key: cord-0925260-nxrrjexb
authors: Xiao, Tianshu; Lu, Jianming; Zhang, Jun; Johnson, Rebecca I.; McKay, Lindsay G.A.; Storm, Nadia; Lavine, Christy L.; Peng, Hanqin; Cai, Yongfei; Rits-Volloch, Sophia; Lu, Shen; Quinlan, Brian D.; Farzan, Michael; Seaman, Michael S.; Griffiths, Anthony; Chen, Bing
title: A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent in vitro
date: 2020-09-18
journal: bioRxiv
DOI: 10.1101/2020.09.18.301952
sha: 54907e15d728b246236d3870253151cd4cf5e658
doc_id: 925260
cord_uid: nxrrjexb

Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase that forms a dimer and serves as the cellular receptor for SARS-CoV-2. It is also a key negative regulator of the renin-angiotensin system (RAS), conserved in mammals, which modulates vascular functions. We report here the properties of a trimeric ACE2 variant, created by a structure-based approach, with binding affinity of ~60 pM for the spike (S) protein of SARS-CoV-2, while preserving the wildtype peptidase activity as well as the ability to block activation of angiotensin II receptor type 1 in the RAS. Moreover, the engineered ACE2 potently inhibits infection of SARS-CoV-2 in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.

The current COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 29 million people worldwide, leading to over 900 thousand deaths, with devastating socio-economic impacts. Effective intervention strategies are urgently needed to control the pandemic.

Since the outbreak of the virus, several therapeutic approaches have been evaluated in the hope of providing a viable treatment for COVID-19. First, convalescent sera from individuals recovered from the infection were used with encouraging results 1-3 , but also some drawbacks (e.g., batch variations, possible blood-borne pathogens and blood-type matching). Second, patient-derived, potently neutralizing monoclonal antibodies have been isolated, which could provide a more powerful passive immunotherapy than convalescent sera [4] [5] [6] [7] . Third, structure-guided design of peptide-based viral entry inhibitors has yielded promising leads in in vitro assays 8, 9 ; their efficacy requires further clinical evaluation. Fourth, known drugs or drug candidates, including remdesivir, favipiravir and ribavirin (viral RNA polymerase inhibitors); lopinavir and ritonavir (viral protease inhibitors); as well as hydroxychloroquine, corticosteroids and interferons (with more complicated antiviral mechanisms), have been repurposed as COVID-19 therapuetics 10, 11 .

Among them, remdesivir has received Emergency Use Authorizations (EUA) from the U.S. Food and Drug Administration (FDA), while hydroxychloroquine has been shown to be ineffective 12 . Finally, many new therapeutic candidates are in various stages of development (ref 13, 14 ; https://www.bio.org/policy/human-health/vaccinesbiodefense/coronavirus/pipeline-tracker). While the infection resolves on its own in most asymptomatic and mild cases over time, in severe cases appears to progress in two phases -initial active viral replication in the respiratory system and subsequent excessive immune responses leading to multiple organ failure and possible death 15 . Thus, antivirals alone may be insufficient to change the course of disease progression for the population that needs intervention the most if administrated too late.

Human angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for SARS-CoV-promote viral entry into the host cells and initiate infection 16, 17 . It is a type I membrane glycoprotein containing an extracellular ectodomain that has metallopeptidase activity. Its neck domain near the transmembrane anchor mediates dimerization 18 . ACE2 is also a key negative regulator of the renin-angiotensin system (RAS) -a major hormone system, conserved in mammals and some other vertebrate animals, for modulating vascular function 19, 20 . The RAS controls extracellular fluid volume and blood pressure homeostasis by regulating the levels of renin and angiotensins in the circulation. Renin cleaves angiotensinogen to release angiotensin I (Ang I), which can be further processed by angiotensin-converting enzyme (ACE) into angiotensin II (Ang II) -a vasoconstrictive peptide that raises blood pressure and increases the extracellular fluid volume in the body by activating the angiotensin II receptors, including angiotensin II receptor type I (AT1R) 21 . ACE2 primarily converts Ang II to angiotensin-(1-7) (Ang 1-7), which is a vasodilator, thereby counter-balancing the effect of ACE/Ang II and playing critical roles in preventing hypertension and tissue damages 22 .

