bims-cepepe Biomed News
on Cell-penetrating peptides
Issue of 2025–02–09
fifteen papers selected by
Henry Lamb, Queensland University of Technology
  1. Active- and Allosteric-Site Cyclic Peptide Inhibitors of Secreted M. tuberculosis Chorismate Mutase.
  2. New Trends in Brain Shuttle Peptides.
  3. Innovative cyclic peptide disrupts IL-17RB-MLK4 interaction for targeted pancreatic cancer therapy.
  4. Applications of cell penetrating peptide-based drug delivery system in immunotherapy.
  5. Correction: Peptide macrocyclisation via intramolecular interception of visible-light-mediated desulfurisation.
  6. Redox-Responsive Side Chain Structural Changes in a Seven-Membered Cyclic α,α-Disubstituted α-Amino Acid with a Disulfide Bond Enable Reversible Conformational Changes in Peptides.
  7. Tying the Knot: In Silico Design of Foldable Lasso Peptides.
  8. Peptide Design for Enhanced Anti-Melanogenesis: Optimizing Molecular Weight, Polarity, and Cyclization.
  9. Novel chimeric peptides of endomorphin-2 and the active fragments of ghrelin exhibit blood-brain barrier permeability and central antinociceptive effects with reduced opioid-related side effects.
  10. Application of Intramolecular O-to-N Phosphoryl Transfer Reaction to Design Fluorogenic Probes to Detect Activities of Enzymes That Metabolize Short Peptides and Acylamino Acids.
  11. Efficient Delivery of Oncolytic Peptide LTX-315 by ZIF-8: pH-Responsive Release, Improved Stability, and Reduced Hemolysis.
  12. Cyclization of Short Peptides Designed from Late Embryogenesis Abundant Protein to Improve Stability and Functionality.
  13. Biased agonism in peptide-GPCRs: A structural perspective.
  14. Twin-Tail Tadpole-Shaped Ce6-Peptide Conjugate for Enhanced Photodynamic Cancer Therapy.
  15. Development of Novel Peptide-Based Radiotracers for Detecting FGL1 Expression in Tumors.
  1. ACS Infect Dis. 2025 Feb 04.
    Active- and Allosteric-Site Cyclic Peptide Inhibitors of Secreted M. tuberculosis Chorismate Mutase.
    Renier H P van Neer, Patricia K Dranchak, Mahesh Aitha, Lijun Liu, Emma K Carlson, Isabella E Jacobsen, Kevin Battaile, Yuhong Fang, Dingyin Tao, Ganesha Rai, Janak Padia, Scott Lovell, Hiroaki Suga, James Inglese.
      The secreted Chorismate mutase enzyme of Mycobacterium tuberculosis (*MtbCM) is an underexplored potential target for the development of new antitubercular agents that are increasingly needed as antibiotic resistance rises in prevalence. As an enzyme suspected to be involved in virulence and host-pathogen interactions, disruption of its function could circumvent the difficulty of treating tuberculosis-infected granulomas. Drug development, however, is limited by novel ligand discovery. Currently, *MtbCM activity is measured by using a low throughput acid/base-mediated product derivatization absorbance assay. Here, we utilized an RNA-display affinity selection approach enabled by the Random Peptides Integrated Discovery (RaPID) system to screen a vast library of macrocyclic peptides (MCP) for novel *MtbCM ligands. Peptides identified from the RaPID selection, and analogs thereof identified by analyzing the selection population dynamics, produced a new class of *MtbCM inhibiting MCPs. Among these were two noteworthy "chorismides", whose binding modes were elucidated by X-ray crystallography. Both were potent inhibitors of the CM enzyme activity. One was identified as an allosteric binding peptide revealing a novel inhibition approach, while the other is an active-site binding peptide that when conjugated to a fluorescent probe allowed for the development of a series of alternative fluorescence-based ligand-displacement assays that can be utilized for the assessment of potential *MtbCM inhibitors.
