Extracts from the George Mason University’s Brilacidin complete presentation.
The purpose of this extract is to simply the reading while highlighting the most important points. I wish it known that I have added definitions and notes to increase readability. Ronald A Phillips
The manuscript introduction.
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the newly emergent causative agent of coronavirus disease-19 (COVID-19), has resulted in more than two million deaths worldwide…. There is a critical global need for therapeutic intervention strategies that can be deployed to safely treat COVID-19 disease and reduce associated morbidity and mortality.
Increasing evidence shows that both natural and synthetic antimicrobial peptides (AMPs), also referred to as Host Defense Proteins/Peptides (HDPs), can inhibit SARS-CoV-2, paving the way for the potential clinical use of these molecules as therapeutic options.
The purpose of this manuscript
In this manuscript, we describe the potent antiviral activity exerted by brilacidin in 4 major ways:
A.) Brilacidin is a new and freshly designed synthetic small molecule that captures the biological properties of Host Defense Proteins/Peptides HDPs—on SARS-CoV-2 in a human lung cell line (Calu-3)…..
B.) These data suggest that SARS-CoV-2 inhibition in these cell culture models is likely to be a result of the impact of brilacidin on viral entry and its disruption of viral integrity.
C.). Brilacidin demonstrated synergistic antiviral activity when combined with remdesivir.
D.) Collectively, our data demonstrate that brilacidin exerts potent inhibition of SARS-CoV-2 against different strains of the virus in cell culture.
1. Manuscript Introduction:
(As of this writing.) The global coronavirus disease-19 (COVID-19) pandemic resulting from infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the novel coronavirus, has resulted in excess of 100 million infections and 2.1 million deaths worldwide, including over 25 million cases and 425,000 fatalities in the United States [1].
Approximately 15% of COVID-19 patients will develop lung injury, including severe respiratory distress that can progress to Acute Respiratory Distress Syndrome (ARDS), often requiring prolonged ventilator support and leading to death. Intensive care units, hospitals, and health care systems risk becoming overwhelmed by critically ill COVID-19 patients.
( note: what follows is the main reason C19 is a killer )
COVID-19 itself is characterized by a heightened inflammatory component, with several proinflammatory cytokines,
(Note: Cytokines are small proteins that are crucial in controlling the growth and activity of other immune system cells and blood cells. When released, they signal the immune system to do its job. Cytokines affect the growth of all blood cells and other cells that help the body's immune and inflammation responses.)
The prevalence of secondary bacterial infections which can occur in up to 20% of cases among hospitalized patients, reinforces the need for a multipronged treatment approach that can address the complexities of COVID-19.
To date, only two vaccines have received Emergency Use Authorization from the FDA to prevent SARS-CoV-2 infection, alongside few modestly effective therapies to treat COVID-19, with remdesivir the only FDA-approved treatment.
Therapies are predominately being developed to treat acute viral infections in hospital settings versus being evaluated for their prophylactic potential. It is likely multiple interventional strategies will be required to prevent and treat COVID-19 particularly given the emergence of drug- and vaccine-resistant variants, with SARS-CoV-2 possibly becoming an endemic viral infection.
Drugs shown to exhibit broad spectrum antiviral activity might help address COVID-19 and potential future viral pandemics.
Natural and synthetic antimicrobial peptides (AMPs), also called Host Defense Proteins/Peptides (HDPs), comprise potentially effective countermeasures against COVID-19, having shown inhibitory activity against multiple Integral components of the innate immune response.
HDPs are typically small (12–80 amino acids) proteins and peptides expressed widely in the animal kingdom that serve as the “first line of defense” against foreign pathogens and potential subsequent infection and related inflammation. HDPs have been recognized as potential sources for promising therapeutics. In mammals, HDPs are found within granules of neutrophils and in secretions from epithelial cells covering skin and mucosal surfaces.
Despite a variety of chemical sequences, as well as secondary and tertiary structures, most HDPs share an amphiphilic topology.
( note: Amphiphilic" is used to describe the properties of a certain group of chemicals. A chemical substance being amphiphilic possesses both hydrophilic and lipophilic functional groups. As a result, amphiphilic compounds show good affinity for both water and oil. )
Due to these differences, HDPs can selectively and variously interact with pathogens, increasing their susceptibility to proteolysis and degradation.
SARS-CoV-2 infection has been shown to suppress defensins, a type of HDP, suggesting that the innate immunity provided by defensins may be compromised .
