bims-cepepe 2025-02-23 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2025–02–23
twelve papers selected by
Henry Lamb, Queensland University of Technology

Molecular Chimera in Cancer Drug Discovery: Beyond Antibody Therapy, Designing Grafted Stable Peptides Targeting Cancer.
Antimicrobial cyclic peptides effectively inhibit multiple forms of Borrelia and cross the blood-brain barrier model.
Strong Membrane Permeabilization Activity Can Reduce Selectivity of Cyclic Antimicrobial Peptides.
Cyclic Peptides Targeting Granzyme B: Potential Applications as PET Imaging Agents.
Deciphering optimal molecular determinants of non-hemolytic, cell-penetrating antimicrobial peptides through bioinformatics and Random Forest.
Optimizing the discovery bioanalysis strategy for macrocyclic peptides.
Discovery and characterization of potent macrocycle inhibitors of ubiquitin-specific protease-7.
Promoting transcellular traversal of the blood-brain barrier by simultaneously improving cellular uptake and accelerating lysosomal escape.
De Novo Design of Proteins That Bind Naphthalenediimides, Powerful Photooxidants with Tunable Photophysical Properties.
Improving the Activity and Selectivity of a Scorpion-Derived Peptide, A3a, against Acinetobacter baumannii through Rational Design.
Characterization of an Iterative Halogenase Acting on Ribosomal Peptides Underlies the Combinatorial Biosynthesis Logic of Lasso Peptides.
The structural integrity of human TFF1 under reducing conditions.

Int J Pept Res Ther. 2025 ;31(3): 38

Molecular Chimera in Cancer Drug Discovery: Beyond Antibody Therapy, Designing Grafted Stable Peptides Targeting Cancer.

Arpan Chowdhury, Prajesh Shrestha, Seetharama D Jois.

   Background: Several cancer therapies are being developed, and given the variability of different cancer types, the goal of these therapies is to remove the invasive tumor from the body, kill the cancer cells, or else retard the growth. These include chemotherapeutic agents and targeted therapy using small molecules and antibodies. However, antibodies can generate an immune response upon repeated administration, and producing antibodies could be expensive.
Purpose: The purpose of this review is to describe different therapeutic approaches utilized for cancer therapy, the current therapeutic approaches, and their limitations. As a novel strategy to combat cancer, designing new stable peptide scaffolds such as cyclotides and sunflower trypsin inhibitors (SFTI) is described.
Results: Stable peptides that can target proteins can be used as therapeutic agents. Here, we review the utilization and amalgamation of plant-based peptides with biological epitopes in designing molecules called "Molecular Chimeras" using a grafted peptide strategy. These cyclic peptides can bind to target receptors or modulate protein-protein interactions as they bind with high affinity and selectivity. Grafted peptides also possess better serum stability owing to the head-to-tail cyclization and other structural modifications.
Conclusion: Stable cyclic peptides outweigh the other biologicals in terms of stability and manufacturing process. Peptides and peptidomimetics can be used as therapeutic agents, and these molecules provide alternatives for biologicals and small molecule inhibitors as drugs.

Keywords:  Cancer; Cyclotides; Small molecules; Sunflower trypsin inhibitor; Targeted therapy

DOI:  https://doi.org/10.1007/s10989-025-10690-6
Sci Rep. 2025 Feb 20. 15(1): 6147

Antimicrobial cyclic peptides effectively inhibit multiple forms of Borrelia and cross the blood-brain barrier model.

Evelína Mochnáčová, Katarína Bhide, Katarína Kucková, Jana Jozefiaková, Tomáš Maľarik, Mangesh Bhide.

  Infection caused by neuroinvasive Borrelia often manifests long-term CNS disorders and is difficult to treat as most antibiotics fail to attain an effective concentration within the brain or cannot kill the persister forms of Borrelia (cysts and round bodies). Thus, this study focused on developing antimicrobial cyclic peptides (AMPs) from a combinatorial phage display library that target phosphatidylcholine of the borrelial cell membrane. Isolated cyclic peptides with anti-Borrelia properties were then fused with the CNS homing peptide developed in this study (designated as O-BBB) to facilitate AMP transport across the blood-brain barrier. Among all O-BBB fused AMPs, Bor-18 had half maximal effective concentration (EC50) 0.83 µM when tested against spirochetal Borrelia. Bor-16, Bor-18, and Bor-26 inhibited the cystic form with EC50 0.83 µM, while Bor-11 had EC50 0.41 µM. Within an hour, all four peptides caused a permeability breach in the borrelial cell membrane, causing depolarization of the membrane. Bor peptides did not inhibit eukaryotic cell metabolism or proliferation, nor did they cause erythrocyte lysis. Peptides were stable in serum, could cross the BBB in-vitro, and remained effective against Borrelia. Cyclic AMPs fused with a CNS homing moiety, the Bor peptides, deserve further investigation for their potential use in neuroborreliosis therapy.

