bims-cepepe 2024-08-25 papers

bims-cepepe

Biomed News

on Cell-penetrating peptides

Issue of 2024–08–25
ten papers selected by
Henry Lamb, Queensland University of Technology

First-Generation Radiolabeled Cyclic Peptides for Molecular Imaging of Platelet-Derived Growth Factor Receptor α.
Oligoarginine peptide structure and its effect on cell penetration in ocular drug delivery.
Structure-Based Design of Bicyclic Helical Peptides That Target the Oncogene β-Catenin.
Machine learning trims the peptide drug design process to a sweet spot.
Preclinical evaluation of [225Ac]Ac-crown-TATE - An alpha-emitting radiopharmaceutical for neuroendocrine tumors.
Unified and Versatile Multiplex Platform for Expedited Method Development and Comprehensive Characterization of Therapeutic Peptides.
The first-in-class pro-apoptotic peptide PEP-010 is effective in monotherapy and in combination with paclitaxel on resistant ovarian adenocarcinoma cell models.
The Missing Link(er): A Roadmap to Macrocyclization in Drug Discovery.
Impact of N-Terminal PEGylation on Synthesis and Purification of Peptide-Based Cancer Epitopes for Pancreatic Ductal Adenocarcinoma (PDAC).
Pore Formation by Amyloid-like Peptides: Effects of the Nonpolar-Polar Sequence Pattern.

Mol Pharm. 2024 Aug 17.

First-Generation Radiolabeled Cyclic Peptides for Molecular Imaging of Platelet-Derived Growth Factor Receptor α.

Susan Pike, Melinda Wuest, Ana Lopez-Campistrous, Mi Yao Hu, Ratmir Derda, Frank Wuest, Todd McMullen.

  Occult nodal spread and metastatic disease require longstanding imaging and biochemical assessments for thyroid cancer, a disease that has a propensity for diffuse, small-volume disease. We have developed a 64Cu-labeled platelet-derived growth factor receptor α (PDGFRA) antibody for immuno-PET of PDGFRA in metastatic papillary thyroid cancer (PTC). The present work describes the discovery of small cyclic PDGFRA-targeting peptides, their binding features, and radiolabeling with positron emitter gallium-68 (68Ga) for in vitro and in vivo characterization in thyroid cancer models. Phage-display technology with two separate libraries and seven different cell lines was used through three rounds of biopanning as well as flow cytometry and comparative analysis with recombinant protein to select specific peptide sequences. Phenotypic binding analysis was completed by using phosphorylation and cell migration assays. In vitro protein binding was analyzed with thermophoresis and flow cytometry using the fluorescent-labeled PDGFRA peptide. Peptide candidates were modified with the NOTA chelator for radiolabeling with 68Ga. In vitro cell uptake was studied in various thyroid cancer cell lines. In vivo studies of 68Ga-labeled peptides included metabolic stability and PET imaging. From the original library (1013 compounds), five different peptide groups were identified based on biopanning experiments with and without the α subunit of PDGFR, leading to ∼50 peptides. Subsequent phenotypic screening revealed two core peptide sequences (CP16 and CP18) that demonstrated significant changes in the level of PDGFRA phosphorylation and cell migration. Alanine scan sublibraries were created from these two lead peptide sequences, and peptides were radiolabeled using 68Ga-GaCl3 at pH 4.5, resulting in RCP > 95% within 34-40 min, including SPE purification. Cyclic peptide CP18.5 showed the strongest effects on cell migration, flow cytometry, and binding by visual interference color assay. 68Ga-labeled PDGFRA-targeting peptides showed elevated cell and tumor uptake in models of thyroid cancer, with 68Ga-NOTA-CP18.5 being the lead candidate. However, metabolic stability in vivo was compromised for 68Ga-NOTA-CP18.5 vs 68Ga-NOTA-CP18 but without impacting tumor uptake or clearance profiles. First-generation radiolabeled cyclic peptides have been developed as novel radiotracers, particularly 68Ga-NOTA-CP18.5, for the molecular imaging of PDGFRA in thyroid cancer.

