Cyclic ⁶⁸ Ga-Labeled Peptides for Specific Detection of Human Angiotensin-Converting Enzyme 2

Abstract

In this study, we developed angiotensin-converting enzyme 2 (ACE2)–specific, peptide-derived ⁶⁸Ga-labeled radiotracers, motivated by the hypotheses that ACE2 is an important determinant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) susceptibility and that modulation of ACE2 in coronavirus disease 2019 (COVID-19) drives severe organ injury. <b>Methods:</b> A series of NOTA-conjugated peptides derived from the known ACE2 inhibitor DX600 were synthesized, with variable linker identity. Since DX600 bears 2 cystine residues, both linear and cyclic peptides were studied. An ACE2 inhibition assay was used to identify lead compounds, which were labeled with ⁶⁸Ga to generate peptide radiotracers (⁶⁸Ga-NOTA-PEP). The aminocaproate-derived radiotracer ⁶⁸Ga-NOTA-PEP4 was subsequently studied in a humanized ACE2 (hACE2) transgenic model. <b>Results:</b> Cyclic DX-600–derived peptides had markedly lower half-maximal inhibitory concentrations than their linear counterparts. The 3 cyclic peptides with triglycine, aminocaproate, and polyethylene glycol linkers had calculated half-maximal inhibitory concentrations similar to or lower than the parent DX600 molecule. Peptides were readily labeled with ⁶⁸Ga, and the biodistribution of ⁶⁸Ga-NOTA-PEP4 was determined in an hACE2 transgenic murine cohort. Pharmacologic concentrations of coadministered NOTA-PEP (blocking) showed a significant reduction of ⁶⁸Ga-NOTA-PEP4 signals in the heart, liver, lungs, and small intestine. Ex vivo hACE2 activity in these organs was confirmed as a correlate to in&nbsp;vivo results. <b>Conclusion:</b> NOTA-conjugated cyclic peptides derived from the known ACE2 inhibitor DX600 retain their activity when N-conjugated for ⁶⁸Ga chelation. In vivo studies in a transgenic hACE2 murine model using the lead tracer, ⁶⁸Ga-NOTA-PEP4, showed specific binding in the heart, liver, lungs and intestine—organs known to be affected in SARS-CoV-2 infection. These results suggest that ⁶⁸Ga-NOTA-PEP4 could be used to detect organ-specific suppression of ACE2 in SARS-CoV-2–infected murine models and COVID-19 patients.

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