Cyclic Peptide Research Articles

Discovery of Novel Peptide Antagonists Targeting GPR55 for Liver Inflammation and Fibrosis.

Liver fibrosis is a condition characterized by aberrant proliferation of connective tissue in the liver resulting from diverse etiological factors. G protein-coupled receptor GPR55 has recently been identified as a regulator of liver diseases. Herein, we report the discovery of a cyclic peptide P1-1 that antagonizes GPR55 and suppresses collagen secretion in hepatic stellate cells. The alanine scanning and docking study was carried out to predict the binding mode and allowed for further structural optimization of peptide antagonists for GPR55. The subsequent in vivo study demonstrated that P1-1 ameliorates CCl4-induce and MCD-diet-induce acute liver inflammation and fibrosis. Further study indicates that P1-1 reduces reactive oxygen species (ROS) production, attenuates ER stress, and inhibits mitochondria-associated hepatocyte apoptosis. In this work, we provided the first successful example of antagonizing GPR55 for liver inflammation and fibrosis, which validates GPR55 as a promising target for the treatment of liver fibrosis and affords a high-potent GPR55 antagonist P1-1 as a potential therapeutic candidate.

Exploring the Potential of Cyclic Peptidyl Antitumor Agents Derived from Natural Macrocyclic Peptide Phakellistatin 13.

The exploration of novel anticancer compounds based on natural cyclopeptides has emerged as a pivotal paradigm in the contemporary advancement of macrocyclic pharmaceuticals. Phakellistatin 13 is a cycloheptapeptide derived from the brown snubby sponge and exhibits remarkable antitumor activity. In this study, we have designed and synthesized a series of chiral cyclopeptides incorporating the rigid isoindolinone moiety at various sites within the natural cycloheptapeptide Phakellistatin 13, with the aim of investigating conformationally constrained cyclopeptides as potential antitumor agents. Cyclopeptide 3, comprising alternating l-/d-amino acid residues, exhibited promising antihepatocellular carcinoma effects. Detailed biological experiments have revealed that Phakellistatin 13 analogs effectively inhibit the proliferation of tumor cells and induce apoptosis and autophagy, while also causing cell cycle arrest through the modulation of the p53 and mitogen-activated protein kinase (MAPK) signaling pathway. This study not only provides valuable insights into chemical structural modifications but also contributes to a deeper understanding of the biological mechanisms underlying the development of natural cyclopeptide-based drugs.

Trifluoroacetic Acid Mediated Additive-Free Late-Stage Native Peptide Cyclization to Form Disulfide Mimetics via Thioketalization with Ketones.

Peptide cyclization is often used to introduce conformational rigidity and to enhance the physiological stability of the peptide. This study presents a novel late-stage cyclization method for creating thioketal cyclic peptides from bis-cysteine peptides and drugs. Symmetrical cyclic ketones and acetone were found to react with bis-cysteine unprotected peptides efficiently to form thioketal linkages in trifluoroacetic acid (TFA) without any other additive. The attractive features of this method include high chemoselectivity, operational simplicity, and robustness. In addition, TFA as the reaction solvent can dissolve any unprotected peptide. As a showcase, the dimethyl thioketal versions of lanreotide and octreotide were prepared and evaluated, both of which showed much improved reductive stability and comparable activity.

Palmitoylation enhances short polar peptide permeation across stratum corneum lipid bilayer: A molecular dynamics study

Designing chemical molecules that target the skin for non-invasive transdermal drug delivery is of significant interest for both wound healing and skincare applications. These skin-targeting molecules must permeate the outermost protective layer of the skin, the stratum corneum (SC), which consists of dead corneocytes embedded in a lipid matrix, to fulfill their biological functions. Adsorption onto and diffusion through the lipid matrix in the SC represent two key steps for the successful permeation of a skin-targeting molecule across the SC into the underlying skin layers. Here we compare the effects of cyclization and palmitoylation on the adsorption and diffusion of a short polar peptide across a model SC lipid bilayer using molecular dynamics simulations. The cyclized peptide showed slightly better binding to the SC lipid bilayer and similar interaction energies with SC lipids compared to the unmodified peptide. In contrast, the palmitoylated peptide exhibited much stronger interaction with SC lipids via insertion of its attached fatty acid tail into the SC lipid bilayer. The average diffusivity of the cyclized peptide across the SC lipid bilayer was approximately twice that of the unmodified peptide, whereas the palmitoylated peptide’s diffusivity was about 2.7 times higher. Thus, palmitoylation appears to be a promising strategy for enhancing the binding and permeability of short polar peptides across the SC lipid matrix.

