key: cord-1029316-iz5kx50b
authors: Zhang, Guofang; Cong, Yalin; Cao, Guoli; Li, Liang; Yu, Peng; Song, Qingle; Liu, Ke; Qu, Jing; Wang, Jing; Xu, Wei; Liao, Shumin; Fan, Yunping; Li, Yufeng; Wang, Guocheng; Fang, Lijing; Chang, Yanzhong; Zhao, Yuliang; Boraschi, Diana; Li, Hongchang; Chen, Chunying; Wang, Liming; Li, Yang
title: Spike Protein Targeting “Nano-Glue” that Captures and Promotes SARS-CoV-2 Elimination
date: 2021-04-14
journal: bioRxiv
DOI: 10.1101/2021.04.13.439641
sha: 18d8cc54ca166766dc6852ac0beccb2caac814b8
doc_id: 1029316
cord_uid: iz5kx50b

The global emergency caused by the SARS-CoV-2 pandemics can only be solved with adequate preventive and therapeutic strategies, both currently missing. The electropositive Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein with abundant β-sheet structure serves as target for COVID-19 therapeutic drug design. Here, we discovered that ultrathin 2D CuInP2S6 (CIPS) nanosheets as a new agent against SARS-CoV-2 infection, which also able to promote viral host elimination. CIPS exhibits extremely high and selective binding capacity with the RBD of SARS-CoV-2 spike protein, with consequent inhibition of virus entry and infection in ACE2-bearing cells and human airway epithelial organoids. CIPS displays nano-viscous properties in selectively binding with spike protein (KD < 1 pM) with negligible toxicity in vitro and in vivo. Further, the CIPS-bound SARS-CoV-2 was quickly phagocytosed and eliminated by macrophages, suggesting CIPS could be successfully used to capture and facilitate the virus host elimination with possibility of triggering anti-viral immunization. Thus, we propose CIPS as a promising nanodrug for future safe and effective anti-SARS-CoV-2 therapy, as well as for use as disinfection agent and surface coating material to constrain the SARS-CoV-2 spreading.

The Corona Virus Disease 2019 (COVID-19), induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), broke out since December 2019 and has become a worldwide health crisis 1 . The US FDA granted Remdesivir and hydroxychloroquine with Emergency Use Authorization to treat COVID-19, based on earlier reports of their capacity to inhibit SARS-CoV-2 2-4 , but revoked the use of hydroxychloroquine for COVID-19 5,6 , due to lacking effectiveness in clinical use 7, 8 .

Although Remdesivir has been approved for COVID-19 treatment in October 22 nd , 2020, by US FDA, the interim results of the WHO Solidarity trial showed no significant effect [9] [10] [11] . Several mutations of SARS-CoV-2 on Spike (S) protein have been recently identified, which apparently cause increased viral infectivity 12 . The mutated SARS-CoV-2 variants have raised concerns regarding the effectiveness of the currently approved anti-SARS-CoV-2 drugs, neutralizing antibodies and vaccines 13 .

Further, the resource-consuming logistics of cold-chain products risks to fail stopping SARS-CoV-2 transmission and pandemic spread 14 . Thus, despite a powerful effort in drug repurposing for the identification of new antiviral compounds 15, 16 , novel compounds with effective antiviral activity are an urgent need for the treatment and containment of the SARS-CoV-2 pandemic infection 17 .