Keywords:   Borrelia ; Antimicrobial peptides; Blood-brain barrier; CNS homing peptide; Combinatorial phage display; Phosphatidylcholine

DOI:  https://doi.org/10.1038/s41598-025-90605-z
J Phys Chem B. 2025 Feb 19.

Strong Membrane Permeabilization Activity Can Reduce Selectivity of Cyclic Antimicrobial Peptides.

Katharina Beck, Janina Nandy, Maria Hoernke.

Selectivity is a key requirement for membrane-active antimicrobials to be viable in therapeutic contexts. Therefore, the rational design or suitable selection of new compounds requires adequate mechanistic understanding of peptide selectivity. In this study, we compare two similar cyclic peptides that differ only in the arrangement of their three hydrophobic tryptophan (W) and three positively charged arginine (R) residues, yet exhibit different selectivities. This family of peptides has previously been shown to target the cytoplasmic membrane of bacteria, but not to act directly by membrane permeabilization. We have systematically studied and compared the interactions of the two peptides with zwitterionic phosphatidylcholine (PC) and negatively charged phosphatidylglycerol/phosphatidylethanolamine (PG/PE) model membranes using various biophysical methods to elucidate the mechanism of the selectivity. Like many antimicrobial peptides, the cyclic, cationic hexapeptides investigated here bind more efficiently to negatively charged membranes than to zwitterionic ones. Consequently, the two peptides induce vesicle leakage, changes in lipid packing, vesicle aggregation, and vesicle fusion predominantly in binary, negatively charged PG/PE membranes. The peptide with the larger hydrophobic molecular surface (three adjacent W residues) causes all these investigated effects more efficiently. In particular, it induces leakage by asymmetry stress and/or leaky fusion in zwitterionic and charged membranes, which may contribute to high activity but reduces selectivity. The unselective type of leakage appears to be driven by the more pronounced insertion into the lipid layer, facilitated by the larger hydrophobic surface of the peptide. Therefore, avoiding local accumulation of hydrophobic residues might improve the selectivity of future membrane-active compounds.

DOI: https://doi.org/10.1021/acs.jpcb.4c05019
ACS Med Chem Lett. 2025 Feb 13. 16(2): 180-181

Cyclic Peptides Targeting Granzyme B: Potential Applications as PET Imaging Agents.

Qi-Long Hu, Steven H Liang.

This patent application pertains to macrocyclic peptides and their radiolabeled derivatives, generally represented by Formula I. These compounds exhibit selective binding to Granzyme B (GzmB) and hold the potential for imaging disease or disorders associated with elevated GzmB levels. Such conditions include cancer immunotherapy, autoimmunity, wound healing, and inflammation.

DOI: https://doi.org/10.1021/acsmedchemlett.4c00608
Brief Bioinform. 2024 Nov 22. pii: bbaf049. [Epub ahead of print]26(1):

Deciphering optimal molecular determinants of non-hemolytic, cell-penetrating antimicrobial peptides through bioinformatics and Random Forest.

Ashok Kumar, Sonia Chadha, Mradul Sharma, Mukesh Kumar.

