Library Optimization of Fluorine-Containing FC131 Analogues as CXCR4-Targeted Peptide Radiopharmaceuticals

Introduction: The CXCR4 receptor is highly expressed in numerous tumour types, thus the development of targeted imaging agents could play an imperative role in the diagnosis and treatment of various cancers. In recent years, several ligands have been found to target CXCR4, including the peptide FC131, but these have not yet been developed into clinical ¹⁸F-PET imaging agents. The objective is to create a library of fluorine-containing FC131 analogues with optimization through structural modifications of stereochemistry, N-methylation, side-chain length, and linker composition.High affinity library leads will be conjugated with a ¹⁸F-labelled prosthetic group using strain-promoted alkyne-azide cycloaddition (SPAAC) and evaluated.

Methods: A fluoro-azadibenzocyclooctyne (¹⁹F-ADIBO) was synthesized and conjugated to the peptides through SPAAC.^{<w:sdt docpart="A6BDF94BFF0E03449AA04E9D48F44B01" id="71640632" sdttag="MENDELEY_CITATION_v3_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">1</w:sdt>} FC131 analogues were synthesized using standard Fmoc solid-phase peptide synthesis, purified by preparative HPLC, and characterized by high-resolution mass spectrometry. Binding affinity towards CXCR4 was evaluated through a competitive radioligand displacement binding assay using U87 CD4+CXCR4+ cells.^{<w:sdt docpart="1DF03B440900304AA47E2740B09D7711" id="-1914853633" 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The azadibenzocyclooctyne prosthetic group precursor was synthesized to produce ¹⁸F-ADIBO as a prosthetic group for radiofluorination of lead peptides.

Results: A library of FC131 peptides conjugated to a ¹⁹F-ADIBO imaging moiety was synthesized. Exhibited by previous FC131 investigations, the Arg³ position was chosen to append the imaging moiety as it has been shown to be effectively modified without receptor affinity interference.^{<w:sdt docpart="D42F1385BD24D041A9F6A9B8AADA85A2" id="688261670" sdttag="MENDELEY_CITATION_v3_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">3</w:sdt>} Initial library analogues demonstrated that direct attachment of the imaging moiety through a triazole ring resulted in loss of affinity. This attachment disrupted essential ligand-receptor binding interactions, irrespective of amino acid stereochemistry or length, as well as backbone N-methylation. Efforts to extend the space to F-ADIBO through a 4-(azidomethyl)benzoic acid linker improved binding affinity. The addition of a linker region that incorporated hydrogen bonding/positively charged amino acids resulted in lead peptides with high receptor affinity. Library leads are subsequently being labelled with the ¹⁸F-analogue of the cyclooctyne prosthetic group to be translated into PET cancer imaging probes.

Conclusions: A library of fluorine-containing FC131 peptides was synthesized and screened against the CXCR4 receptor, a cancer biomarker of interest. Optimization of the attachment site of F-ADIBO resulted in peptides with significantly improved binding affinity. Library leads are being directly translated into PET cancer imaging probes through the substitution of the ¹⁹F-ADIBO prosthetic group with its ¹⁸F analogue to be evaluated in vitro and in vivo.

Acknowledgements

Funding: National Sciences and Engineering Research Council of Canada (NSERC). Molecular Imaging Collaborative Program, University of Western Ontario. The following reagents were obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH: U87 CD4+CXCR4+ cells, ARP-4036, contributed by Drs. HongKui Deng and Dan R. Littman.