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

  1. Matthew F. L. Parker
  2. Joseph Blecha
  3. Oren Rosenberg
  4. Michael Ohliger
  5. Robert R. Flavell
  6. David M. Wilson

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Abstract

In this study, we developed ACE2-specific, peptide-derived 68 Ga-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 68 Ga to generate peptide radiotracers ([ 68 Ga]NOTA-PEP). The aminocaproate-derived radiotracer [ 68 Ga]NOTA-PEP4 was subsequently studied in a humanized ACE2 (hACE2) transgenic model.

Results

Cyclic DX-600 derived peptides had markedly lower IC 50 ’s than their linear counterparts. The three cyclic peptides with triglycine, aminocaproate, and polyethylene glycol linkers had calculated IC 50 ’s similar to, or lower than the parent DX600 molecule. Peptides were readily labeled with 68 Ga, and the biodistribution of [ 68 Ga]NOTA-PEP4 was determined in a hACE2 transgenic murine cohort. Pharmacologic concentrations of co-administered NOTA-PEP (“blocking”) showed significant reduction of [ 68 Ga]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 68 Ga chelation. In vivo studies in a transgenic hACE2 murine model using the lead tracer [ 68 Ga]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 [ 68 Ga]NOTA-PEP4 could be used to detect organ-specific suppression of ACE2 in SARS-CoV-2 infected murine models and COVID-19 patients.

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    SciScore for 10.1101/2020.12.15.412809: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Free gallium migrates to the solvent front (∼90 mm) and bound gallium remains at the origin (∼20 mm). μPET/CT imaging: All animal procedures were approved by the UCSF Institutional Animal Care and Use Committee, and all studies were performed in accordance with UCSF guidelines regarding animal housing, pain management, and euthanasia.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Experimental Models: Organisms/Strains
    SentencesResources
    Humanized ACE2 recombinant mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J, 034860, Jackson Laboratory) were obtained from Jackson Labs, aged 6-10 weeks38–40.
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    Software and Algorithms
    SentencesResources
    All statistical analysis was performed using Microsoft Excel.
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    In other words, high background signal due to the normal excretion pathway of [68Ga]NOTA-PEP4 may represent a limitation of this method to detect ACE2 activity in the kidney. In the future, hACE2 expression-specific [68Ga]NOTA-PEP4 signals versus background excretion needs to be further clarified, perhaps using koACE2 animals50 in addition to the inhibitory studies described in this manuscript. The in vivo studies performed also reflect a limitation of most academic centers in the United States; specifically, few facilities have a biosafety level 3 (BSL-3) 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 studies51. 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 rise52. 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.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 24. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.12.15.412809 on bioRxiv