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

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. 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-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. TOC figure For Table of Contents use only


Introduction:
The novel coronavirus SARS-CoV-2 has had profound effects on global health, especially in the United States, the country with the largest number of confirmed COVID-19 cases, and associated deaths. Many of these patients progress to Acute Respiratory Distress Syndrome (ARDS), respiratory failure with widespread injury of the lungs. The underlying mechanisms include diffuse alveolar damage, surfactant dysfunction, and immune cell activation 1-3 . Of note, many pathologic conditions can cause this convergent picture, including both bacterial and viral infections. These causes of ARDS likely share dysfunction of the renin-angiotensin system, especially loss of angiotensin converting enzyme II (ACE2) function [4][5][6][7][8] . ACE2 is a transmembrane protein that functions as angiotensin receptor (ATR) chaperone. The roles of ACE2, ACE, and angiotensin II are highlighted in Figure 1A which describes dual functions of the renin-angiotensin system with opposing effects on cardiovascular biology 9 . In this pathway, ACE2 performs an important regulatory role, converting angiotensin II to angiotensin 1,7 which causes vasodilatation and has anti-inflammatory effect, unlike activation of ATR which will lead to vasoconstriction, higher blood pressure and inflammation (potentially ARDS) [10][11][12][13] .
Although several recent papers suggest that other mammalian transmembrane proteins (for example CD147 and CD26) allow SARS-C0V-2 to infect different cell types 14,15 , ACE2 is the main point of entry of the virus into host cells ( Figure 1B). This process depends on this receptor as well as on its spike (S) protein, with cryo-EM and Xray crystal structures of the complex recently described, as well as characterization of the complex via atomic force microscopy ( Figure 1C) [16][17][18] . This protein has 2 subunits-S1 containing receptor binding domains (RBDs), and S2, which is responsible for membrane fusion. The RBDs can mimic the ACE2 interaction with ATR (hydrophobic and strong electrostatic interactions, including pi-pi, and cation-pi) and gain entry via strong non-covalent attachment to ACE2 in the ATR binding site 19 . Three recent cryo-EM studies demonstrated that SARS-CoV-2 spike protein directly binds to ACE2 and that the SARS-CoV-2 spike protein likely recognizes human ACE2 with even higher binding affinity than spike from SARS-CoV [20][21][22] . This binding was suggested to alter virus configuration and expose a cleavage site on S2, resulting in host protease cleavage (mainly by transmembrane protease/serine subfamily member 2 -TMPRSS2), allowing the virus to enter the cell 23 . This mechanism was recently supported by a cryo-EM post fusion analysis that showed structural and conformational rearrangements of the Sprotein compared to its pre-fusion structure 24 .
To investigate SARS-CoV-2 susceptibility, and organ-specific suppression of ACE2 in COVID-19, new ACE2-specific imaging methods would be profoundly helpful.
However, ACE2-specific small molecules and peptides developed following the original 2003 SARS-CoV outbreak (this virus also depends on ACE2 for viral entry) offer clues as to how active-site targeted, high affinity ligands might be developed. A large number of ACE2-specific ligands have been reported, generally characterized by their ACE2 IC 50 's, and including the peptide DX600 discovered via phage display [25][26][27][28][29][30] . The DX600 sequence is shown in Figure 1D. In this manuscript, we report development of ACE2specific PET radiotracers ([ 68 Ga]NOTA-PEP) derived from this sequence. We anticipate that ACE2-specific PET could help evaluate which systems are most targeted by SARS-CoV infection, the timing of disease, and how ACE2 modulation correlates with ARDS susceptibility and other organ injury. Recent work has highlighted the role of ACE2 in a large number of organs beyond the lungs, including the heart, kidneys, and gastrointestinal system [31][32][33][34][35][36] . We therefore believe that the information gleaned from [ 68 Ga]NOTA-PEP4 or some other in vivo ACE2 sensor will potentially be helpful in COVID-19 treatment, either via exogenous ACE2 4,37 or some other therapy.

Materials and Methods:
Peptides: The DX600-derived peptides studied were obtained from AnaSpec (Fremont, CA) as a custom synthesis, fully characterized by HPLC and mass spectrometry. These peptides were radiolabeled without additional modification.   Ex vivo analyses of mice: Upon completion of imaging, mice were sacrificed and biodistribution analysis performed. Gamma counting of harvested tissues was performed using a Hidex Automatic Gamma Counter (Turku, Finland). Organs were also harvested from a separate cohort of mice for an ACE2 activity assay. The tissues were homogenized and aliquots were used for protein concentration using a standard Bradford assay. Additional tissue aliquots were used as the source of ACE2 in a commercially available ACE2 assay (AnaSpec, Fremont, CA). The initial velocities were normalized relative to muscle tissue. Relative activities are reported as the relative initial velocity/g of protein. Data were analyzed using an unpaired two-tailed Student's t-test. All graphs are depicted with error bars corresponding to the standard error of the mean.

