Abstract Cyclic peptides represent a very useful modality in modern drug discovery. However, there remain substantial technical challenges in the de novo biosynthesis of complex cyclic peptides particularly in an intracellular manner. Herein, we demonstrate that an isothiocyanate (ITC) group encoded via genetic code expansion could selectively and efficiently crosslink to proximal N‐terminal Pro (P‐ITC crosslinking) and affords cyclic peptides without obvious limitations related to amino acid composition (including Lys and Cys) or sequence length. Notably via P‐ITC crosslinking, a disulfide‐bridged 13‐mer bicyclic peptide is facilely constructed in E. coli , thus lending us the ability to intracellularly construct and select complex cyclic peptides.

Cyclic peptides represent a very useful modality in modern drug discovery. However, there remain substantial technical challenges in the de novo biosynthesis of complex cyclic peptides particularly in an intracellular manner. Herein, we demonstrate that an isothiocyanate (ITC) group encoded via genetic code expansion could selectively and efficiently crosslink to proximal N-terminal Pro (P-ITC crosslinking) and affords cyclic peptides without obvious limitations related to amino acid composition (including Lys and Cys) or sequence length. Notably via P-ITC crosslinking, a disulfide-bridged 13-mer bicyclic peptide is facilely constructed in E. coli, thus lending us the ability to intracellularly construct and select complex cyclic peptides.

Abstract 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 peptides, with two cyclic substructures, have emerged as a powerful tool for modulating challenging targets such as protein-protein interactions. Meanwhile, DNA-encoded library technology (DELT) provides a powerful platform for hit discovery. The unity of both fields has the potential to identify potent bicyclic ligands for the targets of interest. Therefore, there is a high demand to develop an efficient way to construct bicyclic peptide libraries. Herein, we describe a novel and efficient approach to the synthesis of DNA-encoded bicyclic peptides via a cysteine-promoted cyclization and amide condensation reaction. This strategy proceeds smoothly under mild conditions and can generate a wide range of bicyclic peptides with various peptide sequences and ring sizes in good conversions.

Abstract Bicyclic peptides have emerged as privileged scaffolds in chemical biology and drug discovery owing to their high target affinity, enhanced metabolic stability, and rigid conformational structure. Conventional bicyclization strategies often face challenges related to efficiency and chemoselectivity, which can limit their broader applicability. Here, we present the first application of thiol–ene photoclick chemistry for the bicyclization of unprotected peptides, employing TAIC as a trifunctional crosslinker. This photochemical approach enables rapid, high‐yield, and highly selective cyclization under mild, biocompatible conditions and is compatible with all proteinogenic amino acids. The reaction reaches completion within 6 minutes and is fully compatible with phage display, allowing high‐throughput generation and selection of genetically encoded bicyclic peptide libraries. Using this platform, we identified two submicromolar ligands targeting cyclophilin A, both of which showed substantially improved binding affinity compared to their linear counterparts. These findings establish thiol–ene photoclick chemistry as a robust and versatile platform for the discovery of conformationally constrained peptide ligands, particularly suited for addressing therapeutically challenging protein–protein interactions.

Bicyclic peptides have emerged as privileged scaffolds in chemical biology and drug discovery owing to their high target affinity, enhanced metabolic stability, and rigid conformational structure. Conventional bicyclization strategies often face challenges related to efficiency and chemoselectivity, which can limit their broader applicability. Here, we present the first application of thiol-ene photoclick chemistry for the bicyclization of unprotected peptides, employing TAIC as a trifunctional crosslinker. This photochemical approach enables rapid, high-yield, and highly selective cyclization under mild, biocompatible conditions and is compatible with all proteinogenic amino acids. The reaction reaches completion within 6 minutes and is fully compatible with phage display, allowing high-throughput generation and selection of genetically encoded bicyclic peptide libraries. Using this platform, we identified two submicromolar ligands targeting cyclophilin A, both of which showed substantially improved binding affinity compared to their linear counterparts. These findings establish thiol-ene photoclick chemistry as a robust and versatile platform for the discovery of conformationally constrained peptide ligands, particularly suited for addressing therapeutically challenging protein-protein interactions.