Suppression of LL37, a human cathelicidin (another type of HDP) shown to possess immunomodulatory and antiviral properties, has also been observed in COVID-19 patients. Increased expression of defensins and cathelicidins (e.g., LL37) can decrease both the viral and inflammatory load in the context of several respiratory viral infections further supporting the protective role of HDPs. Numerous recently published reports support the antiviral activity of HDPs in the context of coronaviruses, including SARS-CoV-2.
Brilacidin (PMX-30063) is a synthetic, nonpeptidic, small-molecule mimetic of HDPs. Building on “first principles” in medicinal chemistry, rational design tools—leveraging sophisticated informatics to fine-tune physicochemical properties and structure–activity relationships enabled brilacidin (an arylamide foldamer ) to overcome the shortcomings and challenges that have complicated the clinical development of natural HDPs .
These include proteolytic degradation, toxicity, lack of efficacy, malabsorption, and high cost to produce. In contrast to natural HDPs, as well as other HDP analogs, brilacidin was designed de novo to be much smaller, more stable, more potent, more selective, and more economical to manufacture.
Brilacidin has already been successfully tested in clinical trials and shown to exhibit potent antibacterial activity in Phase 2 clinical trials for treatment of Acute Bacterial Skin and Skin Structure Infections (ABSSSI), and anti-inflammatory activity, as supported in Phase 2 clinical trials for treatment of Ulcerative Proctitis/Ulcerative Proctosigmoiditis.
As such, brilacidin has already demonstrated HDP-like therapeutic properties in the clinic.
2. Materials and Methods
2.1. Cell Culture. ….. for details see full report
2.2. Inhibitors ( for synergy analysis )
Brilacidin (as brilacidin tetrahydrochloride) was provided by Innovation Pharmaceuticals Inc. (Wakefield, MA, USA) and dissolved in dimethyl sulfoxide (DMSO) (Fisher Scientific, BP231). Hydroxychloroquine sulfate (SelleckChem, S4430, Houston, TX, USA), remdesivir (MedChemExpress, HY-104077, Monmouth Junction, NJ, USA), and favipiravir (FisherScientific, NC1312443) were obtained and dissolved in DMSO.
2.3. Toxicity Screens
2.4. SARS-CoV-2 Infections
SARS-CoV-2 (Washington strain 2019-nCoV/USA-WA1/2020) was obtained from BEI Resources (NR-52281) and was used for all infections, unless otherwise specified.
….. (details extracted )…. . After this incubation, the treated inoculum was overlaid on cells for 1 h. Conditioned media containing inhibitor or standard media were added to cells after removal of virus. For synergy experiments, fresh media containing inhibitor(s) were added to cells after removal of virus.
2.5. Plaque Assay
(for details read full report )
2.6. RNA Extraction and RT-PCR
(for details read full report )
2.7. Statistical Analyses
Statistical analyses and significance was determined using One-Way ANOVA with Dunnett’s Post Test in Prism 7 (Graph Pad) unless otherwise stated. … (Significant values are presented in the full report )
3. Results
3.1. Brilacidin Inhibits SARS-CoV-2 Replication in Vero Cells
As a first step, the potential of brilacidin to exert an antiviral activity against SARS- CoV-2 was assessed using Vero cells as an infection model.
Toxicity assessment of brilacidin in Vero cells was initially performed by incubating the cells with increasing concentrations of the compound for 24 h,
The effect of brilacidin treatment on SARS-CoV-2 viral replication was then evaluated in Vero cells by plaque assay. …..
Infection was allowed to progress for 1 h, ….
The data demonstrate that brilacidin treatment resulted in a dose-dependent decrease in infectious viral titer….
3.2. Brilacidin Appears to Disrupt the Integrity of the SARS-CoV-2 Virion in a Manner That Interferes with Entry
The potential for brilacidin to interfere directly with the virus prior to cell attachment was assessed.
The outcomes of this experiment revealed a higher inhibition of SARS-CoV-2 (72% inhibition), alluding to an inhibitory activity exerted upon the virus directly which demonstrated a 29% decrease in the viral genomic copies with brilacidin treatment; this extent of inhibition of intracellular RNA copies assessed at later timepoints in infection is not unexpected for an inhibitor with likely activity exerted during the early entry and postentry steps.
