Development of Macrocyclic Neurotensin Receptor Type 2 (NTS2) Opioid-Free Analgesics.

The opioid crisis has highlighted the urgent need to develop non-opioid alternatives for managing pain, with an effective, safe, and non-addictive pharmacotherapeutic profile. Using an extensive structure-activity relationship approach, here we have identified a new series of highly selective neurotensin receptor type 2 (NTS2) macrocyclic compounds that exert potent, opioid-independent analgesia in various experimental pain models. To our knowledge, the constrained macrocycle in which the Ile12 residue of NT(7-12) was substituted by cyclopentylalanine, Pro7 and Pro10 were replaced by allyl-glycine followed by side-chain to side-chain cyclization is the most selective analog targeting NTS2 identified to date (Ki 2.9 nM), showing 30,000-fold selectivity over NTS1. Of particular importance, this macrocyclic analog is also able to potentiate the analgesic effects of morphine in a dose- and time-dependent manner. Exerting complementary analgesic actions via distinct mechanisms of nociceptive transmission, NTS2-selective macrocycles can therefore be exploited as opioid-free analgesics or as opioid-sparing therapeutics, offering superior pain relief with reduced adverse effects to pain patients.

Rational design of glycosaminoglycan binding cyclic peptides using cPEPmatch.

Cyclic peptides present a robust platform for drug design, offering high specificity and stability due to their conformationally constrained structures. In this study, we introduce an updated version of the Cyclic Peptide Matching program (cPEPmatch) tailored for the identification of cyclic peptides capable of mimicking protein-glycosaminoglycan (GAG) binding sites. We focused on engineering cyclic peptides to replicate the GAG-binding affinity of antithrombin III (ATIII), a protein that plays a crucial role in modulating anticoagulation through interaction with the GAG heparin. By integrating computational and experimental methods, we successfully identified a cyclic peptide binder with promising potential for future optimization. MD simulations and MM-GBSA calculations were used to assess binding efficacy, supplemented by umbrella sampling to approximate free energy landscapes. The binding specificity was further validated through NMR and ITC experiments. Our findings demonstrate that the computationally designed cyclic peptides effectively target GAGs, suggesting their potential as novel therapeutic agents. This study advances our understanding of peptide-GAG interactions and lays the groundwork for future development of cyclic peptide-based therapeutics.

First-Generation Radiolabeled Cyclic Peptides for Molecular Imaging of Platelet-Derived Growth Factor Receptor α.

Occult nodal spread and metastatic disease require longstanding imaging and biochemical assessments for thyroid cancer, a disease that has a propensity for diffuse, small-volume disease. We have developed a 64Cu-labeled platelet-derived growth factor receptor α (PDGFRA) antibody for immuno-PET of PDGFRA in metastatic papillary thyroid cancer (PTC). The present work describes the discovery of small cyclic PDGFRA-targeting peptides, their binding features, and radiolabeling with positron emitter gallium-68 (68Ga) for in vitro and in vivo characterization in thyroid cancer models. Phage-display technology with two separate libraries and seven different cell lines was used through three rounds of biopanning as well as flow cytometry and comparative analysis with recombinant protein to select specific peptide sequences. Phenotypic binding analysis was completed by using phosphorylation and cell migration assays. In vitro protein binding was analyzed with thermophoresis and flow cytometry using the fluorescent-labeled PDGFRA peptide. Peptide candidates were modified with the NOTA chelator for radiolabeling with 68Ga. In vitro cell uptake was studied in various thyroid cancer cell lines. In vivo studies of 68Ga-labeled peptides included metabolic stability and PET imaging. From the original library (1013 compounds), five different peptide groups were identified based on biopanning experiments with and without the α subunit of PDGFR, leading to ∼50 peptides. Subsequent phenotypic screening revealed two core peptide sequences (CP16 and CP18) that demonstrated significant changes in the level of PDGFRA phosphorylation and cell migration. Alanine scan sublibraries were created from these two lead peptide sequences, and peptides were radiolabeled using 68Ga-GaCl3 at pH 4.5, resulting in RCP > 95% within 34-40 min, including SPE purification. Cyclic peptide CP18.5 showed the strongest effects on cell migration, flow cytometry, and binding by visual interference color assay. 68Ga-labeled PDGFRA-targeting peptides showed elevated cell and tumor uptake in models of thyroid cancer, with 68Ga-NOTA-CP18.5 being the lead candidate. However, metabolic stability in vivo was compromised for 68Ga-NOTA-CP18.5 vs 68Ga-NOTA-CP18 but without impacting tumor uptake or clearance profiles. First-generation radiolabeled cyclic peptides have been developed as novel radiotracers, particularly 68Ga-NOTA-CP18.5, for the molecular imaging of PDGFRA in thyroid cancer.

