key: cord-325348-yi6yu5l1
authors: Zhang, G.; Pomplun, S.; Loftis, A. R.; Tan, X.; Loas, A.; Pentelute, B. L.
title: Investigation of ACE2 N-terminal fragments binding to SARS-CoV-2 Spike RBD
date: 2020-06-17
journal: bioRxiv
DOI: 10.1101/2020.03.19.999318
sha: 
doc_id: 325348
cord_uid: yi6yu5l1

Coronavirus disease 19 (COVID-19) is an emerging global health crisis. With over 7 million confirmed cases to date, this pandemic continues to expand, spurring research to discover vaccines and therapies. SARS-CoV-2 is the novel coronavirus responsible for this disease. It initiates entry into human cells by binding to angiotensin-converting enzyme 2 (ACE2) via the receptor binding domain (RBD) of its spike protein (S). Disrupting the SARS-CoV-2-RBD binding to ACE2 with designer drugs has the potential to inhibit the virus from entering human cells, presenting a new modality for therapeutic intervention. Peptide-based binders are an attractive solution to inhibit the RBD-ACE2 interaction by adequately covering the extended protein contact interface. Using molecular dynamics simulations based on the recently solved cryo-EM structure of ACE2 in complex with SARS-CoV-2-RBD, we observed that the ACE2 peptidase domain (PD) α1 helix is important for binding SARS-CoV-2-RBD. Using automated fast-flow peptide synthesis, we chemically synthesized a 23-mer peptide fragment of the ACE2 PD α1 helix (SBP1) composed entirely of proteinogenic amino acids. Chemical synthesis of SBP1 was complete in 1.5 hours, and after work up and isolation >20 milligrams of pure material was obtained. Bio-layer interferometry (BLI) revealed that SBP1 associates with micromolar affinity to insect-derived SARS-CoV-2-RBD protein obtained from Sino Biological. Association of SBP1 was not observed to an appreciable extent to HEK cell-expressed SARS-CoV-2-RBD proteins and insect-derived variants acquired from other vendors. Moreover, competitive BLI assays showed SBP1 does not outcompete ACE2 binding to Sino Biological insect-derived SARS-CoV-2-RBD. Further investigations are ongoing to gain insight into the molecular and structural determinants of the variable binding behavior to different SARS-CoV-2-RBD protein variants.

A novel coronavirus (SARS-CoV-2) from Wuhan, China, has caused over 7 million 54 confirmed cases and over 400,000 deaths globally, according to the COVID-19 situation report 55 from WHO on June 10, 2020 (https://www.who.int/emergencies/diseases/novel-coronavirus-56 2019/situation-reports/), and the number is continually growing. Similar to the SARS-CoV 57 outbreak in 2002, SARS-CoV-2 causes severe respiratory problems. Coughing, fever, difficulties 58 in breathing and/or shortage of breath are the common symptoms. Aged patients with pre-existing 59 medical conditions are at most risk with a mortality rate ~1.5% or even higher in some regions. 60

Moreover, human-to-human transmission can occur rapidly by close contact. To slow this 61 pandemic and treat infected patients, rapid development of specific antiviral drugs is of the highest 62

The closely-related SARS-CoV coronavirus invades host cells by binding the angiotensin-64 converting enzyme 2 (ACE2) receptor on human cell surface through its viral spike protein (S) [1-65 4] . It was recently established that SARS-CoV-2 uses the same receptor for host cell entry and 66 binds ACE2 with an affinity comparable with the corresponding spike protein of SARS-CoV [5, 6] . 67

Recent cryo-electron microscopy (cryo-EM) structural studies of the SARS-CoV-2 spike protein 68 receptor binding domain (RBD) in complex with full-length human ACE2 receptor revealed key 69 amino acid residues at the contact interface between the two proteins and estimated the binding 70 affinity at ~15 nM [7, 8] . These studies provide valuable information that can be leveraged for the 71 development of disruptors specific for the SARS-CoV-2/ACE2 protein-protein interaction (PPI). 72

Small-molecule inhibitors are often less effective at disrupting extended protein binding interfaces 73 [9] . Peptides, on the other hand, offer a synthetically accessible solution to disrupt PPIs by binding 74 at interface regions containing multiple contact "hot spots" [10] . 75

