The Ras superfamily of small GTPases are challenging targets for therapeutic inhibition, partially due to a lack of pockets amenable to small molecule inhibition. Our previous work identified high-affinity cyclized peptide binders of Cdc42, a member of the Rho family of small GTPases, capable of inhibiting activity. To further optimize these Cdc42 inhibitors, we have engineered modifications to the best sequence available from the original maturation and screened the ability of these third-generation peptides to compete with Cdc42-effector interactions. Improvements in affinity were achieved by single amino acid substitutions at several residue positions. We present the structure of one of these nanomolar affinity, cyclized peptides in complex with Cdc42. The structure reveals that the peptide binds in a β-hairpin conformation to create an extension of the β-sheet of the GTPase Rossman fold, acting as a structural mimic of native Cdc42 effectors. We additionally elucidate the NMR structures of four unbound C-terminal alanine variants and employ both the bound and unbound structures to inform the rational design of substituted peptide inhibitors. Overall, this study expands our understanding of how Ras GTPases can be targeted, by demonstrating a rare example of an inhibitor binding contiguously with a surface of β-strand of the small G protein, which illustrates an innovative avenue for noncovalent therapeutic design.

Nα-isophthalic acid 1 or Nα-isophthaloyl dichloride 2 has been coupled with the proper sarcosine methyl esters to create several linear and macrocyclic pentapeptide derivatives. The analogous Nα-isophthaloyl-bis-[sarcosine methyl ester], 3, was obtained by coupling 1 or 2 with sarcosine methyl ester. The equivalent acid 4, Nα-isophthaloyl-bis-[sarcosine], was subsequently obtained by hydrolyzing this with methanolic sodium hydroxide. The corresponding tetrapeptide ester 5, Nα-isophthaloyl-bis [sarcosyl-sarcosine methyl ester], was created by coupling the latter product 4 with another molecule of sarcosine methyl ester. This compound was then hydrolyzed with methanolic sodium hydroxide to obtain the corresponding acid 6, Nα-isophthaloyl-bis [sarcosyl - sarcosine]. The equivalent cyclic pentapeptide methyl ester 7, Nα-isophthaloyl)-bis-[sarcosyl - sarcosine]-L-Lys-OMe, was produced by cyclizing tetrapeptide acids with L-lysine methyl ester. Lastly, the corresponding acids 8 and 9, cyclo-(Nα-isophthaloyl)-bis-[sarcosyl - sarcosine]-L-Ly, and Nα-isophthaloyl)-bis [sarcosyl - sarcosine]-L-Lys-NHNH2, respectively, were obtained by hydrolyzing methyl ester 7 with 1N methanolic sodium hydroxide or hydrazinolyzing it with hydrazine hydrate. KEY WORDS: Nα-isophthaloyl dichloride, Amino acids, Linear peptides, Cyclic pentapeptides Bull. Chem. Soc. Ethiop. 2026, 40(2), 341-351 DOI: https://dx.doi.org/10.4314/bcse.v40i2.6

Side chain-to-side chain peptide macrocyclization or stapling is a chemical modification that is frequently used to increase the metabolic stability, the cell permeability, and/or the binding affinity of peptide drugs. Interestingly, it was recently reported that α-helical stapling can also protect the amyloidogenic peptide hormone islet amyloid polypeptide (IAPP) from aggregation and amyloid-associated toxicity. IAPP is the major component of insoluble amyloid deposits found in diabetic patients, and its derivatives constitute potential therapeutic candidates to treat metabolic disorders. Herein, we investigated the effects of macrocyclization chemistry on amyloid formation and cytotoxicity by comparing different stapling strategies: lactamization, azide-alkyne click chemistry, and formation of thioether link. The (i, i + 4) intramolecular macrocyclization of IAPP between positions 13 and 17 imposed, or not for some derivatives, a local stability of the helical secondary structure, modulating the propensity of the peptide to self-assemble into amyloid fibrils. The helically constrained derivatives inhibited the aggregation of unmodified IAPP and showed a reduced capacity to perturb the cell plasma membrane and to induce cell death. This study offers key molecular insights into the use of stapling strategies as a chemical approach to prevent the aggregation of peptide therapeutics and to inhibit the cytotoxicity of amyloidogenic peptides associated with protein misfolding disorders.

Structural insights into frog skin-derived cyclic peptides as selective matriptase inhibitors.