[1-7-NαC]-Crocaorb A1 and A2, orbitides from the latex of Croton campanulatus.

Two new heptapeptides, [1-7-NαC]-crocaorbs A1 (1) and A2 (2), were isolated from the latex of Croton campanulatus. Their structures were determined using NMR spectroscopic techniques, ESI-HRMS data, Marfey's method, and further refined using molecular dynamics with simulated annealing (MD/SA). Molecular dynamics calculations of peptides 1 and 2 demonstrated greater stability in simulations using a biological solvent compared to those using DMSO. Compound 1, the most abundant peptide in latex, was assessed for NO production, antiplasmodial and cytotoxicity activities. The peptide significantly increased nitric oxide (NO) production at concentrations of 40, 20 or 10 µM (17.932 ± 1.1, 18.270 ± 0.9, 18.499 ± 0.7, respectively). Its antiplasmodial activity exhibited limited efficacy, with only 5% inhibition of Plasmodium falciparum 3D7 growth at a concentration of 50 µM. Also, it exhibited no cytotoxic effects in the J774A.1 murine macrophages cell line. This study represents the first report of a phytochemical investigation of the species C. campanulatus, which showed orbitides with distinctive sequences in contrast to other peptides described for the genus Croton and contributes to the study of structural diversity within this particular class of compounds.

Small Natural Cyclic Peptides from DBAASP Database.

Antimicrobial peptides (AMPs) are promising tools for combating microbial resistance. However, their therapeutic potential is hindered by two intrinsic drawbacks-low target affinity and poor in vivo stability. Macrocyclization, a process that improves the pharmacological properties and bioactivity of peptides, can address these limitations. As a result, macrocyclic peptides represent attractive drug candidates. Moreover, many drugs are macrocycles that originated from natural product scaffolds, suggesting that nature offers solutions to the challenges faced by AMPs. In this review, we explore natural cyclic peptides from the DBAASP database. DBAASP is a comprehensive repository of data on antimicrobial/cytotoxic activities and structures of peptides. We analyze the data on small (≤25 AA) ribosomal and non-ribosomal cyclic peptides from DBAASP according to their amino acid composition, bonds used for cyclization, targets they act on, and mechanisms of action. This analysis will enhance our understanding of the small cyclic peptides that nature has provided to defend living organisms.

Modular, Scalable Total Synthesis of Lapparbin with a Noncanonical Biaryl Linkage.

This work reports the development of a novel synthetic approach for the highly strained atrop-Tyr C-6-to-Trp N-1' linkage, which can be executed on a decagram scale using a modular strategy involving Pd-catalyzed C-H arylation followed by Larock macrocyclization. Moreover, the first total synthesis of lapparbin (1) was achieved by applying this synthetic strategy. Furthermore, the modular synthesis utilizing C-H arylation and Larock macrocyclization, discovered in the total synthesis of lapparbin (1), was demonstrated to be applicable to various arbitrary biaryl linkages, including non-natural types.

CYCLOPEp Builder: Facilitating cyclic peptide and nanotube research through a user-friendly web platform.

