Angiotensin II Converting Enzyme (ACE2) Inhibitor Screening Kit (BN00569)

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BN00569
€708

Description

Angiotensin II Converting Enzyme (ACE2) Inhibitor Screening Kit

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 to convert it into Angiotensin 1-7. It also cleaves Angiotensin I to produce Ang 1-9, of unknown function. ACE2 is a receptor of human coronaviruses, such as SARS and HCoV-NL63. It is expressed on the vascular endothelial cells of heart and kidney. The inhibitors of ACE2 could be able to regulate hypertension by changing vascular permeability. Screening for small molecule and peptide inhibitors might also help in finding treatment for coronavirus mediated infection. 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.

Figure: Inhibition of ACE2 activity by ACE2 Inhibitor, IC 50 = 33.0 ± 0.6 nM (n = 3). Assay was performed following the kit protocol.

Key Information Description

Product SKU

BN00569

Size

100 assays

Detection Method

Fluorescence (Ex/Em = 320/420 nm)

Applications

Screening/characterizing/studying ACE2 inhibitors/activators

Features and Benefits
  • Simple and reliable test to screen ACE2 inhibitors/activators
  • High-throughput compatible
  • Includes Inhibitor Control

Kit Components
  • ACE2 Assay Buffer
  • ACE2 Dilution Buffer
  • ACE2 Enzyme
  • ACE2 Substrate
  • ACE2 Inhibitor (0.5 mM)

Storage Conditions

-20°C

Shipping Conditions

Gel Pack

USAGE

For Research Use Only! Not For Use in Humans.

ACE2 function

Carboxypeptidase which converts angiotensin I to angiotensin 1-9, a peptide of unknown function, and angiotensin II to angiotensin 1-7, a vasodilator (PubMed:10969042, PubMed:10924499, PubMed:11815627). Also able to hydrolyze apelin-13 and dynorphin-13 with high efficiency (PubMed:11815627). By cleavage of angiotensin II, may be an important regulator of heart function (PubMed:10969042, PubMed:10924499). By cleavage of angiotensin II, may also have a protective role in acute lung injury (By similarity). Plays an important role in amino acid transport by acting as binding partner of amino acid transporter SL6A19 in intestine, regulating trafficking, expression on the cell surface, and its catalytic activity (PubMed:18424768, PubMed:19185582).

ACE2 & the coronavirus

Angiotensin-converting enzyme 2 (ACE2) has been determined to be a functional receptor which facilitates the uptake of SARS-CoV-2 into host cells. (Source) ACE2 is expressed in multiple locations and cell types, notably including alveolar epithelial cells, surface enterocytes within the small intestine, and heart and kidney endothelial cells.

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

Increased binding affinity to human ACE2 has been correlated with an increase in transmission efficiency in previous outbreaks of SARS-CoV. (Source) 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. (Source) 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. (Source)

ACE2 Key information

Uniprot

Q9BYF1

Gene name

ACE2

Alternative names

Angiotensin-converting enzyme 2, ACE-related carboxypeptidase, Angiotensin-converting enzyme homolog, ACEH, Metalloprotease MPROT15

Subcellular location

Extracellular region or secreted, cell membrane, cytoplasm

Tissue Specificity

Expressed in endothelial cells from small and large arteries, and in arterial smooth muscle cells (at protein level) (PubMed:15141377). Expressed in lung alveolar epithelial cells, enterocytes of the small intestine, Leydig cells and Sertoli cells (at protein level) (PubMed:15141377). Expressed in the renal proximal tubule and the small intestine (at protein level) (PubMed:18424768). Expressed in heart, kidney, testis, and gastrointestinal system (PubMed:10969042, PubMed:10924499, PubMed:15231706, PubMed:12459472, PubMed:15671045).

Induction

Up-regulated in failing heart.

ACE 2 Activity Assay Kit - Protocol

Step Procedure

ACE2 Enzyme Working Solution Preparation:

Add 198 µl of ACE2 Dilution Buffer to the ACE2 enzyme vial. The diluted enzyme can
be stored at -20°C in aliquots. For each well (Enzyme Control-EC, Sample-S, Inhibitor Control-IC and Solvent Control-SC), prepare 50 μl of ACE2 Enzyme Working Solution:

EC, S, SC and IC

  • 48 μl ACE2 Assay Buffer
  • 2 μl Diluted ACE2 enzyme solution

Background Control (BC)

  • 50 μl ACE2 Assay Buffer

Mix well and add 50 μl/well into desired wells in a 96-well microtiter plate

Screening Compounds, Inhibitor Control & Enzyme Control Preparations:

Dissolve candidate inhibitors at 100X highest final test concentration using preferred solvent. Dilute to 10X the desired test concentration with ACE2 Assay Buffer. Add 10 μl test inhibitors (Sample, S) or ACE2 Assay Buffer (EC and BC). Prepare Inhibitor Control by adding 50 µl ACE2 Assay Buffer to the vial containing ACE2 Inhibitor, mix. For Inhibitor Control (IC), add 10 μl ACE2 Inhibitor into ACE2 enzyme containing well(s). Incubate at Room temperature (RT) for 15 min.

ACE2 Substrate Mix:

Prepare enough reagents for the number of assays to be performed. For each well, prepare 40 μl of the Substrate Mix:

  • 38 μl ACE2 Assay Buffer
  • 2 μl ACE2 Substrate

Mix & add 40 µl of ACE2 Substrate Mix into Background Control, Enzyme Control/Solvent Control, Inhibitor Control & Sample (S) wells. Mix well.

Measurement:

Measure fluorescence (Ex/Em = 320/420 nm) in a kinetic mode for 1 hr at room temperature.
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