N(G)-Functionalized Arginines as Arginine Substitutes: Preparation and Biological Characterization of Novel-Type Fluorescence and Radiolabelled Peptidic Receptor Ligands

• Applicant: Professor Dr. Max Keller
• Subject Area: Pharmacy
• Term: from 2013 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 233221909

• Final Report Year: 2024

Final Report Abstract

Radiolabeling and fluorescence labeling of bioactive peptides gives access to molecular tools, which play an important role in pharmaceutical, biochemical and medical research. Proteinogenic amino acids, allowing an efficient and diverse conjugation of peptides, are lysine, cysteine and unmasked N-terminal amino acids. However, the modification of peptides usually affects their biological activity and many peptides are devoid of lysine, cysteine and/or a free N-terminus. Therefore, a new, widely applicable labeling strategy for peptides, based on the replacement of the natural amino acid arginine by an Nω-carbamoylated amino-functionalized arginine, was introduced within the first funding period. The amino-functionalized arginine allows for a versatile peptide conjugation and labeling. This concept was demonstrated by the preparation of radiolabeled and fluorescence labeled arginine-containing peptides addressing neurotensin, angiotensin II (AngII) und neuropeptide Y (NPY) receptors with high binding affinity. During the second funding period, the aforementioned labeling strategy was extended by the introduction of an alkyne-functionalized arginine building block, also being compatible with solidphase peptide synthesis. The alkyne-functionalized arginine enables, after incorporation into a peptide, a regioselective “bioorthogonal” conjugation based on “click chemistry” even if the peptide contains, e.g. lysine residues. Furthermore, new fluorescence-labeled peptidic ligands for the neurotensin receptor 1 (NTS1R) and the AngII receptor 1 were prepared and characterized. Moreover, the suitability of the Nω-carbamoylated arginines for peptide cyclization via arginine side chains was demonstrated for peptidic NPY Y4 receptor ligands. Within the second and third funding period, NTS1R PET ligands (PET = positron emission tomography), being superior to previously reported NTS1R PET ligands with respect to in vivo stability and receptor affinity, were developed based on the new labeling strategy. These 18F- or 68Ga-labeled PET ligands were successfully applied in preclinical tumor imaging studies using tumor-bearing mice. Moreover, in the third funding period, dually labeled (radioactive [tritium] + fluorescence) NTS1R ligands were prepared using the aforementioned labeling strategy. Together with their non-tritiated analogues, the dually labeled peptides served as molecular tools for a systematic comparison of radiochemical and fluorescence-based receptor-ligand binding assays. Finally, prompted by the discovery of high-affinity cyclic peptidic Y4 receptor ligands (N-terminus to arginine side-chain cyclization), new fluorescent Y4 receptor ligands were developed and introduced as tool compounds useful for fluorescence-based Y4 receptor binding studies. Concludingly, the projects enabled a broadening of the portfolio of strategies that can be used for the preparation of labeled bioactive peptides.

Publications

• Frontiers in Medicinal Chemistry (GDCh, DPhG), Tübingen March 15-19th 2014, poster and oral poster presentation „NG-Carbamoylated Arginines: New Building Blocks for Solid Phase Synthesis Enabling Radio- and Fluorescence-Labeling of Biologically Active Peptides”
  Max Keller
  
• Habilitanden-Workshop DPhG-Fachgruppe Pharmazeutische/Medizinische Chemie, Tübingen March 19th 2014, oral presentation „A Novel Approach to Peptide Labeling via Arginine Residues”
  Max Keller
  
• Frontiers in Medicinal Chemistry (GDCh, DPhG), Marburg March 15-18th 2015, poster presentation „Incorporation of NG-carbamoylated arginines in peptides retains affinity and enables radiolabeling: neurotensin(8-13) and angiotensin II analogs as examples”
  Max Keller
  
• Nω-Carbamoylation of the Argininamide Moiety: An Avenue to Insurmountable NPY Y1 Receptor Antagonists and a Radiolabeled Selective High-Affinity Molecular Tool ([3H]UR-MK299) with Extended Residence Time. Journal of Medicinal Chemistry, 58(22), 8834-8849.
  Keller, Max; Weiss, Stefan; Hutzler, Christoph; Kuhn, Kilian K.; Mollereau, Catherine; Dukorn, Stefanie; Schindler, Lisa; Bernhardt, Günther; König, Burkhard & Buschauer, Armin
  
• 34th European Peptide Symposium and 8th International Peptide Symposium, Leipzig September 4-9th 2016, poster and oral poster presentation „A novel appraoch to peptide labeling via Nω-carbamoylated arginines”
  Max Keller
  
• 8th Summer School Medicinal Chemistry, Regensburg September 21st-23rd 2016, poster and oral poster presentation „Conjugation and labeling of bioactive peptides via Nωcarbamoylated arginines”
  Max Keller
  