    Keywords:  Mycobacterium tuberculosis; allosteric binding; assay development; cyclic peptides; isomerase; ligand displacement
    DOI:  https://doi.org/10.1021/acsinfecdis.4c00798
  2. Mol Pharm. 2025 Feb 03.
    New Trends in Brain Shuttle Peptides.
    Roger Prades, Meritxell Teixidó, Benjamí Oller-Salvia.
      The pharmacological treatment of central nervous system diseases faces significant challenges due to the presence of the blood-brain barrier (BBB). This barrier naturally protects the brain and prevents therapeutics from reaching their targets efficiently. However, the BBB allows the passage of nutrients and other molecules that guarantee brain homeostasis through selective transport mechanisms present at the BBB. These mechanisms provide an opportunity for delivering therapeutic agents into the central nervous system using brain shuttles. Here we review the progress of brain shuttle peptide development from 2015 until 2025. We highlight the most utilized peptides and describe trends in strategies to develop new shuttles and enhance their transport efficiency. Additionally, we compared them with other types of brain shuttles and emphasize the progress of peptide shuttles toward clinical translation.
    Keywords:  Blood−brain barrier; brain shuttle; central nervous system therapeutics; drug delivery; peptide shuttle
    DOI:  https://doi.org/10.1021/acs.molpharmaceut.4c01327
  3. Biomed Pharmacother. 2025 Feb 05. pii: S0753-3322(25)00086-1. [Epub ahead of print]184 117892
    Innovative cyclic peptide disrupts IL-17RB-MLK4 interaction for targeted pancreatic cancer therapy.
    Chun-Mei Hu, Sui-Chih Tien, Yung-Chen Lo, Ching-Hsuan Huang, Yi-Ling Ko, Dan-Ni Wu, Jiin Horng Lee, Ying-Ta Wu, Hui-Ming Yu, Kuo-Ging Lin, Lee Zong-You, Wei-Chieh Cheng.
      The IL-17B/IL-17RB oncogenic signaling axis promotes pancreatic cancer progression through interaction with mixed-lineage kinase 4 (MLK4). Here, we improved the effectiveness of a therapeutic peptide (TAT-IL17RB403-416, loop peptide) that disrupted IL-17RB/MLK4 interaction by converting its linear structure into a cyclic form. The modified cyclic peptide with higher uptake efficiency inhibited pancreatic cancer cell growth and metastasis, outperforming the original linear peptide both in vitro and in an orthotopic mouse model. At the molecular level, cysteine 408 in IL-17RB was important for mediating interactions with arginine 216 within MLK4 kinase domain. This interaction was fundamental to the efficacy of the cyclic peptide. Additionally, lysine 410 in IL-17RB was essential for maintaining the structural integrity of the cyclic peptide as a protein-protein disruptor These findings provide a deeper understanding of the IL-17RB-MLK4 interaction, offering insights for developing therapeutic agents targeting this pathway in pancreatic cancer.
    Keywords:  Cyclic peptide; IL-17B; IL-17RB; MLK4; Pancreatic cancer; Protein–protein disruptor
    DOI:  https://doi.org/10.1016/j.biopha.2025.117892
  4. Front Immunol. 2025 ;16 1540192
    Applications of cell penetrating peptide-based drug delivery system in immunotherapy.
    Jing-Jing Du, Ru-Yan Zhang, Shangchi Jiang, Shanshan Xiao, Yiting Liu, Yongheng Niu, Wen-Xiang Zhao, Dongyuan Wang, XianShi Ma.
      Cell penetrating peptides (CPPs) are usually positive charged peptides and have good cell membrane permeability. Meanwhile, CPPs are facile to synthesize, and can be functionalized to satisfy different demands, such as cyclization, incorporating unnatural amino acids, and lipid conjugation. These properties have made them as efficient drug-delivery tools to deliver therapeutic molecules to cells and tissues in a nontoxic manner, including small molecules, DNA, siRNA, therapeutic proteins and other various nanoparticles. However, the poor serum stability and low tumor targeting ability also hindered their broad application. Besides, inappropriate chemical modification can lead to membrane disruption and nonspecific toxicity. In this paper, we first reviewed recent advances in the CPP applications for cancer therapy via covalent or non-covalent manners. We carefully analyzed the advantages and disadvantages of each CPP modifications for drug delivery. Then, we concluded the recent progress of their clinical trials for different diseases. Finally, we discussed the challenges and opportunities CPPs met to translate into clinical applications. This review presented a new insight into CPPs for drug delivery, which could provide advice on the design of clinically effective systemic delivery systems using CPPs.
