key: cord-0318157-3pbe2uy1
authors: Jin, Can; Feng, Bo; Pei, Rongjuan; Ding, Yaqi; Li, Meixia; Chen, Xia; Du, Zhenyun; Ding, Yangxiao; Huang, Chunfan; Zhang, Bo; Chen, Xinwen; Zang, Yi; Li, Jia; Ding, Kan
title: Novel pectin from crude polysaccharide of Syzygium aromaticum against SARS-CoV-2 activities by targeting 3CLpro
date: 2021-10-29
journal: bioRxiv
DOI: 10.1101/2021.10.27.466067
sha: a2f6e3b2ba23f947a51501e3e53f521aa5267811
doc_id: 318157
cord_uid: 3pbe2uy1

To date, COVID-19 is still a severe threat to public health, hence specific effective therapeutic drugs development against SARS-CoV-2 is urgent needed. 3CLpro and PLpro and RdRp are the enzymes required for the SARS-CoV-2 RNA synthesis. Therefore, binding to the enzyme may interfere the enzyme function. Before, we found that sulfated polysaccharide binding to 3CLpro might block the virus replication. Hence, we hypothesize that negative charged pectin glycan may also impede the virus replication. Here we show that 922 crude polysaccharide from Syzygium aromaticum may near completely block SARS-CoV-2 replication. The inhibition rate was 99.9% (EC50 : 0.90 μM). Interestingly, 922 can associates with 3CLpro, PLpro and RdRp. We further show that the homogeneous glycan 922211 from 922 may specifically attenuate 3CL protease activity. The IC50s of 922 and 922211 against 3CLpro are 4.73 ± 1.05 µM and 0.18 ± 0.01 µM, respectively. Monosaccharide composition analysis reveals that 922211 with molecular weight of 78.7 kDa is composed of rhamnose, galacturonic acid, galactose and arabinose in the molar ratio of 8.21 : 37.81 : 3.58 : 4.49. The structure characterization demonstrated that 922211 is a homogalacturonan linked to RG-I pectin polysaccharide. The linear homogalacturonan part in the backbone may be partly methyl esterified while RG-I type part bearing 1, 4-linked α-GalpA, 1, 4-linked α-GalpAOMe and 1, 2, 4-linked α-Rhap. There are four branches attached to C-1 or C4 position of Rhamnose glycosyl residues on the backbone. The branches are composed of 1, 3-linked β-Galp, terminal (T)-linked β-Galp, 1, 5-linked α-Araf, T-linked α-Araf, 4-linked α-GalpA and/or 4-linked β-GalpA. The above results suggest that 922 and 922211 might be a potential novel leading compound for anti-SARS-CoV-2 new drug development.

injected in each run (60 min for each run). The eluate was monitored with an RI (Keep in 25 ℃) 160 and a UV detector, and the column temperature was kept at 35 ℃. 161

The monosaccharide composition was analyzed using PMP pre-column derivatization 163 based on the previous reported (J. Dai et al., 2010) . In briefly, 922211 (2 mg) was hydrolyzed 164 with 4 mL of 2 M TFA (trifluoroacetic acid), followed by PMP derivation. 10 μL of the 165 derivative solution was analyzed by high performance liquid chromatography (HPLC) to 166 understand the sugar composition. 167

For NMR analysis, 922211 (30 mg) was deuterium-exchanged and dissolved by 0.5 mL 169 D2O (99.8% D), and then lyophilized and redissolved in 0.5 mL D2O (99.8% D). The 1 H, 13 C 170 NMR and 2D NMR spectra (COSY, HSQC and HMBC) were measured at 25 °C with acetone 171 as internal standard (δH = 2.29, δC = 31.5). NMR spectra were recorded on a Bruker AVANCE 172 III NMR spectrometer. 173

The dried polysaccharide (10 mg) was methylated for 3-4 times based on previous 175 methods ( HAKOMORI, 1964) . The methylated polysaccharide was hydrolyzed and then 176 reduced with sodium borohydride and acetylated. The partially methylated alditol acetates were 177 examined by gas chromatography-mass spectrometry (GC-MS). Mass spectra of the 178 derivatives were analyzed using Complex Carbohydrate Structural Database of Complex

The approach of uronic acid reduction was based on the reported method (Taylor & Conrad, 183 1972). In brief, 40 mg polysaccharide was dissolved in 40 mL H2O. CMC (600 mg) was added 184 and pH was kept at 4.75 with 0.01 M HCl for 2 h. Then 2 M fresh aqueous sodium borohydride 185 (16 mL) was added slowly to the mixture (Sodium borohydride solution shall be added within 186 30-60 min) and maintained pH at 7 with 4 M HCl for 2 h at room temperature. The mixture was 187 dialyzed (1,000 mL × 4) for 24 h at room temperature. Then the retentate was lyophilized to 188 achieve carboxyl reduced polysaccharide, followed by monosaccharide composition and 189 glycosyl residues analyses. 190

The enzyme activity and inhibition assays of SARS-CoV-2 3CLpro have been described 

To determine the glycosyl linkage type, 922211 was methylated, hydrolyzed, reduced and 252 acetylated to produce the partially methylated alditol acetates (PMAA), which was analyzed by 253 gas chromatography-mass spectrometry (GC-MS) ( Table 2) . For 922211, the linkage pattern 254 of the galactose residues included 1, 3-linked Gal (7.81%) and Terminal (T)-linked Gal 255 (12.67%). The arabinose residues in the 922211 were consisted of Terminal (T)-linked Ara 256 (16.46%) and 1, 5-linked Ara (4.80%). The rhamnose residues contained 1, 2, 4-linked Rha 257 (19.07%). 922211 had a mass of galacturonic acid (Table 1) , which could not be methylated 258 successfully. Hence, the carboxyl group of galacturonic acid needs to be reduced to hydroxyl 259 to acetylate and produce the PMAA successfully to then analyze the linkage type of 

