Development and Synthesis of Bombesin-Based Radiopharmaceutical Precursors Modified with Knottin

Bombesin receptors on the cell surface are of great interest as a target for targeted cancer therapy. One of the strategies of targeting bombesin receptors involves the use of tropic short peptides. However, the main limitation for the wide application of peptides as drugs is their low stability in vivo due to their sensitivity to extreme conditions of the internal body environment such as temperature and action of enzymes. In our work, a short bombesin peptide, taken as a basis, was modified with a knottin, a toxin with an inhibitor cystine knot, increasing thereby the stability of the short peptide under various conditions.

The aim of the investigation is to study the chemical and radiochemical stability of the structure based on the short bombesin peptide and knottin, as well as the ability of the obtained structure to bind to tumor cells.

Materials and Methods. The work analyzed the chemical and radiochemical stability of the synthesized peptide labeled with a lutetium radioisotope using high-performance liquid chromatography. A fluorescent-labeled peptide, obtained by a solid-phase peptide synthesis, was used to analyze binding to cultures expressing bombesin receptors.

Results. The analysis has shown increased chemical and radiochemical stability of the knottin-modified peptide, as compared to the commercial analog, and maintenance of a high ability to bind to receptors on the surface of cancer cells.

Conclusion. The structure created on the basis of a short bombesin peptide and knottin possesses increased stability and retains the ability to bind to cancer cells. All this allows us to consider the creation of these structures as a strategy for fabricating stabilizing scaffolds for short peptides for a peptide-receptor therapy.

