What is the clinical evidence from this study?

Study design: Other. Intervention: DX600 vs. 1 μM DX600 or uninhibited ACE2. Primary outcome: Half maximal inhibitory concentration (IC50) of DX600 for ACE2.

What question did this study set out to answer?

To evaluate species differences in ACE2 sensitivity to the inhibitor DX600 and establish an optimized enzymatic activity assay for rodent tissues.

September 1, 2011AJP Regulatory Integrative and Comparative PhysiologyOpen Access

Species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ACE2) and its implication for ACE2 activity assays

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Key result

Human ACE2 is highly sensitive to DX600 inhibition (IC50 ~0.09 μM), whereas rodent ACE2 is much less sensitive (IC50 ~7 μM), necessitating optimized assay conditions with 10 μM DX600 at pH 6.5 to accurately quantify endogenous rodent ACE2.

Population

Recombinant human, mouse, and rat ACE2 overexpressed in cell lines and endogenous ACE2 from tissues of 3…

Comparison

DX600 at varying concentrations and pH conditions vs Absence of DX600

Design

Preclinical

Authors

KPKim Brint PedersenLouisiana State University Health Sciences Center New Orleans SSSrinivas SriramulaUniversidade Federal de Minas Gerais KCKavaljit H. ChhabraUniversity of Kentucky

Discussion

Member takes

Implication

Urges caution extrapolating recombinant ACE2 assays to endogenous activity in mice; leaves open validated protocols for cardiovascular research.

Key Points

To evaluate species differences in ACE2 sensitivity to the inhibitor DX600 and establish an optimized enzymatic activity assay for rodent tissues.
Assessed DX600 inhibitory sensitivity across human, mouse, and rat ACE2 overexpressed in cell lines using quenched fluorescent substrate hydrolysis.
Optimized assay conditions (pH, substrate concentration, and DX600 concentration) and validated endogenous ACE2 detection in wild-type and ACE2 knockout mouse tissues (kidney, brain, heart, and plasma).
DX600 inhibited ACE2 across human, mouse, and rat orthologs, but the half maximal inhibitory concentration (IC50) for human ACE2 was 78-fold lower than for rodent ACE2.
Accurate quantification of mouse and rat ACE2 required 10 μM DX600 (>95% inhibition), whereas the standard 1 μM concentration yielded inadequate inhibition (<75%).

Structured PICO

Population

In vitro and ex vivo methodology study evaluating the species-specific sensitivity of human, mouse, and rat ACE2 to the inhibitor DX600 using cell lines and mouse tissues.

Intervention

DX600 (ACE2-specific inhibitor) at varying concentrations (up to 10 μM) and pH conditions (6.5 to 8.0)

Comparator

Absence of DX600

Outcome

ACE2 enzymatic activity measured by hydrolysis of quenched fluorescent substrates (Mca-YVADAPK(Dnp) and Mca-APK(Dnp))surrogate

Rodent ACE2 is significantly less sensitive to the inhibitor DX600 than human ACE2, requiring optimized assay conditions (pH 6.5, 10 μM DX600) for accurate quantification of endogenous murine ACE2 activity.

Limitations

Inability to reproduce other groups' findings of near-complete inhibition of rodent ACE2 with 1 μM DX600
DX600 contains free cysteine residues and tends to polymerize, requiring fresh preparation
In vivo tissue concentrations and binding affinity of DX600 are unknown

Cite This Study

Pedersen et al. (2011) studied this question. DX600 vs. 1 μM DX600 or uninhibited ACE2 was evaluated on Half maximal inhibitory concentration (IC50) of DX600 for ACE2. Human ACE2 is highly sensitive to DX600 inhibition (IC50 ~0.09 μM), whereas rodent ACE2 is much less sensitive (IC50 ~7 μM), necessitating optimized assay conditions with 10 μM DX600 at pH 6.5 to accurately quantify endogenous rodent ACE2.

synapsesocial.com/papers/6a6bb8e978a09e5ab7b3b595 https://doi.org/10.1152/ajpregu.00339.2011

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Implication

Urges caution extrapolating recombinant ACE2 assays to endogenous activity in mice; leaves open validated protocols for cardiovascular research.

Species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ACE2) and its implication for ACE2 activity assays

Discussion

Member takes

Implication

Key Points

Structured PICO

Limitations

Cite This Study

Species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ACE2) and its implication for ACE2 activity assays

Discussion

Member takes

Implication

Key Points

Structured PICO

Limitations

Cite This Study

Also Consider

Also Consider