The IPIX Preprint of Brilacidin for COVID-19 (B-CV
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Posted On: 11/01/2020 2:33:54 AM
The IPIX Preprint of Brilacidin for COVID-19 (B-CV19) has such a wealth of information it is difficult to try to summarize it in a single post. As I read through the Preprint it became crystal clear that Brilacidin has unique characteristics which differentiate it vs all other CV19 treatments. The superior properties and expansive method of action will elevate Brilacidin as a high demand stand-alone treatment and has significant synergistic properties, especially in use with Remdesivir which will also make it a high demand combination therapy for CV19. I tried to pull as much summary information from the Preprint and highlighted key points that I felt were the most noteworthy. A couple of key points that are reiterated in the below Preprint verbiage:
Brilacidin exhibits potent inhibition of SARS-CoV-2 in a human cell line in nontoxic concentrations resulting in 95% and 97% reduction of infectious viral load and has a direct inhibitory effect on the virus in a manner similar to the neutralization of antibodies, potentially by disrupting viral integrity and thus impairing the virion’s ability to complete the viral entry process.
Selectivity Index of 426 – quantum mechanical molecular screening study of 11,522 compounds identified strongly support brilacidin’s treatment potential to achieve positive antiviral outcomes in humans. A vast majority of other drugs being evaluated as potential COVID-19 treatments, including repurposed drugs, have SIs that are much lower than that achieved by brilacidin, with most drugs failing to show anti-SARS-CoV-2 potency. Note that the formula uses for the SI is well below clinically-achievable concentrations based on pharmacokinetics observed in a Phase 2b clinical trial of brilacidin for the treatment of Acute Bacterial Skin and Skin Structure Infections (ABSSSI).
Brilacidin with remdesivir reduced the viral infectious titer by >99%, providing a highly effective inhibition profile and achieving greater inhibition than with either compound alone. Brilacidin appears to act primarily by disrupting viral integrity and blocking viral entry, combining the drug with antiviral treatments that have a different mechanism of action may result in synergistic inhibition when administered in combination.
Brilacidin directly interferes with the integrity of the virion, and hence restricts the virion’s ability to interact with the ACE2 receptor to facilitate the entry process. This mechanism of inhibition may be akin to that achieved by neutralizing antibodies that may interact with specific exposed epitopes on the surface of virions. While brilacidin’s mechanism of action appears primarily to be extracellular, it may also impact intracellular viral replication.
Any reduction in inflammatory mediators are likely to be a direct consequence of decrease in replicating virus. Moreover, ARDS and associated organ failure observed in the context of COVID-19 are typically later-stage manifestations of disease that are not directly related to, or dependent on, a mounting viral load. Consequently, use of a drug strategy that exhibits intrinsic anti-inflammatory activity will add value to controlling later onset inflammatory damage in COVID-19 patients, potentially occurring beyond the time of active viral multiplication. Along similar lines, it remains to be determined if the combination of convalescent serum—a current COVID-19 treatment that has received FDA Emergency Use Authorization— with brilacidin may confer a protective advantage to patients.
Clearly, an effective COVID-19 therapeutic (or therapeutics in combination) ideally would control both viral load and the corresponding inflammatory damage due to SARS-CoV-2, and mitigate bacterial co-infections. Exhibiting three-in-one properties—antiviral, immuno/anti-inflammatory, and antibacterial—brilacidin is being developed for the intravenous treatment of COVID-19 in hospitalized patients and may be able to address different disease parameters within the one therapeutic treatment. Brilacidin has been successfully tested in 8 clinical trials across multiple indications providing established safety and efficacy data on over 460 subjects.
Additional formulation work is planned for the inhaled delivery of brilacidin for prophylactic use, toward controlling infection in the nasal passage and lungs and if successful, may enable brilacidin to emerge as a particularly effective and differentiated antiviral by preventing and/or decreasing early infectivity due to SARS-CoV-2.
Destabilizing viral integrity is a desirable antiviral property, especially in relation to pan-coronavirus agents, as the viral membrane is highly conserved and similar in construct across different coronavirus strains. Moreover, drugs that can directly disrupt viral integrity would be less prone to resistance due to mutation, unlike many antivirals, antibody-based treatments and vaccines that are currently in use and in development.
Additional testing planned in other lethal coronaviruses (MERS-CoV, SARS-CoV), toward assessing the potential of brilacidin as a broad spectrum inhibitor of coronaviruses.
==============================
Summary statement from page 2 of Preprint:
These data suggest that SARS-CoV-2 inhibition in these cell culture models is primarily a result of the impact of brilacidin on viral entry and its disruption of viral integrity. Brilacidin has demonstrated synergistic antiviral activity when combined with remdesivir. Collectively, our data demonstrate that brilacidin exerts potent inhibition of SARS-CoV-2 and thus supports brilacidin as a promising COVID-19 drug candidate.
