extracellular PPIs, but not intracellular ones, due to their lack of cell permeability. Bicyclic peptides have demonstrated the capacity for binding to PPI targets with antibody-like affinity and specificity and represent an exciting modality for targeting both extracellular and intracellular PPIs. Powerful combinatorial library technologies have recently been developed to rapidly synthesize and screen large bicyclic peptide libraries for ligands against enzymes and PPI targets. Bicyclic peptide ligands against extracellular targets have already advanced into the clinic, while examples of cell-permeable bicyclic peptide inhibitors against intracellular PPIs have recently emerged. This chapter provides a summary of the recent developments in the synthesis and applications of bicyclic peptides as PPI inhibitors.

Bicyclic peptides are an attractive molecule format for the development of therapeutics and research tools. Our laboratory is specialized on the development of bicyclic peptide ligands by phage display. In brief, libraries of randompeptides each containing three cysteines are displayed on the surface of a phage and cyclized with a trivalent thiol-reactive chemical reagent. Ligands of a target of interest are isolated by affinity selection and their sequence identified by DNA sequencing. The first goal of my thesis was to test if more diverse bicyclic peptide libraries can be generated if linear peptides on phage are cyclized in parallel with multiple different chemical cyclization linkers. Towards this end, we cyclized peptide libraries of the format XCXnCXnCX (X = random amino acid, C = cysteine, n = 3 or 4) with three different chemicals and panned the library against the protease urokinase-type plasminogen activator (uPA). Interestingly, different peptide sequences were enriched when the phage peptide library was cyclized with the different chemical linkers, indicating that a larger diversity of bicyclic peptides is generated. Screening the larger library generated binders with a higher binding affinity. In the second project I carried out, I isolated bicyclic peptide ligands to a protein of therapeutic interest, b-catenin. b-catenin is the main translational co-activator of theWnt cell signaling pathway. Pathological upregulation of b-catenin is associated with the majority of colorectal cancer. Blocking b-catenin activity is thus an attractive therapeutic approach. However, so far the development of b-catenin inhibitors has been challenging due to the flat and featureless surface of the protein, lacking binding pockets for synthetic ligands. Screening a phage display library comprising >12 billion bicyclic peptides yielded binders for different protein epitopes. The binding site of four peptides was identified to correspond to that of ICAT (inhibitor of b-catenin and Tcf), which is a prime target site on b-catenin for therapeutic intervention and to which no synthetic ligands could be isolated so far. In the third project, we isolated and characterized bicyclic peptides binding to globular actin (G-actin). G-actin constitutes the fundamental building block of microfilaments (also named F-actin) in eukaryotic cells. Beyond this structural function, G-actin has a role in gene transcription and chromatin remodeling. While specific F-actin probes are available for in vitro applications, G-actin probes are lacking. To fulfil this demand, we screened a library of bicyclic peptides for G-actin binders and isolated ligands with dissociation constants in the low nanomolar range. The isolated peptides were found to bind to the same surface region as thymosin b4 and a range or marine toxins. The bicyclic peptide G-actin ligands allowed measurement of the binding affinity of natural G-actin ligands in fluorescence anisotropy competition assays.