• High Affinity Agonists of the Neuropeptide Y (NPY) Y4 Receptor Derived from the C-Terminal Pentapeptide of Human Pancreatic Polypeptide (hPP): Synthesis, Stereochemical Discrimination, and Radiolabeling. Journal of Medicinal Chemistry, 59(13), 6045-6058.
  Kuhn, Kilian K.; Ertl, Thomas; Dukorn, Stefanie; Keller, Max; Bernhardt, Günther; Reiser, Oliver & Buschauer, Armin
  
• Mimicking of Arginine by Functionalized Nω-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples. Journal of Medicinal Chemistry, 59(5), 1925-1945.
  Keller, Max; Kuhn, Kilian K.; Einsiedel, Jürgen; Hübner, Harald; Biselli, Sabrina; Mollereau, Catherine; Wifling, David; Svobodová, Jaroslava; Bernhardt, Günther; Cabrele, Chiara; Vanderheyden, Patrick M. L.; Gmeiner, Peter & Buschauer, Armin
  
• In Search of NPY Y4R Antagonists: Incorporation of Carbamoylated Arginine, Aza-Amino Acids, or d-Amino Acids into Oligopeptides Derived from the C-Termini of the Endogenous Agonists. ACS Omega, 2(7), 3616-3631.
  Kuhn, Kilian K.; Littmann, Timo; Dukorn, Stefanie; Tanaka, Miho; Keller, Max; Ozawa, Takeaki; Bernhardt, Günther & Buschauer, Armin
  
• 8th Austrian Peptide Symposium, December 13th 2018, poster presentation „Structural modifications of NT(8-13) to increase metabolic stability: N-methylation, N-terminal acylation and Ile/Tle exchange”
  Lisa Schindler
  
• 9th Summer School Medicinal Chemistry, Regensburg September 19-20th 2018, lecture „Radio- and fluorescence labeled molecular tools for muscarinic acetylcholine, neurotensin, angiotensin II and neuropeptide Y receptors”
  Max Keller
  
• Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor. Nature, 556(7702), 520-524.
  Yang, Zhenlin; Han, Shuo; Keller, Max; Kaiser, Anette; Bender, Brian J.; Bosse, Mathias; Burkert, Kerstin; Kögler, Lisa M.; Wifling, David; Bernhardt, Guenther; Plank, Nicole; Littmann, Timo; Schmidt, Peter; Yi, Cuiying; Li, Beibei; Ye, Sheng; Zhang, Rongguang; Xu, Bo; Larhammar, Dan ... & Wu, Beili
  
• 9th Austrian Peptide Symposium, Vienna December 5th 2019, poster and oral poster presentation poster presentation „N-terminus to side chain cyclization of linear peptidic neuropeptide Y Y4 receptor ligands results in picomolar receptor affinities”
  Adam Konieczny
  
• 9th Austrian Peptide Symposium, Vienna December 5th 2019, poster and oral poster presentation „Metabolically stabilized neurotensin(8-13) derivatives: 68Ga-labeling via carbamoylated arginines gives NTS1R PET ligands with high receptor affinity”
  Lisa Schindler
  
• An Alkyne-functionalized Arginine for Solid-Phase Synthesis Enabling “Bioorthogonal” Peptide Conjugation. ACS Medicinal Chemistry Letters, 11(3), 334-339.
  Spinnler, Katrin; von Krüchten, Lara; Konieczny, Adam; Schindler, Lisa; Bernhardt, Günther & Keller, Max
  
• DPhG-Jahrestagung, Heidelberg September 2nd-4th 2019, short lecture „Functionalized Nω-carbamoylated arginines give access to labeled peptide receptor ligands”
  Max Keller
  
• Fluorescence Labeling of Neurotensin(8–13) via Arginine Residues Gives Molecular Tools with High Receptor Affinity. ACS Medicinal Chemistry Letters, 11(1), 16-22.
  Keller, Max; Mahuroof, Shahani A.; Hong, Yee Vivyanne; Carpenter, Jessica; Schindler, Lisa; Littmann, Timo; Pegoli, Andrea; Hübner, Harald; Bernhardt, Günther; Gmeiner, Peter & Holliday, Nicholas D.
  
• Frontiers in Medicinal Chemistry (GDCh, DPhG), Würzburg March 24-27th 2019, poster presentation „Labeling of peptide receptor ligands via Nω-carbamoylated arginines”
  Max Keller
  
• Modifications at Arg and Ile Give Neurotensin(8–13) Derivatives with High Stability and Retained NTS1 Receptor Affinity. ACS Medicinal Chemistry Letters, 10(6), 960-965.
  Schindler, Lisa; Bernhardt, Günther & Keller, Max
  
• 58th Annual meeting of the German Society for Nuclear Medicine, July 7-9th 2020 (online conference), poster presentation (Lisa Schindler) „Metabolically stabilized neurotensin(8-13) derivatives: 68Ga-labeling via carbamoylated arginines gives NTS1R PET ligands with high receptor affinity”
  Lisa Schindler
  
