key: cord-0769554-r2lcjcun
authors: Li, Jinwen; Zhang, Yantao; Pang, Huimin; Jie Li, Shu
title: Heparin interacts with the main protease of SARS-CoV-2 and inhibits its activity
date: 2021-11-15
journal: Spectrochim Acta A Mol Biomol Spectrosc
DOI: 10.1016/j.saa.2021.120595
sha: 8131f921f2b73e1a1f64466d7eacabda1c28b5a8
doc_id: 769554
cord_uid: r2lcjcun

The ability of SARS-CoV-2 to replicate in host cells is dependent on its main protease (M(pro), also called 3CLpro) that cut the viral precursor polyproteins and is a major target for antiviral drug design. Here, we showed that heparin interacts with the M(pro) of SARS-CoV-2 and inhibits its activity. Protein fluorescence quenching showed that heparin strongly binds to the M(pro) protein with dissociation constants K(D) of 16.66 and 31.60 μM at 25 and 35 (o)C, respectively. From thermodynamic parameters of the interaction, there are hydrophobic and hydrogen bond interactions between them. Fluorescence resonance energy transfer (FRET) assay demonstrated that heparin inhibits the proteolytic activity of M(pro) with an inhibition constant Ki of 6.9 nM and a half maximal inhibitory concentrations (IC(50)) of 7.8±2.6 nM. Furthermore, molecular docking analysis revealed that the recognition and binding groups of heparin within the active site of SARS-CoV-2 M(pro) provide important new information for the characteristics of the interactions of heparin with the protease. Our finding suggested that heparin might have a potential role in inhibiting SARS-CoV-2 infection through inhibiting M(pro) activity of SARS-CoV-2.

Coronavirus infects humans and other animals and causes a variety of highly prevalent and severe diseases, including severe acute respiratory syndrome (SARS) and

Middle East respiratory syndrome (MERS) [1] . A novel coronavirus, called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the pathogen of the COVID-19 pandemic of 2019-2021 [2, 3] .

SARS-CoV-2 is an RNA virus from the Betacoronavirus genus [2, 3] . The genome of this virus is about 88% homologous to the bat coronavirus, but only 79% to SARS-CoV and 50% to MERS-CoV [4] . SARS-CoV-2 has a typical coronavirus gene sequence, and the genome contains about 30,000 nucleotides. About two thirds of the genome is occupied by two open reading frames ORF1a and ORF1ab that encode the non-structural proteins, while the remainder of the region next to the 3' end encodes structural proteins [4] . Orf1ab is translated into two polyproteins PP1a and PP1ab by host ribosomes, which are processed by the virus' main protease (M pro , also called 3CL pro ) and a papain-like protease (PL pro ). PL pro cleaves the first three sites at the Nterminus and M pro cuts at the remaining 11 sites at the C-terminus, which form 15 nonstructural proteins (NSP) [3, 4] . The M pro of SARS-CoV-2 contains three domains (domains I to III) and has a 6-stranded β-barrel chymotrypsin-like fold with a homology to the monomeric picornavirus 3C protease, which forms a homodimer and whose active site contains a cysteine-histidine catalytic dyad [5, 6] . The sequence recognized by Mpro contains Leu-Gln-(Ser/Ala/Gly) with a cleavage site occurring after the Gln residue [5, 6] . Because Mpro of SARS-CoV-2 is necessary for the viral replication and transcription and no human homologues [3] , it is one of the most characteristic and ideal antiviral targets in the virus [7, 8] . Heparin is a sulfated polysaccharide widely used in clinic as an intravenous anticoagulant [9, 10] , which is mainly composed of a tri-sulfated disaccharide repeating unit, 2-O-sulfo-α-L-iduronic acid 1-4 linked to 6-O-sulfo-N-sulfo-α-D-glucosamine ( Fig. 1 ) [11] [12] [13] . In addition to tri-sulfated disaccharides, heparin also contains disaccharides with a lower degree of sulfation, resulting in a heterogeneous structure, named heparin sulfate (HS) (Fig. 1) [12, 13] . HS forms the precursor polysaccharide which is transformed by a series of enzymes into the main repeating unit of heparin [13] . The structural distinction between heparin and HS is subtle; Over 70% of heparin consists of the disaccharide shown in the top panel of Fig. 1 , but HS contains much less of the main repeating unit of heparin, which has a higher proportion of the many intermediate structures resulting from incomplete action of the postpolymerization enzymes [12, 13] .