Nerve regeneration scaffolds often consist of soft hydrogels modified with extracellular matrix (ECM) proteins or fragments, as well as linear and cyclic peptides. One of the commonly used integrin-mediated cell adhesive peptide sequences is Arg-Gly-Asp (RGD). Despite its straightforward coupling mechanisms to artificial extracellular matrix (aECM) constructs, linear RGD peptides suffer from low stability towards degradation and lack integrin selectivity. Cyclization of RGD improves the affinity towards integrin subtypes but lacks selectivity. In this study, a new class of short bicyclic peptides with RGD in a cyclic loop and ‘random screened’ tri-amino acid peptide sequences in the second loop is investigated as a biochemical cue for cell growth inside three-dimensional (3D) synthetic poly(ethylene glycol) (PEG)-based Anisogels. These peptides impart high integrin affinity and selectivity towards either αvβ3 or α5β1 integrin subunits. Enzymatic conjugation of such bicyclic peptides to the PEG backbone enables the formulation of an aECM hydrogel that supports nerve growth. Furthermore, different proteolytic cleavable moieties are incorporated and compared to promote cell migration and proliferation, resulting in enhanced cell growth with different degradable peptide crosslinkers. Mouse fibroblasts and primary nerve cells from embryonic chick dorsal root ganglions (DRGs) show superior growth in bicyclic RGD peptide conjugated gels selective towards αvβ3 or α5β1, compared to monocyclic or linear RGD peptides, with a slight preference to αvβ3 selective bicyclic peptides in the case of nerve growth. Synthetic Anisogels, modified with bicyclic RGD peptides and containing short aligned, magneto-responsive fibers, show oriented DRG outgrowth parallel to the fibers. This report shows the potential of PEG hydrogels coupled with bicyclic RGD peptides as an aECM model and paves the way for a new class of integrin selective biomolecules for cell growth and nerve regeneration.

DNA-Encoded Library Technology (DELT) After a Quarter Century

Conformationally constrained peptide ligands offer an attractive format for the development of therapeutics. They can bind with high affinity and selectivity to protein targets, they are small enough to diffuse into tissues, they can be synthesized chemically, and their degradation products are not toxic. In recent years, a number of novel techniques were innovated to develop peptide-based ligands. One such technique is the generation of bicyclic peptides by phage display. This technique is well established in our laboratory and has led to the isolation of high-affinity antagonist for a range of important therapeutic targets. In a first project I aimed at developing bicyclic peptide ligands of the epidermal growth factor receptor Her2. Aberrant expression of Her2 has been implicated in various malignancies including breast cancer. In the treatment of this cancer type several Her2 specific antibodies were proved to be efficient. Due to their small size, bicyclic peptides could potentially offer advantages over antibodies such as ease of synthesis and conjugation, higher molecule-permass ratios, and better tumor penetration. I panned a large bicyclic peptide phage library against the extra-cellular domain of Her2 and obtained a range of peptide sequences binding to Her2 in a specific manner. After affinity maturation, a bicyclic peptide that bound Her2 with a Kd of 304 nM could be obtained. This peptide ligand offers a valuable starting point for further improvement and the development of high-affinity Her2 binders with potential application for tumor imaging and therapy. A second project was triggered by my observation that it is relatively difficult to generate high-affinity bicyclic peptide ligands to proteins with flat and featureless surfaces such as Her2. The major reason for the limited binding affinity of bicyclic peptides was supposed to lie in the lack of a defined secondary structure in solution and the resulting entropic penalty upon binding. To overcome this problem, I proposed to evolve ligands based on a-helices. Chemically stabilized a-helices, also named stapled peptides, were previously developed by rational design. I proposed to evolve a-helical peptides by phage display wherein the helix, is stabilized in a chemical reaction prior to phage panning. I tested this strategy by affinity maturing an alpha-helical peptide binding to beta-catenin. The project resulted with a 250-fold improved ligand of beta-catenin. In a third project, I developed a novel constrained peptide format that I dubbed “helix-loop” motif. A stabilized alpha-helix was expanded with a peptide loop in order to increase the number of amino acids that can formcontacts with a target. The loop was linked to either the N- or C-terminal end of the helix and via a cysteine residue to the chemical linker stabilizing the alpha-helix. Libraries were created adding randomized loops with different length to either side of the peptide. Ligands with improved affinity were isolated against the cancer target beta-catenin. In summary, I have exploited the existing bicyclic peptide format to evolve bicyclic peptide ligands of Her2. In addition, I have developed a new approach to affinity mature stabilized helical peptides by phage display as well as a new constrained peptide format. This new approach should be applicable for affinity maturation of any stabilized alpha-helix. The new peptide format promises the generation of high affinity ligands to any protein target, including Her2.