The study of cyclic peptides (CPs) and self-assembling cyclic peptide nanotubes (SCPNs) is pivotal in advancing applications in diverse fields such as biomedicine, nanoelectronics, and catalysis. Recognizing the limitations in the experimental study of these molecules, this article introduces CYCLOPEp Builder, a comprehensive web-based application designed to facilitate the design, simulation, and visualization of CPs and SCPNs. The tool is engineered to generate molecular topologies, essential for conducting Molecular Dynamics simulations that span All-Atom to Coarse-Grain resolutions. CYCLOPEp Builder's user-friendly interface simplifies the complex process of molecular modeling, providing researchers with the ability to readily construct CPs and SCPNs. The platform is versatile, equipped with various force fields, and capable of producing structures ranging from individual CPs to complex SCPNs with different sequences, offering parallel and antiparallel orientations among them. By enhancing the capacity for detailed visualization of molecular assemblies, CYCLOPEp Builder improves the understanding of CP and SCPN molecular interactions. This tool is a step forward in democratizing access to sophisticated simulations, offering an invaluable resource to the scientific community engaged in the exploration of supramolecular structures. CYCLOPEp is accessible at http://cyclopep.com/.

Macrocyclic peptides derived from AcPHF6* and AcPHF6 to selectively modulate the Tau aggregation.

Ten macrocyclic peptides, each comprising 14 amino acids, were designed and synthesized based on the Tau aggregation model hexapeptides AcPHF6* and AcPHF6. The design took into account the aggregation tendencies of each residue in AcPHF6* and AcPHF6, their aggregation models, while employing peptide-based structural design principles including N-methylation to promote turns and to block hydrogen bond propagation and elongation of the aggregation chain. NMR analysis supported that all these peptides adopted an antiparallel ß-sheet conformation. Self-aggregation studies characterized the aggregation properties of these peptides, identifying two peptides with the highest (P3) and lowest (P8) aggregation tendencies. In cross-aggregation studies with the parent peptides AcPHF6* and AcPHF6, P3 and P8 were found to promote and reduce aggregation, respectively. Furthermore, P3 and P8 demonstrated an enhancement and diminution effect on the aggregation of K18wt, indicating their capacity to modulate aggregation even at the macromolecular level. Thus, the two simple peptides, P3 and P8 selectively exhibit pro- or anti-aggregation effects on PHF peptides and Tau. This study, has thus developed structurally well-defined non-complex peptides, derived from AcPHF6* and AcPHF6, to modulate Tau aggregation as desired, offering applications in Tau model studies and the development of Tau aggregation inhibitors or promoters.

Destruxin E backbone modification effects on osteoclast Morphology: Synthesis and SAR study of N-Desmethyl and N-Methyl analogs.

The design and synthesis of N-desmethyl and N-methyl destruxin E analogs have been demonstrated. The X-ray single crystal structure of destruxin E (1a) revealed a stable three-dimensional (3D) structure, including a s-cis amide bond at the MeVal-MeAla moiety and two intramolecular hydrogen bonds between NH(ß-Ala) and OC(Ile) and between NH(Ile) and OC(ß-Ala). N-Desmethyl analogs 2a (MeAla â†’ Ala) and 2b (MeVal â†’ Val) were synthesized through macrolactonization similar to our previously reported synthesis of 1a. Conversely, for the synthesis of N-methyl analogs 2c (Ile â†’ MeIle) and 2d (ß-Ala â†’ Meß-Ala), macrolactonization did not proceed; therefore, cyclization precursors 10c and 10d were designed to maintain the intramolecular hydrogen bonds described above during their cyclization. The macrolactamization proceeded despite the presence of a less reactive N-methylamino group at the N-terminus in both cases. Analog 2a, which exhibits multiple conformers in solutions, was inactive at 50 µM, whereas analog 2b, which exhibits a conformation similar to that of 1a in solutions, exhibited morphological changes against osteoclast-like multinuclear cells at 1.6 µM. The activity of the MeIle analog 2c, which cannot take the intramolecular hydrogen bond (Ile)NH•••OC(ß-Ala) in 1a, was markedly diminished compared with that of 1a, and that of the Meß-Ala analog 2d, which cannot take the intramolecular hydrogen bond (ß-Ala)NH•••OC(Ile) in 1a, was further reduced to one-fourth of that of 2c. The overall results indicate that both the s-cis amide bond at the MeVal-MeAla moiety and two intramolecular hydrogen bonds (ß-Ala)NH•••OC(Ile) and (Ile)NH•••OC(ß-Ala) are important for constraining the conformation of the macrocyclic peptide backbone in destruxin E, thereby exhibiting its potent biological activity.