To independently assess the impact of brilacidin on the virion and thus add support to the role of brilacidin as a potential inhibitor of viral entry, a virus inhibition assay was conducted akin to virus neutralization observed in the presence of antibodies…… Standard pre- and post-treatment conditions were run alongside as controls to compare the impact of brilacidin treatment on the virus alone. The infectious virus titer in the supernatant was quantified by plaque assay, which revealed marked reductions of virus titer The level of inhibition observed at entry was only slightly lower than that observed when the cells were pre- and post-treated with brilacidin concomitantly. This is supporting the concept that brilacidin has an 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.
3.3. Brilacidin Inhibits SARS-CoV-2 in Calu-3 Cells .. (human lung cells)..
To further ascertain that brilacidin can elicit anti-SARS-CoV-2 activity in an ACE2- positive human lung cell, additional experiments were conducted in the
Calu-3 .. (human lung cells) infection model. …..
However, when the experiment was modified to include a brilacidin- treated inoculum—with preincubation of the virus with brilacidin for 1 h 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 10 and 20 µM concentration of the compound, respectively.
Quantification of intracellular viral RNA by semiquantitative RT-PCR at 24 h postinfection (for 10 µM brilacidin) demonstrated a 33% decrease in the viral genomic copies upon brilacidin treatment.
Brilacidin illustrations show Brilacidin exhibits potent inhibition of SARS-CoV-2 in an ACE2-positive human lung cell line (Calu-3 cells). (A) Calu-3 cells were pretreated for 2 h with 10 or 20 μM brilacidin and infected with SARS-CoV-2 at MOI 0.1 nondirectly or (B,C) directly with brilacidin for 1 h as described in Materials and Methods. Cells were post-treated with media containing brilacidin, and at 24 hpi, viral supernatants were evaluated by plaque assay (A,
or intracellular RNA extracted and viral RNA quantified by RT-PCR (C) as described in Materials and Methods. (D) Calu-3 cells were pretreated for 2 h with 10 μM of brilacidin, infected with SARS-CoV-2 at MOIs of 0.1, 0.05, 0.01, or 0.001 directly with brilacidin at 10 μM for 1 h, and post-treated with media containing brilacidin as described in Materials and Methods. At 24 hpi, viral supernatants were evaluated by plaque assay as described in Materials and Methods. Statistical analyses for varying MOIs was determined using Two-Way ANOVA with Sidak’s multiple comparisons test. Significance for all other graphs were determined as described in Materials and Methods. Graphs are representative of one independent experiment performed in technical triplicates (n = 3). ** p < 0.0021, *** p < 0.0002, **** p < 0.0001, ns = not significant.
The inhibition of infectious virus titer as a variable of viral load was assessed by quantifying inhibition at lower multiplicities of infection. Interestingly, the inhibitory potential of brilacidin The inhibition exerted at the MOIs of 0.1 and 0.05 were comparable to each other.
3.4. Selectivity Index Determination for Brilacidin against SARS-CoV-2 in Calu-3 Cells
The Selectivity Index, a ratio that compares a drug’s cytotoxicity and antiviral activity, is a measure of how likely a drug is to be safe and effective when translated to human testing in the clinic.
The test values suggested brilacidin was extremely well tolerated . Quantification of the inhibitory response,
when the virus was directly preincubated with brilacidin prior to infection; cells were treated prior to infection; brilacidin was present during infection; and infected cells were maintained in the presence of brilacidin postinfection —— test here demonstrated that brilacidin achieved 90% inhibition.
Brilacidin exhibits potent inhibition of SARS-CoV-2 in Calu-3 cells. .. (human lung cells)..
3.5. Brilacidin in Combination with Other Antiviral Treatments: Synergistic Activity against SARS-CoV-2 in Combination with Remdesivir in Calu-3 Cells .. (human lung cells)..
As brilacidin appears to act primarily by disrupting viral integrity and inhibiting 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 .. (human lung cells).. cell line at 24 h post-treatment. No apparent toxicity could be detected up to a 10 µM concentration of each of the drugs in the combination regimen. To evaluate the efficacy of combining remdesivir or favipiravir with brilacidin, the cells were pretreated with brilacidin for 2 h. The virus inoculum was also independently preincubated with brilacidin for 1 h, and then the treated inoculum was overlaid on cells and the infection allowed to proceed for 1 h in the presence of brilacidin. Postinfection, the inoculum was removed and media containing both brilacidin and remdesivir or favipiravir or each drug alone for efficacy comparison were added to the infected cells. Supernatants were obtained at 24 h postinfection and infectious titer was 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.