Results:
NOTA-conjugated, cyclic peptides targeting the ACE2 active site retain their potency relative to the DX600 parent compound. Based on our hypothesis that potent peptide-derived ACE2 inhibitors, modified with linkers/chelating groups will retain their activity and specificity, several NOTA-modified peptide-derived ACE2 inhibitors derived from the DX600 sequence 29 (K i = 2.8 nM, K d = 10.8 nM) were synthesized and screened for ACE2 inhibition. These were synthesized via Fmoc-protected linkers and N-capping NOTA reagents (Figure 2A,B). The general structure pursued was a NOTA-linkerpeptide with three different linkers used, conferring varying degrees of hydrophobicity and hydrogen bonding: triglycine, PEG, or caproic acid. These were synthesized using standard Fmoc solid-phase synthesis 41 (AnaSpec, Fremont CA) with purity and identity confirmed by HPLC and mass spectrometry. Because DX600 contains two cysteine residues, a cyclized set of peptides were also synthesized via disulfide bridge formation 42 . When these compounds were compared to the parent DX600 peptide in a commercially available fluorometric ACE2 inhibition assay (AnaSpec), all three cyclic peptides (NOTA-PEP2, NOTA-PEP4, NOTA-PEP6) showed ACE2 inhibition nearly identical to DX600 (Figure 2C,D,E). In other words, the N-terminal modification caused no loss of inhibitory activity when compared to the parent peptide, and in fact the cyclic peptide NOTA-PEP4 was a slightly better ACE2 inhibitor than DX600. In contrast, the linear derivatives showed much lower activity, which may result from a solution confirmation for which the NOTA interferes with ACE2 active site binding. To further evaluate this loss of potency, we studied ACE2 inhibition using a cyclic NOTA-PEP with and without addition of the reducing agent tris(2-carboxyethyl)phosphine (TCEP) which is expected to reduce the disulfide bridge in the cyclic peptide (producing the linear NOTA-PEP5) (Figure 2F. Supp. Fig. 1). As anticipated, addition of TCEP markedly increased the observed ACE2 IC 50 .

[ 68 Ga]NOTA-PEP4 signals in the lungs, heart, small intestine and liver of hACE2 transgenic mice are attenuated with co-administration of inhibitory cyclic peptide.
Having developed a radiosynthesis of [ 68 Ga]NOTA-PEP4, we sought to further validate the tracer in a transgenic, humanized ACE2 (hACE2) murine model. The  Figure 3B, Supp. Fig. 3). ROI analysis of the images demonstrated prompt clearance from the blood pool with accumulation in the kidneys, as expected for a small peptide tracer.
Next, we performed an imaging and biodistribution study, to show that [ 68 Ga]NOTA-PEP4 demonstrates specific uptake in tissues with increased expression of ACE2. In order to demonstrate specificity of uptake, blocking with excess cyclic NOTA-PEP inhibitory peptide was employed. With blocking, significant reductions in cyclic [ 68 Ga]NOTA-PEP4 were seen in the heart (p = 0.0203), lung (p <0.0001), liver (p <0.0001) and small intestine (p = 0.0002). ACE2 activity in these organs was subsequently confirmed via harvested organs in a separate hACE2 cohort (N = 3, Supp.   Fig. 4). Taken together, these data demonstrate that [ 68 Ga]NOTA-PEP4 can specifically bind to tissues with high ACE2 expression.

Discussion:
The (1) by replenishing "protective" ACE2 function and (2) by serving as a "decoy" receptor for the virus. These therapeutic effects, the differential susceptibility of individuals (based on age, co-morbidities) to COVID-19, and the organ-specific effects of SARS-CoV-2 are all potentially addressed by an ACE2-specific imaging method. We therefore sought a PET tracer derived from known inhibitor structures, via modification of the known ACE2 inhibitory peptide DX600 with 68 Ga.
Inhibitor-derived structures modified for PET do not necessarily recapitulate the potency of their parent compounds, so our first efforts focused on the "cold" NOTAconjugated DX600 derived peptides, derived from triglycine, caproic acid, and PEG linkers. Gratifyingly, the DX600-derived cyclic peptides studied all showed ACE2 activity similar to the parent peptide. In contrast, the linear versions were relatively inactive, which may reflect conformational effects. Of note, the calculated IC 50 of DX600 (standard included in AnaSpec assay kit) was > 1 order of magnitude higher than the K i reported by Huang et al. 29 , likely reflecting numerous experimental differences (enzyme concentration and activity, etc.). We therefore considered the IC 50 of the NOTA-derived peptides relative to that of DX600 to be the most important determinant of successful PET probe development. Indeed, our lead cyclic peptide NOTA-PEP4 had an IC 50 lower than that of the DX600 parent, motivating the radiolabeling of NOTA-PEP4 for subsequent imaging studies. compatible μPET-CT imaging system. Future molecular imaging of live SARS-CoV-2 (a BSL-3 organism) and its host effects will therefore require collaborative work with those few centers able to accommodate these studies 51 . Given the history of ACE2 with respect to SARS-CoV (the 2003 SARS coronavirus) and ARDS, we expect that new ACE2-specific PET tools will be relevant beyond the current pandemic. We are partially motivated by data indicating that zoonotic infections especially coronavirus-related are on the rise 52 . The incidence of emerging and re-emerging zoonotic disease is increasing in many parts of the world, with animal viruses able to cross species barriers to infect humans; it appears likely that ACE2 will be relevant in future pandemics. Better understanding ACE2 suppression, and differential susceptibility to SARS-COV-2 will help us better treat COVID-19 and other diseases for which ACE2 plays a critical role.

Conclusions:
Our study shows that the ACE2 active site-targeted inhibitor DX600 can be modified for  force microscopy have elucidated the interaction between the spike protein S1 subunit and ACE2. Of note S1 binds to an ACE2 site remote to its active site, which is targeted by the inhibitory peptides described in this manuscript. Images adapted from Yang et al.    was seen in the heart, lungs, liver, and small intestine, organs implicated in COVID-19.
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