key: cord-0703665-tlc9xblp
authors: Hu, Yanmei; Jo, Hyunil; DeGrado, William F.; Wang, Jun
title: Brilacidin, a COVID‐19 drug candidate, demonstrates broad‐spectrum antiviral activity against human coronaviruses OC43, 229E, and NL63 through targeting both the virus and the host cell
date: 2022-02-02
journal: J Med Virol
DOI: 10.1002/jmv.27616
sha: 9cfd328e70c1987a4aa065f973e2b362d95d8a26
doc_id: 703665
cord_uid: tlc9xblp

Brilacidin, a mimetic of host defense peptides (HDPs), is currently in Phase 2 clinical trial as an antibiotic drug candidate. A recent study reported that brilacidin has antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) by inactivating the virus. In this study, we discovered an additional mechanism of action of brilacidin by targeting heparan sulfate proteoglycans (HSPGs) on the host cell surface. Brilacidin, but not acetyl brilacidin, inhibits the entry of SARS‐CoV‐2 pseudovirus into multiple cell lines, and heparin, an HSPG mimetic, abolishes the inhibitory activity of brilacidin on SARS‐CoV‐2 pseudovirus cell entry. In addition, we found that brilacidin has broad‐spectrum antiviral activity against multiple human coronaviruses (HCoVs) including HCoV‐229E, HCoV‐OC43, and HCoV‐NL63. Mechanistic studies revealed that brilacidin has a dual antiviral mechanism of action including virucidal activity and binding to coronavirus attachment factor HSPGs on the host cell surface. Brilacidin partially loses its antiviral activity when heparin was included in the cell cultures, supporting the host‐targeting mechanism. Drug combination therapy showed that brilacidin has a strong synergistic effect with remdesivir against HCoV‐OC43 in cell culture. Taken together, this study provides appealing findings for the translational potential of brilacidin as a broad‐spectrum antiviral for coronaviruses including SARS‐CoV‐2.

small molecular antiviral drugs are important complements of vaccines to help combat pandemics.

Host defense peptides (HDPs), also called antimicrobial peptides, are typically small peptides (12-50 amino acids) that are expressed in neutrophils and mucosa and serve as the first line of defense against foreign pathogens. 9 HDPs have been extensively explored as antibiotics, 10 antivirals, 11 antifungals, 12 and anticancer agents. 13 Most HDPs share an amphiphilic structure with a positively charged face and a hydrophobic face. 14 It is proposed that HDPs disrupt bacterial cell membranes by interacting with the negatively charged phospholipid headgroups. [15] [16] [17] Brilacidin is a small synthetic HDP mimetic, 18 and has potent antibacterial activity against both Gram-positive and Gramnegative bacteria, 19 and is currently in Phase 2 clinical trials (Clinical Trials NCT01211470, NCT020388, and NCT02324335). The antibacterial mechanisms of action of brilacidin include both membrane disruption and immunomodulation. 20, 21 Brilacidin is also in clinical trial (NCT04784897) as a SARS-CoV-2 antiviral drug candidate for hospitalized COVID-19 patients. A recent study showed that brilacidin exhibited a potent inhibitory effect on SARS-CoV-2 replication (halfmaximal effective concentration, EC 50 = 0.565 μM/50% cytotoxic concentration, CC 50 = 241 μM), and the proposed mechanism of action is through disrupting viral integrity, thereby blocking viral entry. 22 However, the effect of brilacidin on host cells and the antiviral activity of brilacidin against other HCoVs have not been investigated.