    Keywords:  cancer immunotherapy; cell penetration peptide; clinical application; covalent conjugation; non-covalent delivery
    DOI:  https://doi.org/10.3389/fimmu.2025.1540192
  5. Chem Sci. 2025 Jan 31.
    Correction: Peptide macrocyclisation via intramolecular interception of visible-light-mediated desulfurisation.
    Frances R Smith, Declan Meehan, Rhys C Griffiths, Harriet J Knowles, Peiyu Zhang, Huw E L Williams, Andrew J Wilson, Nicholas J Mitchell.
      [This corrects the article DOI: 10.1039/D3SC05865D.].
    DOI:  https://doi.org/10.1039/d5sc90021b
  6. Chempluschem. 2025 Feb 04. e202400772
    Redox-Responsive Side Chain Structural Changes in a Seven-Membered Cyclic α,α-Disubstituted α-Amino Acid with a Disulfide Bond Enable Reversible Conformational Changes in Peptides.
    Makoto Oba, Hikaru Nonaka, Tomohiro Umeno, Takuma Kato, Mitsunobu Doi, Atsushi Ueda, Masakazu Tanaka.
      We report the development of a redox-responsive system that induces reversible conformational changes in peptides through the design of a seven-membered cyclic α,α-disubstituted α-amino acid with a disulfide bond, 5-amino-1,2-dithiepane-5-carboxylic acid (Dtp). Upon reduction, the disulfide bond in Dtp was cleaved to form thiols, converting Dtp into (2-mercaptoethyl)homocysteine (Mhc), and this process was reversed by oxidation. Dtp-containing peptides predominantly adopted 310-helical conformation in solution, whereas Mhc-containing peptides exhibited a mixture of helical and other conformations. This redox-responsive mechanism allows for precise control over peptide secondary structures, making it a promising approach for designing functional helical peptides capable of acting molecular switches in response to intracellular reductive environments.
    Keywords:  conformational change; foldamer; helical conformation; peptide; redox responsiveness
    DOI:  https://doi.org/10.1002/cplu.202400772
  7. bioRxiv. 2025 Jan 22. pii: 2025.01.17.633674. [Epub ahead of print]
    Tying the Knot: In Silico Design of Foldable Lasso Peptides.
    John D M Nguyen, Gabriel C A da Hora, Marcus C Mifflin, Andrew G Roberts, Jessica M J Swanson.
      Lasso peptides are a unique class of natural products with distinctively threaded structures, conferring exceptional stability against thermal and proteolytic degradation. Despite their promising biotechnological and pharmaceutical applications, reported attempts to prepare them by chemical synthesis result in forming the nonthreaded branched-cyclic isomer, rather than the desired lassoed structure. This is likely due to the entropic challenge of folding a short, threaded motif prior to chemically mediated cyclization. Accordingly, this study aims to better understand and enhance the relative stability of pre-lasso conformations-the essential precursor to lasso peptide formation-through sequence optimization, chemical modification, and disulfide incorporation. Using Rosetta fixed backbone design, optimal sequences for several class II lasso peptides are identified. Enhanced sampling with well-tempered metadynamics confirmed that designed sequences derived from the lasso structures of rubrivinodin and microcin J25 exhibit a notable improvement in pre-lasso stability relative to the competing nonthreaded conformations. Chemical modifications to the isopeptide bond-forming residues of microcin J25 further increase the probability of pre-lasso formation, highlighting the beneficial role of non-canonical amino acid residues. Counterintuitively, the introduction of a disulfide cross-link decreased pre-lasso stability. Although cross-linking inherently constrains the peptide structure, decreasing the entropic dominance of unfolded phase space, it hinders the requisite wrapping of the N-terminal end around the tail to adopt the pre-lasso conformation. However, combining chemical modifications with the disulfide cross-link results in further pre-lasso stabilization, indicating that the ring modifications counteract the constraints and provide a cooperative benefit with cross-linking. These findings lay the groundwork for further design efforts to enable synthetic access to the lasso peptide scaffold.