Objectives: Nectin-4 has been successfully used as a target for tumor therapy. Although several bicyclic peptides and antibodies, Nectin-4 positron emission tomography (PET) probes, have been reported for tumor imaging and expression detection, their production costs or pharmacokinetics still need further improvement. This study developed a novel linear peptide PET probe for rapid examination of Nectin-4-related tumors. Methods: [68Ga]Ga-NOTA-SP was prepared by a one-step chelation reaction, and its quality control was carried out by using radio-high-performance liquid chromatography and thin-layer chromatography. Molecular docking was used to predict the predominant binding of NOTA-SP to Nectin-4. Cell experiments using SW780 cells and PET/computed tomography (CT) imaging, using the SW780 tumor model, were performed to assess the specific binding and targeting ability of [68Ga]Ga-NOTA-SP to Nectin-4. Normal BALB/c mice were used to investigate the plasma concentration-time curves. Results: Under optimal labeling conditions, the labeling efficiency of [68Ga]Ga-NOTA-SP can reach above 95%, with a molar-specific activity of 2.45 MBq/nmol and high in vitro stability. The high specificity of [68Ga]Ga-NOTA-SP to Nectin-4 is demonstrated by molecular docking and cell uptake experiment, showing a binding energy of -5.4 kcal/mol and Kd value of 2.483 nM, which was further confirmed by PET-CT imaging. Conclusions: [68Ga]Ga-NOTA-SP using a linear peptide as a vector shows favorable pharmacokinetics and specific targeting ability to Nectin-4, enabling rapid tumor mouse model imaging. It would be a promising PET/CT imaging probe for optimizing Nectin-4-related tumor diagnoses and therapy.

In this work, we develop a platform for multimerizing peptides/miniproteins using various polyvalent thioester cores derived from easy-to-synthesize N-hydroxysuccinimide esters. We employed native chemical ligation to attach multiple copies of peptide/miniprotein around a fixed multi-armed thioester core in a one-pot fashion, leading to the first-generation multimers. Further, using a reverse thioether ligation strategy, we synthesized second-generation branched homo/hetero multimers. The reactions proceed under an aqueous environment that closely mimics physiological conditions, forming multimeric products with yields ranging from 30% to 90%. The general utility of this strategy is showcased by the synthesis of multimeric linear and bicyclic peptides, bicyclic peptide-cell penetrating peptide conjugates, and multimeric miniproteins which show picomolar affinity against SARS-CoV-2 receptor binding domain. We believe that this modular approach of generating multimeric molecules should find broad applicability in peptide chemistry and chemical biology.

No specific vaccines or therapeutics are currently available for the prevention or treatment of Zika virus infections. The viral protease NS2B-NS3 is essential for the replication of Zika and other orthoflaviviruses, making it a target for antiviral drug development. Traditional discovery of competitive inhibitors has relied on substrate recognition sequences, typically yielding multibasic peptides. Herein, a de novo strategy is presented for identifying competitive inhibitors using peptide phage display in combination with Bi(III)-mediated in situ formation of bicyclic peptides. In an initial screening, phages displaying a library of randomized peptide-bismuth bicycles are eluted by interrupting the phage-target interactions at low pH. This approach yields a small number of peptides biased toward the active site, characterized by dibasic motifs, but only one low-ranking sequence shows modest inhibitory activity. To enhance specificity, a second screening campaign employs competitive phage elution using the dibasic boronate inhibitor CN-714 that covalently binds to the catalytically active serine residue S135 of NS2B-NS3. This strategy enriches a larger pool of competitive inhibitors sharing the characteristic dibasic substrate recognition motif. The most potent peptide-bismuth bicycle identified and synthesized features a completely novel sequence, exhibits an inhibition constant of 3.9 µM and displays remarkable proteolytic stability over 24 h.

