Download Hi-Res ImageDownload to MS-PowerPointCite This:ACS Pharmacol. Transl. Sci. 2020, 3, 6, 1371-1380
Clinically Approved Antiviral Drug in an Orally Administrable Nanoparticle for COVID-19
- Bapurao SurnarBapurao SurnarDepartment of Biochemistry and Molecular Biology, University of Miami, 1011 NW 15th Street, Miami, Florida 33136, United StatesMore by Bapurao Surnar,
- Mohammad Z. KamranMohammad Z. KamranDepartment of Biochemistry and Molecular Biology, University of Miami, 1011 NW 15th Street, Miami, Florida 33136, United StatesMore by Mohammad Z. Kamran,
- Anuj S. ShahAnuj S. ShahDepartment of Biochemistry and Molecular Biology, University of Miami, 1011 NW 15th Street, Miami, Florida 33136, United StatesMore by Anuj S. Shah, and
- Shanta Dhar*Shanta Dhar*Email: [email protected]Department of Biochemistry and Molecular Biology, Sylvester Comprehensive Cancer Center Leonard M. Miller School of Medicine, University of Miami, 1011 NW 15th Street, Miami, Florida 33136, United StatesMore by Shanta Dhar
Abstract
There is urgent therapeutic need for COVID-19, a disease for which there are currently no widely effective approved treatments and the emergency use authorized drugs do not result in significant and widespread patient improvement. The food and drug administration-approved drug ivermectin has long been shown to be both antihelmintic agent and a potent inhibitor of viruses such as Yellow Fever Virus. In this study, we highlight the potential of ivermectin packaged in an orally administrable nanoparticle that could serve as a vehicle to deliver a more potent therapeutic antiviral dose and demonstrate its efficacy to decrease expression of viral spike protein and its receptor angiotensin-converting enzyme 2 (ACE2), both of which are keys to lowering disease transmission rates. We also report that the targeted nanoparticle delivered ivermectin is able to inhibit the nuclear transport activities mediated through proteins such as importin α/β1 heterodimer as a possible mechanism of action. This study sheds light on ivermectin-loaded, orally administrable, biodegradable nanoparticles to be a potential treatment option for the novel coronavirus through a multilevel inhibition. As both ACE2 targeting and the presence of spike protein are features shared among this class of virus, this platform technology has the potential to serve as a therapeutic tool not only for COVID-19 but for other coronavirus strains as well.
Note
This article is made available via the ACS COVID-19 subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.
The COVID-19, a disease caused by a novel coronavirus strain called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a unique challenge to domestic and international public health. As of September 21, 2020, there are over 31.1 million cases worldwide, and nearly 6.9 million cases in the United States alone. This viral strain is highly transmittable and infects respiratory tissue, and can cause flu-like symptoms as well as more severe respiratory issues and death by respiratory failure.(1,2) Until now, over half a million people have died due to this virus in 2020 alone. The SARS-CoV-2 virus surface spike protein interacts with angiotensin-converting enzyme 2 (ACE2) receptors in the lung and facilitates the entry of the virus into host cells. Most of the tissue damage is a product of the immune response and resulting inflammation.(3−11) Therefore, the development of new drugs and treatment modalities is urgently needed to fight the disease.
There are currently at least 15 completed clinical trials conducted around the world to search for various therapeutics to combat COVID-19 infection with no appreciable success (Table S1 in the Supporting Information). Newer studies are focusing more on COVID-19-specific inhibitors from existing natural compounds such as flavonoids.(12,13) Many of the experimental therapeutics are meant only to rescue patients in severe respiratory distress or those already undergoing intubation and mechanical ventilation, rather than patients at an early to moderate stage of infection. Among ongoing clinical trials, one aims to use the “wonder drug” ivermectin (IVM)(14) in combination with aspirin, dexamethasone, and enoxaparin (Table S2). This trial uses IVM via a single dose of 200 μg/kg and may pose issues of toxicity and accelerated clearance from the bloodstream resulting in low effective dose.(15,16) In this article, we report an orally administrable IVM-loaded nanoparticle (NP) and its ability to lower the expression of the ACE2 receptor and the SARS-CoV-2 spike protein.
We recently developed a therapeutic IVM-loaded NP to treat Zika virus infection in the blood.(17) The developed IVM nanoformulation allows the therapeutic to be gradually released into the bloodstream, which maintains its level in the blood at approximately the minimum effective therapeutic dose while keeping it below the maximum tolerated dose. This NP was constructed using poly(lactide-co-glycolide)-b-poly(ethylene glycol)-maleimide (PLGA-b-PEG-Mal) polymer, and was tagged to an Fc immunoglobulin fragment to take advantage of FcRn-driven crossing of the gut epithelial barrier to reach the bloodstream (Figure 1A). Here, we adopted the synthesis and characterization of these NPs and employed the NPs as a treatment option for COVID-19. A key goal of any new agent or nanoformulation should be not only to reduce levels of proteins that contribute to the virus’ infectious nature, but also to exploit mechanisms to prevent viral entry into cells in the first place. ACE2, which is present in high quantities in respiratory epithelia, allows for viral entry and infection of the lung and alveolar cells. ACE2-expressing cells in the lung are involved in key processes such as blood pressure regulation and interferon production. SARS-CoV-2 binding to this receptor can impede on those processes, making it an important possible target to reduce viral infection. Therefore, we hypothesized that if IVM is found to effectively decrease levels of viral spike protein as well as cellular levels of ACE2, it could theoretically be a better therapeutic when delivered in a controlled fashion using our orally administrable NP. IVM itself is known to be toxic, with an EC50 value between 1 and 10 mM. Furthermore, the maximum plasma concentrations of IVM in human after an injected dose of 150 μg/kg is typically only in the range of 9 to 75 ng/mL, but higher doses of the drug may be needed for it to be effective in controlling viral load and transmission.(18) Thus, NP-mediated delivery of IVM will allow the drug to be gradually released into the bloodstream at an effective therapeutic dose while keeping it below the maximum tolerated dose. In particular, IVM-loaded nanoparticles can be engineered to contain a bound Fc immunoglobulin antibody fragment to target FcRn receptors on gut epithelial cells, which should allow for transcytosis of orally delivered nanoparticles into the bloodstream and potential accumulation at respiratory epithelial cells, which are particularly affected by SARS-CoV-2 (Figure 1).
Figure 1
Figure 1. Graphical representation showing that the targeted-Fc-IVM-NPs in the acidic gut lumen bind to FcRn receptors, allowing NPs to transcytose across the intestinal barrier and release at the physiological pH of blood. IVM delivered via T-Fc-IVM-NPs shows the ability to (1) decrease ACE2 receptor levels, (2) decrease SARS-CoV-2 spike protein levels, and (3) decrease levels of the nuclear transport proteins importin α and β1, which leads to (4) an increase in the antiviral activity of infected cells.