Kappa opioid receptor (KOR) antagonists have potential therapeutic applications in the treatment of stress-induced relapse to substance abuse and mood disorders. The dynorphin A analog arodyn (Ac[Phe1,2,3,Arg4,D-Ala8]dynorphin A-(1-11)-NH2) exhibits potent and selective kappa opioid receptor antagonism. Multiple cyclizations in longer peptides, such as dynorphin and its analogs, can extend the conformational constraint to additional regions of the peptide beyond what is typically constrained by a single cyclization. Here, we report the design, synthesis, and pharmacological evaluation of a bicyclic arodyn analog with two constraints in the opioid peptide sequence. The peptide, designed based on structure-activity relationships of monocyclic arodyn analogs, was synthesized by solid-phase peptide synthesis and cyclized by sequential ring-closing metathesis (RCM) in the C- and N-terminal sequences. Molecular modeling studies suggest similar interactions of key aromatic and basic residues in the bicyclic peptide with KOR as found in the cryoEM structure of KOR-bound dynorphin, despite substantial differences in the backbone conformations of the two peptides. The bicyclic peptide's affinities at KOR and mu opioid receptors (MOR) were determined in radioligand binding assays, and its KOR antagonism was determined in the [35S]GTPγS assay in KOR-expressing cells. The bicyclic analog retains KOR affinity and selectivity (Ki = 26 nM, 97-fold selectivity over MOR) similar to arodyn and exhibits potent KOR antagonism in the dynorphin-stimulated [35S]GTPγS assay. This bicyclic peptide represents a promising advance in preparing cyclic opioid peptide ligands and opens avenues for the rational design of additional bicyclic opioid peptide analogs.

Aim: Develop technology to apply bicyclic peptides for discovering covalent inhibitors of proteases and use this technology to create bicyclic peptide—warhead conjugates for targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like (3CL) protease. Enhance the potency of the discovered bicyclic peptides for potential development into anti-SARS-CoV-2 drugs. Methods: Rational design was employed to discover the initial bicyclic peptide—warhead conjugates. Medicinal chemistry optimization was conducted to improve the potency of these peptides. Enzymatic assays and mass spectrometry characterization were performed to validate the covalent inhibition of the target protease. Results: The need for peptide display selection in discovering hit bicyclic peptides was overcome. Active bicyclic peptide—vinyl sulfone inhibitors with nanomolar potency were discovered. Optimization through medicinal chemistry strategies not only improved the potency of the peptides but also revealed residue preferences at individual positions of the bicyclic peptide inhibitors. The most potent bicyclic peptide can inhibit the target with a half-maximal inhibitory concentration (IC50) of 40.46 ± 6.35 nM. Mass spectrometry tests confirmed the covalent inhibition of the target protease by the developed peptides. Conclusions: Bicyclic peptide and vinyl sulfone conjugates are a form of covalent and potent inhibitors for targeting proteases. The rational design of bicyclic peptide ligands is feasible when structural and amino acid preference information is available. Structural information is also crucial for optimizing the potency of bicyclic peptide ligands.

Protein-protein interactions represent a new class of exciting but challenging drug targets, because their large, flat binding sites lack well-defined pockets for small molecules to bind. We report here a methodology for chemical synthesis and screening of large combinatorial libraries of bicyclic peptides displayed on rigid small-molecule scaffolds. With planar trimesic acid as the scaffold, the resulting bicyclic peptides are effective for binding to protein surfaces such as the interfaces of protein-protein interactions. Screening of a bicyclic peptide library against tumor necrosis factor-α (TNFα) identified a potent antagonist that inhibits the TNFα-TNFα receptor interaction and protects cells from TNFα-induced cell death. Bicyclic peptides of this type may provide a general solution for inhibition of protein-protein interactions.

