Evaluation of Angiotensin-Converting\nEnzyme 2 Expression <i>In Vivo</i> with Novel <sup>68</sup>Ga-Labeled Peptides Originated\nfrom the Coronavirus Receptor-Binding Domain

Angiotensin-converting enzyme 2 (ACE2) is not only a\nkey to the\nrenin–angiotensin–aldosterone system and related diseases,\nbut also the main entry point on cell surfaces for certain coronaviruses,\nincluding severe acute respiratory syndrome coronavirus (SARS-CoV)\nand SARS-CoV-2. By analyzing the different key binding sites from\nthe receptor-binding domain (RBD) of SARS-CoV and SARS-CoV-2, nine\nnew ACE2-targeting peptides (A<b>1</b> to A<b>9</b>) were\ndesigned, synthesized and connected with a chelator, 1,4,7-triazacyclononane-<i>N,N′,N’</i>’-triacetic acid (NOTA). NOTA-A<b>1</b>, NOTA-A<b>2</b>, NOTA-A<b>4</b>, NOTA-A<b>5</b>, and NOTA-A<b>8</b> were successfully labeled with\n[<sup>68</sup>Ga]Ga<sup>3+</sup> and were used for biological evaluation.\n[<sup>68</sup>Ga]Ga-NOTA-A<b>2</b>, [<sup>68</sup>Ga]Ga-NOTA-A<b>5</b>, and [<sup>68</sup>Ga]Ga-NOTA-A<b>8</b> showed specific\nbinding to ACE2 via cell assays, and their binding sites and binding\ncapacity were calculated by molecular docking and molecular dynamics\nsimulations. In tumor-bearing mice, A549 tumors were visualized 60\nmin postinjection of [<sup>68</sup>Ga]Ga-NOTA-A<b>2</b>, [<sup>68</sup>Ga]Ga-NOTA-A<b>5</b>, or [<sup>68</sup>Ga]Ga-NOTA-A<b>8</b>. These peptides also accumulated in the organs with high-level\nACE2 expression, confirmed by immunohistochemical stain. Among them,\n[<sup>68</sup>Ga]Ga-NOTA-A<b>5</b> exhibited the highest tumor\nuptake and tumor/background ratio, and it successfully tracked the\nincreased ACE2 levels in mice tissues after excessive Losartan treatment.\nIn a first-in-human study, the distribution of [<sup>68</sup>Ga]Ga-NOTA-A<b>5</b> was evaluated with positron emission tomography/computed\ntomography (PET/CT) in three participants without adverse events. <sup>68</sup>Ga-labeled peptides originated from the coronavirus RBD,\nwith [<sup>68</sup>Ga]Ga-NOTA-A<b>5</b> as a typical representative,\nseem to be safe and effective for the evaluation of ACE2 expression <i>in vivo</i> with PET/CT, facilitating further mechanism investigation\nand clinical evaluation of ACE2-related diseases.