ACE2 Inhibitor Screening Assay Kit

Species Reactivity

Human

Detection Method

Inhibitor Screening Kit

Intended Use

The ACE2 Inhibitor Screening Assay Kit provides a robust and easy-to-use platform for testing potential inhibitors of human ACE2. The assay uses an ACE2-specific fluorogenic substrate, Mca-APK (Dnp). ACE2 cleaves this substrate generating free Mca, which can be easily quantified using a fluorescence plate reader at excitation and emission wavelengths of 320 and 405 nm, respectively.
The potent and reversible ACE2 inhibitor MLN-4760 is included as a positive control. Human ACE2 plays an important role in regulating blood pressure. It is also known to bind SARS-CoV-2 spike. This assay can be used as a counter-screen for potential drug candidates to ensure they do not interfere with the normal physiological functions of ACE2.

Contents of Kit

Kit will arrive packaged as a -80°C kit. After opening kit, store individual components as stated below.
1. ACE2 Assay Buffer (10×), 1vial/10 ml, -20°C
2. ACE2 Enzyme (human, recombinant), 1 vial/20 ul, -80°C
3. ACE2 Substrate(Mca-APK(Dnp)), 1 vial/150 ul, -20°C
4. ACE2 Inhibitor(MLN-4760), 1 vial/40 ul, -20°C
5. Half-Volume 96-Well Solid Plate, 1 plate, RT(black)
6. 96-Well Cover Sheet, 1ea, RT

Storage

This kit will perform as specified if stored as directed in the Reagents And Materials Provided section and used before the expiration date indicated on the outside of the box.

Performance Characteristics

Z' Factor:
Z' factor is a term used to describe the robustness of an assay, which is calculated using the equation below.

The theoretical upper limit for the Z' factor is 1.0. A robust assay has a Z' factor >0.5. The Z' factor for ACE2 Inhibitor Screening Assay Kit was determined to be 0.96.
The data shown here is an example of the data typically produced with this kit; however, your results will not be identical to these. Do not use the data below to directly compare to your samples. Your results could differ substantially.

Figure 2. Inhibition of ACE2 by ACE2 Inhibitor (MLN-4760). Data are plotted as the mean of triplicate measurements ± the standard deviation. The vehicle control (Veh.) represents 100% initial activity. The IC50 value of ACE2 Inhibitor (MLN-4760) is 0.74 nM.

Figure 3. Typical Z' data for the ACE2 Inhibitor Screening Assay Kit. Data are shown from 40 replicates each for vehicle control (Veh.), which represents 100% initial activity, and positive control wells prepared as described in the kit booklet. The calculated Z' factor for this experiment was 0.96. The red dotted lines correspond to three standard deviations from the mean for each control value.
Effects of Solvents:
Compounds may be prepared in organic solvents such as DMSO, DMF, or shortchain alcohols (e.g., MeOH, EtOH), as long as the final concentration of organic solvents in the assay is ≤5% for MeOH, <1% for EtOH, and <1% for DMSO and DMF. A titration of organic solvents showed that the signal decreases with increasing DMSO, DMF, and EtOH concentration so the proper vehicle control should be included in the assay.

Figure 4. The effect of solvent on the readout of ACE2 activity. The data are shown as the mean ± standard deviation for triplicate reactions containing the indicated concentration of solvents.

Precision

Intra-assay precision was determined by analyzing 24 measurements of the background and a vehicle control on the same day. The intra-assay coefficients of variation were 2.9 and 3.1%, respectively. The intra-assay coefficient of variation for the IC50 value of seven inhibition curves performed on the same day was 3.3%.
Inter-assay precision was determined by analyzing inhibition with ACE2 Inhibitor (MLN-4760) in five separate assays on different days. The inter-assay coefficient of variance for the IC50 value was 11.8%.

General Description

Angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase and homolog of ACE1 that is encoded by ACE2 in humans. It is a type I transmembrane protein composed of a cytoplasmic tail and an extracellular domain containing an HEMGH zinc-binding motif, which exhibits carboxypeptidase activity. ACE2 is expressed in vascular endothelial cells where it catalyzes the conversion of angiotensin II to the vasodilatory peptide angiotensin to regulate systemic blood pressure and angiotensin I to angiotensin, a peptide that counter-regulates the function of angiotensin II. It is also expressed in the epithelial cells of the kidney, heart, lung, small intestine, and liver and has roles in fluid homeostasis, cardiac contractility, and amino acid absorption, as well as the prevention of pulmonary fibrosis and hypertension. ACE2 also acts as a functional receptor for severe acute respiratory syndrome coronavirus(SARS-CoV) and SARS-CoV-2 to facilitate viral entry into host cells. Inhibitors of the ACE2 and SARS-CoV-2 interaction may be beneficial against viral infection in the treatment of COVID-19. However, such inhibitors should not affect ACE2's carboxymonopeptidase activity, as doing so could exacerbate COVID-19 comorbidities as seen in Ace2 knockout mouse studies. Ace2-/- mice exhibit increased angiotensin Ill-induced hypertension, susceptibility to atherosclerotic plaques, myocardial dysfunction, and insulin resistance compared with wild-type mice.