The Centers for Disease Control and Prevention (CDC) recently suggested that the pregnant population are at a higher risk for severe complications from COVID-19 as compared to nonpregnant people, and that adverse pregnancy outcomes can happen among pregnant people with COVID-19, a finding supported by other institutions.(19) As a whole, pregnant women are more likely to be admitted to intensive care units and put on mechanical ventilators than are nonpregnant women, placing the fetus at increased risk as well. Thus, it is important to develop therapeutics that can be provided as a method of care to pregnant women without affecting the fetus. Our previous studies have shown that when delivered with the mentioned NP platform, IVM cannot cross the placental barrier.(17) Thus, we envision that this platform can serve as a promising potential therapeutic for pregnant individuals affected by COVID-19. The ability of the NP to reduce ACE2 levels in various cell lines and thus reduce viral uptake, paired with its capacity to decrease spike protein expression, will allow this therapeutic to be utilized against other coronavirus strains, strengthening our preparedness for future viral outbreaks.
Results and Discussion
ARTICLE SECTIONS
Ability of Ivermectin Nanoformulation to Reduce ACE2 and Spike Protein Expression
IVM-loaded PLGA-b-PEG-MAL nanoparticles (IVM-NPs) were synthesized by following a nanoprecipitation method. The NPs were characterized using dynamic light scattering (DLS) and were found to have sizes of approximately 70–80 nm and zeta potential of ∼30 mV with 20% feed of IVM (Figure S1). IVM loading was quantified using high performance liquid chromatography (HPLC) (Figure S1). The Fc immunoglobulin fragment targeting moiety was attached using thiol–ene chemistry, creating the targeted T-Fc-IVM-NPs. The conjugation of the Fc fragment was confirmed and quantified through a bicinchoninic acid (BCA) assay. SARS-CoV-2 is a positive sense single-stranded RNA virus, and one of the most crucial components of its structure is the surface spike protein that allows it to enter and infect cells. Thus, a logical method to model the conditions of viral infection in vitro and study the expression of the spike protein is to transfect cells using a plasmid expressing spike protein. This would mimic infectious conditions and allow for measurements of NP-delivered IVM’s ability to inhibit the viral spike protein without requiring the construction of pseudoviruses or other technologies. To test the therapeutic abilities of the T-Fc-IVM-NPs against both ACE2 and the viral spike proteins, HEK293T human embryonic kidney epithelial cells were transfected with a plasmid containing the SARS-CoV-2 viral spike protein. These cells were subsequently treated with IVM, a nontargeted IVM-loaded nanoparticle, NT-IVM-NP, made from an PLGA-b-PEG–OH polymer, or T-Fc-IVM-NPs. The treatments were with 10 μM of free IVM or the nanoformulations with respect to IVM for a period of 4 h followed by incubation for 20 h. The incubation time and dosing were decided based on the uptake kinetics and the IC50 values for the articles in HEK293T cells (Figure S2 for cellular updake data and Figure S3 for cytotoxicity by the MIT assay). Western blot data revealed that the expressions of the spike protein and ACE2 in the HEK293T cells were significantly decreased by IVM nanoformulations but not by free IVM (Figure 2A,B, Figure S4 for quantification). These results were further confirmed by immunofluorescence studies demonstrating the ability of T-Fc-IVM-NP to reduce ACE2 and spike protein levels (Figure 2B,C). We observed a decrease in spike and ACE2 expression in HEK293T cells when cells were treated with free IVM for 24 h (Figure S5A,B). Similarly, we observed a decrease in spike and ACE2 expression at 24 h by immunofluorescence (Figure S5C). At 4 h treatment, we observed a differential effect of IVM and IVM nanoformulations. However, the 24 h treatment did not show any difference in IVM and IVM-NPs. This may indicate that IVM-loaded NPs are able to be taken up into cells more at earlier time points than free IVM to exert their effects. Furthermore, we have shown that bioavailability of IVM nanoformulations is more than free IVM.(17) In balb/c mice, free IVM showed only 20% of injected dose in the blood as compared to T-Fc-IVM-NP which was >50% in the blood after 24 h. Our studies also revealed that the HEK293T cells have low basal ACE2 expression. To confirm this further, we treated the cells with increased concentrations of angiotensin II (ANG II) and documented that such treatment increases the ACE2 levels in these cells (Figure S6). We also evaluated the effect of free IVM and its nanoformulation on basal ACE2 gene expression at the transcriptional level (Figure 2D). In addition, both ACE2 as well as spike was studied in HEK293T cells transfected with spike plasmid (Figure 2E). Cells were treated with 10 μM of IVM and its nanoformulation for 4 h and real time PCR was carried out. It was observed that T-Fc-IVM-NP significantly inhibited spike mRNA expression. Free IVM and NT-IVM-NP did not have significant inhibitory effects on spike mRNA expression. Similarly, we observed that ACE2 mRNA expression was decreased by T-Fc-IVM-NP. NT-IVM-NP also decreased ACE2 mRNA expression, but this decrease was less compared to that seen in T-Fc-IVM-NP. Taken together, our data suggest that in a cellular context, T-Fc-IVM-NP is more effective than free IVM and NT-IVM-NP in decreasing spike and ACE2 gene expression at the transcriptional level.
Figure 2
Figure 2. (A) Western blot showing expression of ACE2 and spike protein in HEK293T cells transfected with plasmid expressing spike protein with treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 2 or 4 h at a concentration of 10 μM with respect to IVM, after which the media was changed and the cells were further incubated up to a total of 24 h. Immunofluorescence staining showing expression of (B) ACE2 and (C) spike protein in HEK293T cells transfected with a plasmid expressing spike protein with treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 4 h at a concentration of 10 μM with respect to IVM, followed by an additional 20 h incubation. (D) Fold change in mRNA expression level of ACE2 in HEK293T cells upon treatment with articles at a concentration of 10 μM with respect to IVM. (E) Fold change in mRNA expression levels of ACE2 and spike protein in spike-protein expressing HEK293T cells. The cells transfected with spike plasmid were treated with articles at a concentration of 10 μM with respect to IVM. For panels D and E, the treatment time was 4 h, followed by an additional incubation for 20 h.
We also analyzed the effects of T-Fc-IVM-NP in two other ACE2-expressing epithelial cell lines, A549 adenocarcinomic alveolar basal epithelial cells and HeLa malignant epithelial cells, to study the potential impact of the therapeutic on ACE2 and spike protein expression in lung cells and other epithelia that may be infected by SARS-CoV-2. In the A549 cells, ACE2 expression was found to decrease after treatment with IVM and the IVM nanoformulations, and the largest decrease in expression was seen after treatment with the IVM-loaded nanoparticles (Figure 3A, Figure S7A for quantification). Accordingly, the A549 cells were treated with increasing doses of T-Fc-IVM-NPs, and the Western blot revealed a dose-dependent effect of the nanoformulation on ACE2 expression (Figure 3B, Figure S7B for quantification). In HeLa cells, treatment with T-Fc-IVM-NP showed a decrease in the expression of both spike protein and ACE2, and the more evident decrease appeared to be in the cells treated with the nanoparticles (Figure 3C, Figure S7C,D for quantification). Furthermore, immunofluorescence staining in A549 cells revealed a decrease in expression of ACE2 following treatment with IVM, NT-IVM-NPs, and T-Fc-IVM-NPs (Figure 3D). The inhibition of both ACE2 and the viral spike protein in a variety of cell lines shows that the IVM-loaded nanoparticle will be effective in treating many different cell types that may be infected.