Heparin together with HS forms a glycosaminoglycan family, which have a same biosynthesis pathway [11] . COVID-19 is usually accompanied by coagulation dysfunction and disseminated intravascular coagulation (DIC). For these reasons, heparin is used as a therapeutic drug for the treatment of COVID-19 [14] . Heparin can be directly targeted to the surface of the airway lumen by atomization, reducing both the infection of the surface of the airway lumen and the thrombosis in the air sac [15] .

These data suggested that the current use of systemic unfractionated heparin (UFH) in the treatment of patients with COVID-19 in an ICU setting may provide useful antiviral benefits. In addition, various commercially and clinically available UFH have an antiviral effect against live wild-type SARS-CoV-2 through inhibiting the binding of Spike RBD to human ACE2 [16] . However, the interactions between heparin and M pro of SARS-CoV-2 and the effects of heparin on the M pro activity remain unknown.

COVID-19 pandemic is becoming one of the largest in the history of global public health crisis. There are currently no targeted therapies for the disease, and effective treatment options remain very limited. Finding an effective drug to prevent or treat infections is a top priority for health care providers, government officials and the pharmaceutical industries. As a viral protease M pro of the SARS-CoV-2, is a most prominent target for antiviral drug screening [5, 6, 8] . In the present work, we showed that heparin binds with the M pro of SARS-CoV-2 and inhibits the activity of the protease.

Our findings suggested that heparin might have a potential role in inhibiting SARS-CoV-2 infections through inhibiting the viral replication.

Heparin was purchased from Sigma-Aldrich (Shanghai, China), and GC376 from BioChemPartner (Shanghai, China). The fluorescence substrate MCA-AVLQSGFR-Lys(DNP)K-NH2 was synthesized by Nanjing Peptide Bio-tech Ltd. (Nanjing, China).

All the other chemicals and reagents used were of HPLC or analytical grade.

To express the M pro of SARS-CoV-2, the gene encoding the protein (GenBank, accession number MN908947.3) was synthesized and cloned into pGEX6P-1vector

between BamHI and EcoRI sites to create a fused protein with glutathione S-transferase (GST) and a preScission protease cleavage site for the removal of the GST moiety. determined by the BCA method using BSA as a standard [17] .

To assess the interaction of heparin with M pro , fluorescence quenching was carried out at 25 and 35 o C with an RF-5301 PC spectrofluorometer (SHIMADZU, Japan), as previously described with some modifications [18, 19] . In brief, heparin of 66.7 μM was titrated to a cuvette containing 2 mL of 1. [20] is

and the static quenching equation is

where F 0 and F are the fluorescence intensities in the absence and presence of heparin, respectively. K q is the quenching rate constant of the biomolecule; τ 0 is the average lifetime of the molecule without quencher; [Q] is the free concentration of heparin; K SV is the dynamic quenching constant; K A is the formation constant; and K D is the dissociation constant.

In order to determinate the interaction characteristics between heparin and M pro , such as hydrogen bond, van der Waals force, and electrostatic and hydrophobic interaction, the following equations were used:

where, ΔH, ΔG, and ΔS are enthalpy, free energy, and entropy change, respectively.