The host infection by SARS-CoV-2 requires the recognition and binding of the Receptor Binding Domain (RBD) of Spike (S) protein of SARS-CoV-2 to the host cellular ACE (angiotensin converting enzyme) 2 receptor 18,19 . S protein and its RBD thus serve as efficient targets for antiviral drugs. Anti-SARS-CoV-2 neutralizing monoclonal antibodies (nAbs) have been designed for targeted interaction with the RBD of S protein 20,21 or the epitopes of RBD/ACE2 binding site 22-24 , or selected for potent neutralization activity [25] [26] [27] . However, the massive dosage of nAb for effective therapy (10 to 100 mg/kg), their uncertain effectiveness for mutated SARS-CoV-2 variants, and the complex nAb storage and shipping conditions necessary for their long-term integrity and efficacy, push to searching for alternative tools with a comparable therapeutic capacity 28 . In this view, the antimicrobial and antiviral capacity of nanomaterials (NMs) 29,30 has raised particular interest in view of novel anti-SARS-CoV-2 strategies. NMs exhibit good capability in antiviral applications due to large surface area, tunable surface properties, and chemical reactivity. VivaGel, the best known antiviral dendrimer gel, has been used for HIV and HSV prevention by blocking the virus-cells interaction 31 . Metal NMs display antiviral capacity due to the interaction of metallic atoms with virus components, as in the case of silver NM 32 and gold NM 33 . Gold NM with a tunable surface chemistry design that mimics heparan sulfate proteoglycan, effectively binding to viral attachment molecules, has been successfully applied to inhibit the infection of various viruses 30 . Thus, we propose to develop a S protein-targeting viscous nanodrug that could combine high anti-viral efficacy with excellent biocompatibility, easy preparation and convenient storage characteristics, as a promising new tool to prevent/treat COVID-19.

According to the Adaptive Poisson-Boltzmann Solver (APBS) electrostatics calculations 34 and the crystal structure of the RBD and ACE2 interface 35 Its efficacy and excellent biocompatibility suggest CIPS NS as a promising anti-SARS-CoV-2 drug. The viscous flypaper-like and selective binding capacity of CIPS for the SARS-CoV-2 S protein also makes it particularly promising as surface coating material and disinfection agents to contain the SARS-CoV-2 spreading.

CIPS NS was exfoliated from the bulk CIPS ( fig. S4a ) with Li-intercalation by using n-butyl lithium and ultrasound sonication, and its crystalline nature was proven by X-ray diffraction (XRD) characterization ( fig. S4b ). The exfoliated CIPS showed a multi-layer structure with a mean size of ~200 nm determined by SEM ( fig. S4c ) and TEM (Fig. 1a) , and an average thickness of ~3.1 nm by AFM (Fig. 1b-c) . According to extended X-ray absorption fine structure (EXAFS), copper atoms coordinate with sulfur atoms (Fig. 1d, fig. S4d -g and Table S1 ), where Cu has a valence of +1 (Table   S2 ). Sulfur was present as sulfide (S 2-) with a valence of -2 ( fig. S4h- S4j ). The chemical form of In is consistent with crystal structure of CIPS NS where In atoms remain between two different CIPS surface and stably support the structure of CIPS by coordinating with the P and S atoms (Fig. 1e) . The structure of CIPS NS is described in the schematic diagrams in Fig. 1e , which show the Cu, S, and P atoms mainly localized on the surface and the In atoms immersed in the NS.

The anti-SARS-CoV-2 effect of CIPS NS (shorter as CIPS) was investigated with the established pseudoviruses SC2-P (expressing the SARS-CoV-2 S protein) and 

After having excluded possible metallic ion effects ( fig. S8 ), we assessed the interaction of CIPS with SC2-P by TEM (Fig. 4a) , and examined the variations in the CIPS physico-chemical properties ( fig. S9 ). We further detected the interaction of CIPS with SC2-P by examining its S protein in Western blotting (Fig. 4b) . Further, the interaction of CIPS with authentic SARS-CoV-2 virus was also analyzed, indicating that CIPS could effectively adsorb and trap SARS-CoV-2 virus (Fig. 4c ). The capacity of CIPS to adsorb the S protein was also proven by Western blotting (Fig. S10 ). The analysis of CIPS binding to the RBD of the S protein by Biolayer Interferometry (BLI)