Figure 3
Figure 3. (A) Western blot showing expression of ACE2 in A549 adenocarcinomic alveolar basal epithelial cells transfected with plasmid expressing spike protein with treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 4 h at a concentration of 10 μM with respect to IVM, followed by incubation in normal media for an additional 20 h. (B) Western blot showing a dose-dependent decrease in basal ACE2 expression after treatment with varying concentrations of T-Fc-IVM-NPs with respect to IVM in A549 cells. (C) Western blot showing expression of ACE2 in HeLa malignant epithelial cells transfected with plasmid expressing spike protein with treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 4 h at a concentration of 10 μM with respect to IVM, followed by incubation in normal media for an additional 20 h. (D) Immunofluorescence staining showing expression of ACE2 in A549 cells transfected with plasmid expressing spike protein with treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 4 h at a concentration of 10 μM with respect to IVM, followed by incubation in normal media for an additional 20 h.
Pseudovirus Inhibition Study
As we observed a decrease in ACE2 protein expression by T-Fc-IVM-NP, we evaluated if this decrease affects the virus uptake in HEK293T cells by carrying out a red-tagged ACE2 reporter and mNeonGreen pseudo-SARS-CoV-2 based assay. This two-step assay allows for the nuclei of transduced cells to fluorescent green if the virus is taken up in an ACE2-driven process. This experiment was carried out using therapeutic and preventative settings (Figure 4A).
Figure 4
Figure 4. (A) Schematic representation of mNeonGreen pseudovirus reporter protein accumulation in HEK293T cells and the efficacy of T-Fc-IVM-NP showing inhibition of both ACE2 and pseudovirus uptake under (B) therapeutic and (C) preventative treatment methods as measured by the microplate reader. Confocal microscopy images revealing the changes in the expression of red-tagged ACE2 receptor on the cell membrane and mNeonGreen pseudovirus accumulation in the nucleus following the treatment of articles under (D) therapeutic and (E) preventative treatment methods. The article concentration was kept at 10 μM with respect to IVM for 4 h followed by an additional 20 h of incubation.
The therapeutic approach, in which the cells were exposed to the ACE2 reporter and pseudo-SARS-CoV-2 followed by treatment with IVM or its nanoformulation, resulted in significant decrease in the levels of both ACE2 and pseudovirus uptake (Figure 4B). In the preventative approach, in which treatment preceded ACE2 reporter and pseudovirus exposure with the goal of preventing uptake, there were pronounced decreases in both ACE2 expression and pseudovirus uptake after treatment with T-Fc-IVM-NP (Figure 4B). In addition, the effects of the articles were monitored via confocal microscopy, which also revealed decreases in the red and green fluorescence in the cells after treatment with T-Fc-IVM-NP treatment under both the therapeutic (Figure 4C) and preventative (Figure 4D) approaches. The decrease in green fluorescence in cells’ nuclei after the therapeutic approach, in which pseudovirus particles had already entered cells prior to T-Fc-IVM-NP treatment, showed that this nanoformulation may also be effective in decreasing the expression of the viral proteins once they are inside the cells. This assay was also performed in normal human small airway epithelial cells (HSAEC) which are known to get infected by SARS-CoV-2 (Figure S8).
Potential Mechanism of Action of T-Fc-IVM-NP
Though the specific mechanism by which the released IVM could inhibit the replication of the SARS-CoV-2 virus and expression of spike protein is yet to be determined, a possibility could be through the inhibition of the nuclear transport activities mediated through proteins such as importin (IMP) α/β1 heterodimer, as IVM was previously shown to inhibit a similar interaction between IMPα/β1 and the human immunodeficiency virus-1 (HIV-1) integrase protein.(20−25) The IMP α/β1 heterodimer is a key nuclear transport protein and is believed to play a role in transporting viral proteins to the nucleus of infected cells. IMP α and β1 work through the recognition of nuclear localization signals on proteins, and IMP α and β1 have previously been associated with the nuclear transport of other viral proteins such as HIV-1 integrase and dengue virus nonstructural protein 5 (NS5).(24) IMP α and β1 transport of viral proteins to the nucleus allows proteins such as dengue virus’ NS5 protein to diminish cells’ antiviral responses by impacting mRNA splicing and immune signaling.(26) Therefore, investigating the IVM-loaded nanoparticle’s potential inhibitory effect on IMP α and β1 is key to fully characterizing the therapeutic’s antiviral properties. To more closely study the potential inhibitory activity of IVM nanoformulations on IMP α/β1 activity, we conducted a time-dependent treatment study in spike protein-expressing HEK293T cells. Cells were treated with T-Fc-IVM-NP for periods of 2, 4, or 6 h, and then incubated in media for an additional 22, 20, or 18 h, respectively. Western blot analyses revealed that T-Fc-IVM-NP showed inhibition of both IMP α and β1 (Figure 5A). These results suggest that the ability of T-Fc-IVM-NPs to inhibit importin expression might lead to a decrease in viral protein transport to the nucleus. The results may also indicate that uptake of NP-delivered IVM into cells may occur more quickly than the uptake of free IVM, as the effects of free IVM on IMP α and β1 expression only begin to be seen at a later time point. In our studies, we observed that T-Fc-IVM-NP-released IVM has a greater efficiency in inhibiting both IMP α and IMP β1 compared to free IVM or NT-IVM-NP (Figure 5A). The time-dependent studies demonstrated that an incubation time of 4 h is most efficient in bringing such inhibition when the cells are treated with T-Fc-IVM-NP (Figure 5).
Figure 5
Figure 5. (A) Western blot showing the change in expression of IMP α and β1 in HEK293T cells following treatment with IVM and its nanoformulations. Cells were treated with articles for 2, 4, and 6 h at a concentration of 10 μM with respect to IVM, followed by incubation in normal media up to a total of 24 h. (B) Western blot showing dose-dependent changes in IMP α and β1 after treatment with varying concentrations of importazole (IMZ) in HEK293T cells. (C) Efficacy of IMZ in combination with IVM, NT-IVM-NP, and T-Fc-IVM-NP on both ACE2 expression and pseudovirus uptake under preventative treatment method as measured by the microplate reader. (D) Western blot showing the change in expression of ACE2, spike protein, and IMP α and β1 in HEK293T cells following treatment with remdesivir, IVM, or its nanoformulations. Cells were treated with articles for 4 h at a concentration of 10 μM with respect to IVM or remdesivir, followed by incubation in normal media up to a total of 24 h. (E) Western blot (top) and RT-PCR (bottom) data showing the change in expression of MERS-CoV spike protein in HEK293T cells following treatment with remdesivir, IVM, or its nanoformulations. Cells were treated with articles for 4 h at a concentration of 10 μM with respect to IVM or remdesivir, followed by incubation in normal media up to a total of 24 h. (F) Schematic representation of how IVM delivered through T-Fc-IVM-NP inhibits IMP α and β1, thus increasing host cell antiviral activity.