    SIGNIFICANCE: Lasso peptides are a unique class of ribosomally synthesized and post-translationally modified natural products with diverse biological activities and potential for therapeutic applications. Although direct synthesis would facilitate therapeutic design, it has not yet been possible to fold these short sequences to their threaded architecture without the help of biosynthetic enzyme stabilization. Our work explores strategies to enhance the stability of the pre-lasso structure, the essential precursor to de novo lasso peptide formation. We find that sequence design, incorporating non-canonical amino acid residues, and design-guided cross-linking can augment stability to increase the likelihood of lasso motif accessibility. This work presents several strategies for the continued design of foldable lasso peptides.
    DOI:  https://doi.org/10.1101/2025.01.17.633674
  8. Drug Des Devel Ther. 2025 ;19 645-670
    Peptide Design for Enhanced Anti-Melanogenesis: Optimizing Molecular Weight, Polarity, and Cyclization.
    Selvi Apriliana Putri, Rani Maharani, Iman Permana Maksum, Teruna J Siahaan.
      Melanogenesis is a biochemical process that regulates skin pigmentation, which is crucial role in protecting against ultraviolet radiation. It is also associated with hyperpigmentation conditions such as melasma and age spots, which negatively impact aesthetics and self-confidence. Tyrosinase (TYR), a key enzyme in the melanogenesis pathway, catalyzes the biosynthesis of melanin in the skin. Inhibition of tyrosinase particularly by blocking its active site and preventing the binding of natural substrates such as tyrosine, can reduce melanin production, making it a promising therapeutic target for treating hyperpigmentation. Peptides have emerged as promising therapeutics to regulate melanogenesis by minimizing the side effects associated with conventional skin whitening therapeutics. This review is designed to offer a comprehensive analysis of current strategies in peptide design aimed at optimizing anti-melanogenic activity, by focusing on the role of molecular weight, polarity, and cyclization strategies in enhancing peptide efficacy and stability. It was found that optimal peptide size was within the range of 400-600 Da. The balance between hydrophilic and hydrophobic properties in peptides is crucial for effective TYR inhibition, as higher hydrophilicity enhances affinity for the TYR active site and stronger catalytic inhibition, while hydrophobicity can contribute through alternative mechanisms. Cyclization of peptides enhances their structural stability, serum resistance, and binding affinity while reducing toxicity. This process increases resistance to enzymatic degradation and improves target specificity by limiting conformational flexibility. Additionally, the rigidity and internal hydrogen bonding of cyclic peptides can aid in membrane permeability, making them more effective for therapeutic use. Peptide optimizations through size modification, polarity change, and cyclization strategies have been shown to be promising as reliable and safe agents for melanin inhibition. Future studies exploring specific amino acid in peptide chains are required to improve efficacy and potential clinical applications of these anti-melanogenic peptides as a hyperpigmentation treatment.
    Keywords:  TYR; amino acid; anti-hyperpigmentation; anti-melanogenesis; tyrosinase
    DOI:  https://doi.org/10.2147/DDDT.S500004
  9. Neuropharmacology. 2025 Feb 02. pii: S0028-3908(25)00030-9. [Epub ahead of print] 110324
    Novel chimeric peptides of endomorphin-2 and the active fragments of ghrelin exhibit blood-brain barrier permeability and central antinociceptive effects with reduced opioid-related side effects.
    Yongling Liu, Biao Xu, Songxia Cheng, Yan Wang, Jiali Ding, Xiaoyu Shen, Bing Wu, Liangquan Xu, Jie Wei.
