Discovery and Hit to Lead Optimization of Macrocyclic Peptides as Novel Tropomyosin Receptor Kinase A Antagonists.

Tropomyosin receptor kinases (Trks) are receptor tyrosine kinases activated by neurotrophic factors, called neurotrophins. Among them, TrkA interacts with the nerve growth factor (NGF), which leads to pain induction. mRNA-display screening was carried out to discover a hit compound 2, which inhibits protein-protein interactions between TrkA and NGF. Subsequent structure optimization improving phosphorylation inhibitory activity and serum stability was pursued using a unique process that took advantage of the peptide being synthesized by translation from mRNA. This gave peptide 19, which showed an analgesic effect in a rat incisional pain model. The peptides described here can serve as a new class of analgesics, and the structure optimization methods reported provide a strategy for discovering new peptide drugs.

Structure-activity relationships of middle-size cyclic peptides, KRAS inhibitors derived from an mRNA display.

Cyclic peptides are attracting attention as therapeutic agents due to their potential for oral absorption and easy access to tough intracellular targets. LUNA18, a clinical KRAS inhibitor, was transformed-without scaffold hopping-from the initial hit by using an mRNA display library that met our criteria for drug-likeness. In drug discovery using mRNA display libraries, hit compounds always possess a site linked to an mRNA tag. Here, we describe our examination of the Structure-Activity Relationship (SAR) using X-ray structures for chemical optimization near the site linked to the mRNA tag, equivalent to the C-terminus. Structural modifications near the C-terminus demonstrated a relatively wide range of tolerance for side chains. Furthermore, we show that a single atom modification is enough to change the pharmacokinetic (PK) profile. Since there are four positions where side chain modification is permissible in terms of activity, it is possible to flexibly adjust the pharmacokinetic profile by structurally optimizing the side chain. The side chain transformation findings demonstrated here may be generally applicable to hits obtained from mRNA display libraries.

A Bicyclic Analog of the Linear Peptide Arodyn Is a Potent and Selective Kappa Opioid Receptor Antagonist.

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.

Accessing diverse bicyclic peptide conformations using 1,2,3-TBMB as a linker.

Bicyclic peptides are a powerful modality for engaging challenging drug targets such as protein-protein interactions. Here, we use 1,2,3-tris(bromomethyl)benzene (1,2,3-TBMB) to access bicyclic peptides with diverse conformations that differ from conventional bicyclisation products formed with 1,3,5-TBMB. Bicyclisation at cysteine residues under aqueous buffer conditions proceeds efficiently, with broad substrate scope, compatibility with high-throughput screening, and clean conversion (>90%) for 96 of the 115 peptides tested. We envisage that the 1,2,3-TBMB linker will be applicable to a variety of peptide screening techniques in drug discovery.

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.

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.

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.

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.

Supramolecular Motion Enables Chondrogenic Bioactivity of a Cyclic Peptide Mimetic of Transforming Growth Factor-ß1.

Transforming growth factor (TGF)-ß1 is a multifunctional protein that is essential in many cellular processes that include fibrosis, inflammation, chondrogenesis, and cartilage repair. In particular, cartilage repair is important to avoid physical disability since this tissue does not have the inherent capacity to regenerate beyond full development. We report here on supramolecular coassemblies of two peptide amphiphile molecules, one containing a TGF-ß1 mimetic peptide, and another which is one of two constitutional isomers lacking bioactivity. Using human articular chondrocytes, we investigated the bioactivity of the supramolecular copolymers of each isomer displaying either the previously reported linear form of the mimetic peptide or a novel cyclic analogue. Based on fluorescence depolarization and 1H NMR spin-lattice relaxation times, we found that coassemblies containing the cyclic compound and the most dynamic isomer exhibited the highest intracellular TGF-ß1 signaling and gene expression of cartilage extracellular matrix components. We conclude that control of supramolecular motion is emerging as an important factor in the binding of synthetic molecules to receptors that can be tuned through chemical structure.

Chemical cyclization of tyrosine-containing peptides via in situ generated triazolinedione peptides.

Tyr-derived cyclic peptide natural products are formed by enzymatic manifolds that oxidatively cross-link embedded phenolic side chains of tyrosine (Tyr) and 4-hydroxyphenylglycine residues during their controlled production. Bioactive Tyr-derived cyclic peptides, such as the arylomycins and vancomycins, continue to motivate the development of enzymatic and chemical strategies for their de novo assembly and modification. However, chemical access to these structurally diverse natural cycles can be challenging and step intensive. Therefore, we developed an oxidative procedure to selectively convert Tyr-containing N4-substituted 1,2,4-triazolidine-3,5-dione peptides (urazole peptides) into stable Tyr-linked cyclic peptides. We show that Tyr-containing urazole peptides are simple to prepare and convert into reactive N4-substituted 1,2,4-triazoline-3,5-dione peptides by oxidation, which then undergo spontaneous cyclization under mildly basic aqueous conditions to form a cross-linkage with the phenol side chain of embedded Tyr residues. Using this approach, we have demonstrated access to over 25 Tyr-linked cyclic peptides (3- to 11-residue cycles) with good tolerance of native residue side chain functionalities. Importantly, this method is simple to perform, and product formation can be quickly confirmed by mass spectrometric and 1H NMR spectroscopic analyses.

