key: cord-0313546-8b4q2342 authors: Huang, Jiahao; Wang, Jing; Wang, Ziyuan; Chu, Ming; Wang, Yuedan title: Tuftsin: a natural molecule against SARS-CoV-2 infection date: 2022-01-17 journal: bioRxiv DOI: 10.1101/2022.01.10.475746 sha: fdb0b847f3a8d12dab94b770eae4ed460dd517cf doc_id: 313546 cord_uid: 8b4q2342 Coronavirus disease 2019 (COVID-19) continuously proceeds despite the application of a variety of vaccines. It is still urgent to find effective ways to treat COVID-19. Recent studies indicate that NRP1, an important receptor of the natural peptide tuftsin, facilitates SARS-CoV-2 infection. Importantly, tuftsin is a natural human molecule released from IgG. Here, we found 91 overlapping genes between tuftsin targets and COVID-19-associated genes. Bioinformatics analyses indicated that tuftsin could also target ACE2 and exert some immune-related functions to treat COVID-19. Using surface plasmon resonance (SPR) analysis, we confirmed that tuftsin can bind ACE2 and NRP1 directly. Moreover, tuftsin effectively impairs the binding of SARS-CoV-2 S1 to ACE2. Thus, tuftsin is an attractive drug against COVID-19. And tuftsin as natural immunostimulating peptide in human, we speculate that tuftsin may has crucial roles in asymptomatic carriers or mild cases of COVID-19. Introduction 32 cells in the presence of ACE2 8 . It is worth noting that NRP1 is an important receptor 48 of tuftsin 9,10 . Tuftsin, a natural phagocytosis-stimulating peptide, was found by Victor 49 Najjar et al. in 1970 11 . Tuftsin is released from the Fc fragment of IgG by an 50 endocarboxy-peptidase in the spleen and a leukokininase on the outer membrane of 51 neutrophilic leukocytes 11, 12 . Furthermore, tuftsin is a tetrapeptide that consists of Thr-52 Lys-Pro-Arg, located at amino acid residues 289 to 292 of the heavy chain of IgG. 53 Tuftsin has a broad spectrum of activities mainly associated with immune system 54 functions and exerts effects on phagocytic cells, especially macrophages. These 55 functions of Tuftsin briefly include cell phagocytosis, motility, immunogenic response, 56 and bactericidal and tumoricidal activities 13, 14 . It was reported that tuftsin activity is 57 inversely correlated with splenectomy function and is significantly lower in patients 58 with AIDS, cirrhosis, intestinal failure and some infectious diseases 12,15,16 . Moreover, 59 it was demonstrated that tuftsin has stability and low toxicity in vitro and in vivo 14,17,18 . Compound profiling and disease-related gene identification 67 The structure of tuftsin was found in PubChem (https://pubchem.ncbi.nlm.nih.gov/). The 3D structure of tuftsin was built using Chem3D. Afterward, the target proteins 69 corresponding to tuftsin screened from the Pharmmapper database and PubMed 70 database were standardized in UniProt (http://www.uniprot.org/). Finally, Cytoscape 71 3.8.2 was used to determine the drug-target network. COVID-19-related genes were 72 mined from the GeneCards database. All of the disease gene targets were normalized 73 with R software using the Bioconductor package when redundancy was deleted 19 . Network establishment 76 Screening for drug-disease crossover genes was performed. Based on previous steps, 77 two sets of target lists were prepared: drug targets and disease-related genes. The 78 crossover genes were filtered with R software using the Venn Diagram package. The 79 STRING 11.5 database (http://string-db.org/) was used to analyse the intersecting 80 protein-protein interactions (PPIs), and the common targets were counted with R 81 software. Enrichment analysis 84 The proteins with overlapping expression patterns were evaluated by bioinformatics 85 annotation with R software using the Bioconductor package, including a panther The results were analysed using Student's t test with SPSS software and R 4.1.0. 128 4 a molecular simulation assay (Fig. 1A) . In addition to the reported receptors of tuftsin, 131 the potential targets of tuftsin in humans were also predicted through the PharmMapper 132 database. Together, 284 targets of tuftsin were collected ( Fig. 1B and data S1). 