The rising incidence of multidrug-resistant (MDR) bacteria demands the creation of innovative antimicrobial agents characterized by enhanced stability and efficacy. In this study, a series of peptide-heterocycle conjugates was systematically designed and synthesized using solid-phase peptide synthesis (SPPS), featuring octapeptide-triazole conjugates modified with a triazole-based moiety. Non-proteinogenic amino acids, specifically 2-aminoisobutyric acid (Aib) and 2-naphthylalanine (2-Nal), were used to add structural diversity. SPPS is a relatively easy way to synthesize these compounds, which ensures accuracy in the sequence. The synthesized peptides were subjected to an antimicrobial activity assessment following a molecular docking study. In comparison to the standard drug, compound 7e exhibited promising antibacterial activity against E. coli, with the MIC value of 12 μg/mL, whereas compound 7a showed enhanced activity against P. aeruginosa with the MIC value of 12 μg/mL. Compound 7a exhibited better to moderate antifungal activity against C. albicans, with the MIC values of 25 μg/mL.
This study describes the supramolecular behaviour and gelation of heterochiral, aliphatic dipeptides d-Ile-l-Xaa (Xaa = Ala, Val, Leu or Ile) in various solvents and buffered water at neutral pH. In the case of metastable gels, the consequent rapid transition towards crystals enabled X-ray diffraction analysis to unveil the dipeptides' packing modes in amphipathic layers.
α-Synuclein (αSyn) is a major component of pathogenic Lewy bodies and Lewy neurites and is closely associated with Parkinson's disease. Among the various posttranslational modifications of αSyn, several have been implicated in the degeneration of dopaminergic neurons and are thought to promote Parkinson's disease through enhanced misfolding, aggregation, and accumulation of αSyn. Two such modifications, phosphorylation at Ser129 (S129Phos) and hydroxylation at Tyr136 (Y136DOPA), exert distinct effects on αSyn aggregation: S129Phos has been reported to either inhibit or promote aggregation, whereas Y136DOPA induces the formation of short oligomeric species. To gain insight into the molecular basis underlying the initiation of αSyn multimerization, we semisynthesized αSyn carrying either S129Phos or Y136DOPA and prepared recombinant unmodified full-length αSyn as a control. Vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy revealed that these αSyn variants in their monomeric states possessed essentially identical secondary structures. These results suggest that the modifications themselves do not induce significant secondary structural changes in monomeric αSyn.
Solid tumors, the most prevalent form of malignancy, pose therapeutic challenges distinct from hematologic malignancies due to their complex biology, including high tumor heterogeneity, a dense extracellular matrix (ECM), an immunosuppressive tumor microenvironment (TME), and multifaceted drug resistance. Peptide drugs have emerged as a focal point in precision oncology, combining the deep tissue penetration of small molecules with the high target specificity, low immunogenicity, and sequence designability of antibodies. This review systematically summarizes advancements in peptide-based therapeutics for solid tumors from 2020 to 2025. These agents are categorized by function into five classes: tumor-homing peptides, surface receptor antagonist/inhibitory peptides, interfering peptides, peptide vaccines, and cell-penetrating peptides as delivery tools. We also highlight the transformative role of artificial intelligence (AI) in peptide design and discovery. Finally, we discuss outcomes from clinical trials of peptide drugs in solid tumors, underscoring their potential as multifunctional agents in this setting.
Candida albicans is a critical-priority fungal pathogen due to its global burden of infection, biofilm-forming ability, and increasing drug resistance. Previously, the tick-derived antimicrobial peptide (AMP), Os-C, demonstrated antifungal, antioxidant, and nitric oxide (NO) scavenging activity. Subsequent tryptophan end-tagging produced Os-C(W5) which improved antifungal activity and reduced salt sensitivity; however, the proteolytic susceptibility of Os-C(W5) remained a limitation. Researchers have shown that N-terminal tryptophan tagging of a gold binding peptide generated gold nanoparticle (GNPs) of a clinically relevant size. We successfully applied this method with minor modifications to generate GNPs coated with Os-C(W5). GNP@Os-C(W5) exhibited an average diameter of 14.2 ± 0.32 nm, with preserved peptide disorder in peptide secondary structure after conjugation. Compared with free Os-C(W5), GNP@Os-C(W5) activity against planktonic C. albicans was reduced, but antibiofilm activity was maintained, associated with a reduction in biofilm biomass. Ultrastructural changes to planktonic C. albicans included roughened and irregular surfaces, membrane indentations, and extracellular debris indicating cell lysis. Samples treated with Os-C(W5) and GNP@Os-C(W5) showed visibly less dense biofilm architecture and fewer apparent hyphal structures relative to the untreated control. Both Os-C(W5) and GNP@Os-C(W5) were non-cytotoxic to HaCat cells. Most importantly, resistance to the protease trypsin was increased. However, antioxidant properties of Os-C(W5) were lost with GNP conjugation, whereas both Os-C(W5) and GNP@Os-C(W5) compared with Os-C lacked NO scavenging activity. Overall, this method of GNP conjugation provides a viable strategy to achieve enhanced AMP stability, retained antifungal activity, although the ability of GNP@Os-C(W5) to reduce oxidative stress was compromised.