In this study, we showed that brilacidin inhibits SARS-CoV-2 pseudovirus entry into multiple cell lines. However, acetyl brilacidin had no inhibition on SARS-CoV-2 pseudovirus entry, and heparin, a heparan sulfate proteoglycans (HSPGs) mimetic, diminished the inhibitory activity of brilacidin. This result suggests that brilacidin has an additional mechanism of action by binding to HSPGs on the host cell, thereby blocking viral attachment. HSPGs have been reported as an attachment factor for SARS-CoV-2. 23, 24 In addition, we have shown that brilacidin has broad-spectrum antiviral activity against multiple HCoVs including HCoV-229E, HCoV-OC43, and HCoV-NL63. The antiviral mechanism against these viruses similarly involves both virucidal effects and binding to HSPGs. Brilacidin partially loses its antiviral activity against HCoV-229E, HCoV-OC43, HCoV- 30-2003™) . Both media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics. Cells were kept at a cell culture incubator (humidified, 5% CO 2 /95% air, 37°C).

The following reagents were obtained through BEI Resources, NIAID, NIH: HCoV, HCoV-OC43, NR-52725; HCoVs, HCoV-NL63, NR-470.

HCoV-OC43 was propagated in RD cell line; HCoV-NL63 was initially propagated in 293T-ACE2 cell line and accommodated in Vero E6 cell line. HCoV-229E was obtained from Dr. Bart Tarbet (Utah State University) and amplified in Huh-7 or MRC-5 cell lines.

The antiviral activity of brilacidin was tested against HCoV-229E, HCoV-NL63, and HCoV-OC43 in Viral yield reduction (VYR) assays as previously described. [25] [26] [27] [28] Briefly, viruses were first replicated in the presence of serial concentrations of brilacidin (0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 µM). Progeny virions released in the supernatant were collected 24 hour postinfection (hpi) from each concentration of brilacidin and the viral titers were determined by plaque reduction assay. Viruses were serially diluted 10-10 6 folds and infect the cells in a six-well plate. The infected cells were incubated at 33°C or 37°C for 1 h to allow virus adsorption. The viral inoculum was removed and an overlay containing 0.6% Avicel supplemented with 2% FBS in DMEM was added and incubated in the 33°C or 37°C incubators for 4-5 days. The plaque formation was detected by staining the cell monolayer with crystal violet. HCoV-229E and HCoV-OC43 plaque assays were carried out on RD cells and incubated at 33°C, HCoV-NL63 plaque assay was performed on Vero C1008 cells and incubated at 37°C. EC 50 values were determined by plotting the percentage of positive control versus log 10 compound concentrations from best-fit dose-response curves with the variable slope in Prism 8. Viral growth curves were obtained by replicating viruses in the presence or absence of 25 µM brilacidin at the multiplicity of infection (MOI) of 0.1. Viruses in the supernatant were collected at the indicated time point postinfection and viral titers were determined by plaque reduction assay as described in the VYR assay section.

The antiviral activity of brilacidin tested in HCoV-OC43 plaque assay was carried out similarly as described in VYR assay, except that about 100 PFU of HCoV-OC43 virus was used to infect the cells in each well of the six-well plate, and serial concentrations of brilacidin (0, 3.13, 6.25, 12.5, 25, 50, and 100 µM) was included in the Avicel overlay. The plaque areas were quantified using Image J and the EC 50 value was determined by plotting the percentage of plaque area HU ET AL. | 2189 versus log 10 compound concentrations from best-fit dose-response curves with the variable slope in Prism 8.

The antiviral activity of brilacidin against influenza and enterovirus D68 was carried out in plaque assay as previously described. [29] [30] [31] 2.3 | Cytotoxicity assay Cytotoxicity of brilacidin was evaluated in different cell lines using the neutral red uptake assay as previously described. 32 

A pseudotype HIV-1-derived lentiviral particles bearing SARS-CoV-2

Spike and a lentiviral backbone plasmid encoding luciferase as a reporter was produced in HEK293 T cells engineered to express the SARS-CoV-2 receptor ACE2 (293 T-ACE2 cells), as previously described. 35 The pseudovirus was then used to infect Vero C1008 cells, 

The combination antiviral effects of brilacidin and remdesivir were evaluated in HCoV-OC43 plaque assay in cell culture. Brilacidin was mixed with remdesivir at fixed EC 50 ratios of 4:1, 2:1, 1:1, 1:2, 1:4, 