  Antimicrobial peptides (AMPs) are promising molecules for combating resistant pathogens, offering several advantages like broad-spectrum effectiveness and multi-targeted action. While most AMPs exhibit membranolytic activity similar to hemolytic peptides (HPs), some act by entering cells like cell-penetrating peptides (CPPs). The toxicity of AMPs towards the host is the major hurdle in their development and application. Given the peptides' function and toxicity largely depend on their molecular properties, identifying and fine-tuning these factors is imperative for developing effective and safe AMPs. To address these knowledge gaps, we present a study that employs a holistic strategy by investigating the molecular descriptors of AMPs, CPPs, HPs, and non-functional equivalents. The prediction of functional properties categorized datasets of 3697 experimentally validated peptides into six groups and three clusters. Predictive and statistical analyses of physicochemical and structural parameters revealed that AMPs have a mean hydrophobic moment of 1.2, a net charge of 4.5, and a lower isoelectric point of 10.9, with balanced hydrophobicity. For cluster AC-nHPs containing peptides with antimicrobial, cell-penetrating, and non-hemolytic properties, disordered conformation and aggregation propensities, followed by amphiphilicity index, isoelectric point, and net charge were identified as the most critical properties. In addition, this work also explains why most AMPs and HPs are membrane-disruptive, while CPPs are non-membranolytic. In conclusion, the study identifies optimal molecular descriptors and offers valuable insights for designing effective, non-toxic AMPs for therapeutic use.

Keywords:  Random Forest; antimicrobial peptide; cell-penetrating; hemolytic peptides; machine learning; physicochemical parameters

DOI:  https://doi.org/10.1093/bib/bbaf049
Anal Bioanal Chem. 2025 Feb 15.

Optimizing the discovery bioanalysis strategy for macrocyclic peptides.

Xing Zhang, Stephanie Dale, Yusi Cui, Joe Napoli, Huy Nguyen, Jingwei Cai, Brian Dean.

  Macrocyclic peptides (MCPs) have remained a compelling modality in drug discovery and development, with many successful marketed drugs. Their unique molecular structure and ADME properties have posed bioanalytical challenges that cannot be fully addressed with conventional small molecule LC-MRM assays. In this work, we developed and optimized a high-throughput discovery bioanalytical strategy for MCPs with 16 marketed MCP drugs. By evaluating ten different sample extraction methods based on the recovery and matrix effect, we identified that the protein precipitation extraction with MeOH/ACN (1/1 v/v) with 0.5% FA outperformed the other sample extraction methods, achieving 80% recovery for 80% of the MCP drugs and 90% matrix effect for 90% of the MCP drugs. By assessing the sensitivity of the targeted-selected ion monitoring (t-SIM) and parallel reaction monitoring (PRM) on the Orbitrap HRMS and comparing with the conventional LC-MRM, we concluded that the t-SIM provided comparable sensitivity with MRM (LOQ at 1~3 ng/mL for the majority of the MCP drugs), with the extra benefits of minimal method development and high post-acquisition flexibility in data processing. The optimized bioanalytical strategy was applied to various biological matrices and displayed performance that met the quantitation requirements for discovery bioanalysis.

Keywords:  Bioanalytical methods; Macrocyclic peptides; Orbitrap high-resolution MS; Quantitative analysis; Sample preparation

DOI:  https://doi.org/10.1007/s00216-025-05781-8
Structure. 2025 Feb 18. pii: S0969-2126(25)00021-8. [Epub ahead of print]

Discovery and characterization of potent macrocycle inhibitors of ubiquitin-specific protease-7.

Rafael Miranda, Francesca Anson, Shannon T Smith, Mark Ultsch, Connie A Tenorio, Lionel Rougé, Brennan Farrell, Emel Adaligil, Jeffrey K Holden, Seth F Harris, Erin C Dueber.

  The ubiquitin-specific protease (USP) family of deubiquitinases (DUBs) are regulators of Ub signaling that share a common catalytic-domain fold. The dynamic nature of this domain is important for controlling the function of USPs, with inter- and intramolecular interactions often influencing the structure and enzymatic activity of these DUBs. This conformational flexibility, in combination with the high sequence conservation of the USP active site, has made it challenging to readily identify potent and selective inhibitors for individual USPs. Here, we demonstrate how a naive, macrocycle-mRNA display selection rapidly yielded high-affinity binders to USP7 that specifically inhibit the DUB with nanomolar half-maximal inhibitory concentration (IC50) values. Structural analysis of the macrocycles bound to USP7 revealed a variety of binding modes and identified inhibition hotspots on the enzyme that mirror those used by small-molecule inhibitors. Together, these data suggest that initial macrocyclic hits could serve as pivotal tools in developing USP-specific inhibitors and probing USP biology.

Keywords:  DUB; USP7; allostery; chemical biology; inhibition; mRNA display; macrocycle

DOI:  https://doi.org/10.1016/j.str.2025.01.021
Nanoscale. 2025 Feb 18.

Promoting transcellular traversal of the blood-brain barrier by simultaneously improving cellular uptake and accelerating lysosomal escape.