To further probe the mechanism of action of the IVM-loaded targeted NP through the IMPα/β1 system, spike protein-expressing cells were treated with importazole, a commercially available IMPβ1 inhibitor, before pseudovirus infection, with the goal of inhibiting IMPβ1 and observing whether pseudovirus protein transport to the nucleus was diminished. A concentration-dependent study indicated that 25 μM of importazole resulted in a significant decrease in IMPβ1 expression (Figure 5B), but not significant decrease in the nuclear transport of the pseudovirus protein (Figure S9). Subsequent studies conducted by pretreating the cells with 20 μM of importazole followed by T-Fc-IVM-NP treatment indicated that there was still a concurrent decrease in ACE2 and spike protein expression despite importazole treatment (Figure 5C), demonstrating that this NP may have multiple molecular targets and may be inhibiting pseudovirus expression and nuclear transport through other mechanisms, such as by lowering surface ACE2, inhibiting spike protein, and impacting other processes in addition to IMPα/β1 inhibition.
To compare the efficacy of the T-Fc-IVM-NP treatment against an FDA-approved COVID-19 treatment, remdesivir, the expression levels of ACE2, spike protein, and IMP α and β1 were observed after treatment with either remdesivir, IVM, or the IVM nanoformulations in spike expressing HEK293T cells (Figure 5D for Western blot, Figure S10 for RT-PCR). While remdesivir did not show the ability to decrease the levels of these proteins key to SARS-CoV-2 cell entry and virulence, the IVM nanoformulations were able to lower expression of ACE2, spike protein, and IMP β1. This resulting difference in expression may occur because treatments such as remdesivir specifically target the virus’ RNA polymerase, while IVM has multiple molecular targets.
To further test the applicability of T-Fc-IVM-NP treatment against other spike-containing members of the coronavirus family, we observed the ability of T-Fc-IVM-NP to decrease both protein and gene expression of the spike protein of MERS-CoV, a previous 2012 pandemic-causing coronavirus strain (Figure 5E, top for protein expression and bottom for RT-PCR). Remdesivir was unable to lower MERS-CoV spike protein levels. T-Fc-IVM-NPs lowered the expression of MERS-CoV spike at the both protein and gene levels indicating that the IVM nanoformulation’s spike-inhibiting capacity may extend across multiple other coronavirus strains. While the molecular target of remdesivir, the viral RNA polymerase, is known to frequently mutate across coronavirus strains, the structural similarities of spike protein across coronavirus strains means that the IVM nanoformulation might have the potential to serve as treatment for future spike-containing viruses as well. These results altogether indicate that the T-Fc-IVM-NPs are able to be taken up into cells, and not only decrease ACE2 expression and viral spike protein expression, but also inhibit the IMP α/β1 heterodimer (Figure 5F). Because of the IMP α/β1 proteins’ extensive role in the transport of viral proteins to the nucleus for other viruses, the results suggest that the T-Fc-IVM-NP-mediated reduction in transport of SARS-CoV-2 proteins to the nucleus may improve the cells’ antiviral responses.
Mitochondrial Functions and Inflammation of Spike-Infected Cells and Effects of T-Fc-IVM-NP
SARS-CoV-2 has been found to impact host mitochondrial functions through ACE2 regulation and open-reading frames that can allow for increased viral replication and evasion of host cell immunity.(27) The mitochondrial effects of spike protein expression and the mitochondrial toxicity of treatment using IVM and its nanoformulations was tested using a mitostress assay in HEK293T cells transfected with spike plasmid. Initially, spike protein expression within the HEK293T cells was found to slightly impact mitochondrial bioenergetics through the decrease of basal and maximum respiration as well as ATP production (Figure 6A,B). This effect was further compounded by treatment with free IVM, which more significantly decreased these three metrics and led to further mitochondrial dysfunction. However, the NT-IVM-NP and T-Fc-IVM-NP treatments did not lead to further decreases in basal respiration, maximum respiration, and ATP production over what had already been caused by spike protein, indicating nanoparticle-delivered IVM is less toxic and may even slightly reverse the toxic effects of spike protein on mitochondria (Figure 6). The improvement of these indicators of mitochondrial health supports the use of the therapeutic nanoparticle over free IVM as an antiviral agent to treat SARS-CoV-2 infection. Seeing as the therapeutic nanoparticle increased respiration and ATP production close to the levels of normal untreated cells, future time-dependent treatments using IVM-loaded NPs may show that higher doses of T-Fc-IVM-NPs allow for a full increase of mitochondrial health back up to the levels of control cells (Figure 6A,B). Our preliminary analyses of cytochrome c oxidase and mitochondrial complex V activity in spike expressing HEK293T cells and subsequent treatment with IVM and its T/NT NPs did not demonstrate any significant differences (Figure S11).
Figure 6
Figure 6. (A) Cellular toxicity of IVM and IVM-loaded NPs as measured by mitochondrial respiration profiles in spike protein-expressing HEK293T cells using the Seahorse analyzer and MitoStress assay: oligomycin, ATP synthase inhibitor; FCCP-carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, an ionophore; rotenone, an inhibitor of mitochondrial complex I; and antimycin A, an inhibitor of mitochondrial complex III. (B) Basal respiration and ATP production from the MitoStress assay. (C) Expression of cytokines IL-6, IL-1β, and TNFα in the media of spike protein-expressing HEK293T cells treated with IVM, NT-IVM-NP, or T-Fc-IVM-NP.
Conclusions
ARTICLE SECTIONS
As a whole, these results paint a positive picture for the development of an IVM-loaded nanoparticle therapeutic to treat COVID-19. Future in vivo testing and other toxicity studies need to be conducted, but the ability of an orally deliverable therapeutic to both decrease the infectious capabilities of SARS-CoV-2 through inhibition of its spike protein as well as decrease its entry rates through downregulation of ACE2 expression makes the nanoformulation a promising possible treatment for COVID-19. Considering that the gut is a potential route of entry for SARS-CoV-2, the gut-to-bloodstream entry of our ivermectin-loaded nanoformulation is ideal, as ivermectin is released gradually once in the bloodstream. Furthermore, in the bloodstream the nanoparticles will eventually be able to reach the lung epithelia as part of normal circulation and treat SARS-CoV-2 infection there, a process that will be augmented due to COVID-19-associated inflammation in the lung and resulting leaky endothelia that will allow nanoparticles to accumulate at the infected sites. The nanoparticles are not particularly immunogenic, so their accumulation at infected lung alveolar cells will not result in additional immune cell activation.
