Designed variants of ACE2-Fc that decouple anti-SARS-CoV-2 activities from unwanted cardiovascular effects

Abstract

Angiotensin-converting enzyme 2 (ACE2) is the entry receptor for SARS-CoV-2, and recombinant ACE2 decoys are being evaluated as new antiviral therapies. We designed and tested an antibody-like ACE2-Fc fusion protein, which has the benefit of long pharmacological half-life and the potential to facilitate immune clearance of the virus. Out of a concern that the intrinsic catalytic activity of ACE2 may unintentionally alter the balance of its hormonal substrates and cause adverse cardiovascular effects in treatment, we performed a mutagenesis screening for inactivating the enzyme. Three mutants, R273A, H378A and E402A, completely lost their enzymatic activity for either surrogate or physiological substrates. All of them remained capable of binding SARS-CoV-2 and could suppress the transduction of a pseudotyped virus in cell culture. This study established new ACE2-Fc candidates as antiviral treatment for SARS-CoV-2 without potentially harmful side effects from ACE2's catalytic actions toward its vasoactive substrates.

Keywords: ACE2-Fc; COVID-19; Mutagenesis; SARS-CoV-2.

Fig. 1

Mutagenesis strategies for catalytic inactivation of ACE2. A. Schematic representation of chimeric ACE2-Fc. The ectodomain sequence from amino acid 19 to 740 of human ACE2 is fused to the N-terminus of Fc domain of human IgG1, which forms a dimer via disulfide bridges (blue lines). The overall plan was to make individual point mutations (denoted as stars) of the catalytic site to inactivate the ACE2 peptidase activity. B. Mutant ACE2-Fc proteins, each carries a single alanine substitution of a selected residue, were produced by HEK-293 cells. As predicted, the proteins ran above the 250 kDa marker under nonreducing condition. C. The cocrystal structure (PDB: 6M0J) shows SARS-CoV-2 spike RBD binding of ACE2. ACE2 exists in a clam shell-like configuration holding a catalytic cleft between its proximal and distal lobes. A zinc ion resides within the proximal lobe of the cleft void. D. In an inhibitor (MLN-4760)-bound structure of ACE2 (PBD: 1R4L), the inhibitor induced a conformational change of the catalytic cleft to adapt a ‘closed’ configuration [41]. E. Three proximal lobe residues H374, H378 and E402 formed interactions with the zinc ion. F. The side chains of six proximal and distal lobe residues, E145, R273, H345, P346, D368 and H505 (in green) formed direct interactions with inhibitor MLN-4760 (in magentas).

Fig. 2

Substrate-dependent inactivation of ACE2-Fc peptidase among ACE2-Fc mutants. Three peptide substrates were tested in catalytic reactions with ten individual variants of ACE2-Fc (wild-type and 9 mutants). The reactions were carried out in two different ways. Left panels: the reactions were performed using a high amount of purified ACE2-Fc enzyme (100 ng) with varying concentrations of the substrates between 0.39 μM and 200 μM (x-axis). Right panels: a lower dose of 10 ng ACE2-Fc was incubated with a fixed amount of 2 nmole of Mca-APK(Dnp) or 10 nmole of AngII/Apelin-13. Reactions proceeded for a standard length of time of 20 min. A. Surrogate fluorogenic substrate Mca-AP↓K(Dnp) was tested (↓: cleavage site). ACE2-Fc peptidase activities were compared between wild-type and mutants. Seven out of the nine mutants showed a completely loss-of-activity. B. When AngII was used in reactions with the ACE-2-Fc panel, DRVYIHP↓F of AngII sequence was cleaved by ACE2, releasing a Phe/F. The assay detected the generation of amino acid Phe/F as the results of ACE2-Fc activity. C. Similar to AngII, Apelin-13 was cleaved by ACE2 between the proline(P)-phenylalanine (F) bond in its sequence, Pyr-RPRLSHKGPMP↓F. The rates of Phe/F release from the reactions were detected. Data are shown as the mean ± SD from triplicate experiments.

Fig. 3

Binding affinities of individual ACE2-Fc variants to RBD of SARS-CoV-2 spike protein. A. A fixed amount of recombinant viral RBD protein was coated to an ELISA plate (buffer coated well were used as controls). Wild-type and nine mutants of ACE2-Fc were added to the wells at varying concentrations between 0.5 ng/mL and 1200 ng/mL (x-axis). Binding was determined by the difference in signal intensity between the RBD-coated and the corresponding control wells. B. While all variants of ACE2-Fc exhibited affinity to viral RBD protein, there were differences in their calculated EC₅₀ values. Data are shown as the mean ± SD from triplicate experiments.

Fig. 4

Inhibition of a pseudotyped virus by wild-type ACE2-Fc and ∆ACE2-Fc variants. A. The transduction activity of a pseudotyped virus expressing SARS-CoV-2 spike protein to HEK293 cells expressing receptor ACE2 was measured through a firefly luciferase reporter. The cell transduction assays were performed in the presence of various concentrations of individual ACE2-Fc variants. B. IC₅₀ values were calculated based on calculated ACE2-Fc concentrations needed to inhibit 50% reporter activity. Data are shown as the mean ± SD from triplicate experiments.

Fig. 5

Pharmacokinetics of wild-type ACE2-Fc and ∆ACE2-Fc variants. A. After a bolus i.v. injection of the listed ACE2-Fc variants in mice, drug concentration in blood was monitored over a period time. B. t_(1/2) values of individual biologics were calculated by GraphPad Prism software. Data are shown as the mean ± SD (n = 3).