The protective roles of ACE2 in acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) have been demonstrated in animal models [23] [24] [25] 

To facilitate design of ACE2-based viral fusion inhibitors, we first determined, by cryo-EM, the structures of a monomeric soluble ACE2 (residue 18-615) in complex with a stabilized soluble SARS-CoV-2 S protein trimer ( Fig. S1 ; ref 27 ). We prepared the complex by mixing the two proteins because the monomeric ACE2 dissociates from S trimer very rapidly 28 . After 3D classification ( Fig. S2 and S3), we found four distinct classes that represent ACE2-free S trimer in the one-RBD-up conformation, one ACE2 bound S trimer, two ACE2 bound S trimer, and three ACE2 bound S trimer, respectively ( Fig. 1) . Consistent with previous findings with ACE2 binding to SARS-CoV S protein as well as a recent SARS-CoV-2 study 29 , ACE2 interacts with the RBD in its up conformation. While the NTD (N-terminal domain) of S1 shifts outwards slightly, the S2 portion remains largely unchanged upon ACE2 binding, even when compared to our recently published structure of the full-length S protein in the closed prefusion conformation 28 . The structure of the complex with three ACE2 bound is not symmetrical, as the distances between the C-termini of the three ACE2s (residue Tyr613) are 107Å, 109Å and 120Å, respectively (Fig. S4 ). This distance in the complex with two ACE2s bound is 110Å. These observations suggest that there is a modest degree of freedom for the up conformation of RBD when ACE2 is bound. All the substrate binding sites of the bound ACE2s face away from the threefold axis of the S trimer (Fig. S4) , incompatible with the structure of the full length ACE2 dimer in complex with the amino acid transporter B 0 AT1, in which the two active sites of the two protomers are facing each other 18 . If the B 0 AT1-bound ACE2 dimer is indeed the form recognized by SARS-CoV-2, it appears that only one ACE2 protomer in the dimer can bind one RBD in an S trimer unless there are unexpectedly large structural rearrangements in either ACE2 or S.

Measurements of the binding kinetics of soluble monomeric ACE2 (ACE2 615 ; Fig. 2 ) to the SARS-CoV-2 S trimer shows a relatively fast dissociation rate 28 , limiting its ability to compete with the membrane bound ACE2 on the surface of a target cell. We therefore sought to enhance the effective affinity by creating a trimeric form of soluble ACE2. We fused a trimerization foldon tag, derived from bacteriophage T4 fibritin 30 , to the Cterminal end of the ACE2 peptidase domain (residue 615) through a 11-residue flexible linker, a construct we refer to as ACE2 615 -foldon (Fig. 2) . We have also created another version (ACE2 615 -LL-foldon) with a slightly longer linker (LL) with 13 residues between ACE2 and the foldon tag to assess its impact on binding. To further strengthen the interaction between ACE2 and the RBD, we introduced mutations guided by high-resolution structures ( Fig. S5 ; ref 31 ), at three different positions in the ACE2-RBD interface -T27, H34 and K353, respectively. Substitution with a bulky hydrophobic residue at each of these sites may enhance hydrophobic interactions between the two proteins and slow the dissociation (Fig. S5) . We designed five mutants, T27Y, T27W, H34W, K353Y and K353W, in the ACE2 615 -foldon background. Finally, for comparison, we also include two versions of dimeric forms, ACE2m 615 -Fc and ACE2 740 -Fc, both fused to an Fc domain of an immunoglobulin G ( Fig. 2A) . ACE2m615-Fc contains H374N and H378N mutations at its peptidase active site and ACE2740-Fc includes the neck domain that mediates dimerization in the full length ACE2 18 .