The IVM-loaded nanoparticle presents the opportunity for treatment not only of SARS-CoV-2, but of other coronavirus strains as well, due to the main two molecular targets—spike protein and the ACE2 receptor. These targets are common among other strains of coronavirus, which may vary in disease severity but have the potential to respond to IVM nanoformulation treatment. Currently, many treatments such as remdesevir and steroids are used to treat patients in critical condition, many of whom are experiencing severe respiratory distress, but these treatments are not available to or reasonable for the vast majority of less severe cases. Whereas treatments such as remdesivir target the virus’ RNA polymerase, are given intravenously, and may not be applicable to other diseases, the orally delivered IVM nanoformulation can be tested against a variety of coronavirus strains and other respiratory viruses.
Materials and Methods
ARTICLE SECTIONS
Description of materials and methods used, cell lines, chemicals, and biochemical are described in the Supporting Information.
Statistics
All the statistical analyses and graphical representations were performed using Prism (GraphPad). As the tests of statistical significance do not provide an estimate of the magnitude of the difference between groups, all levels of significance were described as either significant or not significant within the text of this report. The two-tailed statistical analyses were conducted at significance level P = 0.05.
Supporting Information
ARTICLE SECTIONS
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.0c00179.
Description of materials and methods, cell lines, chemicals, biochemicals, tables showing completed and ongoing clinical trial for different therapeutic options of COVID-19, and additional data (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Author Information
ARTICLE SECTIONS
- Shanta Dhar - Department of Biochemistry and Molecular Biology, Sylvester Comprehensive Cancer Center Leonard M. Miller School of Medicine, and , University of Miami, 1011 NW 15th Street, Miami, Florida 33136, United States;
http://orcid.org/0000-0003-3042-5272;
- Bapurao Surnar - Department of Biochemistry and Molecular Biology and , University of Miami, 1011 NW 15th Street, Miami, Florida 33136, United States;http://orcid.org/0000-0001-5997-3120
S.D. conceptualized the research; S.D., B.S., and A.S.S. designed the research; S.D. supervised experiments, provided directions and resources for all experiments; B.S., M.Z.K., and A.S.S. performed research; B.S. and M.Z.K. contributed reagents; B.S., M.Z.K., A.S.S., and S.D. analyzed the data. All authors discussed the results and commented on the manuscript; B.S., M.Z.K., A.S.S., and S.D. wrote the manuscript.
Acknowledgments
ARTICLE SECTIONS
This work was supported by the Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine. We thank Shrita Sarkar for her help for the illustration of Figure 1 and Dr. Nagesh Kolishetti for helpful discussion.
References
ARTICLE SECTIONS
This article references 27 other publications.
- 1Li, X., Geng, M., Peng, Y., Meng, L., and Lu, S. (2020) Molecular immune pathogenesis and diagnosis of COVID-19. J. Pharm. Anal. 10 (2), 102– 108, DOI: 10.1016/j.jpha.2020.03.001[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB38zksVygtg%253D%253D&md5=a0f4031c4fb4a0ddb72c96905fb0a8f2Molecular immune pathogenesis and diagnosis of COVID-19Li Xiaowei; Geng Manman; Peng Yizhao; Meng Liesu; Lu Shemin; Li Xiaowei; Geng Manman; Peng Yizhao; Meng Liesu; Lu SheminJournal of pharmaceutical analysis (2020), 10 (2), 102-108 ISSN:.Coronavirus disease 2019 (COVID-19) is a kind of viral pneumonia which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The emergence of SARS-CoV-2 has been marked as the third introduction of a highly pathogenic coronavirus into the human population after the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV) in the twenty-first century. In this minireview, we provide a brief introduction of the general features of SARS-CoV-2 and discuss current knowledge of molecular immune pathogenesis, diagnosis and treatment of COVID-19 on the base of the present understanding of SARS-CoV and MERS-CoV infections, which may be helpful in offering novel insights and potential therapeutic targets for combating the SARS-CoV-2 infection.
- 2Yuki, K., Fujiogi, M., and Koutsogiannaki, S. (2020) COVID-19 pathophysiology: A review. Clin. Immunol. 215, 108427, DOI: 10.1016/j.clim.2020.108427[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXosFWitr0%253D&md5=8c97d9785e6bfbf2a1885e12400cc55fCOVID-19 pathophysiology: A reviewYuki, Koichi; Fujiogi, Miho; Koutsogiannaki, SophiaClinical Immunology (Amsterdam, Netherlands) (2020), 215 (), 108427CODEN: CLIIFY; ISSN:1521-6616. (Elsevier B.V.)A review. In Dec. 2019, a novel coronavirus, now named as SARS-CoV-2, caused a series of acute atypical respiratory diseases in Wuhan, Hubei Province, China. The disease caused by this virus was termed COVID-19. The virus is transmittable between humans and has caused pandemic worldwide. The no. of death tolls continues to rise and a large no. of countries have been forced to do social distancing and lockdown. Lack of targeted therapy continues to be a problem. Epidemiol. studies showed that elder patients were more susceptible to severe diseases, while children tend to have milder symptoms. Here we reviewed the current knowledge about this disease and considered the potential explanation of the different symptomatol. between children and adults.
- 3Gheblawi, M., Wang, K., Viveiros, A., Nguyen, Q., Zhong, J., Turner, A. J., Raizada, M. K., Grant, M. B., and Oudit, G. Y. (2020) Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin System. Circ. Res. 126 (10), 1456– 1474, DOI: 10.1161/CIRCRESAHA.120.317015[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXptVaksrY%253D&md5=f240902dd9bf0f970ad227a1c5d4f037Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin System: Celebrating the 20th Anniversary of the Discovery of ACE2Gheblawi, Mahmoud; Wang, Kaiming; Viveiros, Anissa; Nguyen, Quynh; Zhong, Jiu-Chang; Turner, Anthony J.; Raizada, Mohan K.; Grant, Maria B.; Oudit, Gavin Y.Circulation Research (2020), 126 (10), 1456-1474CODEN: CIRUAL; ISSN:0009-7330. (Lippincott Williams & Wilkins)A review. ACE2 (angiotensin-converting enzyme 2) has a multiplicity of physiol. roles that revolve around its trivalent function: a neg. regulator of the renin-angiotensin system, facilitator of amino acid transport, and the severe acute respiratory syndrome-coronavirus (SARS-CoV) and SARS-CoV-2 receptor. ACE2 is widely expressed, including, in the lungs, cardiovascular system, gut, kidneys, central nervous system, and adipose tissue. ACE2 has recently been identified as the SARS-CoV-2 receptor, the infective agent responsible for coronavirus disease 2019, providing a crit. link between immunity, inflammation, ACE2, and cardiovascular disease. Although sharing a close evolutionary relationship with SARS-CoV, the receptor-binding domain of SARS-CoV-2 differs in several key amino acid residues, allowing for stronger binding affinity with the human ACE2 receptor, which may account for the greater pathogenicity of SARS-CoV-2. The loss of ACE2 function following binding by SARS-CoV-2 is driven by endocytosis and activation of proteolytic cleavage and processing. The ACE2 system is a crit. protective pathway against heart failure with reduced and preserved ejection fraction including, myocardial infarction and hypertension, and against lung disease and diabetes mellitus. The control of gut dysbiosis and vascular permeability by ACE2 has emerged as an essential mechanism of pulmonary hypertension and diabetic cardiovascular complications. Recombinant ACE2, gene-delivery of Ace2, Ang 1-7 analogs, and Mas receptor agonists enhance ACE2 action and serve as potential therapies for disease conditions assocd. with an activated renin-angiotensin system. RhACE2 (recombinant human ACE2) has completed clin. trials and efficiently lowered or increased plasma angiotensin II and angiotensin 1-7 levels, resp. Our review summarizes the progress over the past 20 years, highlighting the crit. role of ACE2 as the novel SARS-CoV-2 receptor and as the neg. regulator of the renin-angiotensin system, together with implications for the coronavirus disease 2019 pandemic and assocd. cardiovascular diseases.