Proteolytic activity of M pro was determined by a fluorescence resonance energy transfer (FRET) assay using a peptide substrate MCA-AVLQ↓SGFR-Lys(DNP)K-NH 2 in which MCA is a fluorescence Donor and DNP is a fluorescent receptor or known as a quencher [5] . The two groups are closely enough to produce a FRET, and the fluorescence from MCA is very small and almost not detected, when the substrate not to be digested by the M pro . When the substrate is digested by the protease, the separation of the two groups induces an increase in MCA fluorescence and the more substrate is 

All statistics were performed using GraphPad Prism 8 software. Data were represented as mean ± SD. Comparison of the mean between groups was performed by t test. P values < 0.05 were considered significant.

The M pro protein was expressed with a GST-tag and purified with Glutathi-one-Sepharose 4B affinity column and gel filtration column to high purity with a molecular weight about 35 kDa, which is consistent with 34.436 kDa of the theoretical molecular weight ( Fig. 2A) . To investigate whether there is an interaction between heparin and the M pro of SARS-CoV-2, the fluorescence quenching of the M pro protein was employed.

As observed in Fig. 2B , C and D, the fluorescence intensity of M pro was gradually decreased with an increase in the concentrations of heparin, demonstrating that the tryptophan fluorescence of the M pro protein was quenched by heparin and an interaction between the heparin and M pro occurred. Fluorescence quenching includes dynamic and static modes, in which the static mode the fluorophore and quencher form a complex [18, 19] . As shown in Fig. 2E , the Stern-Volmer curves were linear, and the slope decreased with increasing temperature.

The Stern-Volmer quenching constant K SV is equal to K q τ 0 according to Stern-Volmer equation [18, 20] , in which the average lifetime of the enzyme without heparin τ 0 is 10 −8 s [22] . Therefore, the quenching constant K q for heparin can be obtained from the slope of the Stern-Volmer curves (Fig. 2E) . The maximum collision quenching constant of various quenchers with protein is 2.0 × 10 10 l mol −1 s −1 [23] . In our case, the rate constant of the quenching process of M pro induced by heparin is greater than the K q of the dispersion process, so the quenching is not caused by dynamic collisions, but by the formation of a complex between heparin and M pro . Hence, the static quenching equation is suitable for the present work [18] , and the dissociation constants (K D ) can be derived from the slopes of the curves (Fig. 2F) (5), as our previous described [18, 19] . From the values of ΔH, ΔS and ΔG, the interactions between heparin and M pro are mainly hydrogen bond and van der Waals force (Table   2 ), which is in accordance with the result from in the presence of higher salt concentration. 

To determine the M pro activity, a commercially available FRET based substrate was used [5] . To calculate K cat , a standard curve was produced and the fluorescence intensities were converted into the amount of cleaved substrate according to the standard curve (Fig. 3A) . On the basis of establishing the experimental conditions for detecting protease activity, the inhibitory effects of different concentrations of heparin on the protease activity were investigated. Heparin was pre-incubated with M pro in the reaction buffer to ensure the full interaction between them before the addition of the FRET substrate.

As shown in Fig. 3B , in the absence of the inhibitor, the fluorescence of the substrate was gradually increased with time, indicating an increase in the amount of cleaved substrate with time. However, in the presence of the inhibitor, we can clearly observe that the fluorescence of the substrate remarkably attenuated, suggesting that heparin has a prominent intervention effect on the activity of M pro (Fig. 3C) . When the concentration of heparin is high enough, the fluorescence of the substrate was almost completely suppressed (Fig. 3D ). In the presence of a variety of concentrations of heparin, the fluorescence intensities at a wavelength of 393 nm excited by a wavelength of 325 nm proceeding over time up to 50 and 20 min were shown in Fig. 4A and B, respectively. When the substrate hydrolysis reaction exceeded 20 min, significant substrate consumption was observed. Therefore, the reaction progress curve in the first 20 min is selected for fitting (Fig. 4B) . The slope ratio in the presence and absence of the inhibitor reflects the inhibition rate of the inhibitor to the enzyme. The curve of the enzyme activities under different heparin concentrations was plotted, and fitted to use the Morrison equation (Fig. 4C and D) . Carbon atoms of the heparin are in magenta, except for the sulfonic groups, which is in red and yellow; oxygen atoms are red, nitrogen blue, and sulfur yellow. Hydrogen bonds are indicated by dashed green lines.