Production of constrained L-cyclo-tetrapeptides by epimerization-resistant direct aminolysis.

The synthesis of constrained 12-membered rings is notably difficult. The main challenges result from constraints during the linear peptide cyclization. Attempts to overcome constraints through excessive activation frequently cause peptidyl epimerization, while insufficient activation of the C-terminus hampers cyclization and promotes intermolecular oligomer formation. We present a ß-thiolactone framework that enables the synthesis of cyclo-tetrapeptides via direct aminolysis. This tactic utilizes a mechanism that restricts C-terminal carbonyl rotation while maintaining high reactivity, thereby enabling efficient head-to-tail amidation, reducing oligomerization, and preventing epimerization. A broad range of challenging cyclo-tetrapeptides ( > 20 examples) are synthesized in buffer and exhibits excellent tolerance toward nearly all proteinogenic amino acids. Previously unattainable macrocycles, such as cyclo-L-(Pro-Tyr-Pro-Val), have been produced and identified as µ-opioid receptor (MOR) agonists, with an EC50 value of 2.5 nM. Non-epimerizable direct aminolysis offers a practical solution for constrained peptide cyclization, and the discovery of MOR agonist activity highlights the importance of overcoming synthetic challenges for therapeutic development.

Tetrazine cyclized peptides for one-bead-one-compound library: Synthesis and sequencing.

While most FDA-approved peptide drugs are cyclic, robust cyclization chemistry of peptides and the deconvolution of the cyclic peptide sequences using tandem mass spectrometry render cyclic peptide drug discovery difficult. In this chapter, the protocol for the successful synthesis of tetrazine-linked cyclic peptide library in solid phase, which shows both robust cyclization and easy sequence deconvolution, is described. The protocol for the linearization and cleavage of cyclic peptides from the solid phase by simple UV light irradiation, followed by accurate sequencing using tandem mass spectrometry, is described. We describe the troubleshooting for this dithiol bis-arylation reaction and for the successful cleavage of the aryl cyclic peptide into linear form. This method for efficient solid-phase macrocyclization can be used for the rapid production of loop-based peptides and screening for inhibition of protein-protein interactions, by using the covalent inverse electron-demand Diels Alder reaction to supplement the non-covalent interaction between a protein and its peptide binder, isolating highly selective peptides in the process.

Electrochemical synthesis of peptide aldehydes via C‒N bond cleavage of cyclic amines.

Peptide aldehydes are crucial biomolecules essential to various biological systems, driving a continuous demand for efficient synthesis methods. Herein, we develop a metal-free, facile, and biocompatible strategy for direct electrochemical synthesis of unnatural peptide aldehydes. This electro-oxidative approach enabled a step- and atom-economical ring-opening via C‒N bond cleavage, allowing for homoproline-specific peptide diversification and expansion of substrate scope to include amides, esters, and cyclic amines of various sizes. The remarkable efficacy of the electro-synthetic protocol set the stage for the efficient modification and assembly of linear and macrocyclic peptides using a concise synthetic sequence with racemization-free conditions. Moreover, the combination of experiments and density functional theory (DFT) calculations indicates that different N-acyl groups play a decisive role in the reaction activity.

Polypeptide Preparation by ß-Lactone-Mediated Chemical Ligation.

Native chemical ligation (NCL) represents a cornerstone strategy in accessing synthetic peptides and proteins, remaining one of the most efficacious methodologies in this domain. The fundamental requisites for achieving a proficient NCL reaction involve chemoselective coupling between a C-terminal thioester peptide and a thiol-bearing N-terminal peptide. However, achieving coupling at sterically congested residues remains challenging. In addition, while most NCLs proceed without epimerization, ß-branched (e.g., Ile, Thr, Val) and Pro-derived C-terminal thioesters react slowly and can be susceptible to significant epimerization and hydrolysis. Herein, we report an epimerization-free NCL reaction via ß-lactone-mediated native chemical ligation which constructs sterically congested Thr residues. The constrained ring from the ß-lactone allows rapid peptide ligation without detectable epimerization. The method has a broad side-chain tolerance and was applied to the preparation of cyclic peptides and polypeptidyl thioester, which could be difficult to obtained otherwise.

PepExplainer: An explainable deep learning model for selection-based macrocyclic peptide bioactivity prediction and optimization.