- 4Li, G., He, X., Zhang, L., Ran, Q., Wang, J., Xiong, A., Wu, D., Chen, F., Sun, J., and Chang, C. (2020) Assessing ACE2 expression patterns in lung tissues in the pathogenesis of COVID-19. J. Autoimmun. 112, 102463, DOI: 10.1016/j.jaut.2020.102463[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnt1WnsLY%253D&md5=9f7532dfbe5c0d07277a7d290b2761cdAssessing ACE2 expression patterns in lung tissues in the pathogenesis of COVID-19Li, Guoping; He, Xiang; Zhang, Lei; Ran, Qin; Wang, Junyi; Xiong, Anying; Wu, Dehong; Chen, Feng; Sun, Jinlyu; Chang, ChristopherJournal of Autoimmunity (2020), 112 (), 102463CODEN: JOAUEP; ISSN:0896-8411. (Elsevier Ltd.)It has been reported that SARS-CoV-2 may use ACE2 as a receptor to gain entry into human cells, in a way similar to that of SARS-CoV. Analyzing the distribution and expression level of ACE2 may therefore help reveal underlying mechanisms of viral susceptibility and post-infection modulation. In this study, we utilized previously uploaded information on ACE2 expression in various conditions including SARS-CoA to evaluate the role of ACE2 in SARS-CoV and extrapolate that to COVID-19. We found that the expression of ACE2 in healthy populations and patients with underlying diseases was not significantly different. However, based on the elevated expression of ACE2 in cigarette smokers, we speculate that long-term smoking may be a risk factor for COVID-19. Anal. of ACE2 in SARS-CoV infected cells suggests that ACE2 is not only a receptor but is also involved in post-infection regulation, including immune response, cytokine secretion, and viral genome replication. Moreover, we constructed Protein-protein interaction (PPI) networks and identified hub genes in viral activity and cytokine secretion. Our findings may help clinicians and researchers gain more insight into the pathogenesis of SARS-CoV-2 and design therapeutic strategies for COVID-19.
- 5McGonagle, D., Sharif, K., O’Regan, A., and Bridgewood, C. (2020) The Role of Cytokines including Interleukin-6 in COVID-19 induced Pneumonia and Macrophage Activation Syndrome-Like Disease. Autoimmun. Rev. 19 (6), 102537, DOI: 10.1016/j.autrev.2020.102537[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXms1ylt78%253D&md5=d05b5c11ac442ff5e1573fc14db7921cThe Role of Cytokines including Interleukin-6 in COVID-19 induced Pneumonia and Macrophage Activation Syndrome-Like DiseaseMcGonagle, Dennis; Sharif, Kassem; O'Regan, Anthony; Bridgewood, CharlieAutoimmunity Reviews (2020), 19 (6), 102537CODEN: ARUEBU; ISSN:1568-9972. (Elsevier B.V.)A review. Severe COVID-19 assocd. pneumonia patients may exhibit features of systemic hyper-inflammation designated under the umbrella term of macrophage activation syndrome (MAS) or cytokine storm, also known as secondary haemophagocytic lymphohistocytosis (sHLH). This is distinct from HLH assocd. with immunodeficiency states termed primary HLH -with radically different therapy strategies in both situations. COVID-19 infection with MAS typically occurs in subjects with adult respiratory distress syndrome (ARDS) and historically, non-survival in ARDS was linked to sustained IL-6 and IL-1 elevation. We provide a model for the classification of MAS to stratify the MAS-like presentation in COVID-19 pneumonia and explore the complexities of discerning ARDS from MAS. We discuss the potential impact of timing of anti-cytokine therapy on viral clearance and the impact of such therapy on intra-pulmonary macrophage activation and emergent pulmonary vascular disease.
- 6Merad, M. and Martin, J. C. (2020) Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages. Nat. Rev. Immunol. 20 (6), 355– 362, DOI: 10.1038/s41577-020-0331-4[Crossref], [PubMed], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXoslOqt7s%253D&md5=d38b037fed1fcc469e6c8caa23777fc7Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophagesMerad, Miriam; Martin, Jerome C.Nature Reviews Immunology (2020), 20 (6), 355-362CODEN: NRIABX; ISSN:1474-1733. (Nature Research)A review. Abstr.: The COVID-19 pandemic caused by infection with SARS-CoV-2 has led to more than 200,000 deaths worldwide. Several studies have now established that the hyperinflammatory response induced by SARS-CoV-2 is a major cause of disease severity and death in infected patients. Macrophages are a population of innate immune cells that sense and respond to microbial threats by producing inflammatory mols. that eliminate pathogens and promote tissue repair. However, a dysregulated macrophage response can be damaging to the host, as is seen in the macrophage activation syndrome induced by severe infections, including in infections with the related virus SARS-CoV. Here we describe the potentially pathol. roles of macrophages during SARS-CoV-2 infection and discuss ongoing and prospective therapeutic strategies to modulate macrophage activation in patients with COVID-19.
- 7Ou, X., Liu, Y., Lei, X., Li, P., Mi, D., Ren, L., Guo, L., Guo, R., Chen, T., Hu, J., Xiang, Z., Mu, Z., Chen, X., Chen, J., Hu, K., Jin, Q., Wang, J., and Qian, Z. (2020) Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat. Commun. 11, 1620, DOI: 10.1038/s41467-020-15562-9[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlvFyjt78%253D&md5=6b0b1ef5a68f4a35da4aabecb0f99544Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoVOu, Xiuyuan; Liu, Yan; Lei, Xiaobo; Li, Pei; Mi, Dan; Ren, Lili; Guo, Li; Guo, Ruixuan; Chen, Ting; Hu, Jiaxin; Xiang, Zichun; Mu, Zhixia; Chen, Xing; Chen, Jieyong; Hu, Keping; Jin, Qi; Wang, Jianwei; Qian, ZhaohuiNature Communications (2020), 11 (1), 1620CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biol. of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are crit. for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.