Keywords:  PDGFRA; PET; cyclic peptides; thyroid cancer

DOI:  https://doi.org/10.1021/acs.molpharmaceut.4c00549
Heliyon. 2024 Aug 15. 10(15): e35109

Oligoarginine peptide structure and its effect on cell penetration in ocular drug delivery.

Stefana Duca, Naa Dei Nikoi, Madeline Berrow, Lois Barber, Louise N Slope, Anna F A Peacock, Felicity de Cogan.

  Oligoarginine cell-penetrating peptides (CPPs) are short peptides that can enhance drug delivery into cells and are of particular interest in ocular topical formulations for age-related macular degeneration (AMD) treatments. The length and structural characteristics of these peptides are considered crucial for drug delivery. This study investigates how oligoarginine length (Rn) affects their penetration mechanism, drug delivery capabilities, and antimicrobial properties, providing insights into their potential roles in AMD treatment delivery. In this study, oligoarginine peptides showed limited pore-forming abilities in a carboxyfluorescein-containing liposomal model, with R9 being the only oligoarginine length recording a significant pore-formation level. Their antibacterial efficacy depended on both the CPP length and bacterial class, with longer peptides exhibiting stronger antibacterial effects. Importantly, oligoarginine was found nontoxic to relevant mammalian cells for ocular delivery. The membrane translocation abilities of oligoarginine were consistent regardless of cargo presence. Additionally, cargo delivery by oligoarginine across in vitro cellular models for ocular delivery was dependent on peptide length and cell type, with longer chains being more effective at cargo uptake in a corneal epithelium cell line, and with shorter chains proving more effective for cargo delivery in a retinal epithelium cell line. This proposes that the chain length of oligoarginine could be used as a strategic tool in the formulation process to selectively target distinct regions of the eye. Overall, this study expands our understanding of how oligoarginine CPPs can be applied as penetration enhancers to improve the delivery of therapeutics in an ocular topical formulation within the clinical context of AMD.

Keywords:  Antibacterial; Cell-penetrating peptides; Ocular drug delivery; Oligoarginine

DOI:  https://doi.org/10.1016/j.heliyon.2024.e35109
Angew Chem Int Ed Engl. 2024 Aug 21. e202411749

Structure-Based Design of Bicyclic Helical Peptides That Target the Oncogene β-Catenin.

Alejandro Yeste-Vázquez, Felix M Paulussen, Mathias Wendt, Rasmus Klintrot, Clemens Schulte, Kerstin Wallraven, Lieke van Gijzel, Boris Simeonov, Maurice van der Gaag, Alan Gerber, Hans M Maric, Sven Hennig, Tom N Grossmann.

  The inhibition of intracellular protein-protein interactions is challenging, in particular, when involved interfaces lack pronounced cavities. The transcriptional co-activator protein and oncogene β‑catenin is a prime example of such a challenging target. Despite extensive targeting efforts, available high-affinity binders comprise only large molecular weight Inhibitors. This hampers the further development of therapeutically useful compounds. Herein, we report the design of a considerably smaller peptidomimetic scaffold derived from the α-helical β‑catenin-binding motif of Axin. Sequence maturation and bicyclization provided a stitched peptide with an unprecedented crosslink architecture. The binding mode and site were confirmed by a crystal structure. Further derivatization yielded a β-catenin inhibitor with single-digit micromolar activity in a cell-based assay. This study sheds a light on how to design helix mimetics with reduced molecular weight thereby improving their biological activity.

Keywords:  Helixmimetic; Peptidomimetic; Stapled peptide; Structure-based Design; protein-protein interaction

DOI:  https://doi.org/10.1002/anie.202411749
Nat Chem. 2024 Aug 16.

Machine learning trims the peptide drug design process to a sweet spot.

Chloe E Markey, Daniel Reker.