The GDF15-GFRaL-RET signaling complex is involved in a broad range of disease states, with agonistic action of GDF15 affecting metabolism and body weight control, while inhibition is indicated in cancer and wasting disorders like cachexia. Here, we describe the discovery of the peptide inhibitors of the GDF15-GFRaL protein-protein interaction to prevent RET-induced signaling using both a structure-guided design and a phage display approach. Phage display provided bicyclic peptide hits with high affinity for GFRaL, and these were dimerized to mimic the bidentate interaction of homodimeric GDF15. Guided by structural data, the monomeric peptides were converted into tandem Bicycle molecules with picomolar affinities, similar to that of the endogenous GDF15 ligand. These dimerized protein mimetics inhibited cell signaling in a functional assay and showed improved pharmacokinetic properties compared with their monomeric counterparts. This is the first example of a homodimeric Bicycle molecule inhibiting receptor complex formation, thereby antagonizing the intracellular signaling response.

The cell adhesion protein nectin-4 emerged as a valid therapeutic target for antibody- and peptide-drug conjugates in cancer. To support patient stratification for such targeted therapies, there is a clinical need for molecular imaging agents capable of quantifying nectin-4 levels noninvasively in vivo. For this purpose, we developed 64Cu- and 68Ga-labeled ligands derived from bicyclic peptide-drug conjugate BT8009. A library of peptides was prepared with a major focus on the bioisosteric replacement of the original methionine residue due to its susceptibility to oxidation. The peptides were characterized for their binding behavior to nectin-4, and radiopharmacological characterization of selected radioligands was performed using urothelial carcinoma cell lines and tumor xenograft models derived thereof. The suitability of the most promising ligand from the preclinical studies, NECT-224, for PET imaging purposes was also demonstrated in a first-in-human application using [68Ga]Ga-NECT-224. The results suggest its further clinical development, but also that of [64Cu]Cu-NECT-224.

The lipopolysaccharide (LPS) transport (Lpt) system in Gram-negative bacteria maintains the integrity of the asymmetric bacterial outer membrane (OM). LPS biogenesis systems are essential in most Gram-negative bacteria, with LptDE responsible for the delivery of LPS to the outer leaflet of the OM. As an externally accessible, essential protein, LptDE offers a promising target for inhibitor development without the need for cellular penetration. However, there are no direct inhibitors of E. coli LptDE, and drug discovery is made challenging since it is a membrane target without a conventional active site. Here, the bicycle phage display platform was used in combination with cryogenic-electron microscopy (cryo-EM) and surface plasmon resonance to identify and map bicyclic peptide binders to Shigella flexneri LptDE (SfLptDE). Four distinct epitopes with unique bicycle molecule binding motifs were identified across the SfLptD β-barrel. This method represents a streamlined workflow for the identification and prioritization of hit molecules against LptDE.

Tag-like cyclic peptides: Modular affinity elements for protein integration and bioengineering applications.

In this work, the previously reported SeODR strategy was used successfully for the synthesis of bicyclic peptides (BPs). BPs with different rings bearing various thioether linkages were prepared through a one-pot reaction; 15 BPs with satisfactory yields were achieved. Cross-linkers including xylylene dibromide (xylyl), 2,6-bis(bromomethyl)pyridine (BBP), 4,4'-bis(bromomethyl)biphenyl (BBMP), 1,3-dichloroacetone (DCA), and dichloro-s-tetrazine (DCTZ) are all compatible with the SeODR approach used for deprotection of the S-acetamidomethyl (Acm) group in peptides. Moreover, four novel monosaccharide-based cross-linkers were prepared through the conjugation of m-xylyl with glucose, mannose, and galactose by the click reaction. Consequently, seven bicyclic glycopeptide conjugates (BGPs) were first synthesized by the use of the SeODR strategy in a one-pot manner, and good yields were all achieved. In these syntheses, monosaccharide and triazole groups in the monosaccharide-based cross-linkers were all compatible with the SeODR approach. Finally, a tricycle glycopeptide conjugate (TGP) based on conotoxin mr3e bearing three different monosaccharide cross-linkers was successfully synthesized by a combination of the SeODR strategy with the synthetic strategy of the monosaccharide-based cross-linkers. Therefore, multiple cyclic peptides with a large structural diversity can be synthesized conveniently by the combined strategies, with the multiple cyclic peptides being glycosylated by different monosaccharides.