1. Lu X., Lu C., Yang Y., Shi X., Wang H., Yang N., Yang K., Zhang X. Current status and trends in peptide receptor radionuclide therapy in the past 20 years (2000–2019): a bibliometric study. Front Pharmacol 2021; 12: 624534, https://doi.org/10.3389/fphar.2021.624534.
2. Sriram K., Insel P.A. G protein-coupled receptors as targets for approved drugs: how many targets and how many drugs? Mol Pharmacol 2018; 93(4): 251–258, https://doi.org/10.1124/mol.117.111062.
3. Jensen R.T., Battey J.F., Spindel E.R., Benya R.V. International Union of Pharmacology. LXVIII. Mammalian bombesin receptors: nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states. Pharmacol Rev 2008; 60(1): 1–42, https://doi.org/10.1124/pr.107.07108.
4. Moody T.W., Merali Z. Bombesin-like peptides and associated receptors within the brain: distribution and behavioral implications. Peptides 2004; 25(3): 511–520, https://doi.org/10.1016/j.peptides.2004.02.012.
5. Ramos-Álvarez I., Moreno P., Mantey S.A., Nakamura T., Nuche-Berenguer B., Moody T.W., Coy D.H., Jensen R.T. Insights into bombesin receptors and ligands: highlighting recent advances. Peptides 2015; 72: 128–144, https://doi.org/10.1016/j.peptides.2015.04.026.
6. Hoppenz P., Els-Heindl S., Beck-Sickinger A.G. Peptide-drug conjugates and their targets in advanced cancer therapies. Front Chem 2020; 8: 571, https://doi.org/10.3389/fchem.2020.00571.
7. Liolios C., Buchmuller B., Bauder-Wüst U., Schäfer M., Leotta K., Haberkorn U., Eder M., Kopka K. Monomeric and dimeric 68Ga-labeled bombesin analogues for positron emission tomography (PET) imaging of tumors expressing gastrin-releasing peptide receptors (GRPrs). J Med Chem 2018; 61(5): 2062–2074, https://doi.org/10.1021/acs.jmedchem.7b01856.
8. Engel J.B., Keller G., Schally A.V., Halmos G., Hammann B., Nagy A. Effective inhibition of experimental human ovarian cancers with a targeted cytotoxic bombesin analogue AN-215. Clin Cancer Res 2005; 11(6): 2408–2415, https://doi.org/10.1158/1078-0432.ccr-04-1670.
9. Reubi J.C., Fleischmann A., Waser B., Rehmann R. Concomitant vascular GRP-receptor and VEGF-receptor expression in human tumors: molecular basis for dual targeting of tumoral vasculature. Peptides 2011; 32(7): 1457–1462, https://doi.org/10.1016/j.peptides.2011.05.007.
10. Fleischmann A., Waser B., Reubi J.C. High expression of gastrin-releasing peptide receptors in the vascular bed of urinary tract cancers: promising candidates for vascular targeting applications. Endocr Relat Cancer 2009; 16(2): 623–633, https://doi.org/10.1677/erc-08-0316.
11. Moody T.W., Moreno P., Jensen R.T. Neuropeptides as lung cancer growth factors. Peptides 2015; 72: 106–111, https://doi.org/10.1016/j.peptides.2015.03.018.
12. Schulz S., Röcken C., Schulz S. Immunohistochemical detection of bombesin receptor subtypes GRP-R and BRS-3 in human tumors using novel antipeptide antibodies. Virchows Arch 2006; 449(4): 421–427, https://doi.org/10.1007/s00428-006-0265-7.
13. Li M., Liang P., Liu D., Yuan. F., Chen G.C. Zhang L. Liu Y., Liu H. Bombesin receptor subtype-3 in human diseases. Arch Med Res 2019; 50(7): 463–467, https://doi.org/10.1016/j.arcmed.2019.11.004.
14. Erspamer V., Falconieri Erspamer G., Inselivini M., Negri L. Occurrence of bombesin and alytesin in extracts of the skin of three European discoglossid frogs and pharmacological actions of bombesin on extravascular smooth muscle. Br J Pharmacol 1972; 45(2): 333–348, https://doi.org/10.1111/j.1476-5381.1972.tb08087.x.
15. Minamino N., Kangawa K., Matsuo H. Neuromedin C: a bombesin-like peptide identified in porcine spinal cord. Biochem Biophys Res Commun 1984; 119(1): 14–20, https://doi.org/10.1016/0006-291x(84)91611-5.
16. McDonald T.J., Jörnvall H., Nilsson G., Vagne M., Ghatei M., Bloom S.R., Mutt V. Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue. Biochem Biophys Res Commun 1979; 90(1): 227–233, https://doi.org/10.1016/0006-291x(79)91614-0.
17. Xiao C., Reitman M.L. Bombesin-like receptor 3: physiology of a functional orphan. Trends Endocrinol Metab 2016; 27(9): 603–605, https://doi.org/10.1016/j.tem.2016.03.003.
18. Iurova E., Beloblov I., Beloborodov E., Rastorgueva E., Pogodina E., Tazintseva E., Fomin A. PSMA-specific peptide with inhibitor cystine knot for prostate cancer treatment. Sys Rev Pharm 2020; 11(9): 187–194.
19. George S.C., Samuel E.J.J. Developments in 177Lu-based radiopharmaceutical therapy and dosimetry. Front Chem 2023; 11: 1218670, https://doi.org/10.3389/fchem.2023.1218670.