• DPhG-Jahrestagung, Leipzig September 29th-October 1st 2021, short lecture „Hexapeptides as neuropeptide Y Y4 receptor ligands: cyclization results in increased potency and incorporation of Trp in position 4 turns agonism into antagonism”
  Max Keller
  
• N-Terminus to Arginine Side-Chain Cyclization of Linear Peptidic Neuropeptide Y Y4 Receptor Ligands Results in Picomolar Binding Constants. Journal of Medicinal Chemistry, 64(22), 16746-16769.
  Konieczny, Adam; Conrad, Marcus; Ertl, Fabian J.; Gleixner, Jakob; Gattor, Albert O.; Grätz, Lukas; Schmidt, Maximilian F.; Neu, Eduard; Horn, Anselm H. C.; Wifling, David; Gmeiner, Peter; Clark, Timothy; Sticht, Heinrich & Keller, Max
  
• 11th Austrian Peptide Symposium, Vienna December 1st 2022, poster presentation „UR-JG114 - a fluorescent oligopeptidic NPY Y4R ligand with high binding affinity” and poster presentation (Fabian Ertl) „Synthesis and characterization of fluorescently labelled NT(8- 13) derivatives”
  Fabian Ertl
  
• Development of a Neurotensin-Derived 68Ga-Labeled PET Ligand with High In Vivo Stability for Imaging of NTS1 Receptor-Expressing Tumors. Cancers, 14(19), 4922.
  Schindler, Lisa; Moosbauer, Jutta; Schmidt, Daniel; Spruss, Thilo; Grätz, Lukas; Lüdeke, Steffen; Hofheinz, Frank; Meister, Sebastian; Echtenacher, Bernd; Bernhardt, Günther; Pietzsch, Jens; Hellwig, Dirk & Keller, Max
  
• DPhG-Jahrestagung, Marburg September 14-16th 2022, invited short lecture „Receptor-specific sets of tool compounds: 3H, 18F, 68Ga and fluorescently labeled neuropeptide Y Y1 and neurotensin NTS1 receptor ligands” and poster presentation (Lisa Schindler) „Development of a peptidic 68Ga-labeled PET ligand with high in vivo stability for NTS1R- mediated tumor imaging”
  Max Keller
  
• Neurotensin analogs by fluoroglycosylation at Nω-carbamoylated arginines for PET imaging of NTS1-positive tumors. Scientific Reports, 12(1).
  Schindler, Lisa; Wohlfahrt, Katrin; Gluhacevic, von Krüchten Lara; Prante, Olaf; Keller, Max & Maschauer, Simone
  
• Characterization of Fluorescent Dyes Frequently Used for Bioimaging: Photophysics and Photocatalytical Reactions with Proteins. The Journal of Physical Chemistry B, 127(44), 9532-9542.
  Archipowa, Nataliya; Wittmann, Lukas; Köckenberger, Johannes; Ertl, Fabian J.; Gleixner, Jakob; Keller, Max; Heinrich, Markus R. & Kutta, Roger Jan
  
• Spring School Medicinal Chemistry, Regensburg March 22nd-24th 2023, poster presentation „UR-JG114 - a fluorescent oligopeptidic NPY Y4R ligand with high binding affinity
  Jakob Gleixner
  
• Dually Labeled Neurotensin NTS1R Ligands for Probing Radiochemical and Fluorescence-Based Binding Assays. Journal of Medicinal Chemistry, 67(18), 16664-16691.
  Ertl, Fabian J.; Kopanchuk, Sergei; Dijon, Nicola C.; Veikšina, Santa; Tahk, Maris-Johanna; Laasfeld, Tõnis; Schettler, Franziska; Gattor, Albert O.; Hübner, Harald; Archipowa, Nataliya; Köckenberger, Johannes; Heinrich, Markus R.; Gmeiner, Peter; Kutta, Roger J.; Holliday, Nicholas D.; Rinken, Ago & Keller, Max
  
• Frontiers in Medicinal Chemistry (GDCh, DPhG), München March 17-20th 2024, poster presentation „UR-JG114 - a novel fluorescent tool compound for luminescencebased neuro-peptide Y Y4R binding studies” and poster presentation (Fabian Ertl) „Synthesis and characterization of a dually labelled NTS1R ligand”
  Jakob Gleixner
  
• Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools. ACS Pharmacology & Translational Science, 7(4), 1142-1168.
  Gleixner, Jakob; Kopanchuk, Sergei; Grätz, Lukas; Tahk, Maris-Johanna; Laasfeld, Tõnis; Veikšina, Santa; Höring, Carina; Gattor, Albert O.; Humphrys, Laura J.; Müller, Christoph; Archipowa, Nataliya; Köckenberger, Johannes; Heinrich, Markus R.; Kutta, Roger Jan; Rinken, Ago & Keller, Max