DOI: https://doi.org/10.1038/s41557-024-01610-0
Nucl Med Biol. 2024 Jul 31. pii: S0969-8051(24)00070-2. [Epub ahead of print]138-139 108944

Preclinical evaluation of [225Ac]Ac-crown-TATE - An alpha-emitting radiopharmaceutical for neuroendocrine tumors.

Aidan Ingham, Luke Wharton, Helena Koniar, Helen Merkens, Scott McNeil, Sathiya Sekar, Maryam Osooly, Cristina Rodríguez-Rodríguez, François Bénard, Paul Schaffer, Hua Yang.

   BACKGROUND: Targeted alpha therapy (TAT) of somatostatin receptor-2 (SSTR2) positive neuroendocrine tumors (NETs) involving Ac-225 ([225Ac]Ac-DOTA-TATE) has previously demonstrated improved therapeutic efficacy over conventional beta particle-emitting peptide receptor radionuclide therapy agents. DOTA-TATE requires harsh radiolabeling conditions for chelation of [225Ac]Ac3+, which can limit the achievable molar activities and thus therapeutic efficacy of such TAT treatments. Macropa-TATE was recently highlighted as a potential alternative to DOTA-TATE, owing to the mild radiolabeling conditions and high affinity toward [225Ac]Ac3+; however, elevated liver and kidney uptake were noted as a major limitation and a suitable imaging radionuclide is yet to be reported, which will be required for patient dosimetry studies and assessment of therapeutic benefit. Previously, [155Tb]Tb-crown-TATE has shown highly effective imaging of NETs in preclinical SPECT/CT studies, with high tumor uptake and low non-target accumulation; these favourable properties and the versatile coordination behavior of the crown chelator may therefore show promise for combination with Ac-225 for TAT.
METHODS: Crown-TATE was labeled with Ac-225, and radiochemical yield was analyzed as the function of crown-TATE concentration. LogD7.4 was measured as the indication of hydrophilicity. Free [225Ac]Ac3+ release from [225Ac]Ac-crown-TATE in human serum was studied. Biodistribution studies of [225Ac]Ac-crown-TATE in mice bearing AR42J tumors was evaluated at 1, 4, 24, 48, and 120 h, and the absorbed dose to major organs calculated. Therapy-monitoring studies with AR42J tumor bearing mice were undertaken using 30 kBq and 55 kBq doses of [225Ac]Ac-crown-TATE and compared to controls treated with PBS or crown-TATE.
RESULTS: [225Ac]Ac-crown-TATE was successfully prepared with high molar activity (640 kBq/nmol), and characterized as a moderately hydrophilic radioligand (LogD7.4 = -1.355 ± 0.135). No release of bound Ac-225 was observed over 9 days in human serum. Biodistribution studies of [225Ac]Ac-crown-TATE showed good initial tumor uptake (11.1 ± 1.7% IA/g at 4 h) which was sustained up to 120 h p.i. (6.92 ± 2.03% IA/g). Dosimetry calculations showed the highest absorbed dose was delivered to the tumors. Therapy monitoring studies demonstrated significant (log-rank test, P < 0.005) improved survival in both treatment groups compared to controls.
CONCLUSIONS: This preclinical study demonstrated the therapeutic efficacy of [225Ac]Ac-crown-TATE for treatment of NETs, and highlights the potential of using crown chelator for stable chelation of Ac-225 under mild conditions.

Keywords:  Actinium-225; Crown chelator; Neuroendocrine tumors; Peptide-receptor radionuclide-therapy; Targeted alpha therapy; Therapy-monitoring

DOI:  https://doi.org/10.1016/j.nucmedbio.2024.108944
Anal Chem. 2024 Aug 17.

Unified and Versatile Multiplex Platform for Expedited Method Development and Comprehensive Characterization of Therapeutic Peptides.

Alexandre Goyon, Shirley Wang, Kate Hofmann, Daniel Ngoc Nguyen, Peter Yehl, Kelly Zhang.