133 Furthermore, we collected 2572 disease-associated genes of COVID-19 from the 134 GeneCards database (data S2). We surprisingly found 91 intersecting proteins of tuftsin 135 targets and COVID-19-associated genes through intersection analysis (Fig. 1C) . It is 136 intriguing that the overlapping proteins account for nearly one-third of tuftsin targets. Moreover, the protein-protein interaction network of the overlapping proteins was 138 established, and it showed that JAK2, STAT1 and AKT1 are core molecules in the 139 network ( Fig. 1D ). Furthermore, we performed enrichment analysis for the 91 140 intersecting genes. GO annotation revealed that the expressed tuftsin-COVID-19 141 crossover proteins were mainly associated with immune functions such as neutrophil 142 activation, neutrophil-mediated immunity and cytokine receptor binding. Moreover, the 143 KEGG pathway enrichment analysis showed that the COVID-19 pathway was the most 144 significantly enriched. In addition, many target genes were strongly associated with 145 some immunologic pathways, such as Th17 cell differentiation, the IL-17 signaling 146 pathway and the immune checkpoint pathway (Fig. 1E ). In the COVID-19 pathway, 147 the SARS-CoV-2 receptors ACE2 and NRP1 were targets of tuftsin. Moreover, IL-2, 148 STAT1 and some complement molecules in the COVID-19 pathway were targets of 149 tuftsin (Fig. 1F) . Together, these results suggest that tuftsin is a promising candidate 150 against COVID-19, owing to its multifaceted pharmacological activities. The interaction of tuftsin with ACE2 and NRP1 analysed by molecular docking 153 It is novel that ACE2 is a potential target of tuftsin, as mentioned above. Thus, 154 molecular docking was performed to determine the potential binding sites and binding 155 affinity between tuftsin and the SARS-CoV-2 receptors ACE2 and NRP1. First, we 156 defined the interaction interface of SARS-CoV-2 S1-RBD with ACE2 as the active sites 157 of ACE2. These interface sites in ACE2 include Q24, M82, N330, and R393, which are 158 mainly located in the N-terminal peptidase domain of ACE2 20 . Then, the docking 159 region was a sphere containing the defined ACE2 active sites (Fig. S1A) . The results 160 showed that the affinity of tuftsin and ACE2 was -6.9 kcal/mol, demonstrating that they 161 could combine spontaneously ( Fig. 2A) . Furthermore, tuftsin could form strong 162 hydrogen bonds to Ser47 and Asp67, carbon hydrogen bonds to His345, Asp67 and 163 Asn51, and salt bridges to Asp67 of ACE2 ( Fig. 2A) . It is worth mentioning that the 164 binding sites were adjacent to the interactional sites of S1-RBD and ACE2, indicating 165 that tuftsin could inhibit S1 binding to ACE2 by covering their binding sites. 166 Meanwhile, the b1b2 domain of NRP1 was prepared, as previous studies showed that 167 the extracellular b1b2 domain of NRP1 mediates binding to CendR peptides 21 . Then, 168 the active sites of NRP1 b1b2 were defined according to the interactional sites of S1-169 RBD and NRP1 b1b2, including D320, E348, Y353 and so on 7 . The docking region 170 was a sphere containing the defined NRP1 b1b2 active sites (Fig. S1B) . The docking 171 results showed that tuftsin and NRP1 b1b2 have a high binding affinity of -8.1 kcal/mol. 172 to Pro398, which are near the interactional sites of S1-RBD and NRP1 b1b2. Moreover, 175 the binding region of tuftsin and NRP1 overlapped with the binding area of NRP1 and 176 S1-RBD in space (Fig. 2B) . Collectively, these results demonstrated that tuftsin could 177 bind ACE2 and NRP1 and inhibit the SARS-CoV-2 S1 binding of ACE2 and NRP1 by 178 covering their interactional sites. Tuftsin impairs the binding of SARS-CoV-2 S1 to ACE2 198 An SPR-based competition assay was employed to determine whether tuftsin could 199 affect the binding of S1 protein with ACE2. We first determined the binding affinity of 200 the S1 protein with ACE2 by SPR assay, which unsurprisingly showed a high affinity. CoV-2 S1 protein as a control. Then, a series of