Peculiarities of laboratory parameters dynamics during helicobacter pylori eradication in patients with rheumatoid arthritis

Helicobacter pylori (H. pylori) is able to participate in the pathogenesis of a number of autoimmune diseases, actively maintains chronic inflammation and stimulates the systemic immune response. The virulence factor of H. pylori is cytotoxin-associated gene A (CagA) is associated with more severe inflammatory reactions, increased risk of poor clinical outcomes and is able to influence the efficacy of infection eradication in patients with rheumatoid arthritis (RA).Purpose of the study. To evaluate laboratory parameters of H. pylori eradication efficacy in RA patients with chronic infection with the strain encoding cytotoxin-associated gene A.Materials and methods. Forty women with RA and confirmed chronic H. pylori infection were included in the study (mean age 55.5±8.7 years; mean disease duration 13.9±9.1 years; DAS-28–3.96±0.56). CagA-IgG associated H. pylori infection was diagnosed in 22 (group I, CagA+) and not diagnosed in 18 (group II, CagA-) patients. All RA patients underwent a course of H. pylori eradication therapy.Results. The process of H. pylori eradication had the most significant effect on laboratory parameters of CagA-negative RA patients (group II). In this group the levels of rheumatoid factor (p=0,028), C-reactive protein (CRP, p=0.001), interleukin-6 (IL-6, p=0.002), tumor necrosis factor alpha (p=0,023), angiopoietinlike protein type 3 (p=0.026) and antibodies to cyclic citrullinated peptide (ACCP, p=0,016) decreased significantly. In patients from group I (CagA+) most parameters remained practically unchanged (p>0.05), except for CRP (p=0.01) and IL-6 (p=0.011). In the short term, the success of eradication in CagA+ patients was significantly lower than in CagA- patients (p=0.033). Moreover, confirmation of successful eradication of H. pylori within the established period of time was extremely rare (p=0.009) in the combination of CagA+ and high titers of ACCP and antibodies to modified citrullinated vimentin.Conclusions. The effectiveness of H. pylori eradication in RA patients depends on the presence of chronic infection with the strain encoding the cytotoxin-associated gene A and the level of antibodies to citrullinated proteins, which should be taken into account when choosing the therapeutic effect on H. pylori in this group.

“Top-down” overexpression optimization of butelase-1 in Escherichia coli and its application in anti-tumor peptides

Butelase-1, the fastest known Asn/Asp-specific peptide ligase capable of catalyzing peptide ligation and cyclization, holds promising application prospects in the fields of food and biology. However, limited research exists on its recombinant expression and potential applications in peptide drugs. In this study, the activity of recombinantly-produced butelase-1 was enhanced by co-expressing it with a molecular chaperone in the SHuffle T7 strain. By introducing single or multiple synonymous rare codons at the beginning of the coding regions of beta-strand or alpha-helix, in combination with ribosomal binding site engineering, the activity of butelase-1 could be further improved. Consequently, the butelase-1 with a specific activity of 386.93 U/mg and a catalytic efficiency of 11,048 M−1 s−1 was successfully prepared in E. coli, resulting in a total activity of 8183.54 U/L and a yield of about 100 mg/L. This optimized butelase-1 was then used to efficiently cyclize the redesigned anti-cancer peptide lunasin, leading to enhanced bioavailability and anti-cancer effects. Overall, this study not only provided valuable biotechnology strategies for improving the recombinant expression of butelase-1 but also demonstrated a successful application for enhancing the biological efficacy of anti-cancer peptides.