ABSTRACT Optical spectroscopic techniques have long been used to determine average secondary structure in proteins and peptides, but these are primarily limited to determination of relative sheet and helix content, with the remainder often designated as “other.” Several decades ago, in cooperation with Claudio Toniolo and co‐workers, our group at UIC was able to show that vibrational circular dichroism (VCD) could be used to distinguish between formation of 3 10 ‐helices and α‐helices in small peptides, as well as from other uniform structures. Conventional electronic CD (ECD) or infrared (IR) spectroscopy did not give unambiguous distinctions between these two right‐handed, internally H‐bonded, helical forms. Theoretical modeling of the VCD spectra showed that the differences between helical forms could not be attributed to just use of Aib residues or of organic solvents, both used to stabilize the 3 10 conformation. Later we showed that, with IR and VCD enhanced by isotopic labeling, it was possible to differentiate site‐specific structures using vibrational spectra interpreted with DFT‐level theoretical modeling. These advances are briefly reviewed here and results of applying them to 3 10 ‐helical structures are discussed.
Jusvinza is an immunomodulatory pharmaceutical drug developed for the treatment of rheumatoid arthritis. Its active ingredient is an altered peptide ligand-derived from a T-cell epitope of the human 60-kDa heat shock protein-referred to as CIGB-814. Jusvinza has also proven effective in reducing hyperinflammation in COVID-19. However, molecular targets of CIGB-814 in patients remain undefined, and this study is aimed at identifying specific plasma proteins that interact with CIGB-814. Affinity chromatography and mass spectrometry analysis revealed apolipoprotein A-I as a CIGB-814-binding partner. Interestingly, no apolipoprotein A-I bands were detected in the affinity matrix of the wild-type peptide (designated as E18-3). In this context, it was relevant to analyze how the substitution of Asp18 with Leu-used to design CIGB-814 from the wild-type peptide-confers its ability to bind apolipoprotein A-I. Docking studies predicted the N-terminal domain of apolipoprotein A-I as the most likely region for peptide interaction and suggested a strong connection between CIGB-814 and apolipoprotein A-I. Further validation by molecular dynamics simulations suggests that CIGB-814 may enhance the flexibility of structural areas in apolipoprotein A-I critical for high-density lipoproteins assembly. Additionally, an affinity enzyme-linked immunosorbent assay (ELISA) confirmed that apolipoprotein A-I has a tenfold greater affinity for CIGB-814 compared to the wild-type peptide. These integrated experimental and computational findings demonstrate that a single residue substitution is critical for apolipoprotein A-I recognition. In addition, this study provides mechanistic insight into CIGB-814 immunomodulatory and metabolic effects, expanding the therapeutic potential of Jusvinza to metabolic diseases associated with lipid homeostasis imbalance.
Excess reactive oxygen species generated by ultraviolet exposure cause photoaging by degrading collagen and inhibiting its synthesis. This study presents a comprehensive strategy connecting the biological stress responses of γ-irradiated microorganisms to the discovery of novel anti-photoaging peptides. We profiled the radiation-regulated transcriptomes of Deinococcus radiodurans and Cryptococcus neoformans, focusing on DNA repair and oxidative stress responses. From these datasets, peptide libraries were generated in silico, filtered for biochemical properties, and prioritized using a seven-classifier machine-learning algorithm. Structural validity was established using Rosetta FlexPepDocking against the KEAP1-NRF2 pocket, which identified 48 docking-positive sequences. We then synthesized the top 21 peptides and subjected them to in vitro validation. Seven of these candidate peptides inhibited collagenase activity at 200 μM. Among them, four peptides dose-dependently increased the procollagen type I C-peptide level in ultraviolet B-induced fibroblasts. Furthermore, these peptides significantly elevated COL1A1 mRNA levels while simultaneously reducing MMP1 and MMP9 transcript and protein levels. In summary, this study provides an integrated strategy that combines omics, machine learning, and docking to discover promising peptide candidates, which were validated through in vitro assessments. This approach offers promising anti-photoaging candidates that can be applied to other oxidative stress pathways and biological resources.