gradient concentrations of tuftsin 203 solutions containing equal concentrations of ACE2 were injected over the immobilized 204 SARS-CoV-2 S1 protein for comparison. We observed that 9 μmol/L tuftsin had a mild 205 inhibitory effect. It is worth noting that the addition of 156 μmol/L tuftsin significantly 206 attenuated the response signal by approximately two-thirds compared to that of ACE2 207 alone over the immobilized S1. Notably, a substantial decrease in the response signal 208 was observed with increasing concentrations of tuftsin. The response single was close 209 to zero when the added concentration of tuftsin was 625 μmol/L. This result indicates 210 that the interaction between S1 and ACE2 was almost completely blocked in the 211 presence of 625 μmol/L tuftsin (Fig. 4) . The experiment was repeated three times 6 independently. In conclusion, the competition binding experiment revealed that tuftsin 213 effectively impairs the binding of SARS-CoV-2 S1 to ACE2 in a dose-dependent 214 manner. At present, vaccination is the most general way to prevent COVID-19; however, the 218 notable problem is the uneven distribution of vaccine resources worldwide 22 . The cost 219 of producing vaccines and neutralizing antibodies is relatively high. It has been reported 220 that the effectiveness of the SARS-CoV-2 vaccine declines significantly during 2021 221 23 . Here, we report that an immune-stimulating peptide, tuftsin, is a potential effective 222 drug for COVID-19. Tuftsin, as a natural tetrapeptide that exists in humans, originates 223 from a special fraction of the parent carrier IgG through enzymatic processing. Accordingly, tuftsin has lower toxicity and fewer side effects than other drugs 24 . There In this research, 9 μM tuftsin had slight inhibitory activity. We observed that when the 234 concentration of tuftsin was 156 μM, the binding affinity of SARS-CoV-2 S1 and 235 ACE2 was reduced significantly. When the concentration reached 625 μ M, the 236 combination of SARS-CoV-2 S1 and ACE2 was completely blocked. It has been 237 confirmed that a 156 μM concentration of tuftsin can exist at a high concentration in 238 the internal environment after intravenous injection 28 . We conceive that tuftsin can be 239 designed as an oral or nasal spray. In this case, the local concentration of tuftsin reached that tuftsin binds at the N-terminus of ACE2, which is the area of S1 protein binding. This indicated that tuftsin can block the binding of S1 protein and ACE2 directly. It is worth noting that there were many asymptomatic and mild infectors during the 249 pandemic. It is clear that innate and adaptive immunity functions during asymptomatic 250 infection; however, the mechanism of the T cell and antibody response is unclear 30,31 . Asymptomatic people seem to clear the virus faster 32 . Tuftsin, a human natural 252 immunostimulating peptide released from IgG, certainly has significant roles related to 253 innate immunity. We reported that tuftsin can target the important receptors of SARS-254 CoV-2 S1, which is similar to adaptive immunity. Thus, we speculated that tuftsin has 255 crucial roles in asymptomatic or mild infection. It is likely that the activity of tuftsin is 256 higher in asymptomatic individuals than in symptomatic individuals. Fig. 4 . Tuftsin inhibits the SARS-CoV-2 S1 binding to ACE2. The binding activity 297 of SARA-CoV-2 S1 to ACE2 in the presence of increasing concentrations of tuftsin. Intensive concentrations of tuftin showed enhanced inhibitory effects. The authors declare that the research was conducted in the absence of any commercial 302 or financial relationships that could be construed as a potential conflict of interest. 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Prevention and treatment of COVID-19 disease by controlled 401 modulation of innate immunity What the data say about asymptomatic COVID infections 316 We thank the State Key Laboratory of Natural and Biomimetic Drugs (Peking 317 University) for their assistance in performing the surface plasmon resonance