Highlights
• Brilacidin potently inhibits SARS-CoV-2 in an ACE2 positive human lung cell line.
• Brilacidin achieved a high Selectivity Index of 426 (CC50=241μM/IC50=0.565μM).
• Brilacidin’s main mechanism appears to disrupt viral integrity and impact viral entry.
• Brilacidin and remdesivir exhibit excellent synergistic activity against SARS-CoV-2
Significance Statement
SARS-CoV-2, the emergent novel coronavirus, has led to the current global COVID-19 pandemic, characterized by extreme contagiousness and high mortality rates. There is an urgent need for effective therapeutic strategies to safely and effectively treat SARS-CoV-2 infection. We demonstrate that brilacidin, a synthetic small molecule with peptide-like properties, is capable of exerting potent in vitro antiviral activity against SARS-CoV-2, both as a standalone treatment and in combination with remdesivir, which is currently the only FDA-approved drug for the treatment of COVID-19.
Brilacidin inhibits SARS-CoV-2 replication (Vero Cells)
The data demonstrate that brilacidin treatment resulted in a dose-dependent decrease in infectious viral titer with a maximum of 53% inhibition of virus observed in the presence of the higher concentration of compound (10µM) that was tested (Figure 2A).
Brilacidin appears to impact entry of SARS-CoV-2 (Vero cells)
The inhibition of pseudovirus (rVSV) attachment and entry into cells was quantified in the context of brilacidin treatment (10µM and 20µM), and hydroxychloroquine was utilized as a control. The data demonstrate that brilacidin treatment inhibited the pseudovirus at both concentrations tested in a comparable manner (Figure 2B). The inhibition observed in the context of the replication-incompetent pseudovirus was supportive of the suggested role of brilacidin as an inhibitor of viral entry and potential early post-entry steps. To further support this observation, confocal microscopy was performed using an antibody directed against the viral spike protein in the presence and absence of brilacidin using the pseudovirus (Figure 2C). Quantification of the confocal images revealed that incubation of SARS-CoV-2 with brilacidin resulted in a decreased intracellular spike protein signal at 1 and 4 hours post infection.
Brilacidin appears to disrupt the integrity of the SARS-CoV-2 virion
The infectious virus titer in the supernatant was quantified by plaque assay, which revealed a dramatic 90% reduction of virus titer (Figure 2E, indicated as [entry]). This inhibition was approximately 25% higher than that observed when the cells were pre- and posttreated with brilacidin concomitantly (Figure 2E) supporting the concept that brilacidin has a direct inhibitory effect on the virus in a manner similar to the neutralization of antibodies, potentially by disrupting viral integrity and thus impairing the virion’s ability to complete the viral entry process.
Brilacidin exhibits potent inhibition of SARS-CoV-2 in a human cell line (Calu-3 cells)
The assay revealed that these concentrations of brilacidin were nontoxic to Calu-3 cells. The inhibitory effect of brilacidin in the Calu-3 cell line was first confirmed by pre-treatment (for 2 hours) and post-infection treatment (for 24 hours) of cells with brilacidin, which demonstrated a dose-dependent decrease of viral load, with the higher concentration of brilacidin providing 61% inhibition of infectious viral titer (Figure 3A). However, when the experiment was modified to include a brilacidin-treated inoculum – with direct pre-incubation of the virus with brilacidin for 1 hour prior to infection, and with infection carried out in the presence of the compound – the extent of inhibition dramatically increased, resulting in 95% and 97% reduction of infectious viral titer at the 10µM and 20µM concentration of the compound respectively (Figure 3B). Quantification of intracellular viral RNA by semi-quantitative RT-PCR at 24 hours post infection (for 10µM brilacidin) demonstrated a 33% decrease in the viral genomic copies upon brilacidin treatment. The inhibition of infectious virus titer as a variable of viral load was assessed by quantifying inhibition at lower multiplicities of infection (MOI) with brilacidin at a fixed concentration of 10µM. Interestingly, the inhibitory potential of brilacidin was best observed at the highest MOIs tested, with inhibition of virus at the lower MOIs (0.01 and 0.001) not showing statistical significance (Figure 3C). The inhibition exerted at the MOIs of 0.1 and 0.05 were extremely comparable to each other.