Pathological mineralization involves the uncontrolled crystallization of calcium phosphate (brushite) and calcium oxalate, leading to renal calculi and ectopic calcifications. Peptides enriched in acidic or phosphorylated residues are potential crystal growth modulators due to their ability to interact with calcium-rich surfaces. This study investigated the in vitro effects of cementum attachment protein-derived peptides, CAP-pi and its phosphorylated analog CAP-pip, on brushite and calcium oxalate crystallization. By isolating these highly anionic motifs, this work introduces a novel biomimetic approach to investigate and modulate the physicochemical mechanisms driving pathological mineral deposition. Assays performed under physiological conditions were analyzed by scanning electron microscopy, Raman spectroscopy, and confocal microscopy, alongside molecular dynamics simulations to examine peptide-calcium oxalate interactions. Both peptides altered crystal growth patterns and lattice organization in a concentration-dependent manner. Peptide treatments induced marked morphological perturbations, promoted irregular and rosette-like habits, and modified vibrational profiles. Furthermore, confocal microscopy revealed selective adsorption onto specific crystallographic regions, whereas molecular dynamics demonstrated enhanced peptide-calcium coordination and interfacial stability for CAP-pip. Collectively, these findings establish phosphorylation as a key determinant of peptide-mineral interactions, providing mechanistic insight into how phosphopeptides regulate crystallization through surface-mediated modulation.
Modification of lysine residues is a common strategy in protein engineering, whether to prevent posttranslational modifications, control bioconjugation, or improve crystallization. The standard genetic approach-replacement with arginine by site-directed mutagenesis-preserves positive charge but alters other physicochemical attributes and cannot address the N-terminal amino group. Here, we characterize reductive methylation as a chemical alternative. This reaction converts every primary amino group to a dimethylamino group rapidly under mild aqueous conditions. Using human ribonuclease 1 and a cytotoxic variant engineered to evade the endogenous ribonuclease inhibitor as model systems, we assess the effects of complete dimethylation on thermostability, enzymatic catalysis, protein-protein interaction, compatibility with bioconjugation, cellular uptake, and intracellular persistence. Dimethylation preserves thermostability and a protein-protein interaction. Enzymatic catalysis, in contrast, is reduced by 102- to 103-fold, consistent with the role of catalytic lysine residues. Dimethylation is fully compatible with bioconjugation chemistry. Dimethylated and unmodified ribonucleases show comparable uptake and persistence in human cells. These findings establish reductive methylation as a practical and conservative strategy for lysine modification in protein and peptide engineering and support its use in applications such as biological proteolysis-targeting chimeras (bioPROTACs).
Brain tumor therapy remains limited by the blood-brain barrier (BBB), which restricts drug access. BBB-penetrating peptides offer a promising strategy for delivering therapeutic and diagnostic payloads. Angiopep-2 is a well-established vector, yet novel radioconjugates based on this vector remain of interest. We report the synthesis and evaluation of DOTA-Angiopep-2 for radiolabeling with Lutetium-177 (177Lu) and Terbium-161 (161Tb). Notably, 177Lu serves as a β- and γ-emitter, whereas 161Tb is an Auger and β-emitter; both are utilized in therapy and SPECT imaging. Peptides were synthesized via solid-phase peptide synthesis. Cytotoxicity assays in T98 glioblastoma cells showed that Angiopep-2 is well-tolerated, maintaining ~100% viability at 20 μM and a moderate decline up to 100 μM. Radiolabeling achieved yields > 95% with excellent radiochemical stability at room temperature for up to 10 days and moderate stability in the presence of human serum. Biodistribution in healthy CFW mice showed a brain-associated radioactivity of 0.24% ± 0.05% IA/g at 5 min p.i. and a 12-fold increase in the brain-to-blood ratio (0.028-0.339) by 60 min p.i. These results support DOTA-Angiopep-2 as a versatile platform for radionuclide delivery and a potential candidate for future glioma-targeted studies. Further studies in tumor-bearing models are ongoing to evaluate therapeutic efficacy and translational potential.