We hypothesized that disruption of the viral SARS-CoV-2-RBD/host ACE2 interaction with 76 peptide-based binders would prevent virus entry into human cells, offering a novel opportunity for RBD was simulated under TIP3P explicit water conditions. Analyzing the simulation trajectory 103 after 200 ns, we found that SBP1 remains on the spike RBD protein surface in a stable 104 conformation ( Fig. 2B ) with overall residue fluctuations smaller than 0.8 nm compared with their 105 starting coordinates ( Fig. 2A) . Per-residue analysis along the 200 ns trajectory showed that the 106 middle residues of SBP1, a 12-mer sequence we termed SBP2, have significantly reduced 107 fluctuations (Fig. 2C, 2D) , indicating key interactions. The results of this MD simulation suggest 108 that SBP1 and SBP2 peptides derived from the ACE-PD α1 helix may alone potentially bind the 109 SARS-CoV-2 spike RBD protein with sufficient affinity to disrupt the associated PPI. 110

Automated fast-flow peptide synthesis yields >95% pure compound 111

The two N-terminal biotinylated peptides, SBP1 and SBP2, derived from the α1 helix were 112 prepared by automated fast-flow peptide synthesis [15, 17] with a total synthesis time of 1.5 h over 113 35 coupling cycles. After cleavage from resin, global deprotection, and subsequent C18 solid-114 phase extraction, the purity of the crude peptides was estimated to be >95% for both biotinylated 115 SBP1 and SBP2 based on LC-MS TIC chromatograms (Supplemental Fig. 1 ). We assessed this 116 purity as acceptable for direct downstream biological characterization. 117 SBP1 peptide binds Sino Biological insect-derived SARS-CoV-2-RBD with micromolar 118 affinity, but does not associate with other commercial sources of SARS-CoV-2-RBD 119

Bio-layer interferometry (BLI) was used to measure the binding affinity of the synthesized 120 peptide SBP1 to glycosylated Sino Biological insect-derived SARS-CoV-2-RBD, Sino Biological 121

AcroBiosystems HEK-expressed SARS-CoV-2-RBD. In all of these assays, biotinylated SBP1 123 was immobilized onto streptavidin (SA) biosensors. After fitting the association and dissociation 124 curves from serial dilutions of the protein, the dissociation constant (KD) of SBP1 to glycosylated 125

Sino Biological insect-derived SARS-CoV-2-RBD was determined to be ~1300 nM using the 126 global fitting algorithm and 1:1 binding model (Fig. 2E ). However, SBP1 did not associate with the 127 other three SARS-CoV-2-RBD proteins studied (Fig. 2E) . Surprisingly, a scrambled sequence of 128 SBP1 exhibited binding to the Sino Biological insect-derived SARS-CoV-2-RBD with comparable 129 association response to SBP1 at 500 nM concentration (Fig. 2E ). SBP1 had no observable 130 binding to a negative control human protein menin (Fig. 2F) . Likewise, no association was 131 observed between the biotinylated 12-mer SBP2 and Sino Biological insect-derived SARS-CoV-132 2-RBD (Fig. 2F) . 133 SBP1 does not compete with biotinylated ACE2 binding to Sino Biological insect-derived 134

Using a competition-format BLI assay, we confirmed that soluble human ACE2 protein 136 could compete with immobilized biotinylated ACE2 (AcroBiosystems) for binding Sino Biological 137 insect-derived SARS-CoV-2-RBD, and that a 5-fold excess of soluble ACE2 (relative to 138 immobilized biotinylated ACE2) abolished nearly all of the initial ACE2/RBD binding interaction 139 ( Fig. 3B, 3D ). However, competition was not observed when using non-biotinylated SBP1 pre-140 mixed in solution with Sino Biological insect-derived SARS-CoV-2-RBD, even with a 1000-fold 141 excess of the peptide (Fig. 3C, 3E ). These data suggest that SBP1 potentially binds SARS-CoV-142 2-RBD at a different site than ACE2, binds SARS-CoV-2-RBD too weakly, or for other unknown 143 reasons cannot disrupt the native ACE2/RBD interaction. 144

Recently published cryo-EM structures of the RBD of SARS-CoV-2 in complex with human 146 ACE2 have identified this PPI as a key step for the entry of SARS-CoV-2 into human cells [7, 8] . 147