shows a very high binding affinity (Fig. 4d , K D <0.001 nM), in contrast with an at least 100x lower affinity for a number of serum proteins and factors ( Fig. 4e and Table   S3 ), suggesting a selective binding capacity for the S protein RBD. A mixture of SC2-P with several fold excess volume of FBS or BSA was used to evaluate the CIPS anti-viral effects in complex media, and shows that CIPS retains a strong binding capacity for the S protein of SC2-P (Fig. 4f) , and that its anti-viral effect is not affected ( Fig. 4g and fig. S11 ). The capacity of other 2D NMs (MoS 2 and GO) to bind RBD (K D 11.7 and 5.2 nM, respectively) was significantly lower than that of CIPS S13 ). The amino acid residues of RBD bound to ACE2 are listed in Table 1 and mostly locate in β -sheet, turn, and random coil stretches. The secondary structure of RBD in the presence and absence of 30 pM CIPS was characterized with circular dichroism (CD) spectra, and shows a majority of β -sheet conformation within the RBD structure ( Fig. 4j-k) . Since β -sheet structures promote protein adsorption on the NS 41 , this may explain the preferential binding of RBD to CIPS. A conformational change in RBD upon CIPS binding was also observed (Fig. 4k) , with a decrease of α -helix (7% to 2%) and turn (23% to 10%), and an increase of β -sheet (39% to 46.5%) and random coil (31% to 41.5%). MD simulation was further used to explore binding interface of RBD to CIPS within 100 ns (fig. S14-S15, Table S4 ), and the typical configuration was shown (Fig. 5a) , with the amino acid residues and the interactive forces contributing to the binding illustrated in colors (Fig. 5a ). Among the 11 amino acid residues of RBD interacting with the ACE2, six showed strong adsorption with CIPS (Table 1 , indicated by *).

Hydrophobic, polar and positively charged residues in RBD mainly contribute to the adsorption of RBD on CIPS (Table S5 ). The functional groups (-NH 3 

NMs can be eliminated by macrophages. Because of the strong interaction of CIPS with SARS-CoV-2, we investigated the possibility that macrophages could efficiently capture and eliminate SARS-CoV-2 when the virus is bound to CIPS. As shown in the figure 6 (a, b), CIPS-treated SC2-P could be effectively phagocytosed by macrophages (differentiated human THP-1 cells) after 24 h incubation, and the virus was effectively eliminated by macrophages during the subsequent degradation process ( Fig.6a-b ). SC2-P was found to be accumulated in macrophages after inhibiting the lysosome function with bafilomycin (BM) (Fig.6a-b) , indicating the majority of SC2-P was degraded in lysosomes. The same findings were obtained with authentic SARS-CoV-2 virus (Fig. 6c-d) . SARS-CoV-2 could be effectively eliminated after remove the extracellular virus (Fig. 6c ). Lysosome inhibition with BM significantly increased the intracellular SARS-CoV-2 levels, implying the SARS-CoV-2 elimination was lysosome-dependent (Fig. 6d) . Similar to the pseudovirus, SARS-CoV-2 phagocytosis was also greatly increased in the presence of CIPS (Fig.   6d ).

SARS-CoV-2 in macrophages has been reported, although in resting/healthy conditions they do not express ACE2 42 . Since a huge amount of SARS-CoV-2 was internalized by macrophages in the presence of CIPS, we examined whether the intracellular virus was able to infect macrophages. With pseudovirus (SC2-P) or authentic SARS-CoV-2 virus, we show that SARS-CoV-2 uptake through CIPS phagocytosis cannot induce macrophage infection ( Fig. 6e-f , SARS-CoV-2 releases in medium) and cause cytopathic or cytotoxic effects (data not shown). This indicates that macrophages still function well with the virus scavenging capacity after massive SARS-COV-2 internalization with CIPS treatment, and avoid viral infection.

We also examined the uptake, accumulation and degradation/clearance of CIPS Table S1 ), suggesting that the intracellular form of CIPS changed from oxidation to degradation, most likely within acidic phagolysosomes.

These results suggest that CIPS can be progressively degraded and metabolized by macrophages.

The intracellular elimination of SARS-CoV-2 in macrophages that promoted by CIPS, may induce the following immunization process such as antigen presentation.

Thus, the expressions of CD86 and HLA-DRA, two molecules important for antigen presentation, were assessed in SARS-CoV-2 treated macrophages and showed greatly up-regulation with CIPS treatment compared to virus alone (Fig. 6k-l) . This antigen presenting capacity that promoted by CIPS was further assessed in dendritic cells (DC), and found CIPS was able to increase the population of mature CD11c + CD86 + DC in the presence of the SC2-P (Fig. 6m) . Thus, the internalization of CIPS by macrophages could facilitate the elimination of the CIPS-trapped SARS-CoV-2, and could further benefit COVID-19 treatment by promoting antigen presentation in the context of MHC-II.

