ACE2 Assays

Angiotensin II converting enzyme (ACE2, EC 3.4.17.23), a carboxypeptidase, is part of the renin-angiotensin system (RAS) that controls regulation of blood pressure by cleaving the C-terminal dipeptide of Angiotensin II (vasoconstrictor) to convert it into Angiotensin 1-7 (vasodilator). It also cleaves Angiotensin I to produce Ang 1-9, a protein hormone of unknown function.

For these reasons, ACE2 has been shown to be an important regulator of heart function. By cleavage of angiotensin II, ACE2 has also been reported to also have a protective role in acute lung injury. As well as this, ACE2 plays an important role in amino acid transport by acting as binding partner of amino acid transporter SL6A19 in the intestine, regulating its expression and catalytic activity.

ACE2 is a receptor of human coronaviruses, such as SARS and HCoV-NL63. It has been determined to be a functional receptor which facilitates the uptake of SARS-CoV-2 into host cells. ACE2 is expressed in multiple locations and cell types, notably including alveolar epithelial cells, surface enterocytes within the small intestine, and vascular endothelial cells of the lung, kidneys and heart.

Coronavirus entry into susceptible cells is mediated by the S1 subunit of the coronavirus transmembrane ‘spike’ glycoprotein (commonly abbreviated ‘S’). S1 contains a receptor binding domain at residues 318–510, which recognises and binds to the LYS341 residue of ACE2 with high affinity. The affinity of CoV-2 is higher than that of CoV.

Increased binding affinity to human ACE2 has been correlated with an increase in transmission efficiency in previous outbreaks of SARS-CoV. Antibodies produced against SARS-CoV-2 target the S2 subunit, which is responsible for the fusion of viral and host membranes. Thus, neutralising the S2 subunit inhibits viral entry into vulnerable cells. Polyclonal antibodies against the S protein of SARS-CoV have been found to significantly attenuate the ability of SARS-CoV-2 to enter cells via human ACE2.

Assay Genie's ACE2 Inhibitor Screening Kit can be used to screen for potent inhibitors of ACE2 activity, it utilizes the ability of an active ACE2 to cleave a synthetic MCA based peptide substrate to release a free fluorophore. The released MCA can be easily quantified using a fluorescence microplate reader. In the presence of an ACE2 specific inhibitor, the enzyme loses its peptidase activity which results in decrease of fluorescence intensity. This assay kit is simple and can be used to identify and characterize ACE2 inhibitors in a high-throughput format.

Assay Genie's ACE2 Activity Assay Kit works along a similar principle; like the ACE2 Inhibitor Screening Kit, a fluorophore is released by cleavage of MCA by ACE2. The released MCA can be easily quantified using a fluorescence microplate reader. In this way, ACE2 activity within a sample may be quantified.

Assay Genie ACE2 assays may therefore have the potential to aid in the discovery of a novel therapy for COVID-19.