Li Zhang, Weibin Li, Zhen Xu, Zhennan Mao, Mengqian Yang, Caixia Wang, Zhihong Liu.

The blood-brain barrier (BBB) impedes the transportation of drugs to the brain, thereby constraining the efficacy of treatments for brain diseases. Here, a pH-sensitive nanocarrier coated with a brain metastatic tumor cell membrane (CA-iRGD-CS@M) is designed to enhance drug delivery across the BBB by simultaneously improving cellular uptake and accelerating lysosomal escape. The cell membrane coating can recognize brain microvessel endothelial cells (BMECs) to improve cellular uptake. The pH-sensitive nanocarrier (CA-iRGD-CS) as the core of CA-iRGD-CS@M undergoes charge reversal triggered by the acidic environment of lysosomes, leading to the disruption of the coated cell membrane and further promoting the escape of the detached core from lysosomes into the brain parenchyma. Facilitated by the targeting ligand iRGD, the detached core containing the photothermal agent (CuS) can target the tumor site and fulfill deep penetration, thereby achieving efficient NIR-II photothermal therapy.

DOI: https://doi.org/10.1039/d4nr05134c
J Am Chem Soc. 2025 Feb 21.

De Novo Design of Proteins That Bind Naphthalenediimides, Powerful Photooxidants with Tunable Photophysical Properties.

Samuel I Mann, Zhi Lin, Sophia K Tan, Jiaqi Zhu, Zachary X W Widel, Ian Bakanas, Jarrett P Mansergh, Rui Liu, Mark J S Kelly, Yibing Wu, James A Wells, Michael J Therien, William F DeGrado.

De novo protein design provides a framework to test our understanding of protein function and build proteins with cofactors and functions not found in nature. Here, we report the design of proteins designed to bind powerful photooxidants and the evaluation of the use of these proteins to generate diffusible small-molecule reactive species. Because excited-state dynamics are influenced by the dynamics and hydration of a photooxidant's environment, it was important to not only design a binding site but also to evaluate its dynamic properties. Thus, we used computational design in conjunction with molecular dynamics (MD) simulations to design a protein, designated NBP (NDI Binding Protein), that held a naphthalenediimide (NDI), a powerful photooxidant, in a programmable molecular environment. Solution NMR confirmed the structure of the complex. We evaluated two NDI cofactors in this de novo protein using ultrafast pump-probe spectroscopy to evaluate light-triggered intra- and intermolecular electron transfer function. Moreover, we demonstrated the utility of this platform to activate multiple molecular probes for protein labeling.

DOI: https://doi.org/10.1021/jacs.4c18151
ACS Omega. 2025 Feb 11. 10(5): 4699-4710

Improving the Activity and Selectivity of a Scorpion-Derived Peptide, A3a, against Acinetobacter baumannii through Rational Design.

Dalton S Möller, Mandelie van der Walt, Carel Oosthuizen, Miruna Serian, June C Serem, Christian D Lorenz, A James Mason, Megan J Bester, Anabella R M Gaspar.

The rise in antimicrobial resistance has led to an increased desire to understand how antimicrobial peptides (AMPs) can be better engineered to kill antibiotic-resistant bacteria. Previously, we showed that C-terminal amidation of a peptide, identified in scorpion Androctonus amoreuxi venom, increased its activity against both Gram-positive and -negative bacteria. Here, we incorporate all-atom molecular dynamics (MD) simulations in a rational design strategy to create analogues of A3a with greater therapeutic potential. We discover two novel AMPs which achieve greater potency against, and selectivity toward, Acinetobacter baumannii ATCC 19606 but via two distinct mechanisms and which are effective in Galleria mellonella models of A. baumannii burn wound infection. While CD spectroscopy indicates A3a adopts an α-helix conformation in the presence of models of the Gram-negative bacterial plasma membrane, MD simulations reveal it adopts a hairpin conformation during initial binding. Three different strategies, designed to stabilize this hairpin conformation, produce substantially different outcomes. Deletion of Ile6 and Ile10 restricts conformational flexibility, characteristic of A3a, during membrane binding, prevents adoption of the α-helix conformation in the steady state, and abrogates the antibacterial activity. In contrast, substitution of arginine 7 to lysine (A3a[R7K]) or isoleucine 14 to tryptophan (A3a[I14W]) does not consistently affect peptide conformations. Both of these new analogues are rapidly bactericidal toward A. baumannii ATCC 19606 but A3a[R7K] also causes rapid permeabilization and while the antibacterial potency and selectivity are increased for both peptides, this is greatest for A3a[I14W]. Integration of atomistic MD simulations into a multidisciplinary approach to understanding antimicrobial peptide mechanism of action is a valuable tool for interpreting the effects of rational design strategies.