Agile analytical approaches are needed for fast and comprehensive characterization of peptide drug candidates. In this study, a unified and versatile multiplex platform was developed to expedite method development and enable the routine determination of multiple quality attributes simultaneously. The platform integrates the automation of size exclusion chromatography (SEC), reversed phase liquid chromatography coupled to reversed phase liquid chromatography (RPLC-RPLC), and hydrophilic interaction liquid chromatography hyphenated to charged aerosol detection (HILIC-CAD). Various therapeutic peptide constructs, including macrocyclic peptides and disulfide constrained peptides, across different lots were studied. The effect of the mobile phase acetonitrile content on the impurity profiles was systematically studied using two SEC columns. A prototype MaxPeak Premier SEC 125 Å column packed with BEH PEO particles achieved the separation of impurities (>2.0% area), whereas no impurities could be observed with an ACQUITY UPLC Protein BEH SEC 125 Å column packed with BEH diol particles. Comprehensive impurity profiling and expedited method development was performed utilizing RPLC-RPLC. Each peptide was analyzed by a combination of 12 conditions in the second dimension, using four columns with octadecyl, phenyl-hexyl, and cyano bonded phases, and three mobile phases with various solvents, modifiers, and pH compositions. Additionally, a HILIC-CAD method was developed for the quantification of TFA, commonly present in peptide products.

DOI: https://doi.org/10.1021/acs.analchem.4c02941
Front Pharmacol. 2024 ;15 1444973

The first-in-class pro-apoptotic peptide PEP-010 is effective in monotherapy and in combination with paclitaxel on resistant ovarian adenocarcinoma cell models.

Aline Lacroix, Rayan Farhat, Aude Robert, Catherine Brenner, Joëlle Wiels, Diego Germini.

  Ovarian adenocarcinoma is the gynecological malignancy with the worst prognosis and the highest mortality rate. In the first stages of treatment, chemotherapy results effective, but its prolonged use and high doses lead to the appearance of resistance to treatments and relapse in most patients, representing a major challenge for clinicians. We developed PEP-010, a cell penetrating proapoptotic peptide disrupting the protein-protein interaction between caspase-9 and protein phosphatase 2A, thereby leading to the recovery of their activity in the apoptotic pathway. MTT assay or Annexin-V/Propidium Iodide staining and flow cytometry analysis were used to assess sensitivity to chemotherapies and apoptosis after treatment with PEP-010 in monotherapy or in combination with paclitaxel in ovarian carcinoma cell lines. DNA damage was assessed by immunofluorescence using γH2AX marker. We show here that PEP-010 effectively induces cell death in monotherapy on in up to 55% of cells from ovarian adenocarcinoma cell models resistant to different chemotherapies. Moreover, when used in combination with paclitaxel, one of the therapeutic options for recurrent ovarian carcinoma, PEP-010 showed a beneficial effect leading to the reduction of the IC50 of paclitaxel of 2.2 times and to apoptosis in 87% of cells. The described results suggest the potential therapeutic interest for PEP-010 and lead to the choice of ovarian adenocarcinoma as one of the major indications of the ongoing clinical trial.

Keywords:  apoptosis; combination therapy; monotherapy; ovarian adenocarcinoma; peptide

DOI:  https://doi.org/10.3389/fphar.2024.1444973
J Med Chem. 2024 Aug 22.

The Missing Link(er): A Roadmap to Macrocyclization in Drug Discovery.

Christian Brudy, Carlo Walz, Moritz Spiske, Johannes K Dreizler, Felix Hausch.

Macrocycles are one of nature's preferred choices to generate large but cell-permeable bioactive molecules. Macrocyclization is increasingly prominent in medicinal chemistry beyond natural products, especially for difficult-to-drug targets. However, strategies to best exploit the potential of macrocycles are only beginning to emerge. Here we survey drug discovery campaigns from the past decade that cumulated in advanced macrocyclic drug-like compounds or drug candidates. Most macrocycles were conceived by ring closing based on U- or C-shaped bioactive conformations observed in co-crystal structures. We focus on the key step from linear precursors to the first macrocycle and the follow-up optimization of the resulting macrocyclic scaffold. Conformational control recurrently emerged as a key factor for macrocycle properties and linkers as an opportunity for optimization. With increasingly challenging drug targets, we expect these trends to become more prominent and relevant.