Blocking this binding interface represents a highly promising therapeutic strategy, as it could 148 potentially hinder cellular uptake of SARS-CoV-2 and intracellular replication. 149

Drugging PPIs is a longstanding challenge in traditional drug discovery and peptide-based 150 approaches might help to solve this problem. Small molecule compounds are unlikely to bind 151 large protein surfaces that do not have distinct binding pockets. Peptides, on the other hand, 152 display a larger surface area and chemical functionalities that can mimic and disrupt the native 153 PPI, as is the case for the clinically approved HIV peptide drug Fuzeon [18, 19] . 154

The identification of a suitable starting point for drug discovery campaigns can be time-155

intensive. During a pandemic such as this one, therapeutic interventions are urgently needed. 156

Peptide-based strategies were developed to target both the spike protein RBD and S2 subunit of 157 the first SARS-CoV virus [11, 12] . Translating these approaches to SARS-CoV-2, inhibitors of the 158 spike protein fusion with the cell membrane and engineered mini-proteins that bind SARS-CoV-2 159 RBD were developed [13, 14] . We aimed to determine the minimum length required of the ACE2 160 N-terminal peptide fragment in order to maintain binding affinity to SARS-CoV-2-RBD and thus 161 potentially deliver a synthetically accessible therapeutic candidate. To rapidly identify potential 162 short peptide binders to the SARS-CoV-2 spike protein, we used molecular dynamics ( and several important interactions with the spike protein were observed consistently with multiple lines of published data [8, 20] . We used this peptide (SBP1) as an experimental starting point for 175 the development of a SARS-CoV-2 spike protein binder. Our rapid automated flow peptide 176 synthesizer enabled the synthesis of tens of milligrams of SBP1 peptide within 1.5 h. The crude 177 purity was determined to be >95% and therefore sufficient for binding validation by BLI. 178

The interaction between N-terminal biotinylated SBP1 and the RBD of glycosylated SARS-179

CoV-2 spike protein was investigated in detail. We performed serial dilutions of the soluble protein 180 to reliably determine the binding affinity of SBP1 to Sino Biological insect-derived SARS-CoV-2-181 RBD. Using a global fitting algorithm, we found that N-terminal biotinylated SBP1 binds Sino 182

Biological insect-derived SARS-CoV-2-RBD with micromolar affinity (KD = 1.3 µM), a value almost 183 100-times higher than the estimated binding affinity of the native ACE2 receptor (KD ~ 15 nM [7]) 184 (Fig. 2E ). The decreased binding affinity relative to ACE2 may partially explain why SBP1 was 185 unable to significantly disrupt ACE2 binding to Sino Biological insect-derived SARS-CoV-2-RBD 186 even at 1000-fold excess in a BLI competition assay (Fig. 3C,E) . In addition, the comparable 187 affinity observed for a scrambled sequence of SBP1 (Fig. 2E) are in progress to gain additional understanding of these molecular processes. 204

In conclusion, a biotinylated peptide sequence derived from human ACE2 was found to 205 bind Sino Biological insect-derived SARS-CoV-2 spike protein RBD with micromolar affinity, but 206 did not associate with SARS-CoV-2-RBD variants obtained from other commercial sources. In 207 spite of this association, competitive BLI data indicates that SBP1, even at 1000-fold excess, did 208 not compete with ACE2 for binding to SARS-CoV-2-RBD. Our preliminary studies highlight the 209 unexpected challenges researchers may encounter while developing peptide-based approaches 210 to disrupt the specific interactions of SARS-CoV-2 with its mammalian cell membrane receptors. 

The cryo-EM structure of ternary complex of SARS-CoV-2-RBD with ACE2-B 0 AT1 (PDB: 6M17) 219 was chosen as the initial structure, which was explicitly solvated in an 87 Å 3 box, to perform a 200 220 ns molecular dynamical (MD) simulation using NAMD on MIT's supercomputing clusters (GPU 221 node). The Amber force field was used to model the protein and peptide. The MD simulation 222 system was equilibrated at 300 K for 2 ns. Periodic boundary conditions were used and long-range electrostatic interactions were calculated with particle mesh Ewald method, with non-224 bonded cutoff set to 12.0 Å. SHAKE algorithm was used to constrain bonds involving hydrogen 225 atoms. Time step is 2 fs and the trajectories were recorded every 10 ps. After simulation 226 production runs, trajectory files were loaded into the VMD software for further analysis. 