In silico Molecular Docking of Cyclic Peptides against TEM-1 Beta-Lactamases for Effective Antimicrobial Drug Development

Targeting the class A Beta lactamases Omega loop is an ideal way to combat drug resistance because of its significant role in the catalytic activity and deacylation process inhibition. Therefore, the molecular docking approach with computerized peptide-based in silico screening has been applied for the identification of inhibitors of TEM-type βLs. Among the subjected 105 peptides, Chrombacin (-47.8 KJ/mol), Gassericin A (-35.7 KJ/mol), Duramycin (-34.1 KJ/mol), Brevinin-1DYa (-34.0 KJ/mol), Amoebapore A (-31.2 KJ/mol), Mundticin ATO6 (-29.0 KJ/mol), Lactocyclicin Q (-26.3 KJ/mol), Cinnamycin (-25.9 KJ/mol showed highest binding energy. Among the peptides that showed the highest docking score Elafin, Cinnamycin, Duramycin interacted with Lys 73 of the α domain of catalytic residues of TEM-1 Beta lactamases, whereas Taromycin A, Gassericin A interacted with Lys 234 of the β domain, depicting a strong inhibition and also exhibited desirable physicochemical properties. Hence further in vitro examination of these cyclic peptides against the resistant strains is warranted to help design further novel inhibitors based on their scaffolds and also for the development of an effective drug combination regime.

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

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

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.

Predicting Peptide Permeability Across Diverse Barriers: A Systematic Investigation.

Peptide-based therapeutics hold immense promise for the treatment of various diseases. However, their effectiveness is often hampered by poor cell membrane permeability, hindering targeted intracellular delivery and oral drug development. This study addressed this challenge by introducing a novel graph neural network (GNN) framework and advanced machine learning algorithms to build predictive models for peptide permeability. Our models offer systematic evaluation across diverse peptides (natural, modified, linear and cyclic) and cell lines [Caco-2, Ralph Russ canine kidney (RRCK) and parallel artificial membrane permeability assay (PAMPA)]. The predictive models for linear and cyclic peptides in Caco-2 and RRCK cell lines were constructed for the first time, with an impressive coefficient of determination (R2) of 0.708, 0.484, 0.553, and 0.528 in the test set, respectively. Notably, the GNN framework behaved better in permeability prediction with larger data sets and improved the accuracy of cyclic peptide prediction in the PAMPA cell line. The R2 increased by about 0.32 compared with the reported models. Furthermore, the important molecular structural features that contribute to good permeability were interpreted; the influence of cell lines, peptide modification, and cyclization on permeability were successfully revealed. To facilitate broader use, we deployed these models on the user-friendly KNIME platform (https://github.com/ifyoungnet/PharmPapp). This work provides a rapid and reliable strategy for systematically assessing peptide permeability, aiding researchers in drug delivery optimization, peptide preselection during drug discovery, and potentially the design of targeted peptide-based materials.

Ribosomal Incorporation of Lithocholic Acid into Peptides for the De Novo Discovery Of Peptide-Lithocholic Acid Hybrid Macrocyclic Peptides.

Peptide-bile acid hybrids offer promising drug candidates due to enhanced pharmacological properties, such as improved protease resistance and oral bioavailability. However, it remains unknown whether bile acids can be incorporated into peptide chains by the ribosome to produce a peptide-bile acid hybrid macrocyclic peptide library for target-based de novo screening. In this study, we achieved the ribosomal incorporation of lithocholic acid (LCA)-d-tyrosine into peptide chains. This led to the construction of a peptide-LCA hybrid macrocyclic peptide library, which enabled the identification of peptides TP-2C-4L3 (targeting Trop2) and EP-2C-4L5 (targeting EphA2) with strong binding affinities. Notably, LCA was found to directly participate in binding to EphA2 and confer on the peptides improved stability and resistance to proteases. Cell staining experiments confirmed the high specificity of the peptides for targeting Trop2 and EphA2. This study highlights the benefits of LCA in peptides and paves the way for de novo discovery of stable peptide-LCA hybrid drugs.

Human estrogen receptor alpha (ERα) targeted cyclic peptides inhibit cell growth and induce apoptosis in MCF-7 cells

