ABSTRACT
In this study, we developed ACE2-specific, peptide-derived 68Ga-labeled radiotracers, based on 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. A commercially available ACE2 inhibition assay was used to identify lead compounds, which were used to synthesize 68Ga-chelated peptide radiotracers ([68Ga]-NOTA-ACE2pep). The aminocaproate-derived radiotracer [68Ga]-NOTA-ACE2pep4 was subsequently studied in a humanized ACE2 (hACE2) transgenic model, with in vivo results correlated with ACE2 activity. Results: Cyclic DX-600 derived peptides had markedly lower IC50s than their linear counterparts, a result confirmed by TCEP reduction of disulfide bridges. The three cyclic peptides derived from triglycine, aminocaproate, and polyethylene glycol linkers had calculated IC50s similar to, or lower than the parent DX600 molecule. Peptides were readily labeled with 68Ga, and the normal biodistribution of [68Ga]-NOTA-ACE2pep4 was determined in a hACE2 transgenic murine cohort. Pharmacologic concentrations of co-administered NOTA-ACE2pep (blocking) showed significant reduction of [68Ga]-NOTA-ACE2pep4 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. Conclusions: 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-ACE2pep4 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-ACE2pep4 may detect organ-specific suppression of ACE2 in SARS-CoV-2 infected murine models and COVID-19 patients.