Selectivitiy Index determination for brilacidin against SARS-CoV-2 in a human cell line (Calu-3 cells)
revealed that the 50% reduction in cell viability was observed at a concentration of 241μM, with 90% viability (CC10) observed at 26.8μM, thus suggesting brilacidin was extremely well tolerated
demonstrated brilacidin achieved 90% inhibition at a concentration of 2.63μM and 50% inhibition at 0.565μM, yielding a Selectivity Index of 426
Brilacidin in combination with other antiviral treatments; synergistic activity against SARS-CoV-2 in combination with remdesivir (Calu-3 cells)
As brilacidin appears to act primarily by disrupting viral integrity and blocking viral entry, combining the drug with antiviral treatments that have a different mechanism of action may result in synergistic inhibition when administered in combination. The potential for brilacidin to exert a synergistic inhibition of SARS-CoV-2 when combined with current frontline COVID-19 antiviral treatments, namely, remdesivir and favipiravir, was assessed. Potential toxicity of combinations of remdesivir or favipiravir with brilacidin were initially assessed in the Calu-3 cell line at 24 hours post treatment. No apparent toxicity could be detected up to 10µM concentration of each of the drugs in the combination regimen. To evaluate the efficacy of combining remdesivir or favipiravir with brilacidin, the cells were pre-treated with brilacidin for 2 hours. The virus inoculum was also independently pre-incubated with brilacidin for 1 hour, and then the treated inoculum was overlaid on cells and the infection allowed to proceed for 1 hour in the presence of brilacidin. Post-infection, the inoculum was removed and media containing both brilacidin and remdesivir or favipiravir or each drug alone for efficacy comparison, was added to the infected cells. Supernatants were obtained at 24 hours post infection and infectious titer quantified by plaque assay. The data revealed that brilacidin and favipiravir independently exerted up to 90% and 80% inhibition respectively and the extent of inhibition did not increase over that exerted by brilacidin alone when the two drugs were used in combination (Figure 5A). In contrast, combination of brilacidin with remdesivir at 10µM and 2.5µM concentrations, respectively, reduced the viral infectious titer by >99%, thus providing a highly effective inhibition profile (Figure 5B) and achieving greater inhibition than with either compound alone. This synergistic inhibition continued to remain higher than 99% when the concentrations of both compounds were equal (2.5µM each) (Figure 5C).
Discussion
Our experiments in the Vero cell line model Brilacidin Antiviral Activity Against SARS-CoV-2 12 demonstrate brilacidin decreases viral load in a robust manner when the virus is pre-incubated with brilacidin (Figure 2D), suggesting brilacidin impacts the virus directly.
In multiple studies, we have attempted to evaluate the capability of brilacidin to decrease viral load in the context of the SARS-CoV-2 infection. Our experiments in the Vero cell line model Brilacidin Antiviral Activity Against SARS-CoV-2 demonstrate brilacidin decreases viral load in a robust manner when the virus is pre-incubated with brilacidin (Figure 2D), suggesting brilacidin impacts the virus directly. This observation was supported by the inhibition seen in the context of a replication incompetent pseudovirus (Figure 2B, Figure 2C), further indicating brilacidin’s inhibition during early stages of viral infection. As the pseudovirus expresses the SARS-CoV-2 spike protein on the surface, the data may also support brilacidin’s ability to interfere with the interaction between the viral spike protein and the cellular ACE2 receptor. Brilacidin’s ability to decrease viral load in an ACE2 positive cell line is demonstrated in Figure 3, Figure 4, and Figure 5, in which Calu-3 cells were used. Additional testing conducted in Caco-2 cells and primary lung fibroblasts obtained from human donors also supported brilacidin’s inhibitory properties in ACE2 positive cell lines (data not shown).
The idea that brilacidin directly interferes with the integrity of the virion, and hence restricts the virion’s ability to interact with the ACE2 receptor to facilitate the entry process, is further supported by the observation that when drug treatment was limited to the virus alone (Figure 2E), with no treatment of host cells, a robust decrease of viral load was still observed. This mechanism of inhibition may be akin to that achieved by neutralizing antibodies that may interact with specific exposed epitopes on the surface of virions. It remains to be determined if the impact of brilacidin on viral membranes is driven by specific viral membrane compositions. Additional studies are planned.
Potent membrane destabilization by brilacidin has been reported in the context of bacteria. These findings indicate brilacidin causes significant cell wall stress and additional internal stress due to the accumulation of misfolded proteins. Additional mechanistic studies of brilacidin analogs against Escherichia coli, in comparison to the antibiotic polymyxin B, further support this class of compounds ability to destabilize bacterial membranes.
While brilacidin’s mechanism of action appears primarily to be extracellular, it may also impact intracellular viral replication. Supportive of this, an in silico quantum mechanical molecular screening study of 11,522 compounds identified brilacidin as a potential inhibitor of SARS-CoV-2 based on the potential of its physico-chemical properties to interfere with the intracellular replication of SARS-CoV-2’s main protease.