Abstract Objectives Human estrogen receptor alpha (ERα) is considered an important target, especially in the treatment of breast cancer, as it has a vital role in cancer development. ERα-targeted therapies generally target the ligand binding domain (LBD) of ERα. However, over time, cells develop resistance to this mechanism alternative approaches to inhibit ERα activity target ERα–DNA or ERα–cofactor interactions. Inhibitors of ERα–cofactor interactions are designed by targeting the hydrophobic hollow region of the receptor box LXXLL motif. Methods In this context, helix-stabilized cyclic peptides (SPs) designed with in silico approaches were obtained by solid phase peptide synthesis. The effects of SPs on MCF-7 cells were examined with MTT and ATP, and qPCR and flow cytometry were used for further analysis. Results Our results demonstrated that the SPs were effective only in MCF-7 cells expressing ERα. In addition, cyclic peptide combinations (SPCs) showed anti-proliferative and toxic effects on MCF-7 cells. The impact of SPCs with the highest inhibitory effect in MCF-7 cells on ERα-related genes and markers of apoptosis was revealed. Moreover, the flow cytometry analysis result used to examine apoptotic cells proved the apoptosis of SPCs in MCF-7 cells. Conclusions These findings suggest that our novel SPs, which inhibit coactivator interactions of ERα, induce apoptosis of MCF-7 cells. Thus, considering this strong effect of SPs in the inhibition of receptors, it is pointed out that they can be further developed as an alternative to current clinical treatments or as an auxiliary approach in the generating of new targeted peptide-based therapies.

Cyclic tachyplesin I kills proliferative, non-proliferative and drug-resistant melanoma cells without inducing resistance

Acquired drug resistance is the major cause for disease recurrence in cancer patients, and this is particularly true for patients with metastatic melanoma that carry a BRAF V600E mutation. To address this problem, we investigated cyclic membrane-active peptides as an alternative therapeutic modality to kill drug-tolerant and resistant melanoma cells to avoid acquired drug resistance. We selected two stable cyclic peptides (cTI and cGm), previously shown to have anti-melanoma properties, and compared them with dabrafenib, a drug used to treat cancer patients with the BRAF V600E mutation. The peptides act via a fast membrane-permeabilizing mechanism and kill metastatic melanoma cells that are sensitive, tolerant, or resistant to dabrafenib. Melanoma cells do not become resistant to long-term treatment with cTI, nor do they evolve their lipid membrane composition, as measured by lipidomic and proteomic studies. In vivo studies in mice demonstrated that the combination treatment of cTI and dabrafenib resulted in fewer metastases and improved overall survival. Such cyclic membrane-active peptides are thus well suited as templates to design new anticancer therapeutic strategies.

Crafting Unnatural Peptide Macrocycles via Rh(III)-Catalyzed Carboamidation.

Contemporary developments in the field of peptide macrocyclization methodology are imperative for enabling the advance of drug design in medicinal chemistry. This report discloses a Rh(III)-catalyzed macrocyclization via carboamidation, reacting acryloyl-peptide-dioxazolone precursors and arylboronic acids to form complex cyclic peptides with concomitant incorporation of noncanonical α-amino acids. The diverse and modular technology allows for expedient access to a wide variety of cyclic peptides from 4 to 15 amino acids in size and features simultaneous formation of unnatural phenylalanine and tyrosine derivatives with up to >20:1 diastereoselectivity. The reaction showcases an expansive substrate scope with 45 examples and is compatible with the majority of standard protected amino acids used in Fmoc-solid phase peptide synthesis. The methodology is applied to the synthesis of multiple peptidomimetic macrocyclic analogs, including derivatives of cyclosomatostatin and gramicidin S.

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.

MDFit: automated molecular simulations workflow enables high throughput assessment of ligands-protein dynamics.

Molecular dynamics (MD) simulation is a powerful tool for characterizing ligand-protein conformational dynamics and offers significant advantages over docking and other rigid structure-based computational methods. However, setting up, running, and analyzing MD simulations continues to be a multi-step process making it cumbersome to assess a library of ligands in a protein binding pocket using MD. We present an automated workflow that streamlines setting up, running, and analyzing Desmond MD simulations for protein-ligand complexes using machine learning (ML) models. The workflow takes a library of pre-docked ligands and a prepared protein structure as input, sets up and runs MD with each protein-ligand complex, and generates simulation fingerprints for each ligand. Simulation fingerprints (SimFP) capture protein-ligand compatibility, including stability of different ligand-pocket interactions and other useful metrics that enable easy rank-ordering of the ligand library for pocket optimization. SimFPs from a ligand library are used to build & deploy ML models that predict binding assay outcomes and automatically infer important interactions. Unlike relative free-energy methods that are constrained to assess ligands with high chemical similarity, ML models based on SimFPs can accommodate diverse ligand sets. We present two case studies on how SimFP helps delineate structure-activity relationship (SAR) trends and explain potency differences across matched-molecular pairs of (1) cyclic peptides targeting PD-L1 and (2) small molecule inhibitors targeting CDK9.