Expression and Subcellular Localization of Lanthipeptides in Human Cells.

Cyclic peptides, such as most ribosomally synthesized and post-translationally modified peptides (RiPPs), represent a burgeoning area of interest in therapeutic and biotechnological research because of their conformational constraints and reduced susceptibility to proteolytic degradation compared to their linear counterparts. Herein, an expression system is reported that enables the production of structurally diverse lanthipeptides and derivatives in mammalian cells. Successful targeting of lanthipeptides to the nucleus, the endoplasmic reticulum, and the plasma membrane is demonstrated. In vivo expression and targeting of such peptides in mammalian cells may allow for screening of lanthipeptide-based cyclic peptide inhibitors of native, organelle-specific protein-protein interactions in mammalian systems.

Comprehensive Study of Artificial Light-Harvesting Systems with a Multi-Step Sequential Energy Transfer Mechanism.

Artificial light-harvesting systems (LHSs) with a multi-step sequential energy transfer mechanism significantly enhance light energy utilization. Nonetheless, most of these systems exhibit an overall energy transfer efficiency below 80%. Moreover, due to challenges in molecularly aligning multiple donor/acceptor chromophores, systems featuring ≥3-step sequential energy transfer are rarely reported. Here, a series of artificial LHSs is introduced featuring up to 4-step energy transfer mechanism, constructed using a cyclic peptide-based supramolecular scaffold. These LHSs showed remarkably high energy transfer efficiencies (≥90%) and satisfactory fluorescence quantum yields (ranging from 17.6% to 58.4%). Furthermore, the structural robustness of the supramolecular scaffold enables a comprehensive study of these systems, elucidating the associated energy transfer pathways, and identifying additional energy transfer processes beyond the targeted sequential energy transfer. Overall, this comprehensive investigation not only enhances the understanding of these LHSs, but also underscores the versatility of cyclic peptide-based supramolecular scaffolds in advancing energy harvesting technologies.

Exploiting Hydrophobic Amino Acid Scanning to Develop Cyclic Peptide Inhibitors of the SARS-CoV-2 Main Protease with Antiviral Activity.

The development of novel antivirals is crucial not only for managing current COVID-19 infections but for addressing potential future zoonotic outbreaks. SARS-CoV-2 main protease (Mpro) is vital for viral replication and viability and therefore serves as an attractive target for antiviral intervention. Herein, we report the optimization of a cyclic peptide inhibitor that emerged from an mRNA display selection against the SARS-CoV-2 Mpro to enhance its cell permeability and in vitro antiviral activity. By identifying mutation-tolerant amino acid residues within the peptide sequence, we describe the development of a second-generation Mpro inhibitor bearing five cyclohexylalanine residues. This cyclic peptide analogue exhibited significantly improved cell permeability and antiviral activity compared to the parent peptide. This approach highlights the importance of optimizing cyclic peptide hits for activity against intracellular targets such as the SARS-CoV-2 Mpro.

HighFold: accurately predicting structures of cyclic peptides and complexes with head-to-tail and disulfide bridge constraints.

In recent years, cyclic peptides have emerged as a promising therapeutic modality due to their diverse biological activities. Understanding the structures of these cyclic peptides and their complexes is crucial for unlocking invaluable insights about protein target-cyclic peptide interaction, which can facilitate the development of novel-related drugs. However, conducting experimental observations is time-consuming and expensive. Computer-aided drug design methods are not practical enough in real-world applications. To tackles this challenge, we introduce HighFold, an AlphaFold-derived model in this study. By integrating specific details about the head-to-tail circle and disulfide bridge structures, the HighFold model can accurately predict the structures of cyclic peptides and their complexes. Our model demonstrates superior predictive performance compared to other existing approaches, representing a significant advancement in structure-activity research. The HighFold model is openly accessible at https://github.com/hongliangduan/HighFold.