*******NOTE: WHY REMDESIVIR AND BRILACIDIN WORK BEST TOGETHE *******
In contrast, combination of brilacidin with remdesivir at 10 and 2.5 µM concentrations, respectively, reduced the viral infectious titer by >99%, thus providing a highly effective inhibition profile 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
Statistical analyses for synergy vs. individual control treatments were determined using Unpaired Two-Tailed Student’s t test
4. Discussion
The ongoing global COVID-19 pandemic powerfully reinforces the need for therapeutic strategies that can safely and effectively address virus- and host-based events elicited during SARS-CoV-2 infection.
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 demonstrate brilacidin decreases viral load in a robust manner when the virus is preincubated with brilacidin, suggesting brilacidin impacts virus integrity.
Brilacidin’s ability to decrease viral load in an ACE2-positive cell line is demonstrable.
All experiments conducted in Vero and Calu-3 cell line models were supportive of an early inhibition exerted by brilacidin on SARS-CoV-2, indicating the drug’s impact on viral integrity.
The idea that brilacidin directly interferes with the integrity of the virion is further supported by the observation that when drug treatment was limited to the virus alone with no treatment of host cells, a robust decrease of viral load was still observed in both the Washington strain and the Italian strain of SARS-CoV-2.
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.
While brilacidin’s mechanism of action appears primarily to be extracellular, it may also impact intracellular viral replication and is planned to be researched further.
The high CC50 (a measure of cytotoxicity) and low IC50 (a measure of potency) values observed for brilacidin in Calu-3 cells … 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,…… tech details extracted ….. even exceed such targets, thereby further supporting the progression of brilacidin to clinical testing for treatment of COVID-19.
After the FDA fast track designation Brilacidin has been tested in numerous human trials (a total of eight) for other clinical indications, providing established safety and efficacy data on over 460 subjects.
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.
Along these lines, we conducted experiments to evaluate the potential of brilacidin to work in conjunction with remdesivir and favipiravir two frontline COVID-19 treatments, which proved supportive of synergistic inhibition between brilacidin and remdesivir. Remdesivir is a SARS-CoV-2 nucleotide analog RNA polymerase inhibitor that impacts the viral RNA synthesis step of the infectious process. By that mechanism, remdesivir may help decrease progeny viral genomes in infected cells but will not be conducive to inhibiting progressive infection of naïve cells once the progeny virions have been released from infected cells.
By combining remdesivir 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. The opportunity that combination treatments with brilacidin could potentially offer in treating COVID-19 requires further exploration, and in vivo animal studies are in planning stages.
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 [72], and mitigate bacterial coinfections. With its HDP mimetic properties—antiviral, immuno/anti-inflammatory, and antibacterial—brilacidin may be able to address the different disease parameters of COVID-19 within the one therapeutic treatment. The results of the planned Phase 2 clinical trial, with intravenous treatment of COVID-19 in addition to standard of care, are highly anticipated.
In this manuscript, we demonstrated
1. brilacidin exhibits robust inhibition of SARS-CoV-2 in Vero cells and Calu-3 cells, and in two strains of the virus. Likely to function as a viral entry inhibitor. the proposed mechanism of action for brilacidin includes affecting the integrity of the viral membrane and interfering with viral entry.
2. Brilacidin also exhibited an excellent synergistic inhibitory profile against SARS-CoV-2 in combination with remdesivir. 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.
Further research will be conducted in the context of other lethal coronaviruses (MERS-CoV, SARS-CoV) toward assessing the potential of brilacidin as a broad-spectrum inhibitor of coronaviruses.
Author Contributions
Conceptualization, A.B., A.N., T.L.C., W.K.W., and J.A.H.; Methodology, A.B., W.K.W., J.A.H.; A.B., K.R., N.B., F.A., and T.L.C. conducted experiments; Visualization, Formal analysis, Data curation, A.B., A.N., W.K.W., T.L.C., and J.A.H.; Writing—original draft preparation, A.B., A.N., W.K.W., and J.A.H.; Writing—review and editing, A.B., A.N., W.K.W., and J.A.H.; Project administration, A.N., W.K.W., and J.A.H.; Funding acquisition, A.N., W.K.W., and J.A.H. All authors have read and agreed to the published version of the manuscript.