A selective RCC1 inhibitor loaded in membrane-coated DNA nanocage for targeted suppression of TNBC progression

BackgroundSome patients with schizophrenia do not respond adequately to current treatments. In 2011, a strong association between the vasoactive intestinal peptide (VIP) receptor 2 (VIPR2) gene duplication and schizophrenia was reported. Therefore, VIPR2-selective inhibitors may help in the development of new treatments for schizophrenia. In 2021, we discovered a bicyclic peptide KS-133, Ac-(CPPYLP[KYLC)D]LI-NH2 (1558.8 g/mol), with a potent and selective VIPR2 antagonist activity and high in vivo stability (Front Pharmacol 12: 751587, 2021). However, the development of KS-133 as an anti-schizophrenia drug has been challenging because KS-133 does not effectively penetrate the blood-brain barrier (BBB) and there is no preclinical proof of concept that VIPR2 inhibition improves symptoms in a rodent model of schizophrenia.Aims & ObjectivesTo overcome these challenges, we combined KS-133 with a reliable brain-targeting technology and investigated in vivo efficacy in a relevant mouse model of schizophrenia.MethodTo facilitate drug transport across the BBB into the brain, we focused on the receptor-mediated transcytosis using low-density lipoprotein receptor-related protein 1 (LRP1). We previously identified KS-487 as a novel LRP1-binding cyclic peptide with high affinity, high stability and high BBB permeability (Biochem Biophys Rep 32: 101367, 2022). Therefore, we attempted to develop a novel drug delivery system by combining KS-487 with KS-133 to enhance brain permeability. All animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Plasma and tissue concentrations of KS-133 were measured using LC-MS/MS.ResultsWe investigated whether the VIPR2 antagonist KS-133 could be transported into the brain when encapsulated in nanoparticles (NPs) displaying KS-487. KS-133 encapsulated in NPs composed of Cremophor EL, a polyethoxylated surfactant, was stably retained at 4 °C but was gradually released from the NPs at 37 °C. Transmission electron microscopy and particle size distribution analysis of KS-133/KS-487 NPs revealed monodisperse particles with diameters of approximately 12 nm. A pharmacokinetic study showed the time-dependent translocation of KS-133 in the brain after subcutaneous administration (3 mg/kg) of KS-133/KS-487 NPs. KS-133 concentrations were consistent in all tissues and peaked 1-2 h after administration. The mean maximum concentration was 8.734 μM in the plasma, 0.054 μmol/kg in the cerebral cortex, 0.058 μmol/kg in the hypothalamus, 0.062 μmol/kg in the hippocampus, 0.044 μmol/kg in the striatum, and 0.103 μmol/kg in the cerebellum. These data suggest that KS-133 is uniformly distributed throughout the brain. The peak concentration of KS-133 in the whole brain was estimated to be approximately 70 nM, which is sufficient for VIPR2 antagonist activity (IC50 = 25 nM). Under these conditions, KS-133/KS-487 or KS-133 NPs (without KS-487) at a dose of 3 mg/kg were administered subcutaneously once daily for 2 weeks to a mouse model of schizophrenia induced by early postnatal treatment with the VIPR2 agonist Ro25-1553. KS-133/KS-487 NPs, but not KS-133 NPs, improved cognitive dysfunction in the novel object recognition test.Discussion & ConclusionsThis is the first study to demonstrate the potential therapeutic efficacy of a multifunctionalized multi-peptide NP with VIPR2 inhibition for the treatment of schizophrenia.

