Cyclic gallium-68 labeled peptides for specific detection of human angiotensin-converting enzyme 2

Abstract

In this study, we developed ACE2-specific, peptide-derived ⁶⁸Ga-labeled radiotracers, motivated by the hypotheses that (1) ACE2 is an important determinant of SARS-CoV-2 susceptibility, and (2) that modulation of ACE2 in 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 two 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. Results: Cyclic DX-600 derived peptides had markedly lower IC50’s than their linear counterparts. The three cyclic peptides with triglycine, aminocaproate, and polyethylene glycol linkers had calculated IC50’s 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 a hACE2 transgenic murine cohort. Pharmacologic concentrations of co-administered NOTA-PEP (“blocking”) showed significant reduction of [⁶⁸Ga]NOTA-PEP4 signals in the 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 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.