—yielding a Selectivity Index (SI) for brilacidin of 426
(CC50=241μM/IC50=0.565μM)—strongly support brilacidin’s treatment potential to achieve positive antiviral outcomes in humans. A vast majority of other drugs being evaluated as potential COVID-19 treatments, including repurposed drugs, have SIs that are much lower than that achieved by brilacidin, with most drugs failing to show anti-SARS-CoV-2 potency. Of note, the IC50 (0.565μM) and IC90 (2.63μM) values for brilacidin observed in the Calu3 cell line are well below clinically-achievable concentrations based on pharmacokinetics observed in a Phase 2b clinical trial of brilacidin for the treatment of Acute Bacterial Skin and Skin Structure Infections (ABSSSI).
A desirable outcome for any potential COVID-19 therapeutic will be its ability to synergize with existing COVID-19 treatments, particularly if the mechanisms of action of the synergistic treatments can impact more than one step of the viral lifecycle. Such combinations are more likely to elicit an additive response, while also reducing the likelihood of viral resistance developing.
Remdesivir and favipiravir are inhibitors that impact the viral RNA synthesis step of the infectious process. These drugs may help decrease progeny viral genomes in infected cells, but they will not be conducive to inhibiting progressive infection of naïve cells once the progeny virions have been released from infected cells.
By combining remdesivir or favipiravir with brilacidin, a two-pronged strategy of inhibiting viral entry and viral RNA synthesis might be successfully leveraged to most effectively control progression of SARS-CoV-2 infection. [/b]Furthermore, remdesivir and favipiravir do not possess intrinsic anti-inflammatory activity, unlike brilacidin. Any reduction in inflammatory mediators, including IL-6 and IL-1β in the context of remdesivir and favipiravir treatment, are likely to be a direct consequence of decrease in replicating virus.
Moreover, ARDS and associated organ failure observed in the context of COVID-19 are typically later-stage manifestations of disease that are not directly related to, or dependent on, a mounting viral load. Consequently, use of a drug strategy that exhibits intrinsic anti-inflammatory activity will add value to controlling later onset inflammatory damage in COVID-19 patients, potentially occurring beyond the time of active viral multiplication. Along similar lines, it remains to be determined if the combination of convalescent serum—a current COVID-19 treatment that has received FDA Emergency Use Authorization— with brilacidin may confer a protective advantage to patients. It is less likely that a synergistic effect may be observed by combining neutralizing antibodies with brilacidin as it appears the dominant mechanism of action in both cases is disruption of viral entry into cells.
Clearly, an effective COVID-19 therapeutic (or therapeutics in combination) ideally would control both viral load and the corresponding inflammatory damage due to SARS-CoV-2, and mitigate bacterial co-infections. Exhibiting three-in-one properties—antiviral, immuno/anti-inflammatory, and antibacterial—brilacidin is being developed for the intravenous treatment of COVID-19 in hospitalized patients and may be able to address different disease parameters within the one therapeutic treatment.
Brilacidin has been successfully tested in 8 clinical trials across multiple indications providing established safety and efficacy data on over 460 subjects. The drug has exhibited potent antibacterial activity in a Phase 2b trial in Acute Bacterial Skin and Skin Structure Infections (ABSSSI) and anti-inflammatory activity, as supported in Phase 2 clinical trials in Ulcerative Proctitis/Ulcerative Proctosigmoiditis and Oral Mucositis (see Supplemental Information). Brilacidin, through modulation of cyclic adenosine monophosphate (cAMP)/cyclic guanosine monophosphate (cGMP) pathway, is postulated to regulate the immune response based largely on its observed inhibition of phosphodiesterases (PDE4 and PDE3). Interestingly, numerous PDE inhibitors have been proposed as potentially promising anti-SARS-CoV-2 treatments by suppressing the heightened inflammatory response.
Additional formulation work is planned for the inhaled delivery of brilacidin for prophylactic use, toward controlling infection in the nasal passage and lungs by leveraging brilacidin’s ability to inhibit SARS-CoV-2 by disrupting viral integrity and impacting viral entry. Such development efforts, if successful, may enable brilacidin to emerge as a particularly effective and differentiated antiviral by preventing and/or decreasing early infectivity due to SARS-CoV-2.
In this manuscript, we demonstrate brilacidin exhibits robust inhibition of SARS-CoV-2 in Vero cells and Calu-3 cells, supporting brilacidin as a promising novel drug candidate for the treatment of COVID-19. Functioning as a viral entry inhibitor, proposed mechanisms of action for brilacidin include affecting the integrity of the viral membrane and preventing viral binding to cells. More detailed mechanistic studies are planned. Destabilizing viral integrity is a desirable antiviral property, especially in relation to pan-coronavirus agents, as the viral membrane is highly conserved and similar in construct across different coronavirus strains. Moreover, drugs that can directly disrupt viral integrity would be less prone to resistance due to mutation, unlike many antivirals, antibody-based treatments and vaccines that are currently in use and in development. Brilacidin exhibits an excellent synergistic inhibitory profile against SARS-CoV-2 in combination with remdesivir. Experiments conducted in endemic human coronaviruses are ongoing, with additional testing planned in other lethal coronaviruses (MERS-CoV, SARS-CoV), toward assessing the potential of brilacidin as a broad spectrum inhibitor of coronaviruses.