Current development of bicyclic peptides

Introduction: Bicycle Therapeutics is developing bicyclic peptide binders, targeting MT1-MMP (a matrix metalloproteinase involved in tumor metastasis) to deliver toxic agents specifically to tumor cells. Due to their high affinity, small size, enhanced tumor penetration, fully synthetic nature and rapid clearance bicyclic peptide drug conjugates (BDCs) may overcome some of the limitations of the antibody drug conjugate (ADC) format. Photoacoustic imaging is an emerging technology that combines the most compelling features of optical imaging and ultrasound, providing both high optical contrast and high ultrasound resolution at depth. Photoacoustic imaging's unique properties make it superior to other modalities in generating information-rich structural and functional images in multiple critical disease areas, including applications within cancer, cardiovascular disease, and inflammation. Here, a fluorescent-labeled Bicycle peptide (Alexa680), and a corresponding antibody (Alexa750), both bind to the same domain (hemopexin) on MMP14, to measure tumor penetration simultaneously. Both bind to the same domain (hemopexin) on MMP14 with similar (low/subnanomolar) affinities. Functional perfusion was measured by Indocyanine Green (ICG) dynamic imaging. By utilizing photoacoustics, we were able to discriminate regions of high and low perfusion in the tumor and measure the amount of each labeled material. Methods: Female Nude mice were implanted with human fibrosarcoma cell line (HT-1080) subcutaneously. HT-1080 is known to express large amounts of MMP14. While anesthesized with isofluorane, a tail vein catheter was introduced and the animal placed into a photoacoustic 3D imager (Nexus 128, Endra Inc. Ann Arbor, MI). The fluore-labeled Bicycle Peptide and Antibody were injected and a 30-minute dynamic scan was acquired for both labels simultaneously. This was followed by a 3-minute ICG scan. Results: Using the ICG scan, regions of high and low perfusion were determined and used to quantify compound penetration. The Bicycle peptide was shown to accumulate into highly perfused regions over the antibody. In the lower perfused regions the bicycle peptide was also shown to amass at higher amounts over the antibody in regions of high vascular perfusion. Conclusion: Using photoacoustics we were able to show regions of low perfusion were accessible by the Bicycle peptide, to a greater extent than the antibody. Relatively low perfusion in localized tumor refund can limit therapeutic effect. The delivery of a therapeutic using a peptide with high tumor affinity and penetration under poor vascular perfusion would overcome such limitations. Citation Format: Christopher Bull, Gavin Bennet, Athena Fletcha, Hellen Harrison, Leonardo Baldassare, Bruce Hamilton. Time-resolved functional perfusion-based analysis of peptide vs. antibody tumor penetration by photoacoustics. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3239. doi:10.1158/1538-7445.AM2015-3239

Bicyclic peptides have greater conformational rigidity and metabolic stability than linear and monocyclic peptides and are capable of binding to challenging drug targets with antibody-like affinity and specificity. Powerful combinatorial library technologies have recently been developed to rapidly synthesize and screen large bicyclic peptide libraries for ligands against enzymes, receptors, and protein-protein interaction targets. Bicyclic peptides have been developed as potential therapeutics against a wide range of diseases, drug targeting agents, imaging/diagnostic probes, and research tools. In this Minireview, we provide a summary of the recent progresses on the synthesis and applications of bicyclic peptides.

A 26-residue peptide BimBH3 binds indiscriminately to multiple oncogenic Bcl2 proteins that regulate apoptosis of cancer cells. Specific inhibition of the BimBH3-Bcl2A1 protein-protein interaction was obtained in vitro and in cancer cells by shortening the peptide to 14 residues, inserting two cyclization constraints to stabilize a water-stable α-helix, and incorporating an N-terminal acrylamide electrophile for selective covalent bonding to Bcl2A1. Mass spectrometry of trypsin-digested bands on electrophoresis gels established covalent bonding of an electrophilic helix to just one of the three cysteines in Bcl2A1, the one (Cys55) at the BimBH3-Bcl2A1 protein-protein interaction interface. Optimizing the helix-inducing constraints and the sequence subsequently enabled electrophile removal without loss of inhibitor potency. The bicyclic helical peptides were potent, cell permeable, plasma-stable, dual inhibitors of Bcl2A1 and Mcl-1 with high selectivity over other Bcl2 proteins. One bicyclic peptide was shown to inhibit the interaction between a pro-apoptotic protein (Bim) and either endogenous Bcl2A1 or Mcl-1, to induce apoptosis of SKMel28 human melanoma cells, and to sensitize them for enhanced cell death by the anticancer drug etoposide. These approaches look promising for chemically silencing intracellular proteins.