      Pharmacological research has showed that multi-targeted drug therapies offer superior efficacy and reduced side effects compared to single-target drug therapies. In this study, we designed and characterized four novel chimeric peptides G(1-5)-EM2, EM2-G(1-5), G(1-9)-EM2 and EM2-G(1-9) which incorporate endomorphin-2 (EM-2) and the active fragments of ghrelin. Calcium mobilization assays revealed that these four chimeric peptides acted as weak mixed agonists for the μ-opioid receptor (MOR), κ-opioid receptor (KOR), and growth hormone secretagogue receptor 1α (GHS-R1α). The results of fluorescence imaging experiments indicated that G(1-5)-EM2 and G(1-9)-EM2 could penetrate the blood-brain barrier (BBB) following intravenous (i.v.) injection. All chimeric peptides induced almost equal antinociceptive effects compared with EM-2 or better antinociceptive effects than EM-2 after intracerebroventricular (i.c.v.) injection in the acute pain in mice. Among them, G(1-5)-EM2 could cross the BBB and enter the brain to induce antinociceptive effect through central opioid receptors after i.v. injection. Our findings demonstrated that the chimeric peptides produced significant antinociception mainly via MOR, DOR and GHS-R1α without inducing antinociceptive tolerance, or with a lower tendency for antinociceptive tolerance after i.c.v. injection in the acute pain in mice. Furthermore, the chimeric peptides mitigated or eliminated the digestive side effects associated with EM-2. The collective results highlight G(1-5)-EM2 as the most promising candidate among the chimeric peptides. The chimeric peptides represent a promising class of potential analgesics for clinical pain management. However, further optimization is necessary to maximize their therapeutic potential.
    Keywords:  Antinociception; Blood-brain barrier; Chimeric peptide; Endomorphin-2; Ghrelin; Side effects of opioids
    DOI:  https://doi.org/10.1016/j.neuropharm.2025.110324
  10. ACS Sens. 2025 Feb 04.
    Application of Intramolecular O-to-N Phosphoryl Transfer Reaction to Design Fluorogenic Probes to Detect Activities of Enzymes That Metabolize Short Peptides and Acylamino Acids.
    Risako Sawayama, Mayano Minoda, Hirotatsu Kojima, Takayoshi Okabe, Yukari Isayama, Kazuki Kato, Hiroshi Nishimasu, Yasuteru Urano, Toru Komatsu.
      We propose the design strategy of fluorogenic probes of proteases/peptidases and acylamino acid hydrolases utilizing an intramolecular O-to-N phosphoryl transfer reaction, in which the main chain of peptides or amino acids is retained from the natural substrate but the side chain was designed to attach the fluorophore. The strategy is useful to design fluorogenic probes for peptidases/proteases that do not prefer the main chain modification and acylamino acid hydrolases. We have developed the fluorogenic substrates for GGT5, GGCT, and PM20D1 and have performed the screening of PM20D1 inhibitors/activators to characterize the compounds that modify the activity of PM20D1.
    Keywords:  chemical biology; enzymes; enzymomics; fluorogenic substrates; high-throughput screening
    DOI:  https://doi.org/10.1021/acssensors.4c03402
  11. Mol Pharm. 2025 Feb 06.
    Efficient Delivery of Oncolytic Peptide LTX-315 by ZIF-8: pH-Responsive Release, Improved Stability, and Reduced Hemolysis.
    Xin-Qi Chen, Su-Su Cui, Yu-Zhen Chen, Cai-Yun Wang, Qing Liu, Yun-Kun Qi, Shan-Shan Du.