To produce the soluble recombinant ACE2 and its variants, we transfected HEK293 cells with the expression constructs of the monomeric and trimeric forms containing a Cterminal his tag and purified the proteins by Ni-NTA and gel filtration chromatography.

The two dimeric forms were purified by protein G resin followed by gel filtration chromatography. While the monomeric and dimeric forms of soluble ACE2 were mostly secreted into cell medium, as judged by western blot, the trimeric ACE2 615 -foldon and its mutants were largely retained inside the cells. We therefore purified the secreted monomer and dimers from the cell supernatants and all the trimers from the cell lysates.

Most proteins eluted from a size-exclusion column as a major symmetrical peak, regardless their secretion status (Fig. S6 ). The ACE2 740 -Fc protein containing the dimerizing neck domain appeared to aggregate substantially more than other constructs.

The ACE2 615 -foldon K353W mutant aggregated completely, and we therefore did not pursue this construct any further. Only the fractions from the major peak for each construct were pooled and used for subsequent analyses. We also compared the secreted ACE2 615 -foldon with the form purified from the cells and their biochemical properties were almost identical (Fig. S7 ).

We next measured binding of these recombinant ACE2 constructs to the stabilized soluble S trimer by bio-layer interferometry (BLI). As shown in Figs. 2B and S8, the monomeric ACE2 615 had a fast dissociation rate and a K D of 77 nM, consistent with the measurement that we reported recently using surface plasmon resonance (SPR; ref 28 ).

The dimeric ACE2m 615 -FC bound slightly more tightly (K D ~22 nM). The dimeric ACE2 740 -Fc also bound more strongly than did the monomer (K D ~12 nM), although the dimer formed by the neck domain is not compatible with two ACE2 peptidase domains interacting with two distinct RBDs in a single S trimer (Fig. S4) . A possible explanation is that the neck domain mediated dimerization is not very strong and that the ACE2 peptidase domains are much more flexible than what the full-length ACE2 structure has indicated 18 , particularly in the absence of B 0 AT1. The trimeric ACE2 615 -foldon interacted with the S trimer much more strongly than any of the monomeric or dimeric forms, with a K D of 1.2 nM. ACE2 615 -LL-foldon with a longer linker between ACE2 and foldon showed an additional modest affinity enhancement (K D ~0.62 nM). The two interface mutants, ACE2 615 -foldon-T27W and ACE2 615 -foldon-T27Y, bound substantially more tightly than did the trimeric wild-type ACE2, with K D s of ~60 and ~90 pM, respectively.

While the H34W afforded a slight affinity increase, the K353Y mutation decreased affinity by more than 25-fold. Overall, these data show that our structure-guided design to increase the affinity of ACE2 to the S trimer, by trimerizing the receptor and by modifying the interface, has indeed been effective.

We performed two independent assays to determine the enzymatic activity of these ACE2 constructs. First, we directly measured the peptidase activity using a synthetic peptide substrate that releases a free fluorophore upon ACE2 cleavage. In Fig. 3A ,

concentrations of all the proteins were normalized based on the number of active sites, and the fluorophore release was monitored continuously up to ~40 min. While all the trimeric forms showed essentially the same specific activity, the monomeric ACE2 615 and the dimeric ACE2 740 -Fc had lower specific activities. The ACE2m 615 -Fc was inactive due to the mutations at the active site. Thus, all these ACE2 constructs with the wildtype sequence at the active site retained their wildtype peptidase activity.

To further support this conclusion, we next tested the ability of the ACE2 constructs to block Ang II-induced activation of AT1R. In Fig. 3B , an Ang II peptide was first directly incubated with various ACE2 proteins and then added to HEK293 cells transfected with an AT1R expression construct. Activation of AT1R was monitored by changes in the intracellular calcium concentration. When the digestion reaction was quenched by EDTA at time 0 as a control, all the mixtures with different ACE2 proteins could efficiently activate AT1R, suggesting that nothing in our protein preparations inhibited Ang IImediated AT1R activation. In contrast, when the digestion was allowed to proceed for 40 min, all AEC2 constructs except for the inactive ACE2m 615 -Fc effectively blocked AT1R activation, presumably by converting Ang II to Ang 1-7, in agreement with the peptidase activity results.