The purpose of this extract is to simply the reading while highlighting the most important points. I wish it known that I have added definitions and notes to increase readability. Ronald A Phillips
The manuscript introduction.
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the newly emergent causative agent of coronavirus disease-19 (COVID-19), has resulted in more than two million deaths worldwide…. There is a critical global need for therapeutic intervention strategies that can be deployed to safely treat COVID-19 disease and reduce associated morbidity and mortality.
Increasing evidence shows that both natural and synthetic antimicrobial peptides (AMPs), also referred to as Host Defense Proteins/Peptides (HDPs), can inhibit SARS-CoV-2, paving the way for the potential clinical use of these molecules as therapeutic options.
The purpose of this manuscript
In this manuscript, we describe the potent antiviral activity exerted by brilacidin in 4 major ways:
A.) Brilacidin is a new and freshly designed synthetic small molecule that captures the biological properties of Host Defense Proteins/Peptides HDPs—on SARS-CoV-2 in a human lung cell line (Calu-3)…..
B.) These data suggest that SARS-CoV-2 inhibition in these cell culture models is likely to be a result of the impact of brilacidin on viral entry and its disruption of viral integrity.
C.). Brilacidin demonstrated synergistic antiviral activity when combined with remdesivir.
D.) Collectively, our data demonstrate that brilacidin exerts potent inhibition of SARS-CoV-2 against different strains of the virus in cell culture.
1. Manuscript Introduction:
(As of this writing.) The global coronavirus disease-19 (COVID-19) pandemic resulting from infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the novel coronavirus, has resulted in excess of 100 million infections and 2.1 million deaths worldwide, including over 25 million cases and 425,000 fatalities in the United States [1].
Approximately 15% of COVID-19 patients will develop lung injury, including severe respiratory distress that can progress to Acute Respiratory Distress Syndrome (ARDS), often requiring prolonged ventilator support and leading to death. Intensive care units, hospitals, and health care systems risk becoming overwhelmed by critically ill COVID-19 patients.
( note: what follows is the main reason C19 is a killer )
COVID-19 itself is characterized by a heightened inflammatory component, with several proinflammatory cytokines,
(Note: Cytokines are small proteins that are crucial in controlling the growth and activity of other immune system cells and blood cells. When released, they signal the immune system to do its job. Cytokines affect the growth of all blood cells and other cells that help the body's immune and inflammation responses.)
The prevalence of secondary bacterial infections which can occur in up to 20% of cases among hospitalized patients, reinforces the need for a multipronged treatment approach that can address the complexities of COVID-19.
To date, only two vaccines have received Emergency Use Authorization from the FDA to prevent SARS-CoV-2 infection, alongside few modestly effective therapies to treat COVID-19, with remdesivir the only FDA-approved treatment.
Therapies are predominately being developed to treat acute viral infections in hospital settings versus being evaluated for their prophylactic potential. It is likely multiple interventional strategies will be required to prevent and treat COVID-19 particularly given the emergence of drug- and vaccine-resistant variants, with SARS-CoV-2 possibly becoming an endemic viral infection.
Drugs shown to exhibit broad spectrum antiviral activity might help address COVID-19 and potential future viral pandemics.
Natural and synthetic antimicrobial peptides (AMPs), also called Host Defense Proteins/Peptides (HDPs), comprise potentially effective countermeasures against COVID-19, having shown inhibitory activity against multiple Integral components of the innate immune response.
HDPs are typically small (12–80 amino acids) proteins and peptides expressed widely in the animal kingdom that serve as the “first line of defense” against foreign pathogens and potential subsequent infection and related inflammation. HDPs have been recognized as potential sources for promising therapeutics. In mammals, HDPs are found within granules of neutrophils and in secretions from epithelial cells covering skin and mucosal surfaces.
Despite a variety of chemical sequences, as well as secondary and tertiary structures, most HDPs share an amphiphilic topology.
( note: Amphiphilic" is used to describe the properties of a certain group of chemicals. A chemical substance being amphiphilic possesses both hydrophilic and lipophilic functional groups. As a result, amphiphilic compounds show good affinity for both water and oil. )
Due to these differences, HDPs can selectively and variously interact with pathogens, increasing their susceptibility to proteolysis and degradation.
SARS-CoV-2 infection has been shown to suppress defensins, a type of HDP, suggesting that the innate immunity provided by defensins may be compromised .