The binding affinity of nAbs with S protein RBD ranges from 0.1 to 100 nM 43,44 , which is similar to the binding affinity between RBD and ACE2 (8.1 nM in our hands, 14.7 nM reported for S protein binding to ACE2) 40 . The comparable affinity suggests that only very few of the currently available nAbs may have sufficient affinity for RBD to efficiently compete with its binding to ACE2 thereby inhibiting cell infection.

Here, we have identified the 2D CIPS NS as an effective nano-viscous material able to capture the S protein and inhibit cell infection by SARS-CoV-2 ( Fig. 3-5) . CIPS strongly binds to the S protein of SARS-CoV-2 and interferes with the ability of the S protein RBD to bind to host ACE2 (Fig. 4) . The binding affinity of CIPS for the S protein RBD is <1 pM, suggesting the formation of very stable complexes and highly efficient inhibition of the virus interaction with ACE2. Computational simulations are consistent with experimental data (Fig. 5, fig. S3 and S12), and show that CIPS/RBD exhibits the lowest interaction energy and the strongest electrostatic attraction (Fig. 5a , d, fig S18， Table S4 ). The effective binding of CIPS with 6 out of the 11 ACE2-binding amino acid residues of the SARS-CoV-2 RBD accounts for the efficient inhibition of viral infectivity displayed by CIPS ( Table 1) showing quick degradation and removal by macrophages (Fig. 6 g-j) . Importantly, the SARS-CoV-2 virus adsorbed on CIPS can be efficiently taken up by macrophages, shuttled to phagolysosomes and completely degraded (Fig. 6 a-d) . This suggests that CIPS could be successfully used to capture the virus in the body and direct it to degradation by facilitating its elimination by macrophages. Another important consequence of the phagocytic virus uptake by macrophages and its intracellular degradation through the endocytic/lysosome pathway, caused by CIPS, is the expected generation of viral peptides to be presented in the context of MHC-II molecules, i.e., the pathway of antigen presentation that induces a strong antibody response (Fig. 6 k-m). Thus, the capacity of CIPS to direct the virus to macrophages has the double advantage of promoting viral destruction (thereby limiting infection) while at the same time triggering anti-viral immunization.

Thus, CIPS is a safe, biocompatible, biodegradable 2D NM capable of inhibiting the infection and promoting the elimination of SARS-CoV-2. The binding between CIPS and the SARS-CoV-2 S protein RBD (K D <1 pM) is 10,000x stronger than the affinity of the virus for ACE2, suggesting that the CIPS-captured virus will not be released and will not infect cells. Notably, CIPS binding is 100x stronger than that described for the best nAbs. In addition, CIPS promotes SARS-CoV-2 uptake and degradation by macrophages through a pathway that promotes virus-specific antibody production. Eventually, its glue-like selective binding capacity makes CIPS applicable both as disinfection agent and in surface coatings, and as a nanodrug candidate for treating COVID-19 (Fig. 7) .

Bulk CuInP 2 S 6 (CIPS) material was synthesized by solid state reaction according to previous publication. 45 CIPS nanosheets (NSs) were exfoliated from the bulk CIPS crystals with Li-intercalation by using n-butyl lithium assisted by ultrasound sonication. After the exfoliation, NSs were centrifuged at 1500 g for 20 min and then rinsed with H 2 O twice to remove free n-butyl lithium in the suspension. The collected suspension was finally lyophilized and stored in dry condition at -20ºC.

The experiments were performed in a biosafety level 3 laboratory with standard 

Vero-E6 cells were seeded in 24-well plate overnight with a density of 5×10 4 cells/well. Cells were challenged with SARS-CoV-2 (12000 pfu) for 2 h, and the 

In a P3 laboratory, the SARS-CoV-2 (6000 pfu) were incubated with CIPS (6 and 12 pM in 200 μ l PBS) for 2h, and centrifuged (3000 rpm for 5 min) to separate the CIPS-trapped SARS-CoV-2 in precipitates. The RNA of the SARS-CoV-2 was extracted for reverse transcription (TAKARA, Japan). The SARS-CoV-2 was quantitative analyzed with real time PCR by targeting S protein.