After peptide synthesis, the peptidyl resin was rinsed with dichloromethane briefly and then dried 237 in a vacuum chamber overnight. Next day, approximately 5 mL of cleavage solution (94% 238 trifluoroacetic acid (TFA), 1% TIPS, 2.5% EDT, 2.5% water) was added into the syringe containing 239 the resin. The syringe was kept at room temperature for 2 h before injecting the cleavage solution 240 into a 50 mL conical tube. Dry-ice cold diethyl ether (~50 mL) was added to the cleavage mixture 241 and the precipitate was collected by centrifugation and triturated twice with cold diethyl ether (50 242 mL). The supernatant was discarded. Residual ether was allowed to evaporate and the peptide 243 was dissolved in water with 0.1% TFA for solid-phase extraction. 244

After peptide cleavage, peptide precipitates were dissolved in water with 0.1% TFA. Agilent Mega 246 TFA, and then equilibrated with 15 mL of water with 0.1% TFA. Peptides were loaded onto the 248 column for binding, followed by washing with 15 mL of water with 0.1% TFA, and finally, eluted 249 with 5 mL of 30/70 water/acetonitrile (v/v) with 0.1% TFA. 250

Liquid chromatography-mass spectrometry (LC-MS) 251

Peptides were dissolved in water with 0.1% TFA followed by LC-MS analysis on an Agilent 6550 252 iFunnel ESI-Q-ToF instrument using an Agilent Jupiter C4 reverse-phase column (2.1 mm × 150 253 mm, 5 μm particle size). Mobile phases were 0.1% formic acid in water (solvent A) and 0.1% 254 formic acid in acetonitrile (solvent B). Linear gradients of 1 to 61% solvent B over 15 minutes (flow 255 rate: 0.5 mL/min) were used to acquire LC-MS chromatograms. 256

A ForteBio Octet® RED96 Bio-Layer Interferometry system (Octet RED96, ForteBio, CA) was 258 used to characterize the in vitro peptide-protein binding affinity at 30 °C and 1000 rpm. Sino Biological HEK-expressed SARS-CoV-2-RBD (solution in phosphate-buffered saline) and 349 (B) glycosylated Sino Biological insect-derived SARS-CoV-2-RBD with associated deconvoluted 350 mass spectra obtained by integration over the protein peak at ~8 min. The broad bands in the TIC 351 chromatograms of (B) are due to additives present in the vendor-formulated solid powder (10% 352 glycerol, 5% trehalose, 5% mannitol and 0.01% tween-80). 353 354

Structure of SARS coronavirus spike receptor-binding 356 domain complexed with receptor

Receptor and viral 359 determinants of SARS-coronavirus adaptation to human ACE2

Lethal infection of 362 K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus

Retroviruses pseudotyped with the severe acute respiratory 366 syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting 367 enzyme 2

SARS-CoV-2 Cell Entry 370 Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Structure, Function, 373 and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

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

Structural basis for the recognition of the 378 SARS-CoV-2 by full-length human ACE2

Features of protein-protein interactions that translate into potent 380 inhibitors: topology, surface area and affinity

mRNA display: from basic principles to macrocycle 382 drug discovery

Identification of critical determinants on ACE2 for 384 SARS-CoV entry and development of a potent entry inhibitor

A pan-coronavirus fusion inhibitor targeting the HR1 domain of human 387 coronavirus spike

Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a 389 highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high 390 capacity to mediate membrane fusion

An engineered stable mini-protein to plug 392 SARS-Cov-2 Spikes

Synthesis of proteins by automated flow chemistry

A fully automated flow-based approach for accelerated peptide synthesis

Peptide-based inhibitors of protein-protein interactions

Enfuvirtide, an HIV-1 fusion inhibitor

Receptor recognition by novel coronavirus 405 from Wuhan: An analysis based on decade-long structural studies of SARS

Site-specific glycan analysis of 408 the SARS-CoV-2 spike

In-solution enrichment 410 identifies peptide inhibitors of protein-protein interactions