Suppression of LL37, a human cathelicidin (another type of HDP) shown to possess immunomodulatory and antiviral properties, has also been observed in COVID-19 patients. Increased expression of defensins and cathelicidins (e.g., LL37) can decrease both the viral and inflammatory load in the context of several respiratory viral infections further supporting the protective role of HDPs. Numerous recently published reports support the antiviral activity of HDPs in the context of coronaviruses, including SARS-CoV-2.
Brilacidin (PMX-30063) is a synthetic, nonpeptidic, small-molecule mimetic of HDPs. Building on “first principles” in medicinal chemistry, rational design tools—leveraging sophisticated informatics to fine-tune physicochemical properties and structure–activity relationships enabled brilacidin (an arylamide foldamer ) to overcome the shortcomings and challenges that have complicated the clinical development of natural HDPs .
These include proteolytic degradation, toxicity, lack of efficacy, malabsorption, and high cost to produce. In contrast to natural HDPs, as well as other HDP analogs, brilacidin was designed de novo to be much smaller, more stable, more potent, more selective, and more economical to manufacture.
Brilacidin has already been successfully tested in clinical trials and shown to exhibit potent antibacterial activity in Phase 2 clinical trials for treatment of Acute Bacterial Skin and Skin Structure Infections (ABSSSI), and anti-inflammatory activity, as supported in Phase 2 clinical trials for treatment of Ulcerative Proctitis/Ulcerative Proctosigmoiditis.
As such, brilacidin has already demonstrated HDP-like therapeutic properties in the clinic.
2. Materials and Methods
2.1. Cell Culture. ….. for details see full report
2.2. Inhibitors ( for synergy analysis )
Brilacidin (as brilacidin tetrahydrochloride) was provided by Innovation Pharmaceuticals Inc. (Wakefield, MA, USA) and dissolved in dimethyl sulfoxide (DMSO) (Fisher Scientific, BP231). Hydroxychloroquine sulfate (SelleckChem, S4430, Houston, TX, USA), remdesivir (MedChemExpress, HY-104077, Monmouth Junction, NJ, USA), and favipiravir (FisherScientific, NC1312443) were obtained and dissolved in DMSO.
2.3. Toxicity Screens
2.4. SARS-CoV-2 Infections
SARS-CoV-2 (Washington strain 2019-nCoV/USA-WA1/2020) was obtained from BEI Resources (NR-52281) and was used for all infections, unless otherwise specified.
….. (details extracted )…. . After this incubation, the treated inoculum was overlaid on cells for 1 h. Conditioned media containing inhibitor or standard media were added to cells after removal of virus. For synergy experiments, fresh media containing inhibitor(s) were added to cells after removal of virus.
2.5. Plaque Assay
(for details read full report )
2.6. RNA Extraction and RT-PCR
(for details read full report )
2.7. Statistical Analyses
Statistical analyses and significance was determined using One-Way ANOVA with Dunnett’s Post Test in Prism 7 (Graph Pad) unless otherwise stated. … (Significant values are presented in the full report )
3. Results
3.1. Brilacidin Inhibits SARS-CoV-2 Replication in Vero Cells
As a first step, the potential of brilacidin to exert an antiviral activity against SARS- CoV-2 was assessed using Vero cells as an infection model.
Toxicity assessment of brilacidin in Vero cells was initially performed by incubating the cells with increasing concentrations of the compound for 24 h,
The effect of brilacidin treatment on SARS-CoV-2 viral replication was then evaluated in Vero cells by plaque assay. …..
Infection was allowed to progress for 1 h, ….
The data demonstrate that brilacidin treatment resulted in a dose-dependent decrease in infectious viral titer….
3.2. Brilacidin Appears to Disrupt the Integrity of the SARS-CoV-2 Virion in a Manner That Interferes with Entry
The potential for brilacidin to interfere directly with the virus prior to cell attachment was assessed.
The outcomes of this experiment revealed a higher inhibition of SARS-CoV-2 (72% inhibition), alluding to an inhibitory activity exerted upon the virus directly which demonstrated a 29% decrease in the viral genomic copies with brilacidin treatment; this extent of inhibition of intracellular RNA copies assessed at later timepoints in infection is not unexpected for an inhibitor with likely activity exerted during the early entry and postentry steps.