Novel CCR3-targeted cyclic peptides as potential therapeutic agents for age-related macular degeneration via inhibiting angiogenesis and reducing retinal photoreceptor damage.

The prolonged intravitreal administration of anti-vascular endothelial growth factor (VEGF) drugs is prone to inducing aberrant retinal vascular development and causing damage to retinal neurons. Hence, we have taken an alternative approach by designing and synthesizing a series of cyclic peptides targeting CC motif chemokine receptor 3 (CCR3). Based on the binding mode of the N-terminal region in CCR3 protein to CCL11, we used computer-aided identification of key amino acid sequence, conformational restriction through different cyclization methods, designed and synthesized a series of target cyclic peptides, and screened the preferred compound IB-2 through affinity. IB-2 exhibits excellent anti-angiogenic activity in HRECs. The apoptosis level of 661W cells demonstrated a significant decrease with the escalating concentration of IB-2. This suggests that IB-2 may have a protective effect on photoreceptor cells. In vivo experiments have shown that IB-2 significantly reduces retinal vascular leakage and choroidal neovascularization (CNV) area in a laser-induced mouse model of CNV. These findings indicate the potential of IB-2 as a safe and effective therapeutic agent for AMD, warranting further development.

Surface Modification of PEEKs with Cyclic Peptides to Support Endothelialization and Antithrombogenicity.

Synthetic polymers are often utilized in the creation of vascular devices, and need to possess specific qualities to prevent thrombosis. Traditional strategies for this include surface modification of vascular devices through covalent attachment of substrates such as heparin, antiplatelet agents, thrombolytic agents, or hydrophilic polymers. One promising prosthetic material is polyether ether ketone (PEEK), which is utilized in various FDA-approved medical devices, including vascular and endovascular prostheses. We hypothesized that surface modification of biologically inert PEEK can help improve its endothelial cell affinity and reduce its thrombogenic potential. To evaluate this, we developed an effective surface-modification approach with unique cyclic peptides, such as CCHGGVRLYC and CCREDVC. We treated the PEEK surface with ammonia plasma, which introduced amine groups onto the PEEK surface. Subsequently, we were able to conjugate these peptides to the plasma-modified PEEKs. We observed that cyclic CCHGGVRLYC conjugated on prosthetic PEEK not only supported endothelialization, but minimized platelet adhesion and activation. This technology can be potentially applied for in vivo vascular and endovascular protheses to enhance their utility and patency.

SARS-CoV-2 Spike Protein-Derived Cyclic Peptides as Modulators of Spike Interaction with GRP78.

The human glucose-regulated protein GRP78 is a human chaperone that translocactes to the cell surface when cells are under stress. Theoretical studies suggested it could be involved in SARS-CoV-2 virus entry to cells. In this work, we used in vitro surface plasmon resonance-based assays to show that human GRP78 indeed binds to SARS-CoV-2 spike protein. We have designed and synthesised cyclic peptides based on the loop structure of amino acids 480-488 of the SARS-CoV-2 spike protein S1 domain from the Wuhan and Omicron variants and showed that both peptides bind to GRP78. Consistent with the greater infectiousness of the Omicron variant, the Omicron-derived peptide displays slower dissociation from the target protein. Both peptides significantly inhibit the binding of wild-type S1 protein to the human protein GRP78 suggesting that further development of these cyclic peptide motifs may provide a viable route to novel anti-SARS-CoV-2 agents.

Broad-spectrum activity of membranolytic cationic macrocyclic peptides against multi-drug resistant bacteria and fungi.