Biolayer Interferometry (BLI) technique was used to measure the binding affinity of CIPS or CIPS-RBD complex to proteins, using an and dissociation rate constants (K off ) were calculated by fitting the curves using the 1:1 kinetic binding model.

The secondary structure of RBD in the presence or absence of CIPS was assessed by Circular Dichroism (CD) (JASCO, J-810). RBD (500 μ l at 200 μ g/ml in 0.01 M phosphate buffer pH 7.4) was mixed with CIPS (15, 30 , and 60 pM) and then added into the liquid well with 1 mm thickness. CD spectra were collected between 190 and 250 nm. Each sample was measured six times and the averaged spectra were obtained.

Spectral data were processed with the CD tool software (available at http://cdtools.cryst.bbk.ac.uk). The baseline (between 235 and 240 nm) was subtracted. Normalized data were analyzed by the web server DICROWEB to calculate the ratio of secondary structure, and the smoothed data are shown.

The THP-1 cells were seeded in a 24-well plate with cover slide at a density of 5x10 4 cell/well (for elimination), or in a 96-well plate at a density of 1x10 4 cell/well (for infection), and treated with 100 ng/ml PMA 24 h for macrophage differentiation.

For elimination experiment, CIPS (12 pM) was used to pre-incubate with SC2-P for 2 h. The SC2-P with/without CIPS pre-treatment was added to the PMA differentiated THP-1 cells for 24 h, named as "phagocytosis". Here after, fresh medium was used to replace the SC2-P containing medium and cultured for another 24 h, named as "degradation". Bafilomycin (BM), the lysosome inhibitor, was added to macrophages 6 h before the end point of each experiment. The macrophages phagocytosis and degradation of SC2-P were detected by IF.

For infection experiment, THP-1 cells were co-cultured with SC2-P (2x10 5 copies) 

The experiments were performed in a biosafety level 3 laboratory with standard regulations. The THP-1 cells were seeded in a 24-well plate at a density of 5x10 4 cell/well, and treated with 100 ng/ml PMA 24 h for macrophage differentiation. Data presented as mean ± SEM (n=3). ***, p<0.005 by Student's t-test. (f) Representative image of H&E staining of cultured tissue cross-sections. Ctrl: uninfected tissue; Virus: tissue infected with SARS-CoV-2; Virus + CIPS: tissue infected with SARS-CoV-2 treated with 24 pM CIPS. (g) The thickness of the human respiratory epithelium section that measured by ImageJ. Data are representative of best epithelium section, and presented as mean±SEM of technical triplicates. *, p<0.05 and **, p<0.01 by Student's t-test. (h) Cell distribution in the human respiratory epithelium after SARS-CoV-2 infection. Cross-section of the human respiratory epithelial tissue in control culture conditions (Ctrl), upon exposure to SARS-CoV-2 (Virus) and exposed to SARS-CoV-2 together with CIPS (Virus+CIPS). Green: tubulin staining the ciliated columnar epithelial cells; Red: MU5AC staining of mucus-producing goblet cells; Blue: DAPI staining of nuclei. Scale bar = 20 μ m. The white arrows show the sites of tissue damage. The upper row shows the bottom view of the amino acid sites of RBD that bind with CIPS, where van der Waals spheres are the residues contacting CIPS (red, polar residues; green, hydrophobic residues; blue, positively charged residues; purple, negative residues; yellow, cysteines containing disulfide bonds). b) Preferred functional groups of RBD amino acids bound to the surface of CIPS, as determined by the interaction energies between CIPS and different functional groups based on MD simulations. c) The interaction energy between RBD and CIPS (black) and the number of contact atoms (with the exception of of H; red) in RBD when approaching the CIPS surface in System 1. d) CIPS binding to RBD interferes with RBD binding to ACE2. Amino acid residues at the RBD binding sites for ACE2 (a, green) and those of RBD to CIPS (at 100 ns in System 1; b, red) are largely overlapping (c, yellow). Fig. 7 . The anti-SARS-CoV-2 capacity of CIPS "nano-glue". The mechanism that possessed by CIPS on SARS-CoV-2 restraining and elimination. The RBD of S protein can be tightly absorbed to CIPS that induced RBD deformation. The selectively binding capacity of CIPS on RBD with a K D < 1 pM that is 1000 times higher than the reported neutralizing antibodies, causes the inhibition of SARS-CoV-2 infection. Further, the CIPS-trapped SARS-CoV-2 can be effectively phagocytized by macrophages and lead to virus elimination in lysosome, which further facilitates COVID-19 treatment.