We used three different assays to assess the neutralization potency of the ACE2 constructs in blocking SARS-CoV-2 infection. The circulating strain during the early days of the pandemic contained a D614 residue in its S protein, but it has subsequently been replaced by an emerging strain harboring a G614 substitution 32 . It has been difficult to generate pseudotyped viruses with the full-length S from the D614 strain. We first used an MLV-based pseudovirus assay with a D614 S construct lacking 19 residues of the cytoplasmic tail, which incorporates efficiently into pseudoviruses. In Furthermore, when they were analyzed by a plaque assay with an authentic SARS-CoV-2, the neutralization pattern was almost identical to that from the MLV-based assay (Fig.   4C ). The IC 50 values for ACE2 615 -foldon-T27W and ACE2 615 -foldon-T27Y were 0.08 and 0.14 µg/ml, respectively. These results indicate that the engineered ACE2 constructs are very potent agents for blocking SARS-CoV-2 infection in cell culture.

A recombinant human ACE2, named APN01, is currently under evaluation as a treatment for COVID-19 in a phase 2 clinical trial (NCT04335136), primarily based on the favorable results from a previous phase 1 safety and tolerability trial (NCT00886353) in a small number of healthy individuals 33 , as well as on the recent evidence that the protein blocks SARS-CoV-2 infection effectively in vitro 26 . APN01 is a soluble ACE2 construct expressing residues 1-740 and probably dimerizes by the neck domain 34 , like ACE2 740 -Fc used in our study. We demonstrate here that our best trimeric ACE2 variant, ACE2 615foldon-T27W, has >200-fold higher binding affinity for the soluble SARS-CoV-2 S trimer, and ~5-fold and ~13-fold higher neutralization potency against pseudoviruses and authentic viruses, respectively, than does ACE2 740 -Fc, while its peptidase activity and ability to block AT1R activation remain essentially unchanged. Using a deep mutagenesis screening approach, a recent study has identified a dimeric ACE2 variant containing multiple mutations, which led to higher affinity binding to the RBD, but also a substantial loss in the catalytic activity (4-8 fold decrease) than the parental construct with the wildtype sequence 35 . One of the mutations from the mutagenesis screening is T27Y, coinciding with our structure-based design. Our approach also distinguishes the S protein binding and the peptidase activity, which can be manipulated separately to maximize the therapeutic benefits of an ACE2 construct.

Although the molecular mechanism by which a soluble ACE2 blocks SARS-CoV-2 infection as a decoy receptor is obvious, its protective role against lung injury -a hallmark of severe COVID-19 cases -appears to be more complicated in humans than in animal models. ACE2 knockout mice have more severe ARDS symptoms than do wildtype mice, while ACE2 overexpression appears to be protective 23 . Moreover, administration of recombinant ACE2 reduces severity of lung injury in mice caused by respiratory syncytial virus or influenza virus 24, 25 . In humans, rhACE2 was well tolerated with a short half-life 33 , but its infusion did not appear to ameliorate ARDS at least in a small number of patients 36 

A synthetic gene encoding an human ACE2 fragment (residues 1-615) fused with a Cterminal 6xHis tag was generated by GenScript (Piscataway, NJ) and cloned into pCMV- 

To prepare cryo grids, 3.5 µl of the freshly prepared mixture of the soluble S trimer and monomeric ACE2 (1:3 molar ratio) at ~1 mg/ml was applied to a 1. Each movie had a total accumulated electron exposure of 50 e/Å2 fractionated in 50 frames of 50 ms. Datasets were acquired using a defocus range of 1.5-2.6 µm.