Brilacidin exhibits potent inhibition of SARS-CoV-2 in a human cell line in nontoxic concentrations resulting in 95% and 97% reduction of infectious viral load and has a direct inhibitory effect on the virus in a manner similar to the neutralization of antibodies, potentially by disrupting viral integrity and thus impairing the virion’s ability to complete the viral entry process.
Selectivity Index of 426 – quantum mechanical molecular screening study of 11,522 compounds identified strongly support brilacidin’s treatment potential to achieve positive antiviral outcomes in humans. A vast majority of other drugs being evaluated as potential COVID-19 treatments, including repurposed drugs, have SIs that are much lower than that achieved by brilacidin, with most drugs failing to show anti-SARS-CoV-2 potency. Note that the formula uses for the SI is well below clinically-achievable concentrations based on pharmacokinetics observed in a Phase 2b clinical trial of brilacidin for the treatment of Acute Bacterial Skin and Skin Structure Infections (ABSSSI).
Brilacidin with remdesivir reduced the viral infectious titer by >99%, providing a highly effective inhibition profile and achieving greater inhibition than with either compound alone. Brilacidin appears to act primarily by disrupting viral integrity and blocking viral entry, combining the drug with antiviral treatments that have a different mechanism of action may result in synergistic inhibition when administered in combination.
Brilacidin directly interferes with the integrity of the virion, and hence restricts the virion’s ability to interact with the ACE2 receptor to facilitate the entry process. This mechanism of inhibition may be akin to that achieved by neutralizing antibodies that may interact with specific exposed epitopes on the surface of virions. While brilacidin’s mechanism of action appears primarily to be extracellular, it may also impact intracellular viral replication.
Any reduction in inflammatory mediators are likely to be a direct consequence of decrease in replicating virus. Moreover, ARDS and associated organ failure observed in the context of COVID-19 are typically later-stage manifestations of disease that are not directly related to, or dependent on, a mounting viral load. Consequently, use of a drug strategy that exhibits intrinsic anti-inflammatory activity will add value to controlling later onset inflammatory damage in COVID-19 patients, potentially occurring beyond the time of active viral multiplication. Along similar lines, it remains to be determined if the combination of convalescent serum—a current COVID-19 treatment that has received FDA Emergency Use Authorization— with brilacidin may confer a protective advantage to patients.
Clearly, an effective COVID-19 therapeutic (or therapeutics in combination) ideally would control both viral load and the corresponding inflammatory damage due to SARS-CoV-2, and mitigate bacterial co-infections. Exhibiting three-in-one properties—antiviral, immuno/anti-inflammatory, and antibacterial—brilacidin is being developed for the intravenous treatment of COVID-19 in hospitalized patients and may be able to address different disease parameters within the one therapeutic treatment. Brilacidin has been successfully tested in 8 clinical trials across multiple indications providing established safety and efficacy data on over 460 subjects.
Additional formulation work is planned for the inhaled delivery of brilacidin for prophylactic use, toward controlling infection in the nasal passage and lungs and if successful, may enable brilacidin to emerge as a particularly effective and differentiated antiviral by preventing and/or decreasing early infectivity due to SARS-CoV-2.
Destabilizing viral integrity is a desirable antiviral property, especially in relation to pan-coronavirus agents, as the viral membrane is highly conserved and similar in construct across different coronavirus strains. Moreover, drugs that can directly disrupt viral integrity would be less prone to resistance due to mutation, unlike many antivirals, antibody-based treatments and vaccines that are currently in use and in development.
Additional testing planned in other lethal coronaviruses (MERS-CoV, SARS-CoV), toward assessing the potential of brilacidin as a broad spectrum inhibitor of coronaviruses.
==============================
Summary statement from page 2 of Preprint:
These data suggest that SARS-CoV-2 inhibition in these cell culture models is primarily a result of the impact of brilacidin on viral entry and its disruption of viral integrity. Brilacidin has demonstrated synergistic antiviral activity when combined with remdesivir. Collectively, our data demonstrate that brilacidin exerts potent inhibition of SARS-CoV-2 and thus supports brilacidin as a promising COVID-19 drug candidate.
Highlights
• Brilacidin potently inhibits SARS-CoV-2 in an ACE2 positive human lung cell line.
• Brilacidin achieved a high Selectivity Index of 426 (CC50=241μM/IC50=0.565μM).
• Brilacidin’s main mechanism appears to disrupt viral integrity and impact viral entry.