Cyclic peptides are increasingly recognised as a therapeutic modality for modulating intracellular protein-protein interactions (PPIs), including those considered ‘undruggable’ by small molecules or biologics. Cyclic gomesin (cGm), an 18-residue β-hairpin peptide containing two disulfide bonds and a cyclised backbone, combines high chemical stability with amphipathic character that promotes selective interaction with negatively charged cancer cell membranes. We previously showed that cGm enters cancer cells at non-toxic concentrations via endocytosis and direct membrane partitioning, outperforming established cell-penetrating peptides, and that it can be engineered to inhibit LDH5 tetramerisation. Here, we further assess its grafting capacity by incorporating bioactive loop sequences of varying size, charge, and hydrophobicity into the cGm framework. Structural and biophysical analyses confirmed that grafted analogues retained the three-dimensional fold, membrane-binding features, anticancer activity, melanoma selectivity, and low toxicity toward non-cancerous cells. These findings demonstrate the tolerance of cGm to sequence variation and support its development as a modular scaffold for designing intracellularly active cyclic peptide therapeutics.

Peptide-based biomaterials have emerged as versatile tools for pharmaceutical drug delivery due to their biocompatibility and tunable sequences, yet a comprehensive overview of their categories, mechanisms, and optimization strategies remains lacking to guide clinical translation. This review systematically collates advances in peptide-based biomaterials, covering peptide excipients (cell penetrating peptides, tight junction modulating peptides, and peptide surfactants/stabilizers), self-assembling peptides (peptide-based nanospheres, cyclic peptide nanotubes, nanovesicles and micelles, peptide-based hydrogels and depots), and peptide linkers (for antibody drug-conjugates, peptide drug-conjugates, and prodrugs). We also dissect sequence-based optimization strategies, including rational design and biophysical optimization (cyclization, stapling, D-amino acid incorporation), functional motif integration, and combinatorial discovery with AI assistance, with examples spanning marketed drugs and research-stage candidates. The review reveals that cell-penetrating peptides enable efficient intracellular payload delivery via direct penetration or endocytosis; self-assembling peptides form diverse nanostructures for controlled release; and peptide linkers achieve site-specific drug release by responding to tumor-associated enzymes or pH cues, while sequence optimization enhances stability and targeting. Peptide-based biomaterials offer precise, biocompatible and tunable solutions for drug delivery, future advancements relying on AI-driven design and multi-functional modification will accelerate their transition from basic research to clinical application.

Cyclotides are plant-derived macrocyclic peptides stabilized by a cystine-knot motif, found in a limited number of angiosperm plants. This study reports the discovery of the cyclotide, Spat1, from Spigelia anthelmia (Loganiaceae), expanding the phylogenetic range of known cyclotide-producing plants. Spat1, a 30-residue bracelet-type cyclotide, was isolated, purified, and sequenced de novo. It demonstrated strong bactericidal activity against the Gram-positive Bacillus subtilis (LC99.9 = 20 μM) via rapid membrane disruption but showed no activity against Staphylococcus aureus or Gram-negative Escherichia coli (LC99.9 > 400 μM). The selective lack of activity against S. aureus is unusual for antimicrobial peptides. The data suggest that Spat1's activity is independent of lipoteichoic acid (LTA) in B. subtilis, suggesting that its mechanism involves interactions with cytoplasmic membrane phospholipids. The lack of phosphatidylethanolamine (PE) in S. aureus membranes and Spat1's weak binding to LTA, combined with its low net positive charge (+1), likely explains its inefficacy against this bacterial species. Structural modeling using AlphaFold AfCycDesign indicated that Spat1 adopts a cyclotide-typical β-sheet architecture and a 310-helix within its loop regions. Overall, Spat1 broadens understanding of cyclotide diversity and evolution, highlighting their functional specialization and the convergent evolutionary pressures that shape their distribution across plant lineages.

Peptide-based therapeutics are increasingly emerging as a promising alternative to small molecules and protein drugs. However, their clinical development still faces significant challenges, particularly in terms of efficacy and safety, which are largely attributed to their suboptimal absorption, distribution, metabolism, and excretion properties and potential toxicity risks (ADMET). To address these challenges, we developed pepADMET (https://pepadmet.ddai.tech), the first publicly accessible AI-driven platform for the systematic and comprehensive assessment of peptide ADMET properties. The platform integrated 36,643 high-quality data entries and covers 19 key ADMET endpoints. By combining molecular graph representations, enzymatic descriptors, and transfer learning with advanced neural architectures such as graph neural networks and relational graph convolutional networks, the platform effectively captures complex molecular and biological features of peptides, thereby substantially enhancing the predictive performance and robustness of the models. Specifically, we introduced MLR-GAT, a novel multilevel framework specifically designed for peptide toxicity prediction, which can hierarchically identify multiple categories of peptide toxicity rather than focusing solely on hemolytic toxicity. Uniquely, pepADMET for the first time simultaneously supports linear, cyclic, modified, and natural peptides, while also accounting for biological variability across species, organs, and cell lines, thereby enabling more precise and biologically relevant ADMET predictions. As a new comprehensive online resource for multiproperty ADMET evaluation of peptides, pepADMET provides a unified, accurate, and intelligent framework to advance peptide drug design and development.