Drift correction for cryo-EM images was performed using MotionCor2 43 , and contrast transfer function (CTF) was estimated by CTFFIND4 44 using motion-corrected sums without dose-weighting. Motion corrected sums with dose-weighting were used for all image processing. CrYOLO 45 was used for particle picking, and RELION3.0.8 46 was used for 2D classification, 3D classification and refinement. A total of 407,761 particles were extracted from 4,292 images. The selected particles were subjected to 2D classification, giving a total of 261,799 good particles. A low-resolution negative-stain reconstruction of the sample was low-pass-filtered to 40Å and used as an initial model for 3D classification in C1 symmetry. One class containing 32,685 particles appeared to represent the free S trimer with no ACE bound was further refined in C1 symmetry, giving a reconstruction at 3.6Å resolution. Another major class with ~49% of the selected particles showing density for ACE2 was refined in C1 symmetry and subsequently subjected to CTF refinement, Bayesian polishing and particle subtraction by masking out the ACE2-RBD density, followed by 3D classification without alignment in six classes.

Whole particles were re-extracted based on the six classes from the masked local classification and refined further, revealing different stoichiometry for ACE2 binding (one ACE2 per S trimer, two ACE2 per S trimer, and three ACE2 per S trimer). Three best maps representing each type of complexes were chosen and further refined in C1 symmetry after CTF refinement and Bayesian polishing, leading to one reconstruction of the complex with one ACE2 bound at 3.6Å resolution from 15,964 particles; another reconstruction of the complex with two ACE2 bound at 3.7Å resolution from 13,515

particles and a third reconstruction of the complex with three ACE2 bound at 3.4Å resolution from 26,298 particles. Reported resolutions are based on the gold-standard Fourier shell correlation (FSC) using the 0.143 criterion. All density maps were corrected from the modulation transfer function of the K3 detector and then sharpened by applying a temperature factor that was estimated using post-processing in RELION. Local resolution was determined using RELION with half-reconstructions as input maps.

The initial templates for model building used the stabilized SARS-CoV-2 S ectodomain trimer structure (PDB ID: 6vyb) and ACE2 from the ACE2-B0AT1 complex structure (PDB ID: 6M17). Several rounds of manual building were performed in Coot. Iteratively, refinement was performed in both Phenix 47 (real space refinement) and ISOLDE 48 , and the Phenix refinement strategy included rigid body fit, minimization_global, local_grid_search, and adp, with rotamer, Ramachandran, and reference-model restraints, using 6vyb and 6M17 as the reference model. The refinement statistics are summarized in Table S1 .

Binding of ACE2 variants to the soluble S trimer was measured using an Control sensors with no S protein were also dipped in the ACE2 solutions and the running buffer as references. Recorded sensorgrams with background subtracted from the references were analyzed using the software Octet Data Analysis HT Version 11.1 (Fortebio). The curves for monomeric ACE2 were fit to a 1:1 binding model, while those for the oligomeric ACE2 variants were fit to a bivalent binding model.

The catalytic activity of the ACE2 variants was measured by detecting a free fluorophore 7-methoxycoumarin-4-acetic acid (MCA) released from a synthetic peptide substrate, using an ACE2 activity kit (BioVision, Milpitas, CA). The ACE2 615 and ACE2 615 -foldon variants were diluted to 0.25 µg/ml using the assay buffer from the kit. The protein, respectively, reaching maximum after the substrates were completely cleaved.

Data from the first 2 minutes within the linear phase with signals less than 10% of the maximum were used for the calculation. The amount of released MCA was derived from the increase of the fluorescence signal divided by the slope of the MCA standard curve.

To treat the Ang II peptide with each ACE2 variant, 2 µl of ACE2 protein at 0. 

Neutralization of HIV-based pseudovirus containing a full-length SARS-CoV-2 S protein was measured using a single-round infection assay in HEK 293T/ACE2 target cells. antibiotic-antimycotic, 2X GlutaMAX (Gibco-Thermo Fisher Scientific) and 10% fetal bovine serum. Plates were incubated at 37°C and 5% CO 2 for 2 days. The monolayers were fixed with 10% neutral buffered formalin and stained with 0.2% aqueous Gentian Violet (RICCA Chemicals, Arlington, TX) in 10% neutral buffered formalin for 30 min, followed by rinsing and plaque counting. The half maximal inhibitory concentrations (IC 50 ) were calculated using GraphPad Prism 8.