B: Two-dimensional LigPlot image of DP4-M pro complex [25] .

To obtain the structural basis about the interactions of heparin with M pro , molecular docking was employed using the Autodock Vina software [21] . The SARS-CoV-2 M pro protein consists of three domains, in which domains I and II have an antiparallel βbarrel structure, and domain III contains five α helices arranged into a largely antiparallel globular cluster [5, 7] . M pro has a Cys-His catalytic dyad, and the substratebinding site is located in a cleft between domain I and domain II. Docking studies using DP4 demonstrated preferred interactions with the substrate-binding pocket (Fig. 5) .

Evaluation of heparin-protein contacts and energy contributions using the in the substrate binding site of the protease through hydrogen bonds [26, 27] . Glu166 residue is a key amino acid involved in the dimerization of M pro and creation of substrate binding pocket. Cys145 and His41 residue forms a catalytic dyad on the active site of the protein essential for its catalytic function. Similar results were obtained from molecular docking with different molecules against the M pro protein [28] .

Coagulation disorder is a major problem in late stage of COVID-19 disease [29] .

Some reports showed that systematic heparin treatment can reduce mortality in hospitalized patients with COVID-19, which is considered to be a consequence of the known anticoagulant effect [30, 31] . The M pro of SARS-CoV-2, as a key enzyme of coronaviruses, plays an essential role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-2 [5, 6, 8] . In this report, we found that heparin binds to the SARS-CoV-2 M pro and inhibits its proteolytic activity in vitro.

Our findings suggested that heparin might inhibit SARS-CoV-2 replication and transcription through inhibiting activity of the SARS-CoV-2 M pro protein.

Heparin was discovered in 1916 and is still in widespread clinical use as an intravenous anticoagulant [9] , which has an average of four negative charges for each disaccharide unit, can interact with a wide range of proteins, with interactions that exhibit a range of specificities [32] and induce several associated biological activities.

These involve plasma or tissue proteins such as heparin cofactor II (HCII), tissue factor plasminogen inhibitor (TFPI), lipoproteinlipase, growth factors and heparinase [33] . As a therapeutic agent, heparin inhibits thrombosis by accelerating the binding of the protease inhibitor, antithrombin III, to thrombin and to other proteases involved in coagulation. Because of its anticoagulant activity, heparin carries a risk of excessive bleeding complications [34] .

Some studies have reported the protective effect of heparin on patients with COVID-19 based on its anticoagulant and anti-inflammatory activities [29] [30] [31] . Heparin interacts with chemokines and DAMPs released during infection, thereby inhibiting the pro-inflammatory activities of these proteins [35] . Clinical data from patients with COVID-19 with coagulopathy have attributed the beneficial effects of LMWH (low-molecular-weight heparin) to its anti-inflammatory effects [30, 31] . The inhibitory effects of UFH and LMWH on antiviral activity against live SARS-CoV-2 have been studied using a plaque inhibition assay with Vero E6 cells, which showed that heparin interacts with the spike protein of SARS-CoV-2 to prevent viral binding to host cellular receptor angiotensin-converting enzyme 2 (ACE2) [16] . Moreover, study showed that the cellular heparin promotes the combination of SARS-CoV-2 spike protein with ACE2 through enhancing the open of the conformation of its receptor-binding domain (RBD) and the spike protein binding to ACE2 depends on both heparan sulfate and ACE2 [35] . In a recently clinical trial, the prophylactic anticoagulation is similar to therapeutic anti-coagulation in the patients with COVID-19 [36] , suggesting that in the actual use of heparin for COVID-19 treatment, it may increase the bleeding of the treated patients is also a problem that needs to be considered in the future. [26] . Recent study showed that sulfated polysaccharides such as fucoidan from brown algae and iotacarrageenan from red algae have an obviously antiviral activity against SARS-CoV-2 [37] . Heparin, as one of the sulfated polysaccharides, is higher sulfurated in each disaccharide unit than HS, fucoidan, iota-carrageenan and chondroitin sulfate [29, 37] .