- 8Qin, C., Zhou, L., Hu, Z., Zhang, S., Yang, S., Tao, Y., Xie, C., Ma, K., Shang, K., Wang, W., and Tian, D. S. (2020) Dysregulation of immune response in patients with COVID-19 in Wuhan, China. Clin. Infect. Dis. 71, 762, DOI: 10.1093/cid/ciaa248[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsF2mt7fF&md5=ff15e4dfcc73998aaa84b06b798cf0a5Dysregulation of immune response in patients with coronavirus 2019 (COVID-19) in Wuhan, ChinaQin, Chuan; Zhou, Luoqi; Hu, Ziwei; Zhang, Shuoqi; Yang, Sheng; Tao, Yu; Xie, Cuihong; Ma, Ke; Shang, Ke; Wang, Wei; Tian, Dai-ShiClinical Infectious Diseases (2020), 71 (15), 762-768CODEN: CIDIEL; ISSN:1537-6591. (Oxford University Press)Background. In Dec. 2019, coronavirus 2019 (COVID-19) emerged in Wuhan and rapidly spread throughout China. Methods. Demog. and clin. data of all confirmed cases with COVID-19 on admission at Tongji Hospital from 10 Jan. to 12 Feb. 2020 were collected and analyzed. The data on lab. examns., including peripheral lymphocyte subsets, were analyzed and compared between patients with severe and nonsevere infection. Results. Of the 452 patients with COVID-19 recruited, 286 were diagnosed as having severe infection. The median age was 58 years and 235 were male. The most common symptoms were fever, shortness of breath, expectoration, fatigue, dry cough, and myalgia. Severe cases tend to have lower lymphocyte counts, higher leukocyte counts and neutrophil-lymphocyte ratio (NLR), as well as lower percentages of monocytes, eosinophils, and basophils. Most severe cases demonstrated elevated levels of infection-related biomarkers and inflammatory cytokines. The no. of T cells significantly decreased, and were more impaired in severe cases. Both helper T (Th) cells and suppressor T cells in patients with COVID-19 were below normal levels, with lower levels of Th cells in the severe group. The percentage of naive Th cells increased and memory Th cells decreased in severe cases. Patients with COVID-19 also have lower levels of regulatory T cells, which are more obviously decreased in severe cases. Conclusions. The novel coronavirus might mainly act on lymphocytes, esp. T lymphocytes. Surveillance of NLR and lymphocyte subsets is helpful in the early screening of crit. illness, diagnosis, and treatment of COVID-19.
- 9Sungnak, W., Huang, N., Becavin, C., Berg, M., Queen, R., Litvinukova, M., Talavera-Lopez, C., Maatz, H., Reichart, D., Sampaziotis, F., Worlock, K. B., Yoshida, M., and Barnes, J. L. (2020) SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes. Nat. Med. 26, 681– 687, DOI: 10.1038/s41591-020-0868-6[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXotVCjurY%253D&md5=cc313df9c190333b98af43951a2146faSARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genesSungnak, Waradon; Huang, Ni; Becavin, Christophe; Berg, Marijn; Queen, Rachel; Litvinukova, Monika; Talavera-Lopez, Carlos; Maatz, Henrike; Reichart, Daniel; Sampaziotis, Fotios; Worlock, Kaylee B.; Yoshida, Masahiro; Barnes, Josephine L.; HCA Lung Biological NetworkNature Medicine (New York, NY, United States) (2020), 26 (5), 681-687CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)We investigated SARS-CoV-2 potential tropism by surveying expression of viral entry-assocd. genes in single-cell RNA-sequencing data from multiple tissues from healthy human donors. We co-detected these transcripts in specific respiratory, corneal and intestinal epithelial cells, potentially explaining the high efficiency of SARS-CoV-2 transmission. These genes are co-expressed in nasal epithelial cells with genes involved in innate immunity, highlighting the cells' potential role in initial viral infection, spread and clearance. The study offers a useful resource for further lines of inquiry with valuable clin. samples from COVID-19 patients and we provide our data in a comprehensive, open and user-friendly fashion at www.covid19cellatlas.org.
- 10Yan, R., Zhang, Y., Li, Y., Xia, L., Guo, Y., and Zhou, Q. (2020) Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 367 (6485), 1444– 1448, DOI: 10.1126/science.abb2762[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlslymsLo%253D&md5=ff4dfdfc646ea878cfb325019160e94aStructural basis for the recognition of SARS-CoV-2 by full-length human ACE2Yan, Renhong; Zhang, Yuanyuan; Li, Yaning; Xia, Lu; Guo, Yingying; Zhou, QiangScience (Washington, DC, United States) (2020), 367 (6485), 1444-1448CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for severe acute respiratory syndrome coronavirus (SARS-CoV) and the new coronavirus (SARS-CoV-2) that is causing the serious coronavirus disease 2019 (COVID-19) epidemic. Here, we present cryo-electron microscopy structures of full-length human ACE2 in the presence of the neutral amino acid transporter B0AT1 with or without the receptor binding domain (RBD) of the surface spike glycoprotein (S protein) of SARS-CoV-2, both at an overall resoln. of 2.9 angstroms, with a local resoln. of 3.5 angstroms at the ACE2-RBD interface. The ACE2-B0AT1 complex is assembled as a dimer of heterodimers, with the collectrin-like domain of ACE2 mediating homodimerization. The RBD is recognized by the extracellular peptidase domain of ACE2 mainly through polar residues. These findings provide important insights into the mol. basis for coronavirus recognition and infection.
- 11Zhang, H., Penninger, J. M., Li, Y., Zhong, N., and Slutsky, A. S. (2020) Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target. Intensive Care Med. 46 (4), 586– 590, DOI: 10.1007/s00134-020-05985-9[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkt1erurY%253D&md5=705f72887420f24bdc15084bb6fdf193Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic targetZhang, Haibo; Penninger, Josef M.; Li, Yimin; Zhong, Nanshan; Slutsky, Arthur S.Intensive Care Medicine (2020), 46 (4), 586-590CODEN: ICMED9; ISSN:0342-4642. (Springer)A review. A novel infectious disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was detected in Wuhan, China, in Dec. 2019. The disease (COVID-19) spread rapidly, reaching epidemic proportions in China, and has been found in 27 other countries. As of Feb. 27, 2020, over 82,000 cases of COVID-19 were reported, with > 2800 deaths. No specific therapeutics are available, and current management includes travel restrictions, patient isolation, and supportive medical care. There are a no. of pharmaceuticals already being tested, but a better understanding of the underlying pathobiol. is required. In this context, this article will brief review the rationale for angiotensin-converting enzyme 2 (ACE2) receptor as a specific target.