To independently assess the impact of brilacidin on the virion and thus add support to the role of brilacidin as a potential inhibitor of viral entry, a virus inhibition assay was conducted akin to virus neutralization observed in the presence of antibodies…… Standard pre- and post-treatment conditions were run alongside as controls to compare the impact of brilacidin treatment on the virus alone. The infectious virus titer in the supernatant was quantified by plaque assay, which revealed marked reductions of virus titer The level of inhibition observed at entry was only slightly lower than that observed when the cells were pre- and post-treated with brilacidin concomitantly. This is supporting the concept that brilacidin has an 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.
3.3. Brilacidin Inhibits SARS-CoV-2 in Calu-3 Cells .. (human lung cells)..
To further ascertain that brilacidin can elicit anti-SARS-CoV-2 activity in an ACE2- positive human lung cell, additional experiments were conducted in the
Calu-3 .. (human lung cells) infection model. …..
However, when the experiment was modified to include a brilacidin- treated inoculum—with preincubation of the virus with brilacidin for 1 h 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 10 and 20 µM concentration of the compound, respectively.
Quantification of intracellular viral RNA by semiquantitative RT-PCR at 24 h postinfection (for 10 µM brilacidin) demonstrated a 33% decrease in the viral genomic copies upon brilacidin treatment.
Brilacidin illustrations show Brilacidin exhibits potent inhibition of SARS-CoV-2 in an ACE2-positive human lung cell line (Calu-3 cells). (A) Calu-3 cells were pretreated for 2 h with 10 or 20 μM brilacidin and infected with SARS-CoV-2 at MOI 0.1 nondirectly or (B,C) directly with brilacidin for 1 h as described in Materials and Methods. Cells were post-treated with media containing brilacidin, and at 24 hpi, viral supernatants were evaluated by plaque assay (A,
or intracellular RNA extracted and viral RNA quantified by RT-PCR (C) as described in Materials and Methods. (D) Calu-3 cells were pretreated for 2 h with 10 μM of brilacidin, infected with SARS-CoV-2 at MOIs of 0.1, 0.05, 0.01, or 0.001 directly with brilacidin at 10 μM for 1 h, and post-treated with media containing brilacidin as described in Materials and Methods. At 24 hpi, viral supernatants were evaluated by plaque assay as described in Materials and Methods. Statistical analyses for varying MOIs was determined using Two-Way ANOVA with Sidak’s multiple comparisons test. Significance for all other graphs were determined as described in Materials and Methods. Graphs are representative of one independent experiment performed in technical triplicates (n = 3). ** p < 0.0021, *** p < 0.0002, **** p < 0.0001, ns = not significant. The inhibition of infectious virus titer as a variable of viral load was assessed by quantifying inhibition at lower multiplicities of infection. Interestingly, the inhibitory potential of brilacidin The inhibition exerted at the MOIs of 0.1 and 0.05 were comparable to each other.
3.4. Selectivity Index Determination for Brilacidin against SARS-CoV-2 in Calu-3 Cells
The Selectivity Index, a ratio that compares a drug’s cytotoxicity and antiviral activity, is a measure of how likely a drug is to be safe and effective when translated to human testing in the clinic.
The test values suggested brilacidin was extremely well tolerated . Quantification of the inhibitory response,
when the virus was directly preincubated with brilacidin prior to infection; cells were treated prior to infection; brilacidin was present during infection; and infected cells were maintained in the presence of brilacidin postinfection —— test here demonstrated that brilacidin achieved 90% inhibition.
Brilacidin exhibits potent inhibition of SARS-CoV-2 in Calu-3 cells. .. (human lung cells)..
3.5. Brilacidin in Combination with Other Antiviral Treatments: Synergistic Activity against SARS-CoV-2 in Combination with Remdesivir in Calu-3 Cells .. (human lung cells)..
As brilacidin appears to act primarily by disrupting viral integrity and inhibiting 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 .. (human lung cells).. cell line at 24 h post-treatment. No apparent toxicity could be detected up to a 10 µM concentration of each of the drugs in the combination regimen. To evaluate the efficacy of combining remdesivir or favipiravir with brilacidin, the cells were pretreated with brilacidin for 2 h. The virus inoculum was also independently preincubated with brilacidin for 1 h, and then the treated inoculum was overlaid on cells and the infection allowed to proceed for 1 h in the presence of brilacidin. Postinfection, the inoculum was removed and media containing both brilacidin and remdesivir or favipiravir or each drug alone for efficacy comparison were added to the infected cells. Supernatants were obtained at 24 h postinfection and infectious titer was 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.