DOI: https://doi.org/10.1021/acs.jmedchem.4c01163
ACS Omega. 2024 Aug 13. 9(32): 34544-34554

Impact of N-Terminal PEGylation on Synthesis and Purification of Peptide-Based Cancer Epitopes for Pancreatic Ductal Adenocarcinoma (PDAC).

Omar F Luna, Yomkippur V Perez, Daniele P Ferrari, Sana S Sayedipour, Miriam Royo, Gerardo A Acosta, Luis J Cruz, Frauke Alves, Erik Agner, Magne O Sydnes, Fernando Albericio.

Peptide-based cancer vaccines have shown promising results in preclinical trials focusing on tumor immunotherapy. However, the presence of hydrophobic amino acid segments within these peptide sequences poses challenges in their synthesis, purification, and solubility, thereby hindering their potential use as cancer vaccines. In this study, we successfully synthesized peptide sequences derived from mesothelin (MSLN), a tumor-associated antigen overexpressed in pancreatic ductal adenocarcinoma (PDAC) by conjugating them with monodisperse polyethylene glycol (PEG). By PEGylating mesothelin epitopes of varying lengths (ranging from 9 to 38 amino acids) and hydrophobicity (60-90%), we achieved an effective method to improve the peptide yield and facilitate the processes of synthesis and purification. PEGylation significantly enhanced the solubility, facilitating the single-step purification of lengthy hydrophobic peptides. Most importantly, PEGylation did not compromise cell viability and had little to no effect on the immunogenicity of the peptides. In contrast, the addition of a palmitoyl group to increase immunogenicity led to reduced yield and solubility. Overall, PEGylation proves to be an effective technique for enhancing the solubility and broadening the range of utility of diverse long hydrophobic peptides.

DOI: https://doi.org/10.1021/acsomega.4c02604
ACS Chem Neurosci. 2024 Aug 22.

Pore Formation by Amyloid-like Peptides: Effects of the Nonpolar-Polar Sequence Pattern.

Warin Rangubpit, Siwaporn Sungted, Jirasak Wong-Ekkabut, Hannah E Distaffen, Bradley L Nilsson, Cristiano L Dias.

  One of the mechanisms accounting for the toxicity of amyloid peptides in diseases like Alzheimer's and Parkinson's is the formation of pores on the plasma membrane of neurons. Here, we perform unbiased all-atom simulations of the full membrane damaging pathway, which includes adsorption, aggregation, and perforation of the lipid bilayer accounting for pore-like structures. Simulations are performed using four peptides made with the same amino acids. Differences in the nonpolar-polar sequence pattern of these peptides prompt them to adsorb into the membrane with the extended conformations oriented either parallel [peptide labeled F1, Ac-(FKFE)2-NH2], perpendicular (F4, Ac-FFFFKKEE-NH2), or with an intermediate orientation (F2, Ac-FFKKFFEE-NH2, and F3, Ac-FFFKFEKE-NH2) in regard to the membrane surface. At the water-lipid interface, only F1 fully self-assembles into β-sheets, and F2 peptides partially fold into an α-helical structure. The β-sheets of F1 emerge as electrostatic interactions attract neighboring peptides to intermediate distances where nonpolar side chains can interact within the dry core of the bilayer. This complex interplay between electrostatic and nonpolar interactions is not observed for the other peptides. Although β-sheets of F1 peptides are mostly parallel to the membrane, some of their edges penetrate deep inside the bilayer, dragging water molecules with them. This precedes pore formation, which starts with the flow of two water layers through the membrane that expand into a stable cylindrical pore delimited by polar faces of β-sheets spanning both leaflets of the bilayer.

Keywords:  amyloid; lipid membrane; membrane damage; permeation; pore formation; sequence pattern

DOI:  https://doi.org/10.1021/acschemneuro.4c00333