Leveraging High-Resolution Ion Mobility-Mass Spectrometry for Cyclic Peptide Soft Spot Identification.

Cyclic peptides are an important class of molecules that gained significant attention in the field of drug discovery due to their unique pharmacological characteristics and enhanced proteolytic stability. Yet, gastrointestinal degradation remains a major hurdle in the discovery of orally bioavailable cyclic peptides. Soft spot identification (SSID) of the regions in the cyclic peptide sequence susceptible to amide hydrolysis by proteases is used in the discovery stage to guide medicinal chemistry design. SSID can be an arduous task, traditionally performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), often resulting in complex and time-consuming manual analysis, particularly when isomeric linear peptide metabolites chromatographically coelute. Here, we present an alternative orthogonal approach that entails a high-resolution ion mobility (HRIM) system based on Structures for Lossless Ion Manipulation (SLIM) technology interfaced with quadrupole time-of-flight (QTOF) mass spectrometry to address some of the challenges associated with SSID. Two strategies were used to resolve linear isomeric peptide metabolites: labeled and label-free, both utilizing the HRIM platform. The label-free strategy leverages negative polarity to ionize the isomers which achieves better separation of the gas phase ions in the ion mobility (IM) dimension as compared to positive polarity, which is a more conventional approach when studying proteins and peptides. The second approach uses an isotope-labeled dimethyl tag on the terminal amine group, acting as a "shift reagent" to influence the mobility of isomers in the positive mode. This method resulted in baseline separation for the isomers of interest and produced unique product ions in the fragmentation spectra for unambiguous soft spot identification. Both label-free and labeled strategies demonstrated the ability to solve the challenges associated with SSID for cyclic peptides.

Hypervalent Iodine Amino Acid Building Blocks for Bioorthogonal Peptide Macrocyclization

AbstractEthynylbenziodoxol(on)es (EB(X)xs) reagents have emerged as useful reagents for peptide/protein modification due to their versatile reactivity and high selectivity. Herein, we report the successful introduction of ethynylbenziodoxoles (EBxs) on different amino acid building blocks (Lys/Orn/Dap), and show their compatibility with both solid phase peptide synthesis (SPPS) and solution phase peptide synthesis (SPS). The selective incorporation of the EBx core into peptide sequences enable efficient macrocyclizations under mild conditions for the synthesis of topologically unique cyclic and bicyclic peptides.

Hypervalent Iodine Amino Acid Building Blocks for Bioorthogonal Peptide Macrocyclization.

Ethynylbenziodoxol(on)es (EB(X)xs) reagents have emerged as useful reagents for peptide/protein modification due to their versatile reactivity and high selectivity. Herein, we report the successful introduction of ethynylbenziodoxoles (EBxs) on different amino acid building blocks (Lys/Orn/Dap), and show their compatibility with both solid phase peptide synthesis (SPPS) and solution phase peptide synthesis (SPS). The selective incorporation of the EBx core into peptide sequences enable efficient macrocyclizations under mild conditions for the synthesis of topologically unique cyclic and bicyclic peptides.

A classic antibiotic reimagined: Rationally designed bacitracin variants exhibit potent activity against vancomycin-resistant pathogens

Bacitracin is a macrocyclic peptide antibiotic that is widely used as a topical treatment for infections caused by gram-positive bacteria. Mechanistically, bacitracin targets bacteria by specifically binding to the phospholipid undecaprenyl pyrophosphate (C55PP), which plays a key role in the bacterial lipid II cycle. Recent crystallographic studies have shown that when bound to C55PP, bacitracin adopts a highly ordered amphipathic conformation. In doing so, all hydrophobic side chains align on one face of the bacitracin-C55PP complex, presumably interacting with the bacterial cell membrane. These insights led us to undertake structure-activity investigations into the individual contribution of the nonpolar amino acids found in bacitracin. To achieve this we designed, synthesized, and evaluated a series of bacitracin analogues, a number of which were found to exhibit significantly enhanced antibacterial activity against clinically relevant, drug-resistant pathogens. As for the natural product, these next-generation bacitracins were found to form stable complexes with C55PP. The structure-activity insights thus obtained serve to inform the design of C55PP-targeting antibiotics, a key and underexploited antibacterial strategy.