To delineate whether brilacidin blocks SARS-CoV-2 viral entry, we generated pseudotyped HIV-1-derived lentiviral particles with SARS-CoV-2 spike protein, 35 which is widely used to study spike-mediated viral entry into host cells in biosafety Level 2 facilities. 40, 41 Brilacidin was tested in SARS-CoV-2 pseudovirus entry assay in several ACE2-expressing cell lines including Vero C1008, Calu-3, Huh-7, Caco-2, and 293T-ACE2. Vero C1008 and 293T-ACE2 express minimal levels of transmembrane serine proteinase 2 (TMPRSS2), therefore the SARS-CoV-2 virus enters into these cell lines mainly through endocytosis and relies on endosomal cathepsin L for viral spike protein activation. 42, 43 In contrast, Calu-3 and Caco-2 endogenously express TMPRSS2, 44 which activates SARS-CoV-2 spike protein on the cell surface so the virus gets into these cell lines through direct cell membrane fusion. Cathepsin L inhibitor E-64d and TMPRSS2 inhibitor camostat mesylate were included as controls. 27 Our results showed that brilacidin inhibited SARS-CoV-2 pseudovirus entry into all cell lines tested with IC 50 values ranging from 12.0 ± 1.7 to 23.0 ± 1.6 µM ( Figure 1 ). Cytotoxicity assays showed that brilacidin was not toxic to all the cell lines tested at the concentrations examined ( Figure 1F ). Overall, brilacidin inhibits SARS-CoV-2 pseudovirus entry into multiple cell lines. These results suggest the antiviral activity of brilacidin is independent of cathepsin L or TMPRSS2 inhibition.

It was recently reported that brilacidin exhibited potent antiviral activity on SARS-CoV-2 replication in both Vero and Calu-3 cells. 22 To test This is probably because higher MOI was used in the pseudovirus assay than in the VYR assay. VYR assay uses live viruses and viruses have multiple cycles of replication during the incubation time, while pseudovirus entry assay is a single cycle assay and high MOI of pseudovirus particle is applied during infection to achieve optimal signal which decreases its sensitivity. To confirm the antiviral activity of brilacidin, we HCoV-229E, respectively ( Figure 2H ). Taken together, these results indicate that brilacidin has potent antiviral activity against HCoVs, but not influenza or enterovirus D68. 

Next, a drug time-of-addition experiment was carried out to de- 

HCoV-OC43, HCoV-229E, and HCoV-NL63 replication in cell culture

It was proposed that brilacidin binds to SARS-CoV-2 spike protein; however, it did not specify which part of the spike protein it binds and no experimental evidence was provided. 27 To test whether brilacidin blocks SARS-CoV-2 pseudovirus entry into host cells through interaction with the spike protein RBD, we tested the direct binding of brilacidin to SARS-CoV-2 spike protein RBD using DSF.

The results demonstrated that brilacidin has no effect on the melting temperature (T m ) of SARS-CoV-2 spike protein RBD up to Figure 5A ). It was found that acetyl brilacidin completely lost inhibitory activity in SARS-CoV-2 pseudovirus entry assay in Vero C1008, Calu-3, and

Caco-2 cells ( Figure 5B-D) . This result suggests that the +4 charge on brilacidin is critical for the antiviral activity, and the antiviral mechanism of action might involve interaction with the binding to

HSPGs.

If brilacidin binds to cell surface HSPGs in cell culture, exogenous addition of HSPG mimetics such as heparin will compete with HSPGs 

Combination therapy is commonly used to slow down drug resistance development and reduce side effects. 25, 50 The antiviral effect of brilacidin and remdesivir in combination therapy was evaluated in HCoV-OC43 plaque assay using the CIs method ( Figure 7) . 51 Remdesivir, a SARS-CoV-2 polymerase inhibitor, is the only Food and Drug Administration (FDA)-approved antiviral for treating COVID-19.