Peptide-derived macrocycles are an emerging class of therapeutics capable of modulating protein-protein interactions that remain inaccessible to small molecules. Genetically encoded library (GEL) platforms such as phage and mRNA display have accelerated macrocyclic ligand discovery by linking peptide sequence to genotype and enabling selections from libraries with up to 1013 members. Efforts to expand the chemical space of GELs have included incorporation of electrophiles, either to generate libraries of true covalent ligands or to enable intramolecular reactions such as peptide cyclization. In the latter case, the electrophile is consumed during library construction, producing transient covalent libraries that enhance stability and diversity but are not designed for direct covalent engagement with targets. By contrast, recent advances have established robust strategies for embedding persistent electrophilic warheads that remain intact during library preparation and selectively react with nucleophilic residues on proteins. These approaches have yielded both reversible and irreversible covalent inhibitors against diverse classes of proteins, while also highlighting challenges in balancing electrophile reactivity with library integrity. Complementary developments in DNA-encoded covalent libraries further underscore the breadth of discovery platforms, though genetically encoded approaches remain uniquely powerful for macrocyclic peptides. Together, these advances define the trajectory of covalent genetically encoded libraries (cGELs) and point toward new opportunities for discovering ligands to historically undruggable targets.

Receptor Interacting Protein Kinase 3 (RIPK3) is a central regulator of necroptosis and a key mediator of inflammatory signaling. Its function is orchestrated through RIP Homotypic Interaction Motif (RHIM)-dependent interactions with other RHIM-containing adapter proteins, forming amyloid-structured necrosomes that trigger kinase activation and subsequently lead to immunogenic cell lysis. Necroptosis eliminates infected or damaged cells and provokes a strong inflammatory response. Dysregulated necroptosis is implicated in chronic inflammatory diseases and associated with ischaemic injury. Despite separate structural elucidation of the isolated RIPK3 kinase domain and the amyloid fibrils formed by the RIPK3 RHIM-containing region, visualizing full-length human RIPK3 in live cells remains challenging due to a lack of specific tools. To address this, we employed Random Non-standard Peptide Integrated Discovery (RaPID) mRNA display to identify cyclic peptides that bind the RHIM-containing region of RIPK3 during its assembly into amyloid fibrils. Three peptides were selected for characterisation and demonstrate utility in visualizing RIPK3 in human cells. These peptides represent promising tools for probing RIPK3 localisation and modulating its function.

CXCR4 is overexpressed in various malignancies and represents an attractive target for PET imaging. However, currently available peptide-based tracers often exhibit rapid clearance and a narrow imaging window, which limit their clinical implication. Here, we report the design and preclinical characterization of a 68Ga-labeled dimeric cyclic peptide, implementing a large-volume linker strategy aimed at achieving an optimized balance between receptor affinity and tumor retention for CXCR4-targeted PET imaging. The tracer was synthesized, radiolabeled with the 68Ga3+ ion, and evaluated for radiochemical purity. In vitro stability, binding affinity, CXCR4-specific cellular uptake, pharmacokinetics, biodistribution, and PET/CT imaging were also assessed. The tracer showed high radiochemical purity and excellent stability. Although its binding affinity was moderate (IC50 = 161.5 nM), the tracer exhibited clear CXCR4-specific uptake and sustained tumor retention, with PET-derived tumor uptake of 3.4-3.8%ID/g between 30 and 240 min and tumor-to-muscle ratios increasing from ∼10 to ∼62 over the same period. However, notable hepatic uptake was observed, which may be attributed to the peptide size and moderate hydrophobicity. Further structural optimization such as PEGylation or scaffold minimization may reduce hepatic uptake and enhance the clinical applicability.

The diverse expression of antigenic subtypes on tumor cells can substantially influence the specific binding and tumor cytotoxicity of antibody-recruiting molecules (ARMs). Therefore, the development of multivalent ARMs with high selectivity and affinity for binding to different subtypes on tumor cells can be expected to improve clinical performance. In this study, multivalent ARMs incorporated with multivalent dinitrophenyl (DNP) haptens and an integrin-specific arginine-glycine-aspartic acid (RGD) macrocyclic peptide were synthesized using a chemoenzymatic approach. The molecules specifically recognized integrin αvβ3-positive tumor cells and exhibited robust antibody recruitment capacity and tumor-killing effects depending on the multivalent effects. Notably, the D3 molecule showed excellent anti-DNP antibody recruitment capacity in the αvβ3-positive tumor cells and antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)-mediated tumor cytotoxicity. Given the variable expression of integrin receptor subtypes among individuals, the multivalent ARMs developed in this study that specifically target αvβ3-positive tumor cells to enhance cancer cytotoxicity represent a promising strategy for tumor immunotherapy.

