key: cord-0719428-cbrmw1fb
authors: Ding, Shilei; Gong, Shang Yu; Grover, Jonathan; Mohammadi, Mohammadjavad; Chen, Yaozong; Vézina, Dani; Beaudoin-Bussières, Guillaume; Verma, Vijay Tailor; Goyette, Guillaume; Richard, Jonathan; Yang, Derek; Smith, Amos B.; Pazgier, Marzena; Côté, Marceline; Abrams, Cameron; Mothes, Walther; Finzi, Andrés; Baron, Christian
title: VE607 Stabilizes SARS-CoV-2 Spike In the “RBD-up” Conformation and Inhibits Viral Entry
date: 2022-02-22
journal: bioRxiv
DOI: 10.1101/2022.02.03.479007
sha: c7adced954cba4b3daa7582c57dd341d9a9780a9
doc_id: 719428
cord_uid: cbrmw1fb

SARS-CoV-2 infection of host cells starts by binding of the Spike glycoprotein (S) to the ACE2 receptor. The S-ACE2 interaction is a potential target for therapies against COVID-19 as demonstrated by the development of immunotherapies blocking this interaction. Here, we present the commercially available VE607, comprised of three stereoisomers, that was originally described as an inhibitor of SARS-CoV-1. We show that VE607 specifically inhibits infection of SARS-CoV-1 and SARS-CoV-2 S-expressing pseudoviral particles as well as authentic SARS-CoV-2. VE607 stabilizes the receptor binding domain (RBD) in its “up” conformation. In silico docking and mutational analysis map the VE607 binding site at the RBD-ACE2 interface. The IC50 values are in the low micromolar range for pseudoparticles derived from SARS-CoV-2 Wuhan/D614G as well as from variants of concern (Alpha, Beta, Gamma, Delta and Omicron), suggesting that VE607 has potential for the development of drugs against SARS-CoV-2 infections.

The COVID-19 pandemic continues to cause widespread morbidity and mortality (Wu et al., 55 2020; Zhu et al., 2020a) . This is largely due to insufficient vaccination levels as vaccines 56 offer good protection against infection and severe disease (Ball, 2021) . The currently used 57 vaccines exploit modified versions of the Spike (S) glycoprotein that is exposed on the 58 surface of viral particles (Krammer, 2020) and infected cells (Ding et al., 2022) . Here we employed differential scanning fluorimetry (DSF) to identify the capacity of 92 the small molecule inhibitor VE607 (Kao et al., 2004) , composed of three stereoisomers, to 93 bind the SARS-CoV-2 RBD. We found that this VE607 mixture of isomers (hereafter referred 94 to as "VE607") is capable of specific inhibition of infection of human cells with pseudoviral 95 particles that express the SARS-CoV-1 or SARS-CoV-2 S-glycoproteins. VE607 was also 96 able to inhibit the infection with authentic SARS-CoV-2 viruses. We found that VE607 97 inhibits the Spike by stabilizing the "up" conformation of the RBD. The mode of binding to 98 RBD was elucidated by in silico docking experiments followed by validation of critical 99 residues through mutagenesis and functional studies. Finally, VE607 remains potent against 100 current variants of concern (VOC) of SARS-CoV-2 suggesting that it may be an interesting 101 lead for the development of drugs for the prevention or treatment of COVID-19 infections. 102

104 Differential scanning fluorimetry and docking suggest that VE607 may bind the RBD 105 106 We tested the ability of previously described SARS-CoV-1 inhibitors VE607 (Kao et al., 107 2004 ) and SSAA09E2 (Adedeji et al., 2013) to bind the SARS-CoV-2 RBD ( Figure 1A ). We 108 used differential scanning fluorimetry (DSF) that, measures the effect of small molecules on 109 the melting temperature of proteins (Mashalidis et al., 2013) . Incubation with VE607 led to a 110 significant decrease of the melting temperature (Tm, -2.3°C) while SSAA09E2 had a 111