To elucidate the detail structure features of the backbone and branches of the 270 polysaccharide, 922211 was subjected firstly to partial acid hydrolysis, followed by dialysis 271 against de-ionized water. The intra-dialysate P2922211I (108 mg, yield: 72%) was obtained. T-, 1, 5-linked Ara. This inference was further evidenced by 1D and 2D NMR data analysis as 294 followed. 295

The 13 C (Fig. 1A , C) and 1 H (Fig. 1B ) NMR spectra were assigned and depicted in Table  297 3 supported by the monosaccharide composition, methylation analysis, partial acid hydrolysis, 298 two-dimension NMR spectra of 922211 and P2922211I (Fig. 2) In 13 C NMR spectrum, the two intense anomeric signals at δ 100.85 and δ 101.08 could be 307 assigned to C-1 of 1, 4-linked α-GalpA (d, d') in different chemical environments, respectively. 308

Signals at δ 101.47 and δ 101.67 could be assigned to C-1 of 1, 4-linked α-6MeOGalpA (e', e) 309 in different chemical environments, respectively. Indeed, the existence of methoxy group was 310 further proved by the appearing signals at δ 172.00 and δ 172.05, which prompting partial 311

GalpA residues might exist methyl ester. Furthermore, signals at δ 54.13 and δ 54.20 indicated 312 that methoxy group was attached to the C-6 position of 1, 4-linked α-6MeOGalpA. Signals at 313 δ 93.52 and δ 97.49 might be attributed to anomeric carbon of 4-linked α-GalpA and 4-linked 314 β-GalpA, respectively. In HSQC (Fig. 2B) indicating that 4-linked GalpA residues are β-and α-configuration, respectively. C-1 signal of 317 T-linked β-Galp (g) was overlapped by C-1 signal of 1, 3-linked β-Galp (f) and assigned to δ 318 104.67. Resonance at δ 100.25 could be assigned to C1 of 1, 2, 4-linked α-Rhap (c) and its 319 correlation with H1 of this residue at δ 5.15 in HSQC spectrum (Fig. 2B) . Arabian residues 320 were too weak to be directly observed in 13 C NMR spectrum. However, combined with the 321 cross peaks at higher chemical shift in HSQC spectrum (Fig. 2B) , resonances at δ 5.28/108.18 322 and δ 5.17/108.61 could be easily assigned to H1 and C1 of T-linked α-Araf (a) and 1, 5-linked respectively. Comparing with 922211, the resonances belonging to T-, 1, 3-linked Gal and T-, 338 1, 5-linked Ara almost vanished in HSQC of P2922211I (Fig. 1C) . Hence, it might be deduced 339 that those vanished glycosyl residues are on side chains. In HMBC spectrum (Fig. 3) 3CLpro activity (Fig. 4A and B) . 4.73 ± 1.05 µM and 0.18 ± 0.01 µM (Fig. 4A and B) used to examine whether 922 and 922211 might disturb the binding between S1 protein in vitro. 460 The results showed that polysaccharide 922 could impede weakly the binding of S1 protein 461 with ACE2 (Fig. 4C) . These results also suggest that anti-SARS-CoV-2 effects of 922 and 462 922211 are more likely to inhibit 3CL protein activity than disturbing the interaction between 463 SARS-CoV-2-S1 and ACE2. 464

The above results implied that 922 might have potential inhibitory effect on SARS-Cov-466 2. Hence, the inhibition effect of native polysaccharide 922 against the virus was examined. 467 Surprisingly, polysaccharide 922 nearly completely blocked SARS-Cov-2 replication in vitro, 468 exhibiting very good anti-SARS-CoV-2 activity in Vero E6 cells. The inhibition rate was 99.9% 469 (Fig. 5A) . The EC50 value was 0.90 µM (or 5.01 µg/mL) (Fig. 5B) . The above results suggested 470 that 922 is the good candidate for anti-SARS-Cov-2 new drug development. Following, we will 471 further explore the antiviral mechanism for 922 and 922211. 2. Unlike other sulfated polysaccharides, which antiviral activity is mainly arisen from 490 competitive inhibition of S1 and ACE2 binding, both 922 and the pectin-like glycan 922211 491 might impede 3CLpro activity, but only tenderly disturb the binding of S1 and ACE2. 492

To further understand whether there some other components in the 922 may interfere 493 3CLpro, PLpro, or RdRp enzyme activity. We firstly pooled the fragment from 922 crude 494 27 / 38 9222P, 9224P, 9221S on 3CLpro was roughly 76.57 μg/mL, 42.12 μg/mL, 17.17 μg/mL, 14.36 500 μg/mL, 22.99 μg/mL (Fig. 6A-E) , respectively. Surprisingly, 92205P, 9222P and 9224P could 501 also potently inhibit RdRp enzyme activity, while 9222P and 9224P might nearly completely 502 block this enzyme activity (Table S1) . However, almost all of these components have no 503 significant effect on the PLpro enzyme (Table S1 ). This imply that polysaccharide 922 inhibit 504 SARS-CoV-2 virus replication at least through disturbing both 3CLpro and RdRp enzymes, but 505 has nothing to do with PLpro enzyme. 506

Although the detail mechanism underlying the action of 922 and 922211 need to be further 507 explored, our study provides evidences for the first time for pectin or pectin-like polysaccharide 508 might be promising candidate for the anti-SARS-CoV-2 virus new drug development. 

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