Abstract Chemical modification and nucleic acid self‐assembly can be used to make protein receptor ligands form specific arrangements. While this property has been extensively exploited for probing of homomultivalent interactions, there has been comparatively little attention paid to the exploration of heteromultivalent interactions. In this study, we investigated the use of readily assemblable DNA duplexes for programming bispecific targeting of specific cell types. In contrast to previous bispecific agents, we leverage the potential of peptide‐based high‐affinity binders of cell surface proteins used in diagnostics/therapeutics. Systematic spatial screening revealed the optimal distance between two (cyclo)peptides required for selectively recognizing cells expressing unique combinations of receptors. The VGFR2/αVβ3 receptor system on HUVECs was tolerant to changes of the distance between two cyclopeptides (L and cyclo(‐RGDf(N‐Me)K‐)) and required that the distance exceeded the equivalent of 20 nucleotides distance. A different distance‐affinity landscape was observed for recognition of EGFR and MET on A549 cells (through GE11 and bicyclic peptide GE‐137). The DNA‐programmed bispecific binders demonstrated specificity and efficient internalization into target cells. Auristatin‐loaded DNA enabled a selective targeting of cytotoxic payload. Of note, the distance‐optimized bispecific DNA‐peptide probes have much lower molecular weight than previously used agents based on DNA nanostructures or antibodies.

Chemical modification and nucleic acid self-assembly can be used to make protein receptor ligands form specific arrangements. While this property has been extensively exploited for probing of homomultivalent interactions, there has been comparatively little attention paid to the exploration of heteromultivalent interactions. In this study, we investigated the use of readily assemblable DNA duplexes for programming bispecific targeting of specific cell types. In contrast to previous bispecific agents, we leverage the potential of peptide-based high-affinity binders of cell surface proteins used in diagnostics/therapeutics. Systematic spatial screening revealed the optimal distance between two (cyclo)peptides required for selectively recognizing cells expressing unique combinations of receptors. The VGFR2/αVβ3 receptor system on HUVECs was tolerant to changes of the distance between two cyclopeptides (L and cyclo(-RGDf(N-Me)K-)) and required that the distance exceeded the equivalent of 20 nucleotides distance. A different distance-affinity landscape was observed for recognition of EGFR and MET on A549 cells (through GE11 and bicyclic peptide GE-137). The DNA-programmed bispecific binders demonstrated specificity and efficient internalization into target cells. Auristatin-loaded DNA enabled a selective targeting of cytotoxic payload. Of note, the distance-optimized bispecific DNA-peptide probes have much lower molecular weight than previously used agents based on DNA nanostructures or antibodies.