Peptide-based nanoparticles (PBN) have emerged as a promising alternative to lipid nanoparticles (LNP) for nucleic acid delivery and efficient cellular uptake. In this study, we evaluated the formulation of WRAP5 (W- and R-rich amphipathic peptide 5)-based PBN using a microfluidic device and assessed the impact of key process parameters, flow rate ratio (FRR), total flow rate (TFR), and mixing channel design, on nanoparticle characteristics. Across 72 formulations encapsulating small interfering RNA (siRNA) or plasmid DNA (pDNA), dynamic light scattering revealed consistent mean sizes ranging from 50 to 70 nm, with a low polydispersity index (PdI < 0.22), independent of FRR, TFR, or mixer type. Stability studies demonstrated that siRNA-loaded PBN exhibited moderate size increases during storage at 4°C, whereas pDNA-loaded PBN remained highly stable for up to 70 days. Biological assays confirmed robust activity: WRAP5:siRNA PBN achieved approximately 50% CDK4 silencing in GIST-T1 cells, and WRAP5:pDNA PBN mediated efficient mCHERRY expression in HeLa cells, regardless of formulation method or storage duration. These findings highlight the robustness and scalability of WRAP5-based PBN, contrasting with LNP systems that require stringent control of FRR and TFR, and partially underscore their potential for nucleic acid delivery applications.
Cationic cell-penetrating peptides (CPPs) are a versatile platform for intracellular cargo delivery into mammalian cells but often suffer from low cellular uptake and endosomal entrapment. Here, we investigated the effect of a single cyclohexylalanine (Cha) residue at the termini of short, conformationally constrained cationic peptides composed of (4S)-guanidiniumproline (Gup). We show that this hydrophobic residue promotes internalization. Our studies also revealed that a hydrophobic residue positioned at the C-terminus enhances cellular uptake more than when at the N-terminus. Comparative studies at different temperatures are consistent with a major entry pathway via direct translocation across the plasma membrane. The findings are useful for the design of CPPs, particularly for enhancing the cellular translocation of otherwise weakly cell-penetrating peptides.
Thioredoxin reductase (TrxR) is a well-known target for gold-based drugs, which typically behave as strong inhibitors of this selenoenzyme. Despite recent evidence that cyclometalated Au(III) complexes inhibit TrxR1 and arylate cysteine (Cys)- and selenocysteine (Sec)-containing systems, there is still limited direct mechanistic evidence at the level of the TrxR C-terminal motif containing a Cys-Sec pair that is essential for catalytic activity. In this study, we investigated the intrinsic reactivity of a cyclometalated Au(III) compound, namely [Au(bnpy)Cl2], toward the 12-mer C-terminal peptide of human TrxR1, at a molecular level, to gain mechanistic insights into the chemoselective arylation of TrxR1. Time-dependent and reagent-ratio studies showed that reductive elimination and liberation of gold(I) is the key step in the process that occurs more rapidly at selenocysteine than at cysteine. Furthermore, comparable extents of arylation were observed under organic and biocompatible conditions, highlighting the efficiency of this transformation in physiologically relevant media. Overall, these findings provide direct mechanistic support for exploiting cyclometalated gold(III) complexes as covalent, Sec-directed agents for targeting thioredoxin reductase and related redox-active proteins. The TrxR C-terminal peptide offers a minimal but informative model to rationalize how scaffold-dependent reactivity can translate into selective and potent inhibition of the full enzyme.
Extrahepatic delivery of small interfering RNA (siRNA) remains a major translational challenge because most nanocarriers preferentially accumulate in the liver, while endosomal sequestration limits productive cytosolic release. Inflammatory macrophages in the spleen are attractive therapeutic targets in systemic inflammation, yet spleen-selective delivery systems with efficient endosomal escape remain underdeveloped. Here, a structure-guided peptide engineering workflow was used to generate histidine-rich, pH-switchable endosomolytic peptides for spleen-selective siRNA delivery. Sequence design integrated pH-dependent charge transition modeling, amphipathic helix prediction, membrane interaction scoring, and safety filtering. Six candidate peptides were synthesized and evaluated for pH-responsive structure, membrane disruption, hemocompatibility, siRNA complexation, serum stability, macrophage uptake, endosomal escape, biodistribution, and anti-inflammatory efficacy. The lead peptide, HSEP-6, showed a predicted net charge increase from +3.1 at pH 7.4 to +7.4 at pH 5.5, helix content increasing from 17% to 56%, and acidic calcein release increasing from 9% to 62%. In inflammatory macrophages and LPS-challenged mice, HSEP-6 enabled efficient siRNA delivery, spleen-selective accumulation, marked Irf5 silencing, reduced TNF-α and IL-6, and no measurable systemic toxicity, supporting histidine-rich pH-switchable peptides as a rational platform for extrahepatic RNA delivery.