      The first-in-class oncolytic peptide LTX-315 has exhibited positive anticancer responses in multiple phase I/II clinical trials. Nevertheless, the linear peptide LTX-315 suffers from poor proteolytic stability and undesired toxicity, especially hemolysis, which may limit its widespread applications. Except for the direct structural modifications, drug delivery systems (DDSs) are expected to protect LTX-315 from degradation and shield its hemolytic properties. Therefore, the LTX-315 and zeolitic imidazolate framework (ZIF-8)-based nanoparticles (NPs) were constructed with a high LTX-315 encapsulation rate of 59.9%, utilizing the biomineralized "one-pot method" in an aqueous system. The release of LTX-315, in vitro anticancer potency, serum stability, anticancer durability, antimigration activity, hemolysis effect, subcellular localization, and the membrane disruption/permeation effects of LTX-315@ZIF-8 NPs were investigated. LTX-315@ZIF-8 NPs exhibited potent cytotoxicity against cancer cells. The serum stability experiment and time-inhibition curve assay indicated that ZIF-8 NPs could effectively improve the stability of LTX-315, prolong the duration of anticancer action, and enhance the cytostatic potency. Especially, the LTX-315@ZIF-8 NPs not only effectively attenuated the hemolytic toxicity of LTX-315 but also achieved the pH-responsive release of LTX-315. The mechanism investigation indicated that LTX-315@ZIF-8 NPs possessed potent membranolytic activity and reduced the mitochondrial membrane potential to trigger cell death. Collectively, this paper not only established a robust strategy to improve the stability and reduce the hemolytic properties of LTX-315 but also provided a reliable reference for the future delivery of oncolytic peptides.
    Keywords:  LTX-315; ZIF-8; anticancer; drug delivery systems; oncolytic peptide
    DOI:  https://doi.org/10.1021/acs.molpharmaceut.4c01144
  12. Chembiochem. 2025 Feb 06. e202401013
    Cyclization of Short Peptides Designed from Late Embryogenesis Abundant Protein to Improve Stability and Functionality.
    Yinghan Wu, Shinya Ikeno.
      LEA peptides, designed based on late embryogenic abundant (LEA) protein sequences, have demonstrated chaperone-like functions, such as improving drought stress tolerance of Escherichia coli (E. coli). Previous studies have focused on the biological functions of linear LEA peptides. However, the function of cyclic LEA peptide is still unknown. This study aimed to explore the cyclic LEA peptides' bio function like enhancing the drought stress tolerance of E. coli by cyclizing the LEA peptide using SICLOPPS (Split Intein Circular Ligation of Peptides and Proteins). The results indicated that cyclization significantly improved the function and extended the potential applications. At the same time, we found that peptides containing numerous lysine residues exhibited reduced performance, possibly due to the exteins' residues affecting the SICLOPPS efficiency.
    Keywords:  LEA peptide; SICLOPPS; cyclization; drought stress
    DOI:  https://doi.org/10.1002/cbic.202401013
  13. Pharmacol Ther. 2025 Jan 29. pii: S0163-7258(25)00018-X. [Epub ahead of print] 108806
    Biased agonism in peptide-GPCRs: A structural perspective.
    Tharindunee Jayakody, Dinath Kavishka Budagoda, Krishan Mendis, Withanage Dhanuka Dilshan, Duvindu Bethmage, Rashmi Dissasekara, Gavin Stewart Dawe.
      G protein-coupled receptors (GPCRs) are dynamic membrane receptors that transduce extracellular signals to the cell interior via a ligand-receptor-effector (ternary) complex that functions via allosterism. Among many signalling molecules, peptides constitute an important class of primary messengers that interact with their cognate GPCRs (peptide-GPCRs). Compared to small-molecule analogues, peptides are more potent and selective at their targets. "Biased agonism", a therapeutically relevant phenomenon exhibited by GPCRs owing to their allosteric nature, has also been observed in peptide-GPCRs, leading to the development of selective therapeutics with fewer side effects. In this review, we have focused on the structural basis of bias by comparing the interactions of biased and balanced peptide ligands at the orthosteric ligand binding sites of peptide-GPCRs of classes A and B, and reviewed the therapeutic relevance of bias at peptide-GPCRs, with the hope of contributing to the discovery of novel biased peptide drugs.
    Keywords:  Biased agonism; G protein; G protein coupled receptor; Peptide; β-arrestin
    DOI:  https://doi.org/10.1016/j.pharmthera.2025.108806
  14. ACS Appl Bio Mater. 2025 Feb 05.
    Twin-Tail Tadpole-Shaped Ce6-Peptide Conjugate for Enhanced Photodynamic Cancer Therapy.