Abstract Background: Zelenectide pevedotin (zele, formerly BT8009) is a highly selective Bicycle® Toxin Conjugate (BTC) comprised of a bicyclic peptide targeting Nectin-4 conjugated to monomethyl auristatin E. BTC® molecules have lower molecular weight and shorter plasma half-life than antibody-drug conjugates, with distinct pharmacokinetics/dynamics, i.e., potential for rapid tumor penetration and minimal healthy tissue exposure. NECTIN4 amp has been shown to be a predictive biomarker for response to Nectin-4 targeted therapy in metastatic urothelial cancer (Klümper et al., 2024). An analysis of NECTIN4 amp in a large, independent sample of 245 breast cancer (BC) pts indicates that amp is common, seen in 19% (30/161), 14% (5/36) and 23% (11/48) of HR+/HER2-, HER2+ and TNBC, respectively (Klümper et al., unpublished data). This initial post-hoc analysis assesses the utility of NECTIN4 amp as a predictor of zele response in heavily pretreated BC pts. Methods: Zele is being evaluated in the ongoing Phase 1/2 study BT8009-100/Duravelo-1 (NCT04561362) for safety and efficacy in pts with advanced solid tumors associated with Nectin-4 expression including BC pts. This analysis focuses on TNBC study pts who had baseline tissue sample available, had consented to optional future research, and tested for NECTIN4 amp by fluorescence in-situ hybridization. NECTIN4 amp positivity was defined as a ratio of NECTIN4: centromere 1 (CEN1) of 2.0 or higher. Assessment of anti-tumor activity was per RECIST v1.1 by investigator. Objective response rate (ORR) is based on the efficacy evaluable population. Results: As of 29 August 2024, 32 heavily pretreated pts with TNBC enrolled in the monotherapy dose escalation and expansion cohorts of BT8009-100. Thirty-one TNBC pts were treated with the zele recommended Phase 2 dose of 5 mg/m2 weekly. At baseline, TNBC pts had a median age of 52 (30-76), median prior lines of therapy of 6 (2-13), and ECOG of 0 or 1 (50.0% each). Of 32 enrolled TNBC pts treated with zele, 30 were efficacy evaluable. Four pts achieved partial response (PR) resulting in an ORR of 13.3% (95% CI: 3.8, 30.7). Nineteen pts were tested for NECTIN4 amp, 6 of whom were positive (31.6%). Of these, 3 achieved a PR with an ORR of 50.0% (95% CI: 11.8, 88.2), and the remaining 3 had stable disease (SD; disease control rate 100.0%). All 3 NECTIN4 amp responders were previously treated with sacituzumab govitecan. Of the NECTIN4 non-amp pts, 1/13 had a PR: ORR of 7.7% (95% CI: 0.2, 36.0). Of note, the non-amp responder pt harbored a polysomy, with NECTIN4 and CEN1 copy number >6 (NECTIN4:CEN1 ratio <2.0). Four HR+ BC pts were tested for NECTIN4 amp. One responder (1/ 4) was also the only one harboring a NECTIN4 amp. Safety and tolerability of zele in the TNBC population was similar to a previously reported cohort of bladder cancer pts (Reig et al., 2024). Grade 3 or higher zele-related adverse events (AEs) occurred in 34.4% of all TNBC pts, and Grade 3 or higher zele-related serious adverse events (SAEs) occurred in 12.5% of all TNBC pts. Zele-related AE frequencies were similar in the NECTIN4 amp pts; however, no NECTIN4 amp pts had a zele-related SAE. Conclusions: NECTIN4 amplification appears to show predictive clinical utility in identifying pretreated TNBC pts with enhanced response to zelenectide pevedotin, with an ORR of 57.1% in NECTIN4 gene amp positive pts including polysomy versus an ORR of 13.3% in biomarker unselected TNBC. Despite the limited sample size, this post-hoc analysis underscores the promising anti-tumor activity of zelenectide pevedotin in pts with NECTIN4 amp TNBC, who continue to have significant unmet medical need for effective, well-tolerated therapy. These findings support further exploration of zele and NECTIN4 amp stratification strategies in BC pts, particularly TNBC. Citation Format: Niklas Klümper, Viktor Grünwald, Johannes Brägelmann, Meredith McKean, Elisa Fontana, Antoine Italiano, Loic Verlingue, Capucine Baldini, Valentina Boni, Alberto J Montero, Thibault de la Motte, Bernard Doger, Jordi Rodón Ahnert, Cecile Vicier, Carly Campbell, Tara Gelb, Sean Santos, Kate Josephs, Cong Xu, Nicholas Keen, Kevin Lee, Santiago Arroyo, Markus Eckstein. Enhanced anti-tumor activity of zelenectide pevedotin in triple negative breast cancer (TNBC) patients (pts) with NECTIN4 gene amplification (amp) [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-10-21.

Rational design of cyclic peptides, with an emphasis on bicyclic peptides.