The emergence of multidrug-resistant (MDR) strains causes severe problems in the treatment of microbial infections owing to limited treatment options. Antimicrobial peptides (AMPs) are drawing considerable attention as promising antibiotic alternative candidates to combat MDR bacterial and fungal infections. Herein, we present a series of small amphiphilic membrane-active cyclic peptides composed, in part, of various nongenetically encoded hydrophilic and hydrophobic amino acids. Notably, lead cyclic peptides 3b and 4b showed broad-spectrum activity against drug-resistant Gram-positive (MIC = 1.5-6.2 µg/mL) and Gram-negative (MIC = 12.5-25 µg/mL) bacteria, and fungi (MIC = 3.1-12.5 µg/mL). Furthermore, lead peptides displayed substantial antibiofilm action comparable to standard antibiotics. Hemolysis (HC50 = 230 µg/mL) and cytotoxicity (>70 % cell viability against four different mammalian cells at 100 µg/mL) assay results demonstrated the selective lethal action of 3b against microbes over mammalian cells. A calcein dye leakage experiment substantiated the membranolytic effect of 3b and 4b, which was further confirmed by scanning electron microscopy. The behavior of 3b and 4b in aqueous solution and interaction with phospholipid bilayers were assessed by employing nuclear magnetic resonance (NMR) spectroscopy in conjunction with molecular dynamics (MD) simulations, providing a solid structural basis for understanding their membranolytic action. Moreover, 3b exhibited stability in human blood plasma (t1/2 = 13 h) and demonstrated no signs of resistance development against antibiotic-resistant S. aureus and E. coli. These findings underscore the potential of these newly designed amphiphilic cyclic peptides as promising anti-infective agents, especially against Gram-positive bacteria.

Identification of "Structural Pin" Interactions and their Significance for the Conformational Control of Macrocyclic Scaffolds.

While peptide macrocycles with rigidified conformations have proven to be useful in the design of chemical probes of protein targets, conformational flexibility and rapid interconversion can be equally vital for biological activity and favorable physicochemical properties. This study introduces the concept of "structural pin", which describes a hydrogen bond that is largely responsible for stabilizing the entire macrocycle backbone conformation. Structural analysis of macrocycles using nuclear magnetic resonance (NMR), molecular modelling and X-ray diffraction indicates that disruption of the structural pin can drastically influence the conformation of the entire ring, resulting in novel states with increased flexibility. This finding provides a new tool to interrogate dynamic behaviour of macrocycles. Identification of structural pins offers a useful conceptual framework to understand positions that can either be modified to give flexible structures or retained to maintain the rigidity of the scaffold.

Synthesis and Functionalization of Azetidine-Containing Small Macrocyclic Peptides.

Cyclic peptides are increasingly important structures in drugs but their development can be impeded by difficulties associated with their synthesis. Here, we introduce the 3-aminoazetidine (3-AAz) subunit as a new turn-inducing element for the efficient synthesis of small head-to-tail cyclic peptides. Greatly improved cyclizations of tetra-, penta- and hexapeptides (28 examples) under standard reaction conditions are achieved by introduction of this element within the linear peptide precursor. Post-cyclization deprotection of the amino acid side chains with strong acid is realized without degradation of the strained four-membered azetidine. A special feature of this chemistry is that further late-stage modification of the resultant macrocyclic peptides can be achieved via the 3-AAz unit. This is done by: (i) chemoselective deprotection and substitution at the azetidine nitrogen, or by (ii) a click-based approach employing a 2-propynyl carbamate on the azetidine nitrogen. In this way, a range of dye and biotin tagged macrocycles are readily produced. Structural insights gained by XRD analysis of a cyclic tetrapeptide indicate that the azetidine ring encourages access to the less stable, all-trans conformation. Moreover, introduction of a 3-AAz into a representative cyclohexapeptide improves stability towards proteases compared to the homodetic macrocycle.

C3-Symmetric ligands in drug design: An overview of the challenges and opportunities ahead.

C3-symmetry is a type of star-shaped molecule consisting of a central core and three symmetrically attached chains. These molecules are used in drug discovery due to their unique three-fold rotational symmetry, which allows for specific binding interactions and improved molecular recognition. In this text, we provide an overview of synthetic approaches with C3-symmetry as a pharmaceutical tool: progress, challenges, and opportunities. C3-symmetric ligands offer both challenges and opportunities in drug design. Their unique symmetry can enhance binding interactions, but careful consideration of rigidity, synthetic complexity, and target compatibility is crucial. Further research and advancements in synthetic methods and modeling tools will likely drive their exploration in drug discovery, leading to the discovery of potent C3-symmetric ligands.