So, the existence of multiple sulphuric acid groups in heparin may be the reason for its binding to M pro and the better inhibitory effect against M pro activity.

In our previous works, we have developed the method of protein fluorescence quenching to investigate the interactions between proteins and other molecules [18] . In the present work, we have determined the dissociation constant (K D ) and interaction characteristics between heparin and M pro protein, using the method. Our results showed that the dissociation constant between heparin and M pro protein is 16.66 μM at 25 o C,

indicating that heparin strongly binds to M pro protein [18, 19] . Thermodynamic parameters calculated from the data of the fluorescence quenching reveals that heparin interacts with M pro protein through hydrogen bond and hydrophobic interactions, but not electrostatic interaction, although having negative charges in heparin due to sulfation. The free binding energy and interaction features obtained from molecular docking are consistent with the results from fluorescence quenching, which provides a structural basis for the interaction between heparin and M pro protein. Nevertheless, to crystalize the heparin-M pro complex and resolve its crystal structure are necessary to elucidate the mechanism that heparin inhibits M pro activity, and the work is developing in our lab.

Heparin belongs to the glycosaminoglycan family and consists of long repeating disaccharide units with variable sulfate groups [11] . The structure and size of the molecules are important to their biological properties. More recently, seven different origins and kinds of heparin including UFH and LMWH from pig and cattle have been studied for antiviral activity against live SARS-CoV-2 [16] . All the kinds of UFH have potent antiviral activities against SARS-CoV-2, with IC 50 values ranging between 25

and 41 μg/ml, whereas the IC 50 values for the kinds of LMWH range from 3.4 to 7.8 mg/ml, less inhibitory effects by ~150-fold, demonstrating that UFH has a significantly strong anti-SARS-CoV-2 activity compared to LMWH. Tree et al. [16] attributed this antiviral activity of UFH to that UFH directly inhibits the binding of spike protein to the human ACE2 protein receptor. In our present work, we showed that UFH binds to M pro protein of SARS-CoV-2 and inhibits its activity in vitro. The processing of the SARS-CoV-2 polyproteins depends on M pro to a great extent [5] . SARS-CoV-2 M pro has higher proteolytic efficiency and can accelerate the life cycle of the virus. Therefore, the anti-SARS-CoV-2 activity of UFH might be contributed by the inhibitory effects of UFH against SARS-CoV-2 M pro activity.

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Conceptualization, Methodology, Software, Formal analysis, Investigation, Data Curation, Writing -Original Draft. Yantao Zhang: Methodology, Software, Formal analysis, Investigation. Huiming Pang: Conceptualization, Methodology, Investigation, Visualization, Supervision, Project administration, Funding acquisition. Shu Jie Li: Conceptualization, Methodology, Validation, Resources, Writing -Review & Editing, Supervision

• Main protease of SARS-CoV-2 is a key enzyme for the viral replication and transcription

• Heparin strongly binds with the main protease of SARS-CoV-2

• Heparin effectively inhibits activity of SARS-CoV-2 main protease

• The findings suggested that heparin might have a potential role in inhibiting SARS-CoV-2 infection through inhibiting the main protease activity of SARS-CoV-2

This work was partly supported by the Science and Technology Program of Jiangsu Province (LYL-SZ201915) and the Natural Science Foundation of Shandong Province (ZR2020MC056).

There are no conflicts of interest.

JWL, HMP and SJL conceived and designed the study. JWL, YTZ and HMP performed the experiments. JWL wrote the paper. SJL and HMP reviewed and edited the manuscript. All authors were involved in data analysis, read and approved the manuscript.

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: