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

In this study, we developed angiotensin-converting enzyme 2 (ACE2)–specific, peptide-derived (68)Ga-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. Methods: 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 (68)Ga to generate peptide radiotracers ((68)Ga-NOTA-PEP). The aminocaproate-derived radiotracer (68)Ga-NOTA-PEP4 was subsequently studied in a humanized ACE2 (hACE2) transgenic model. Results: 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 (68)Ga, and the biodistribution of (68)Ga-NOTA-PEP4 was determined in an hACE2 transgenic murine cohort. Pharmacologic concentrations of coadministered NOTA-PEP (blocking) showed a significant reduction of (68)Ga-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 vivo results. Conclusion: NOTA-conjugated cyclic peptides derived from the known ACE2 inhibitor DX600 retain their activity when N-conjugated for (68)Ga chelation. In vivo studies in a transgenic hACE2 murine model using the lead tracer, (68)Ga-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 (68)Ga-NOTA-PEP4 could be used to detect organ-specific suppression of ACE2 in SARS-CoV-2–infected murine models and COVID-19 patients.