• Brilacidin and remdesivir exhibit excellent synergistic activity against SARS-CoV-2
Significance Statement
SARS-CoV-2, the emergent novel coronavirus, has led to the current global COVID-19 pandemic, characterized by extreme contagiousness and high mortality rates. There is an urgent need for effective therapeutic strategies to safely and effectively treat SARS-CoV-2 infection. We demonstrate that brilacidin, a synthetic small molecule with peptide-like properties, is capable of exerting potent in vitro antiviral activity against SARS-CoV-2, both as a standalone treatment and in combination with remdesivir, which is currently the only FDA-approved drug for the treatment of COVID-19.
Brilacidin inhibits SARS-CoV-2 replication (Vero Cells)
The data demonstrate that brilacidin treatment resulted in a dose-dependent decrease in infectious viral titer with a maximum of 53% inhibition of virus observed in the presence of the higher concentration of compound (10µM) that was tested (Figure 2A).
Brilacidin appears to impact entry of SARS-CoV-2 (Vero cells)
The inhibition of pseudovirus (rVSV) attachment and entry into cells was quantified in the context of brilacidin treatment (10µM and 20µM), and hydroxychloroquine was utilized as a control. The data demonstrate that brilacidin treatment inhibited the pseudovirus at both concentrations tested in a comparable manner (Figure 2B). The inhibition observed in the context of the replication-incompetent pseudovirus was supportive of the suggested role of brilacidin as an inhibitor of viral entry and potential early post-entry steps. To further support this observation, confocal microscopy was performed using an antibody directed against the viral spike protein in the presence and absence of brilacidin using the pseudovirus (Figure 2C). Quantification of the confocal images revealed that incubation of SARS-CoV-2 with brilacidin resulted in a decreased intracellular spike protein signal at 1 and 4 hours post infection.
Brilacidin appears to disrupt the integrity of the SARS-CoV-2 virion
The infectious virus titer in the supernatant was quantified by plaque assay, which revealed a dramatic 90% reduction of virus titer (Figure 2E, indicated as [entry]). This inhibition was approximately 25% higher than that observed when the cells were pre- and posttreated with brilacidin concomitantly (Figure 2E) supporting the concept that brilacidin has a direct inhibitory effect on the virus in a manner similar to the neutralization of antibodies, potentially by disrupting viral integrity and thus impairing the virion’s ability to complete the viral entry process.
Brilacidin exhibits potent inhibition of SARS-CoV-2 in a human cell line (Calu-3 cells)
The assay revealed that these concentrations of brilacidin were nontoxic to Calu-3 cells. The inhibitory effect of brilacidin in the Calu-3 cell line was first confirmed by pre-treatment (for 2 hours) and post-infection treatment (for 24 hours) of cells with brilacidin, which demonstrated a dose-dependent decrease of viral load, with the higher concentration of brilacidin providing 61% inhibition of infectious viral titer (Figure 3A). However, when the experiment was modified to include a brilacidin-treated inoculum – with direct pre-incubation of the virus with brilacidin for 1 hour prior to infection, and with infection carried out in the presence of the compound – the extent of inhibition dramatically increased, resulting in 95% and 97% reduction of infectious viral titer at the 10µM and 20µM concentration of the compound respectively (Figure 3B). Quantification of intracellular viral RNA by semi-quantitative RT-PCR at 24 hours post infection (for 10µM brilacidin) demonstrated a 33% decrease in the viral genomic copies upon brilacidin treatment. The inhibition of infectious virus titer as a variable of viral load was assessed by quantifying inhibition at lower multiplicities of infection (MOI) with brilacidin at a fixed concentration of 10µM. Interestingly, the inhibitory potential of brilacidin was best observed at the highest MOIs tested, with inhibition of virus at the lower MOIs (0.01 and 0.001) not showing statistical significance (Figure 3C). The inhibition exerted at the MOIs of 0.1 and 0.05 were extremely comparable to each other.