20. Tornesello A.L., Buonaguro L., Tornesello M.L., Buonaguro F.M. New insights in the design of bioactive peptides and chelating agents for imaging and therapy in oncology. Molecules 2017; 22(8): 1282, https://doi.org/10.3390/molecules22081282.
21. Baranyai Z., Tircsó G., Rösch F. The use of the macrocyclic chelator DOTA in radiochemical separations. Eur J Inorg Chem 2019; 2020(1): 36–56, https://doi.org/10.1002/ejic.201900706.
22. Khokhlova A., Zolotovskii I., Pogodina E., Saenko Y., Stoliarov D., Vorsina S., Fotiadi A., Liamina D., Sokolovski S., Rafailov E. Effects of high and low level 1265 nm laser irradiation on HCT116 cancer cells. In: Proceedings of the SPIE — International Society for Optical Engineering. Vol. 10861. San Francisco; 2019.
23. Nhàn N.T.T., Yamada T., Yamada K.H. Peptide-based agents for cancer treatment: current applications and future directions. Int J Mol Sci 2023; 24(16): 12931, https://doi.org/10.3390/ijms241612931.
24. Baratto L., Jadvar H., Iagaru A. Prostate cancer theranostics targeting gastrin-releasing peptide receptors. Mol Imaging Biol 2018; 20(4): 501–509, https://doi.org/10.1007/s11307-017-1151-1.
25. Smith C.J., Gali H., Sieckman G.L., Higginbotham C., Volkert W.A., Hoffman T.J. Radiochemical investigations of 99mTc-N3S-X-BBN[7-14]NH2: an in vitro/in vivo structure-activity relationship study where X = 0-, 3-, 5-, 8-, and 11-carbon tethering moieties. Bioconjug Chem 2003; 14(1): 93–102, https://doi.org/10.1021/bc020034r.
26. Van de Wiele C., Dumont F., Vanden Broecke R., Oosterlinck W., Cocquyt V., Serreyn R., Peers S., Thornback J., Slegers G., Dierckx R.A. Technetium-99m RP527, a GRP analogue for visualisation of GRP receptor-expressing malignancies: a feasibility study. Eur J Nucl Med 2007; 27: 1694–1699, https://doi.org/10.1007/s002590000355.
27. Wang L., Zhang Z., Merkens H., Zeisler J., Zhang C., Roxin A., Tan R., Bénard F., Lin K.S. 68Ga-labeled [Leu13ψThz14]bombesin(7–14) derivatives: promising GRPR-targeting PET tracers with low pancreas uptake. Molecules 2022; 27(12): 3777, https://doi.org/10.3390/molecules27123777.
28. Pernot M., Vanderesse R., Frochot C., Guillemin F., Barberi-Heyob M. Stability of peptides and therapeutic success in cancer. Expert Opin Drug Metab Toxicol 2011; 7(7): 793–802, https://doi.org/10.1517/17425255.2011.574126.
29. Shipp M.A., Tarr G.E., Chen C.Y., Switzer S.N., Hersh L.B., Stein H., Sunday M.E., Renherz E.L. CD10/neutral endopeptidase 24.11 hydrolyzes bombesin-like peptides and regulates the growth of small cell carcinomas of the lung. Proc Natl Acad Sci U S A 1991; 88(23): 10662–10666, https://doi.org/10.1073/pnas.88.23.10662.
30. Di L. Strategic approaches to optimizing peptide ADME properties. AAPS J 2015; 17(1): 134–143, https://doi.org/10.1208/s12248-014-9687-3.
31. Silverman A.P., Levin A.M., Lahti J.L., Cochran J.R. Engineered cystine-knot peptides that bind alpha(v)beta(3) integrin with antibody-like affinities. J Mol Biol 2009; 385(4): 1064–1075, https://doi.org/10.1016/j.jmb.2008.11.004.
32. Kimura T. Stability and safety of inhibitor cystine knot peptide, GTx1-15, from the tarantula spider Grammostola rosea. Toxins (Basel) 2021; 13(9): 621, https://doi.org/10.3390/toxins13090621.
33. Accardo A., Galli F., Mansi R., Del Pozzo L., Aurilio M., Morisco A., Ringhieri P., Signore A., Morelli G., Aloj L. Pre-clinical evaluation of eight DOTA coupled gastrin-releasing peptide receptor (GRP-R) ligands for in vivo targeting of receptor-expressing tumors. EJNMMI Res 2016; 6(1): 17, https://doi.org/10.1186/s13550-016-0175-x.
34. Körner M., Waser B., Rehmann R., Reubi J.C. Early over-expression of GRP receptors in prostatic carcinogenesis. Prostate 2014; 74(2): 217–224, https://doi.org/10.1002/pros.22743.
35. Rick F.G., Buchholz S., Schally A.V., Szalontay L., Krishan A., Datz C., Stanlmar A., Aigner E., Perez R., Seitz S., Block N.L., Hohla F. Combination of gastrin-releasing peptide antagonist with cytotoxic agents produces synergistic inhibition of growth of human experimental colon cancers. Cell Cycle 2012; 11(13): 2518–2525, https://doi.org/10.4161/cc.20900.
36. Kalia J., Milescu M., Salvatierra J., Wagner J., Klint J.K., King G.F., Olivera B.M., Bosmans F. From foe to friend: using animal toxins to investigate ion channel function. J Mol Biol 2015; 427(1): 158–175, https://doi.org/10.1016/j.jmb.2014.07.027.

Beloborodov Е.А., Iurova E.V., Fomin А.N., Saenko Yu.V. Development and Synthesis of Bombesin-Based Radiopharmaceutical Precursors Modified with Knottin. Sovremennye tehnologii v medicine 2024; 16(2): 5, https://doi.org/10.17691/stm2024.16.2.01