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. 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 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 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 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.

在这项研究中，我们开发了血管紧张素转换酶 2 (ACE2) 特异性、肽衍生的⁶⁸ Ga 标记放射性示踪剂，其动机是假设 ACE2 是严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 的重要决定因素2019 年冠状病毒病 (COVID-19) 中 ACE2 的调节会导致严重的器官损伤。方法：合成了一系列源自已知 ACE2 抑制剂 DX600 的 NOTA 缀合肽，具有可变的接头身份。由于 DX600 带有 2 个胱氨酸残基，因此对线性肽和环状肽进行了研究。 ACE2 抑制测定用于鉴定先导化合物，这些化合物用⁶⁸ Ga 标记以生成肽放射性示踪剂 ( ⁶⁸ Ga-NOTA-PEP)。随后在人源化 ACE2 (hACE2) 转基因模型中研究了氨基己酸衍生的放射性示踪剂⁶⁸ Ga-NOTA-PEP4。结果：环状 DX-600 衍生肽的半最大抑制浓度明显低于其线性对应物。具有三甘氨酸、氨基己酸酯和聚乙二醇接头的 3 种环肽计算出的半最大抑制浓度类似于或低于母体 DX600 分子。肽很容易用⁶⁸ Ga 标记，并且在 hACE2 转基因小鼠队列中确定了⁶⁸ Ga-NOTA-PEP4 的生物分布。联合给药的 NOTA-PEP（阻断）的药理学浓度显示心脏、肝脏、肺和小肠中的⁶⁸ Ga-NOTA-PEP4 信号显着降低。这些器官中的离体 hACE2 活性被证实与体内结果相关。 结论：源自已知 ACE2 抑制剂 DX600 的 NOTA 缀合环肽在 N 缀合进行⁶⁸ Ga 螯合时保留其活性。使用先导示踪剂⁶⁸ Ga-NOTA-PEP4 在转基因 hACE2 小鼠模型中进行的体内研究显示，在心脏、肝脏、肺和肠道（已知在 SARS-CoV-2 感染中受影响的器官）中具有特异性结合。这些结果表明⁶⁸ Ga-NOTA-PEP4 可用于检测 SARS-CoV-2 感染的小鼠模型和 COVID-19 患者中 ACE2 的器官特异性抑制。