Brilacidin and remdesivir were mixed at different ratios and the corresponding EC 50 values for brilacidin and remdesivir were calculated. CIs versus the EC 50 values of brilacidin and remdesivir at different combination ratios were plotted ( Figure 7A ). The red line indicates additive effect; the right upper area above the red line indicates antagonism, while the left bottom area below the red line indicates synergy. 49 The CIs at all the combination ratios fell below the red line ( Figure 7A ), and the FICI which was used to determine synergistic effects of compounds are less than 0.5 at all combination ratios ( Figure 7B ), suggesting brilacidin has significant synergistic antiviral effect with remdesivir in the combination therapy.

As the COVID19 pandemic keeps ongoing and variants continue to emerge, effective therapeutic interventions are urgently needed.

Although three vaccines are currently available for the prevention of COVID19, there is an urgent need for small molecular antivirals to help combat the pandemic. In this study, we investigated the antiviral activity and mechanism of action of brilacidin against multiple HCoVs.

Our findings include: 1) Brilacidin has broad-spectrum antiviral ac- The proposed antiviral mechanism of brilacidin is summarized in a model illustrated in Figure 8 , which is supported by multiple lines of evidence. Our results showed that brilacidin has a dual antiviral mechanism against HCoVs including blocking viral attachment to host cells through binding to HSPGs and virucidal activity. HCoV-OC43, HCoV-NL63, and HCoV-229E showed a dose-dependent decrease of replication in cells pretreated with brilacidin, and viral particles lose infectivity after incubation with brilacidin ( Figure 3) . Drug time-ofaddition experiment suggested that brilacidin exerted its antiviral activity at two individual steps: viral attachment to host cell and early entry after entering into the host cells ( Figure 4 ). The inhibition of viral attachment by brilacidin was confirmed in the SARS-CoV-2 pseudovirus entry assay (Figure 1 ). DSF assay results demonstrated that brilacidin has no direct binding to SARS-CoV-2 spike protein RBD ( Table 1 ). The competition experiment with heparin indicated that brilacidin binds to host cell surface HSPGs to block viral attachment to host cells. The addition of heparin dose-dependently decreased the inhibition of brilacidin in SARS-CoV-2 pseudovirus entry assay ( Figure 5 ) and the replication of HCoV-OC43, HCoV-NL63, and HCoV-229E in cell culture ( Figure 6 ). The lack of inhibition of brilacidin against EV-D68 was expected as the EV-D68 MO-18947 strain is not known to exploit HSPGs as receptors for cell entry ( Figure 2G ). 52 Similarly, HSPGs are also not required receptors for influenza virus, 53 which explains the lack of antiviral activity of brilacidin against A/California/07/2009 (H1N1) virus ( Figure 2G ).

In summary, our results indicate that brilacidin has a dual antiviral mechanism of action including targeting host cell surface HSPGs to block viral attachment and inactivating viral particles. This dual antiviral mechanism of action might slow down the pace of resistance development. Taken together, the broad-spectrum antiviral activity of brilacidin against coronaviruses and the previously reported immunomodulatory effect and antibacterial activity warrants its further development as a broad-spectrum antiviral for the treatment of not only current COVID-19 but also future emerging coronaviruses.

This study was partially supported by the National Institute of Allergy 

Jun Wang and William F. DeGrado conceived and designed the study.

Yanmei Hu performed the pseudovirus neutralization assay, antiviral assays, time of addition experiment, immunofluorescence assays, thermal shift binding assay, and the combination therapy experiment.

Hyunil Jo provided the brilacidin and acetyl brilacidin samples. Yanmei Hu and Jun Wang wrote the manuscript.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

ORCID Jun Wang http://orcid.org/0000-0002-4845-4621

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COVID-19 drug candidate, demonstrates broadspectrum antiviral activity against human coronaviruses OC43, 229E, and NL63 through targeting both the virus and the host cell