DOI: https://doi.org/10.1021/acsomega.4c09593
J Nat Prod. 2025 Feb 20.

Characterization of an Iterative Halogenase Acting on Ribosomal Peptides Underlies the Combinatorial Biosynthesis Logic of Lasso Peptides.

Jin-Long Lu, Jiao-Jiao Cui, Zhe-Yang Hu, Jin-Ming Di, Yuan-Yuan Li, Jiang Xiong, Yu-Meng Jiao, Kun Gao, Jian Min, Shangwen Luo, Shi-Hui Dong.

Halogenation is commonly utilized in medicinal chemistry for the improvement of drug leads. Flavin-dependent halogenases (FDHs) are ubiquitous across all domains of life, yet iterative FDHs are rare in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). Herein, we characterize a novel iterative FDH, ChlH, which orchestrates nonsequential chlorination of two specific Trp within the core peptide of a lasso precursor containing three Trp. Biochemical and computational studies enable the characterization of ChlH, which employs unique protein-peptide interactions (PPIs) between its distinct N- and C-terminal motifs and a crucial recognition sequence (RS-II) downstream of RS-I in the leader peptide. Previous studies have demonstrated the indispensability of RS-I for lasso peptide biosynthesis, while RS-II was considered to be replaceable. Furthermore, we find that the core peptide substantially contributes to the PPI. Bioinformatic analysis reveals the prevalence of homologous FDHs in the biosynthetic gene clusters (BGCs) of various RiPP classes. Heterologous expression of the chl BGC yields non-, mono-, and dichlorinated lasso peptides, with chlorination, particularly dichlorination, enhancing their antibacterial activity. This study expands the FDH activity spectrum to include iterative catalysis on ribosomal peptides and underscores the significance of RS-II in tailoring enzymes for the combinatorial biosynthesis of lasso peptides.

DOI: https://doi.org/10.1021/acs.jnatprod.4c01199
Redox Biol. 2025 Feb 05. pii: S2213-2317(25)00047-3. [Epub ahead of print]81 103534

The structural integrity of human TFF1 under reducing conditions.

Dilsah Nur Elmaci, Gene Hopping, Werner Hoffmann, Markus Muttenthaler, Matthias Stein.

  The trefoil factor family (TFF) comprises three secretory peptides (TFF1, TFF2, TFF3) that regulate diverse physiological processes to maintain gastrointestinal mucosal integrity and homeostasis. The TFF domain is stabilized by six conserved cysteine residues forming three intramolecular disulfide bonds. In this work, we investigated human TFF1 domain stability against increasing concentrations of the reducing agent tris(2-carboxyethyl)phosphine (TCEP). Experiments revealed high resistance of the disulfide bonds within the TFF1 domain to reduction compared to two reference peptides with similar three-disulfide frameworks, namely the bovine pancreatic trypsin inhibitor (BPTI) and the peptide drug linaclotide. Full reduction of TFF1 was only achieved with a large excess of TCEP (150-fold), and no partially reduced intermediates were observed, supporting a compact TFF1 domain. This TFF1 domain stability was supported by extensive all-atom molecular dynamics simulations for a total of 24 μs of all possible combinatorial states of disulfide bond reduction. Despite minor structural and conformational changes observed upon reduction, the domain substantially retained its overall compactness and solvent exposure when only one or two disulfide bonds were removed. The reduced cysteine residues did not undergo large structural rearrangements and remained buried. The loss of covalent disulfide bonds upon reduction was counterbalanced through persistent non-covalent interactions. These molecular simulations explained why TFF1 could not be partially reduced and alkylated during the experiments despite titrating different TCEP concentrations in the presence of alkylating agents. Our findings provide the first insights into the remarkable stability of the human TFF domain under reducing conditions, supporting its functional resilience upon expression and secretion throughout the human body.

Keywords:  Disulfide bond stability; Molecular dynamics simulation; Reductive stability; Trefoil factor family

DOI:  https://doi.org/10.1016/j.redox.2025.103534