Selectivitiy Index determination for brilacidin against SARS-CoV-2 in a human cell line (Calu-3 cells)
revealed that the 50% reduction in cell viability was observed at a concentration of 241μM, with 90% viability (CC10) observed at 26.8μM, thus suggesting brilacidin was extremely well tolerated
demonstrated brilacidin achieved 90% inhibition at a concentration of 2.63μM and 50% inhibition at 0.565μM, yielding a Selectivity Index of 426
Brilacidin in combination with other antiviral treatments; synergistic activity against SARS-CoV-2 in combination with remdesivir (Calu-3 cells)
As brilacidin appears to act primarily by disrupting viral integrity and blocking viral entry, combining the drug with antiviral treatments that have a different mechanism of action may result in synergistic inhibition when administered in combination. The potential for brilacidin to exert a synergistic inhibition of SARS-CoV-2 when combined with current frontline COVID-19 antiviral treatments, namely, remdesivir and favipiravir, was assessed. Potential toxicity of combinations of remdesivir or favipiravir with brilacidin were initially assessed in the Calu-3 cell line at 24 hours post treatment. No apparent toxicity could be detected up to 10µM concentration of each of the drugs in the combination regimen. To evaluate the efficacy of combining remdesivir or favipiravir with brilacidin, the cells were pre-treated with brilacidin for 2 hours. The virus inoculum was also independently pre-incubated with brilacidin for 1 hour, and then the treated inoculum was overlaid on cells and the infection allowed to proceed for 1 hour in the presence of brilacidin. Post-infection, the inoculum was removed and media containing both brilacidin and remdesivir or favipiravir or each drug alone for efficacy comparison, was added to the infected cells. Supernatants were obtained at 24 hours post infection and infectious titer quantified by plaque assay. The data revealed that brilacidin and favipiravir independently exerted up to 90% and 80% inhibition respectively and the extent of inhibition did not increase over that exerted by brilacidin alone when the two drugs were used in combination (Figure 5A). In contrast, combination of brilacidin with remdesivir at 10µM and 2.5µM concentrations, respectively, reduced the viral infectious titer by >99%, thus providing a highly effective inhibition profile (Figure 5B) and achieving greater inhibition than with either compound alone. This synergistic inhibition continued to remain higher than 99% when the concentrations of both compounds were equal (2.5µM each) (Figure 5C).
Discussion
Our experiments in the Vero cell line model Brilacidin Antiviral Activity Against SARS-CoV-2 12 demonstrate brilacidin decreases viral load in a robust manner when the virus is pre-incubated with brilacidin (Figure 2D), suggesting brilacidin impacts the virus directly.
In multiple studies, we have attempted to evaluate the capability of brilacidin to decrease viral load in the context of the SARS-CoV-2 infection. Our experiments in the Vero cell line model Brilacidin Antiviral Activity Against SARS-CoV-2 demonstrate brilacidin decreases viral load in a robust manner when the virus is pre-incubated with brilacidin (Figure 2D), suggesting brilacidin impacts the virus directly. This observation was supported by the inhibition seen in the context of a replication incompetent pseudovirus (Figure 2B, Figure 2C), further indicating brilacidin’s inhibition during early stages of viral infection. As the pseudovirus expresses the SARS-CoV-2 spike protein on the surface, the data may also support brilacidin’s ability to interfere with the interaction between the viral spike protein and the cellular ACE2 receptor. Brilacidin’s ability to decrease viral load in an ACE2 positive cell line is demonstrated in Figure 3, Figure 4, and Figure 5, in which Calu-3 cells were used. Additional testing conducted in Caco-2 cells and primary lung fibroblasts obtained from human donors also supported brilacidin’s inhibitory properties in ACE2 positive cell lines (data not shown).
The idea that brilacidin directly interferes with the integrity of the virion, and hence restricts the virion’s ability to interact with the ACE2 receptor to facilitate the entry process, is further supported by the observation that when drug treatment was limited to the virus alone (Figure 2E), with no treatment of host cells, a robust decrease of viral load was still observed. This mechanism of inhibition may be akin to that achieved by neutralizing antibodies that may interact with specific exposed epitopes on the surface of virions. It remains to be determined if the impact of brilacidin on viral membranes is driven by specific viral membrane compositions. Additional studies are planned.
Potent membrane destabilization by brilacidin has been reported in the context of bacteria. These findings indicate brilacidin causes significant cell wall stress and additional internal stress due to the accumulation of misfolded proteins. Additional mechanistic studies of brilacidin analogs against Escherichia coli, in comparison to the antibiotic polymyxin B, further support this class of compounds ability to destabilize bacterial membranes.
While brilacidin’s mechanism of action appears primarily to be extracellular, it may also impact intracellular viral replication. Supportive of this, an in silico quantum mechanical molecular screening study of 11,522 compounds identified brilacidin as a potential inhibitor of SARS-CoV-2 based on the potential of its physico-chemical properties to interfere with the intracellular replication of SARS-CoV-2’s main protease.
—yielding a Selectivity Index (SI) for brilacidin of 426
(CC50=241μM/IC50=0.565μM)—strongly support brilacidin’s treatment potential to achieve positive antiviral outcomes in humans. A vast majority of other drugs being evaluated as potential COVID-19 treatments, including repurposed drugs, have SIs that are much lower than that achieved by brilacidin, with most drugs failing to show anti-SARS-CoV-2 potency. Of note, the IC50 (0.565μM) and IC90 (2.63μM) values for brilacidin observed in the Calu3 cell line are well below clinically-achievable concentrations based on pharmacokinetics observed in a Phase 2b clinical trial of brilacidin for the treatment of Acute Bacterial Skin and Skin Structure Infections (ABSSSI).