Macrocyclic peptides possess unique features, making them highly promising as a drug modality. However, evaluating their bioactivity through wet lab experiments is generally resource-intensive and time-consuming. Despite advancements in artificial intelligence (AI) for bioactivity prediction, challenges remain due to limited data availability and the interpretability issues in deep learning models, often leading to less-than-ideal predictions. To address these challenges, we developed PepExplainer, an explainable graph neural network based on substructure mask explanation (SME). This model excels at deciphering amino acid substructures, translating macrocyclic peptides into detailed molecular graphs at the atomic level, and efficiently handling non-canonical amino acids and complex macrocyclic peptide structures. PepExplainer's effectiveness is enhanced by utilizing the correlation between peptide enrichment data from selection-based focused library and bioactivity data, and employing transfer learning to improve bioactivity predictions of macrocyclic peptides against IL-17C/IL-17 RE interaction. Additionally, PepExplainer underwent further validation for bioactivity prediction using an additional set of thirteen newly synthesized macrocyclic peptides. Moreover, it enabled the optimization of the IC50 of a macrocyclic peptide, reducing it from 15 nM to 5.6 nM based on the contribution score provided by PepExplainer. This achievement underscores PepExplainer's skill in deciphering complex molecular patterns, highlighting its potential to accelerate the discovery and optimization of macrocyclic peptides.

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.

Synthesis of the Antimicrobial Peptide Murepavadin Using Novel Coupling Agents.

The problem of antimicrobial resistance is becoming a daunting challenge for human society and healthcare systems around the world. Hence, there is a constant need to develop new antibiotics to fight resistant bacteria, among other important social and economic measures. In this regard, murepavadin is a cyclic antibacterial peptide in development. The synthesis of murepavadin was undertaken in order to optimize the preparative protocol and scale-up, in particular, the use of new activation reagents. In our hands, classical approaches using carbodiimide/hydroxybenzotriazole rendered low yields. The use of novel carbodiimide and reagents based on OxymaPure® and Oxy-B is discussed together with the proper use of chromatographic conditions for the adequate characterization of peptide crudes. Higher yields and purities were obtained. Finally, the antimicrobial activity of different synthetic batches was tested in three Pseudomonas aeruginosa strains, including highly resistant ones. All murepavadin batches yielded the same highly active MIC values and proved that the chiral integrity of the molecule was preserved throughout the whole synthetic procedure.

Computational Design of Novel Cyclic Peptides Endowed with Autophagy-Inhibiting Activity on Cancer Cell Lines.

(1) Autophagy plays a significant role in development and cell proliferation. This process is mainly accomplished by the LC3 protein, which, after maturation, builds the nascent autophagosomes. The inhibition of LC3 maturation results in the interference of autophagy activation. (2) In this study, starting from the structure of a known LC3B binder (LIR2-RavZ peptide), we identified new LC3B ligands by applying an in silico drug design strategy. The most promising peptides were synthesized, biophysically assayed, and biologically evaluated to ascertain their potential antiproliferative activity on five humans cell lines. (3) A cyclic peptide (named Pep6), endowed with high conformational stability (due to the presence of a disulfide bridge), displayed a Kd value on LC3B in the nanomolar range. Assays accomplished on PC3, MCF-7, and A549 cancer cell lines proved that Pep6 exhibited cytotoxic effects comparable to those of the peptide LIR2-RavZ, a reference LC3B ligand. Furthermore, it was ineffective on both normal prostatic epithelium PNT2 and autophagy-defective prostate cancer DU145 cells. (4) Pep6 can be considered a new autophagy inhibitor that can be employed as a pharmacological tool or even as a template for the rational design of new small molecules endowed with autophagy inhibitory activity.

Secondary Structure Stabilization of Macrocyclic Antimicrobial Peptides via Cross-Link Swapping.

Lactam cross-links have been employed to stabilize the helical secondary structure and enhance the activity and physiological stability of antimicrobial peptides; however, stabilization of ß-sheets via lactamization has not been observed. In the present study, lactams between the side chains of C- and N-terminal residues have been used to stabilize the ß-sheet conformation in a short ten-residue analogue of chicken angiogenin-4. Designed using a combination of molecular dynamics simulations and Markov state models, the lactam cross-linked peptides are shown to adopt stabilized ß-sheet conformations consistent with simulated structures. Replacement of the peptide side-chain Cys-Cys disulfide by a lactam cross-link enhanced the broad-spectrum antibacterial activity compared to the parent peptide and exhibited greater propensity to induce proinflammatory activity in macrophages. The combination of molecular simulations and conformational and biological analyses of the synthetic peptides provides a useful paradigm for the rational design of therapeutically active peptides with constrained ß-sheet structures.

Computational Design of Cyclic Peptide Inhibitors of a Bacterial Membrane Lipoprotein Peptidase.

There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.