*******NOTE: WHY REMDESIVIR AND BRILACIDIN WORK BEST TOGETHE *******
In contrast, combination of brilacidin with remdesivir at 10 and 2.5 µM concentrations, respectively, reduced the viral infectious titer by >99%, thus providing a highly effective inhibition profile 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
Statistical analyses for synergy vs. individual control treatments were determined using Unpaired Two-Tailed Student’s t test
4. Discussion
The ongoing global COVID-19 pandemic powerfully reinforces the need for therapeutic strategies that can safely and effectively address virus- and host-based events elicited during SARS-CoV-2 infection.
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 demonstrate brilacidin decreases viral load in a robust manner when the virus is preincubated with brilacidin, suggesting brilacidin impacts virus integrity.
Brilacidin’s ability to decrease viral load in an ACE2-positive cell line is demonstrable.
All experiments conducted in Vero and Calu-3 cell line models were supportive of an early inhibition exerted by brilacidin on SARS-CoV-2, indicating the drug’s impact on viral integrity.
The idea that brilacidin directly interferes with the integrity of the virion is further supported by the observation that when drug treatment was limited to the virus alone with no treatment of host cells, a robust decrease of viral load was still observed in both the Washington strain and the Italian strain of SARS-CoV-2.
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.
While brilacidin’s mechanism of action appears primarily to be extracellular, it may also impact intracellular viral replication and is planned to be researched further.
The high CC50 (a measure of cytotoxicity) and low IC50 (a measure of potency) values observed for brilacidin in Calu-3 cells … 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,…… tech details extracted ….. even exceed such targets, thereby further supporting the progression of brilacidin to clinical testing for treatment of COVID-19.
After the FDA fast track designation Brilacidin has been tested in numerous human trials (a total of eight) for other clinical indications, providing established safety and efficacy data on over 460 subjects.
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.
Along these lines, we conducted experiments to evaluate the potential of brilacidin to work in conjunction with remdesivir and favipiravir two frontline COVID-19 treatments, which proved supportive of synergistic inhibition between brilacidin and remdesivir. Remdesivir is a SARS-CoV-2 nucleotide analog RNA polymerase inhibitor that impacts the viral RNA synthesis step of the infectious process. By that mechanism, remdesivir may help decrease progeny viral genomes in infected cells but will not be conducive to inhibiting progressive infection of naïve cells once the progeny virions have been released from infected cells.
By combining remdesivir 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. The opportunity that combination treatments with brilacidin could potentially offer in treating COVID-19 requires further exploration, and in vivo animal studies are in planning stages.
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 [72], and mitigate bacterial coinfections. With its HDP mimetic properties—antiviral, immuno/anti-inflammatory, and antibacterial—brilacidin may be able to address the different disease parameters of COVID-19 within the one therapeutic treatment. The results of the planned Phase 2 clinical trial, with intravenous treatment of COVID-19 in addition to standard of care, are highly anticipated.
In this manuscript, we demonstrated
1. brilacidin exhibits robust inhibition of SARS-CoV-2 in Vero cells and Calu-3 cells, and in two strains of the virus. Likely to function as a viral entry inhibitor. the proposed mechanism of action for brilacidin includes affecting the integrity of the viral membrane and interfering with viral entry.
2. Brilacidin also exhibited an excellent synergistic inhibitory profile against SARS-CoV-2 in combination with remdesivir. 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.
Further research will be conducted in the context of other lethal coronaviruses (MERS-CoV, SARS-CoV) toward assessing the potential of brilacidin as a broad-spectrum inhibitor of coronaviruses.
Author Contributions
Conceptualization, A.B., A.N., T.L.C., W.K.W., and J.A.H.; Methodology, A.B., W.K.W., J.A.H.; A.B., K.R., N.B., F.A., and T.L.C. conducted experiments; Visualization, Formal analysis, Data curation, A.B., A.N., W.K.W., T.L.C., and J.A.H.; Writing—original draft preparation, A.B., A.N., W.K.W., and J.A.H.; Writing—review and editing, A.B., A.N., W.K.W., and J.A.H.; Project administration, A.N., W.K.W., and J.A.H.; Funding acquisition, A.N., W.K.W., and J.A.H. All authors have read and agreed to the published version of the manuscript.
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Ronald A Phillips