A desirable outcome for any potential COVID-19 therapeutic will be its ability to synergize with existing COVID-19 treatments, particularly if the mechanisms of action of the synergistic treatments can impact more than one step of the viral lifecycle. Such combinations are more likely to elicit an additive response, while also reducing the likelihood of viral resistance developing.
Remdesivir and favipiravir are inhibitors that impact the viral RNA synthesis step of the infectious process. These drugs may help decrease progeny viral genomes in infected cells, but they will not be conducive to inhibiting progressive infection of naïve cells once the progeny virions have been released from infected cells.
By combining remdesivir or favipiravir with brilacidin, a two-pronged strategy of inhibiting viral entry and viral RNA synthesis might be successfully leveraged to most effectively control progression of SARS-CoV-2 infection. [/b]Furthermore, remdesivir and favipiravir do not possess intrinsic anti-inflammatory activity, unlike brilacidin. Any reduction in inflammatory mediators, including IL-6 and IL-1β in the context of remdesivir and favipiravir treatment, are likely to be a direct consequence of decrease in replicating virus.
Moreover, ARDS and associated organ failure observed in the context of COVID-19 are typically later-stage manifestations of disease that are not directly related to, or dependent on, a mounting viral load. Consequently, use of a drug strategy that exhibits intrinsic anti-inflammatory activity will add value to controlling later onset inflammatory damage in COVID-19 patients, potentially occurring beyond the time of active viral multiplication. Along similar lines, it remains to be determined if the combination of convalescent serum—a current COVID-19 treatment that has received FDA Emergency Use Authorization— with brilacidin may confer a protective advantage to patients. It is less likely that a synergistic effect may be observed by combining neutralizing antibodies with brilacidin as it appears the dominant mechanism of action in both cases is disruption of viral entry into cells.
Clearly, an effective COVID-19 therapeutic (or therapeutics in combination) ideally would control both viral load and the corresponding inflammatory damage due to SARS-CoV-2, and mitigate bacterial co-infections. Exhibiting three-in-one properties—antiviral, immuno/anti-inflammatory, and antibacterial—brilacidin is being developed for the intravenous treatment of COVID-19 in hospitalized patients and may be able to address different disease parameters within the one therapeutic treatment.
Brilacidin has been successfully tested in 8 clinical trials across multiple indications providing established safety and efficacy data on over 460 subjects. The drug has exhibited potent antibacterial activity in a Phase 2b trial in Acute Bacterial Skin and Skin Structure Infections (ABSSSI) and anti-inflammatory activity, as supported in Phase 2 clinical trials in Ulcerative Proctitis/Ulcerative Proctosigmoiditis and Oral Mucositis (see Supplemental Information). Brilacidin, through modulation of cyclic adenosine monophosphate (cAMP)/cyclic guanosine monophosphate (cGMP) pathway, is postulated to regulate the immune response based largely on its observed inhibition of phosphodiesterases (PDE4 and PDE3). Interestingly, numerous PDE inhibitors have been proposed as potentially promising anti-SARS-CoV-2 treatments by suppressing the heightened inflammatory response.
Additional formulation work is planned for the inhaled delivery of brilacidin for prophylactic use, toward controlling infection in the nasal passage and lungs by leveraging brilacidin’s ability to inhibit SARS-CoV-2 by disrupting viral integrity and impacting viral entry. Such development efforts, if successful, may enable brilacidin to emerge as a particularly effective and differentiated antiviral by preventing and/or decreasing early infectivity due to SARS-CoV-2.
In this manuscript, we demonstrate brilacidin exhibits robust inhibition of SARS-CoV-2 in Vero cells and Calu-3 cells, supporting brilacidin as a promising novel drug candidate for the treatment of COVID-19. Functioning as a viral entry inhibitor, proposed mechanisms of action for brilacidin include affecting the integrity of the viral membrane and preventing viral binding to cells. More detailed mechanistic studies are planned. Destabilizing viral integrity is a desirable antiviral property, especially in relation to pan-coronavirus agents, as the viral membrane is highly conserved and similar in construct across different coronavirus strains. Moreover, drugs that can directly disrupt viral integrity would be less prone to resistance due to mutation, unlike many antivirals, antibody-based treatments and vaccines that are currently in use and in development. Brilacidin exhibits an excellent synergistic inhibitory profile against SARS-CoV-2 in combination with remdesivir. Experiments conducted in endemic human coronaviruses are ongoing, with additional testing planned in other lethal coronaviruses (MERS-CoV, SARS-CoV), toward assessing the potential of brilacidin as a broad spectrum inhibitor of coronaviruses.
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