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Room-Temperature Crystal-Structure of the Layered Phase CuInP2S6

CIPS incubated with SARS-CoV-2 for 2 h and separated by centrifugation. The SARS-CoV-2 was quantitatively assessed in CIPS precipitates. Data as mean ± SEM of technical triplicates. ***, p<0.005 by Student's t-test. d, e) Binding affinity for CIPS of RBD at different concentrations (nM) (d) and various proteins at 50 nM (e) as determined by Biolayer Interferometry (BLI). f-g) The SARS-CoV-2 inhibiting capacity in complex biological matrices. SC2-P were mixed with FBS at different ratios and incubated with 12 pM CIPS for 2 h. f) WB analysis showing the specific binding of SC2-P to CIPS in the presence of FBS. g) Anti-SARS-CoV-2 activity of CIPS in the presence of FBS. The FBS/SC2-P/CIPS were used to infect ACE2/293T cells for 2 h. Cells were lysed after 40 h incubation and the intracellular SC2-P were detected based on luciferase activity. Data in (g) are from a representative experiment out of three performed, and presented as mean ± SEM of technical triplicates. ***, p<0.005 by Student's t-test. h-i) Inhibition of RBD

SARS-CoV-2 (c-d) in Macrophage-like differentiated THP-1 cells. a-b) The SC2-P were pre-incubated with 12 pM CIPS for 2 h. Macrophage-like differentiated THP-1 cells were treated with SC2-P with/without CIPS pre-incubation for 24 h (Phagocytosis). SC2-P containing medium was replaced by fresh medium and cultured for another 24 h (Degradation). b) Quantitative and statistical analysis with the infected SC2-P of IF data. c) Differentiated THP-1 was challenged with SARS-CoV-2 for 4 h (Phagocytosis). The SARS-CoV-2 challenged THP-1 was continuously cultured in fresh medium for another 48 h (Degradation)

Data presented as mean ± SEM of technical triplicates. ##, **, p<0.01, ###, ***, ∇ ∇ ∇ , p<0.005 by Student's t-test. ND: not detected. g) The uptake and the degradation of CIPS by macrophages as determined by ICP-MS. Intracellular Indium was used to describe the accumulation and degradation of CIPS and related compounds resulting from CIPS degradation. Differentiated THP-1 cells were treated with 12 pM CIPS up to 24 h (Uptake) and further cultured in fresh medium for 24 and 48 h (Degradation). h) Three-dimensional tomographic images showing CIPS intracellular accumulation after 12 h uptake and 48 h degradation. Images were obtained by soft X-ray transmission microscope (Nano-CT). i-j) Cu chemical transformation and degradation from intracellular CIPS during the uptake and degradation phases, as determined by Cu K-edge XANES. (i) Chemical species of Cu in different reference samples and (j) the speciation of Cu from CIPS during the uptake and the degradation processes. The percentage of Cu forms is reported in Table S2. k-l) CD86 and HLA-DRA gene expression in SARS-CoV-2 treated macrophages in the presence or absence of CIPS. SARS-CoV-2 virus with/without CIPS were incubated with PMA-differentiated THP-1 for 48 h. HLA: HLA-DRA. GAPDH was used as housekeeping gene and the relative gene expression was normalized to the virus infection group. Data presented as mean±SEM (n=3). *, p<0.05, **, p<0.01 by Student's t-test. (m) CIPS induced DC maturation after SC2-P incubation. SC2-P, and SC2-P with CIPS were incubated with DC for 24 h. The CD11c and CD86 positive cells were analyzed by flow cytometer

This work was financially supported by the National Natural Science Foundation of 

The authors declare no competing financial interests. Crosby, J. C. et al. COVID