    Mengmeng Jiang, Shaowen Wu, Hongxia Zhang, Menghuan Shen, Yirui Zhang, Zhen Zheng.
      Despite its therapeutic potential, photodynamic therapy faces several key limitations in clinical applications, including poor drug delivery and insufficient tumor selectivity. We engineered RFYFYR-Ce6-RFYFYR (R-Ce6-R), a twin-tail peptide-photosensitizer conjugate that self-assembles into nanostructures for improved cancer treatment. By incorporating arginine-rich peptide sequences, this design not only enhances cellular internalization but also promotes peroxynitrite (ONOO-) formation, amplifying the therapeutic effect. Our studies revealed that R-Ce6-R achieves 33-fold higher potency than unmodified Ce6, with an IC50 of 0.18 μM. The conjugate demonstrated selective accumulation in tumor tissue, robust ROS generation, and complete tumor regression in animal models while maintaining a favorable safety profile. These results establish R-Ce6-R as an innovative approach for advancing photodynamic therapy in cancer treatment.
    Keywords:  arginine-rich peptides; cancer therapy; photodynamic therapy; self-assembly; twin-tail peptide conjugate
    DOI:  https://doi.org/10.1021/acsabm.4c01696
  15. Mol Pharm. 2025 Feb 02.
    Development of Novel Peptide-Based Radiotracers for Detecting FGL1 Expression in Tumors.
    Yue Xu, Jinyuan Zhang, Donghui Pan, Junjie Yan, Chongyang Chen, Lizhen Wang, Xinyu Wang, Min Yang, Yuping Xu.
      A novel immune checkpoint, FGL1, is a potentially viable target for tumor immunotherapy. The development of FGL1-targeted PET probes could provide significant insights into the immune system's status and the evaluation of treatment efficacy. A ClusPro 2.0 server was used to analyze the interaction between FGL1 and LAG3, and the candidate peptides were identified by using the Rosetta peptide derivate protocol. Three candidate peptides targeting FGL1, named FGLP21, FGLP22, and FGLP23, with a simulated affinity of -9.56, -8.55, and -8.71 kcal/mol, respectively, were identified. The peptides were readily conjugated with p-NCS-benzyl-NODA-GA, and the resulting compounds were successfully labeled with 68Ga in approximately 70% yields and radiochemical purity greater than 95%. In vitro competitive cell-binding assay demonstrated that all probes bound to FGL1 with IC50 ranging from 100 nM to 160 nM. Among the probes, PET imaging revealed that 68Ga-NODA-FGLP21 exhibited the best tumor imaging performance in mice bearing FGL1 positive Huh7 tumor. At 60 min p.i., the tumor uptake of 68Ga-NODA-FGLP21 was significantly higher than those of 68Ga-NODA-FGLP22 and 68Ga-NODA-FGLP23, respectively (2.51 ± 0.11% ID/g vs 1.00 ± 0.16% ID/g and 1.49 ± 0.05% ID/g). Simultaneously, the tumor-to-muscle uptake ratios of the former were also higher than those of the latter, respectively (19.40 ± 2.30 vs 9.65 ± 0.62 and 12.45 ± 0.72). In the presence of unlabeled FGLP21, the uptake of 68Ga-NODA-FGLP21 in Huh7 xenograft decreased to 0.81 ± 0.09% ID/g at 60 min p.i., which is similar to that observed in the FGL1 negative U87 MG tumor (0.46 ± 0.03% ID/g). The results were consistent with the immunohistochemical analysis and ex vivo autoradiography. No significant radioactivity was accumulated in normal organs, except for kidneys. In summary, a preclinical study confirmed that the tracer 68Ga-NODA-FGLP21 has the potential to specifically detect FGL1 expression in tumors with good contrast to the background.
    Keywords:  FGL1; immune checkpoint inhibitor; peptide-based radiotracer 68Ga; positron emission tomography (PET); tumor
    DOI:  https://doi.org/10.1021/acs.molpharmaceut.4c01293
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