Peptide-based rotaxanes have attracted interest due to their expected unique physical and functional properties, which are not observed in conventional peptide-based materials. Cyclic peptides, when their primary structure is appropriately designed, are characterized by their ability to form stable host-guest complexes and have emerged as promising candidates as macrocycles that form rotaxanes. However, the effect of the primary structure of cyclic peptides on rotaxane formation remains unclear. Here, we investigated how variations in the primary structure of cyclic peptides affect their interactions with monocationic ammonium threads using umbrella sampling molecular dynamics simulations to elucidate the free energy landscapes of pseudorotaxane formation. Our results revealed that cyclic peptides adopting an all-trans configuration in pseudorotaxanes, such as cyclo(PG)4, facilitated consistent hydrogen bond formation with the thread, leading to greater stability and higher rotaxane yields. In contrast, the presence of bulky side chains or conformational constraints induced by the cis-configuration, as observed in cyclo[GC(ΨMe,MePro)(GP)3], reduced the stability of pseudorotaxanes. Demonstrating the critical role of cyclic peptide conformations and hydrogen bonding in stabilizing pseudorotaxanes provides strategic insights into the rational design of tailored cyclic peptides for rotaxane formation. Given the wide chemical diversity of natural and unnatural amino acids, this simulation method may facilitate the development of peptide-based rotaxanes with novel properties and functions.

Innovative design of CXCR4 cyclic peptide via molecular docking for 99mTc-Labeled SPECT/CT imaging in melanoma.

A new class of supramolecular antimicrobials based on d , l -cyclic peptides containing a dihydroxylated-γ-residue, which was designed to mimic the saccharide component present in certain carbohydrate–peptide hybrids, is described.

Engineered coatings containing cyclic peptides from cyanobacteria delay the development of a stable macrofouling community.

Molecular networking guided discovery of non-limonoids type ingredients from Citrus medica var. Sarcodactylis fruits.

Potent peptide ligands for therapeutically relevant targets are regularly returned from screening trillion-member libraries of ribosomally synthesized peptides containing non-canonical amino acids and macrocyclic architectures. Yet the chemical space explored by these peptides is a fraction of that embodied by natural products and pharmaceuticals, and most peptide leads require exhaustive medicinal chemistry optimization to improve potency and physicochemistry. To address the need for strategies to introduce chemical complexity and conformational control into peptide macrocycles, we report here that linear peptides with a reactive N-terminal β-keto or γ-keto amide can be synthesized ribosomally. Subsequent Friedländer reactions generate quinoline-peptide hybrids, some of which contain stable biaryl atropisomeric axes. We also demonstrate intramolecular Friedländer macrocyclization reactions-sufficiently mild to be employed on unprotected and in vitro-translated peptides-that embed a quinoline pharmacophore directly within the peptide backbone. The introduction of N-terminal ketone motifs into genetically encoded materials and their post-translational derivatization provides a paradigm for the programmed synthesis of peptide-derived materials that more closely resemble complex natural products.

The SETD7-catalysed methylation of lysine 4 in histone 3 (H3K4) plays an important role in the epigenetic control of eukaryotic gene expression. The N-terminal tail of histone H3 binds to SETD7 in a bend-like conformation in which Ala1 and Thr6 are located in close proximity, enabling the Lys4 substrate residue to react with the methyl group of the S-adenosylmethionine cosubstrate. Here, we report a proximity-guided design of H3 peptides stapled between amino acid residues 1 and 6 as potential substrates and inhibitors of human SETD7. Our results demonstrate that most of the appropriately stapled H3 peptides are efficiently methylated by SETD7, outperforming the unstructured, linear histone H3 tail sequence found in nature. The cyclic H3 peptides possessing the lactam linkage are excellent SETD7 substrates, outcompeting the linear H3K4 peptide, as demonstrated by up to 110-fold increase in catalytic efficiencies. The stapled H3 peptides display exclusive substrate selectivity for SETD7 over related H3K4 methyltransferases MLL3 and SETD1A. Inhibition assays show that the norleucine variant of the most efficient 1,6-stapled peptide substrate is a potent inhibitor of human SETD7. Overall, the results highlight a novel approach to selectively modulate the SETD7 activity and emphasise the potential of stapled histone peptides as exceptionally efficient peptidomimetic substrates and inhibitors of epigenetic enzymes.

The impact of bile acid sequestrants on octreotide absorption.