|
HTCC as a highly effective polymeric inhibitor of SARS-CoV-2 and MERS-CoV 2 3
Abstract
The beginning of 2020 brought us information about the novel coronavirus emerging in China. 39 Rapid research resulted in the characterization of the pathogen, which appeared to be a member 40 of the SARS-like cluster, commonly seen in bats. Despite the global and local efforts, the virus 41 escaped the healthcare measures and rapidly spread in China and later globally, officially 42 causing a pandemic and global crisis in March 2020. At present, different scenarios are being 43 written to contain the virus, but the development of novel anticoronavirals for all highly 44 pathogenic coronaviruses remains the major challenge. Here, we describe the antiviral activity 45 of previously developed by us HTCC compound (N-(2-hydroxypropyl)-3-trimethylammonium 46 chitosan chloride), which may be used as potential inhibitor of currently circulating highly 47 pathogenic coronaviruses -SARS-CoV-2 and MERS-CoV. : bioRxiv preprint 65 pneumocytes in human airways, hijacking the angiotensin-converting enzyme 2 (ACE2) to 66 enter the cell, similarly as SARS-CoV and HCoV-NL63. 67 MERS-CoV is related to SARS-CoV-2, but together with some bat viruses forms 68 a separate Merbecovirus subgenus. Bats are believed to serve as an original reservoir also in 69 this case 10 , but camels were identified as the intermediate host 11 . The virus never fully crossed 70 the species border, as the human-to-human transmission is limited, and almost all the cases are 71 associated with animal-to-human transmission. The entry receptor for MERS-CoV is the 72 dipeptidyl peptidase 4 (DPP4) 12,13 . In humans, MERS-CoV causes a respiratory illness with 73 author/funder. All rights reserved. No reuse allowed without permission.
Coronaviruses mainly cause respiratory and enteric diseases in humans, other mammals, 50 and birds. However, some species can cause more severe conditions such as hepatitis, 51 peritonitis, or neurological disease. Seven coronaviruses infect humans, four of which (human pangolins were suggested as such 9 . The virus is associated with a respiratory illness that, in a 63 proportion of cases, is severe. The mortality rate varies between locations, but at present, is 64 estimated to reach 3-4% globally. The virus infects primarily ciliated cells and type II severity varying from asymptomatic to potentially fatal acute respiratory distress [14] [15] [16] . To date, 74 MERS-CoV infection was confirmed in 27 countries, with over 2,000 cases and a mortality rate 75 of ~35%. 76 Currently, there are no vaccines or drugs with proven efficacy to treat coronavirus 77 infection, and treatment is limited to supportive care. However, a range of therapeutics have Consequently, broad-spectrum antivirals are essential in long-term perspective. we dissected the mechanism of the HTCC antiviral activity. We showed that the polymer 96 interacts with the coronaviral Spike (S) protein and blocks its interaction with the cellular 97 receptor 22-24 . Here, we analyzed the HTCC activity against SARS-CoV-2 and MERS-CoV in 98 author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.29.014183 doi: bioRxiv preprint vitro using permissive cell lines and ex vivo, using a model of human airway epithelium (HAE). 99 The study showed that the replication of both viruses was efficiently hampered. Overall, our 100 data show that HTCC polymers are potent broad-spectrum anticoronavirals and may be very 101 promising drug candidates for SARS-CoV-2 and MERS-CoV. 105 Previously, we showed that HTCC with different degrees of substitution (DSs) is a The study on the MERS-CoV using the Vero cells revealed that all HTCC variants 117 inhibit virus replication to a similar extent (~100-1000-time decrease in viral yields), at non-118 toxic concentration (Figure 1A, C) . The inhibition of the SARS-CoV-2 infection in Vero E6 119 cells was even more pronounced, and all HTCC variants inhibited virus replication by ~10,000 120 times at non-toxic concentration (Figure 1B, C) . In this case, the HTCC-63 was arbitrarily 121 selected for further studies on MERS-CoV, while HTCC-77 was selected for SARS-CoV-2. 122 author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the . The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.29.014183 doi: bioRxiv preprint Next, the dose-dependence was tested for the HTCCs. The inhibitory activity of selected 134 polymers was verified for three different concentrations, and obtained data are shown in Figure 135 2.
Based on the data obtained, the basic parameters were calculated and are presented in Table 1 . The parameters observed for the SARS-CoV-2 appear to be favorable with SI above 12. It is 148 also worth to note that HTCC was previously administered by inhalation in rats, and no adverse 149 reactions were observed 25 . In that study, HTCC was used as a carrier for the active substance, 150 and as such, was reported to be promising for local sustained inhalation therapy of pulmonary 151 diseases.
152 author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.29.014183 doi: bioRxiv preprint 153 While the Vero cells constitute a convenient model for antiviral research, it is of utmost 154 importance to verify whether the results obtained are not biased due to the artificial system 155 used. This is especially important for compounds, which activity is based on electrostatic The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.29.014183 doi: bioRxiv preprint 182 Our previous research showed that the HTCC-mediated inhibition of coronaviral The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.29.014183 doi: bioRxiv preprint Next, to verify whether the mechanism of action for the highly pathogenic 219 betacoronaviruses is similar to that observed for alphacoronaviruses 22 , and is based on locking 220 the interaction between the virus and the entry receptor, we analyzed MERS-CoV The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.29.014183 doi: bioRxiv preprint
Taking together, we show here that the previously developed and described polymeric 239 HTCC anticoronaviral compounds based on chitosan are able to efficiently inhibit infection 240 with emerging coronaviruses. We believe that the HTCC can be fine-tuned to target any 241 coronavirus, and this interaction is specific to viruses that belong to the Coronaviridae family.
The active compound 257 The HTCC was prepared in the same manner as previously described 22,23,28 . The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.29.014183 doi: bioRxiv preprint sequence verified that was a gift from Xingchuan Huang. psPAX (Addgene plasmid # 12260) 263 and pMD2G (Addgene plasmid # 12259) was a gift from Didier Trono. Lego-G2 vector 264 (Addgene plasmid #25917) was a gift from Boris Fehs. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.29.014183 doi: bioRxiv preprint Cell viability assay 287 HAE cultures were prepared as described above. Cell viability assay was performed by (TPP) were exposed to MERS-CoV, SARS-CoV-2 or mock at a TCID50 of 400 per ml in the 311 author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.29.014183 doi: bioRxiv preprint presence of tested polymer or control medium. Following a 2 h incubation at 37°C, unbound 312 virions were removed by washing with 100 μl of 1 × PBS and fresh medium containing 313 dissolved respective polymer was added to each well. Samples of cell culture supernatant were 314 collected at day 3 p.i. and analyzed using RT-qPCR. 315 For the ex vivo study, fully differentiated human airway epithelium (HAE) cultures were 316 exposed to the tested polymer or control PBS for 30 min at 37°C, following inoculation with The copyright holder for this preprint (which was not peer-reviewed) is the . (Scientific Volume Imaging B.V., The Netherlands) and processed using ImageJ 1.47v 358 author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.29.014183 doi: bioRxiv preprint (National Institutes of Health, Bethesda, MD, USA). At the time of the study, no antibodies 359 specific to SARS-CoV-2 were available to us. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.29.014183 doi: bioRxiv preprint
|