Effectiveness of convalescent plasma therapy in severe COVID--19 patients

Deployment of convalescent plasma for the prevention and treatment of COVID--19

Treatment of 5 Critically Ill Patients With COVID--19 With Convalescent Plasma

Broad neutralization of SARS--related viruses by human monoclonal antibodies

A human neutralizing antibody targets the receptor binding site of SARS--CoV--2

A neutralizing human antibody binds to the N--terminal domain of the Spike protein of SARS--CoV--2

A noncompeting pair of human neutralizing antibodies block COVID--19 virus binding to its receptor ACE2

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Structure of the RNA--dependent RNA polymerase from COVID--19 virus

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Angiotensin--converting enzyme 2 is a key modulator of the renin--angiotensin system in cardiovascular and renal disease

Angiotensin--converting enzyme 2: the first decade

Angiotensin II receptors and drug discovery in cardiovascular disease

A potential therapeutic role for angiotensin--converting enzyme 2 in human pulmonary arterial hypertension

Angiotensin--converting enzyme 2 protects from severe acute lung failure

Angiotensin--converting enzyme 2 inhibits lung injury induced by respiratory syncytial virus

Angiotensin--converting enzyme 2 protects from lethal avian influenza A H5N1 infections

Inhibition of SARS--CoV--2 Infections in Engineered Human Tissues Using Clinical--Grade Soluble Human ACE2

Stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis

Distinct conformational states of SARS--CoV--2 spike protein

A pH--dependent switch mediates conformational masking of SARS--CoV--2 spike

Foldon, the natural trimerization domain of T4 fibritin, dissociates into a monomeric A--state form containing a stable beta--hairpin: atomic details of trimer dissociation and local beta--hairpin stability from residual dipolar couplings

Structure of the SARS--CoV--2 spike receptor--binding domain bound to the ACE2 receptor

Tracking Changes in SARS--CoV--2 Spike: Evidence that D614G Increases Infectivity of the COVID--19 Virus

Pharmacokinetics and pharmacodynamics of recombinant human angiotensin--converting enzyme 2 in healthy human subjects

Recombinant Expression and Characterization of Human and Murine ACE2: Species--Specific Activation of the Alternative Renin--Angiotensin--System

Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2

A pilot clinical trial of recombinant human angiotensin--converting enzyme 2 in acute respiratory distress syndrome

Evaluation of a mosaic HIV--1 vaccine in a multicentre, randomised, double--blind, placebo--controlled, phase 1/2a clinical trial (APPROACH) and in rhesus monkeys (NHP 13--19)

Phase 1/2 study of COVID--19 RNA vaccine BNT162b1 in adults

Structural basis of homo--and heterotrimerization of collagen I

PEGylation of therapeutic proteins

Cryo--EM structure of the 2019--nCoV spike in the prefusion conformation

Structure, function and antigenicity of the SARS--CoV--2 spike glycoprotein

MotionCor2: anisotropic correction of beam--induced motion for improved cryo--electron microscopy

CTFFIND4: Fast and accurate defocus estimation from electron micrographs

SPHIRE--crYOLO is a fast and accurate fully automated particle picker for cryo--EM

RELION: implementation of a Bayesian approach to cryo--EM structure determination

PHENIX: a comprehensive Python--based system for macromolecular structure solution

ISOLDE: a physically realistic environment for model building into low--resolution electron--density maps

Murine Leukemia Virus (MLV)--based Coronavirus Spike--pseudotyped Particle Production and Infection

Identifying SARS--CoV--2 entry inhibitors through drug repurposing screens of SARS--S and MERS--S pseudotyped particles

We thank Gary Frey for generous advice, Sarah Sterling, Richard Walsh Jr. and Shaun Rawson for technical support, and Stephen Harrison for critical reading of the manuscript. EM data were collected at the Harvard Cryo-EM Center for Structural