Biomineralization is regulated by interactions between inorganic phases and biological macromolecules. Peptides derived from mineral-associated proteins are key modulators due to their ability to bind crystal nuclei and mineral surfaces. However, the molecular features governing peptide-mediated mineralization inhibition remain insufficiently synthesized in current research. This systematic review, conducted following PRISMA and Cochrane standards, evaluated in vitro evidence regarding the physicochemical properties and inhibitory mechanisms of protein- and peptide-based mineral modulators. Comprehensive searches in MEDLINE, LILACS, and Scopus (up to November 2025) identified fifty studies utilizing crystallization assays or mineralizing cell models. A dedicated risk-of-bias assessment tool, adapted from the RoB2 framework, was developed to evaluate methodological quality in in vitro studies. Although both proteins and peptides were investigated, many inhibitory molecules corresponded to peptides derived from proteins, particularly osteopontin and matrix Gla protein. Effective inhibitors shared common features, including enrichment in acidic residues, low isoelectric points (< 4.8), and posttranslational modifications such as phosphorylation. These properties significantly inhibit mineral nucleation and crystal growth, while reducing calcium deposition and alkaline phosphatase activity in cellular models. This review highlights sequence-dependent physicochemical determinants of peptide-mineral interactions and provides a framework for the rational design of peptide-based mineralization inhibitors.
The envelope-anchored trimeric spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates the attachment and entry of the virus within human cells by utilizing the angiotensin converting enzyme 2 (ACE2) as a receptor, present in the epithelial cells of the upper respiratory tract and lungs. The ACE2 interaction interface with the SARS-CoV-2 spike protein was utilized to design an ACE2-derived peptide sequence (FD11) that targeted the viral spike protein. It was found to be non-cell penetrating, while in vivo studies revealed the non-cytotoxic nature of the peptide molecule. The minimum effective concentration of the peptide was found to be 10 μM with 95% cell viability in Vero E6 cell line. The IC50 of the peptide was found to be around 6.4 μM. Treatment of the peptide in SARS-CoV-2 infected Vero E6 cells was found to decrease the infectivity as compared to control. This presents a simple approach where host-pathogen protein interaction-interface derived peptides can be used for binding with substrate macromolecules to target and modulate pathogen infectivity.
Clustering of antimicrobial peptides (AMPs) in bacterial membranes is a key step in the formation of nanoscale pores within them, which may underlie their bactericidal action. Double electron-electron resonance (DEER, also known as PELDOR) is a pulsed electron paramagnetic resonance (EPR) technique that has a unique ability to study distances between spin labels in the nanometer range. DEER has been successfully applied to the study of the conformations of doubly spin-labeled biomolecules; however, when applied to clusters with a number of spin labels N > 2, DEER encounters certain difficulties both in the proper implementation of measurements and in the interpretation of the data. This paper analyzes these emerging issues and provides recommendations on the proper use of the DEER method and the interpretation of its data when studying clusters of spin-labeled molecules. It demonstrates how the results of studies of AMP clusters in model membranes can be reinterpreted.
Peptide-based self-assembled gels are promising soft materials and have applications in biomedicine and material science. While the effect of the substituents on the N-terminal protecting group on the gelation behavior of peptides is often discussed, the role of the C-terminal substitution is seldom addressed. We report a tetrapeptide, P1 (Boc-Ala-Ala-Val-Phe-OMe) with a methyl ester at the C-terminus that forms organogels in various organic solvents. However, hydrolysis of the ester group in P1 to get a carboxylic acid (P2), or preparing a methyl (P3) or an isopropyl (P4) amide converted the peptide into hydrogelators, whereas the dimethyl amide (P5) derivative resulted in the loss of gelation. FE-SEM and AFM studies showed entangled fibrillar networks, driven by the formation of β-sheets, which was confirmed by spectroscopic studies. Rheological measurements demonstrated predominantly elastic behavior for the hydrogels, with P4 exhibiting the highest mechanical strength. To establish the observed effect and to understand the role of aromatic and hydrophobic sidechains, four other peptides were prepared and their gelation behavior was analyzed. These results show how modifications of the C-terminal carboxyl group of a given peptide sequence can affect its gelation and may provide an easier solution in tuning the properties of peptide-based gels.