smaller, yet measurable effect (Tm, -0.7°C) ( Figure 1B ). Since this result suggested binding 112 of VE607 to RBD, we next performed in silico docking against RBD using Glide 113 (Schrödinger, 2020) . We identified moderately favorable potential VE607 binding sites 114 overlapping the ACE2 epitopes in both SARS-CoV-1 and SARS-CoV-2 RBDs ( Figure 1C for pseudoviral particles bearing the VSV-G (IC50 > 100μM, Figure 2A ). To ensure that the 130 inhibitory capacity of VE607 against SARS-CoV-2 was not limited to pseudoviral particles, 131 we evaluated whether the inhibitory capacity of VE607 was maintained against authentic 132 viruses. As shown in Figure 2B , VE607 inhibited authentic SARS-CoV-2 D614G with an 133 IC50 of 2.42 µM. No cell toxicity of VE607 or the three different enantiomers was observed 134 with concentrations up to 100 M on 293T ACE2 cells or Vero-E6 cells ( Figure 2C ). In 135 contrast, SSAA09E2 at concentrations up to 100 M did not inhibit infection of pseudoviral 136 particles (data not shown) and we did not further pursue work with this small molecule. 137 138 As stated above, commercially available VE607 is a mixture of three stereochemical isomers, 139 comprised of the (S,S)-VE607, (R,R)-VE607, and the meso (R,S)-VE607. We observed no 140 differences in the SARS-CoV-2 pseudoviral inhibition among these enantiomers obtained by 141 synthesis, and the commercially available mixture of all three isomers (Figures S1). 142 143 Initial in silico docking identified RBD residues Y505 and Q498 as potential specific 144 contact sites for VE607 ( Figure 1D ). We mutated these residues in the full-length SARS-145

CoV-2 D614G Spike and prepared pseudoviral particles to test whether they affect VE607 146 inhibition. While the Q498V mutation had only a minor effect (IC50 = 1.80 μM), the Y505T 147 mutant was resistant to VE607 inhibition (IC50 > 40 μM, Figure 2D ). These results are in 148 agreement with the in silico analysis, where a strong - interaction between Y505's aromatic 149 side-chain and the central aromatic ring of VE607 is predicted. Alignment of sACE2 on the 150 known ACE2 epitope of the VE607-bound model of RBD displayed significant steric clashes 151 between ACE2 and VE607, suggesting some direct competition for the ACE2 epitope. 152 153 VE607 stabilizes the "up" conformation of the S protein 154 155 We next assessed whether VE607 affects the RBD-ACE2 interaction. Briefly, we measured 156 by flow cytometry the capacity of VE607 to compete with soluble ACE2 (sACE2) for 157 interaction with the full SARS-CoV-2 Spike, expressed at the cell surface, as described 158

previously (Anand et al., 2020) . We observed no competition between VE607 (100 µM) and 159 sACE2 ( Figure 3A ). Since the mode of action of some neutralizing antibodies such as CV3-1 160 involve S1 shedding ( 

180 SARS-CoV-2 is in constant evolution as VOCs keep emerging. VE607 was identified as an 181

inhibitor of SARS-CoV-1, a related Beta-coronavirus, suggesting some inhibitory breadth. 182 Therefore, we tested whether it inhibits pseudoviral particles bearing the Spike glycoproteins 183 from the major VOCs (Alpha, Beta, Gamma, Delta and Omicron). In agreement with its broad 184 virus G (VSV-G)-encoding plasmid (pSVCMV-IN-VSV-G) was previously described (Emi et 338 al., 1991) . Plasmids used to generate SARS-CoV-2 pseudoviral particles for smFRET analysis 339 were described previously (Lu et. al., 2020) . prepare the structures, force field atom types and bond orders were assigned, missing atoms 387 and side-chains were added, protonation states of ionizable amino acid side-chains were 388 determined using PROPKA (Olsson et al., 2011) , water orientations were sampled, and 389 hydrogen bond networks were subsequently optimized by flipping Asn/Gln/His residues and 390 sampling hydroxyl/thiol hydrogen. Constrained energy minimization was then performed 391 using the imperf module from impact (Schrödinger, 2020) to generate the structure to be used 392 in the subsequent modeling calculations. Potential binding sites were explored and 393 characterized using the SiteMap tool (Halgren, 2007; Halgren, 2009 ). VE607 compound was 394 structurally preprocessed using LigPrep (Schrödinger, 2020) to generate multiple states for 395 stereoisomers, tautomers, ring conformations, and protonation states at a selected pH range. index is a measure of the ability of the VE607 treated S1 subunit to remain associated with the 459 trimeric spike (S) protein on the expressing cell relative to that of the mock-treated S1 and 460 was calculated with the following formula: association index = ( To measure the cytotoxicity of VE607 and its stereoisomers on 293T-ACE2 or Vero-E6 cells, 492 a cell viability assay using CellTiter-Glo® One Solution Assay (Promega) was performed. 

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