- 12Pandey, P., Khan, F., Kumar, A., Srivastava, A., and Jha, N. K. (2021) Screening of Potent Inhibitors Against 2019 Novel Coronavirus (Covid-19) from Alliumsativum and Allium cepa: An In Silico Approach. Biointerface Res. Appl. Chem. 11 (1), 7981– 7993, DOI: 10.33263/BRIAC111.79817993
- 13Pandey, P., Khan, F., Rana, A. K., Srivastava, Y., Jha, S. K., and Jha, N. K. (2021) A Drug Repurposing Approach Towards Elucidating the Potential of Flavonoids as COVID-19 Spike Protein Inhibitors. Biointerface Res. Appl. Chem. 11 (1), 8482– 8501, DOI: 10.33263/BRIAC111.84828501
- 14Mastrangelo, E., Pezzullo, M., De Burghgraeve, T., Kaptein, S., Pastorino, B., Dallmeier, K., de Lamballerie, X., Neyts, J., Hanson, A. M., Frick, D. N., Bolognesi, M., and Milani, M. (2012) Ivermectin Is a Potent Inhibitor of Flavivirus Replication Specifically Targeting NS3 Helicase Activity: New Prospects for an Old Drug. J. Antimicrob. Chemother. 67 (8), 1884– 1894, DOI: 10.1093/jac/dks147[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtVeisbzF&md5=a3bf7d857cd11b9bfed8e6780b501b39Ivermectin is a potent inhibitor of flavivirus replication specifically targeting NS3 helicase activity: new prospects for an old drugMastrangelo, Eloise; Pezzullo, Margherita; De Burghgraeve, Tine; Kaptein, Suzanne; Pastorino, Boris; Dallmeier, Kai; de Lamballerie, Xavier; Neyts, Johan; Hanson, Alicia M.; Frick, David N.; Bolognesi, Martino; Milani, MarioJournal of Antimicrobial Chemotherapy (2012), 67 (8), 1884-1894CODEN: JACHDX; ISSN:0305-7453. (Oxford University Press)Objectives Infection with yellow fever virus (YFV), the prototypic mosquito-borne flavivirus, causes severe febrile disease with hemorrhage, multi-organ failure and a high mortality. Moreover, in recent years the Flavivirus genus has gained further attention due to re-emergence and increasing incidence of West Nile, dengue and Japanese encephalitis viruses. Potent and safe antivirals are urgently needed. Methods Starting from the crystal structure of the NS3 helicase from Kunjin virus (an Australian variant of West Nile virus), we identified a novel, unexploited protein site that might be involved in the helicase catalytic cycle and could thus in principle be targeted for enzyme inhibition. In silico docking of a library of small mols. allowed us to identify a few selected compds. with high predicted affinity for the new site. Their activity against helicases from several flaviviruses was confirmed in in vitro helicase/enzymic assays. The effect on the in vitro replication of flaviviruses was then evaluated. Results Ivermectin, a broadly used anti-helminthic drug, proved to be a highly potent inhibitor of YFV replication (EC50 values in the sub-nanomolar range). Moreover, ivermectin inhibited, although less efficiently, the replication of several other flaviviruses, i.e. dengue fever, Japanese encephalitis and tick-borne encephalitis viruses. Ivermectin exerts its effect at a timepoint that coincides with the onset of intracellular viral RNA synthesis, as expected for a mol. that specifically targets the viral helicase. Conclusions The well-tolerated drug ivermectin may hold great potential for treatment of YFV infections. Furthermore, structure-based optimization may result in analogs exerting potent activity against flaviviruses other than YFV.
- 15Heidary, F. and Gharebaghi, R. (2020) Ivermectin: a systematic review from antiviral effects to COVID-19 complementary regimen. J. Antibiot. 73 (9), 593– 602, DOI: 10.1038/s41429-020-0336-z[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtFGgsLbN&md5=5ce416e511fb3575df954eb294ac05cfIvermectin: a systematic review from antiviral effects to COVID-19 complementary regimenHeidary, Fatemeh; Gharebaghi, RezaJournal of Antibiotics (2020), 73 (9), 593-602CODEN: JANTAJ; ISSN:0021-8820. (Nature Research)A review. A reviews. Ivermectin proposes many potentials effects to treat a range of diseases, with its antimicrobial, antiviral, and anti-cancer properties as a wonder drug. It is highly effective against many microorganisms including some viruses. In this comprehensive systematic review, antiviral effects of ivermectin are summarized including in vitro and in vivo studies over the past 50 years. Several studies reported antiviral effects of ivermectin on RNA viruses such as Zika, dengue, yellow fever, West Nile, Hendra, Newcastle, Venezuelan equine encephalitis, chikungunya, Semliki Forest, Sindbis, Avian influenza A, Porcine Reproductive and Respiratory Syndrome, Human immunodeficiency virus type 1, and severe acute respiratory syndrome coronavirus 2. Furthermore, there are some studies showing antiviral effects of ivermectin against DNA viruses such as Equine herpes type 1, BK polyomavirus, pseudorabies, porcine circovirus 2, and bovine herpesvirus 1. Ivermectin plays a role in several biol. mechanisms, therefore it could serve as a potential candidate in the treatment of a wide range of viruses including COVID-19 as well as other types of pos.-sense single-stranded RNA viruses. In vivo studies of animal models revealed a broad range of antiviral effects of ivermectin, however, clin. trials are necessary to appraise the potential efficacy of ivermectin in clin. setting.
- 16Wang, Y. P., Du, W. J., Huang, L. P., Wei, Y. W., Wu, H. L., Feng, L., and Liu, C. M. (2016) The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the Holoenzyme. Front. Microbiol. 7, 124, DOI: 10.3389/fmicb.2016.00124[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28jksVenuw%253D%253D&md5=a0e9889fdb01480d9b5b50ae01ce34c4The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the HoloenzymeWang Yi-Ping; Du Wen-Juan; Huang Li-Ping; Wei Yan-Wu; Wu Hong-Li; Feng Li; Liu Chang-MingFrontiers in microbiology (2016), 7 (), 124 ISSN:1664-302X.Pseudorabies virus (PRV) DNA replication occurs in the nuclei of infected cells and requires the viral DNA polymerase. The PRV DNA polymerase comprises a catalytic subunit, UL30, and an accessory subunit, UL42, that confers processivity to the enzyme. Its nuclear localization is a prerequisite for its enzymatic function in the initiation of viral DNA replication. However, the mechanisms by which the PRV DNA polymerase holoenzyme enters the nucleus have not been determined. In this study, we characterized the nuclear import pathways of the PRV DNA polymerase catalytic and accessory subunits. Immunofluorescence analysis showed that UL42 localizes independently in the nucleus, whereas UL30 alone predominantly localizes in the cytoplasm. Intriguingly, the localization of UL30 was completely shifted to the nucleus when it was coexpressed with UL42, demonstrating that nuclear transport of UL30 occurs in an UL42-dependent manner. Deletion analysis and site-directed mutagenesis of the two proteins showed that UL42 contains a functional and transferable bipartite nuclear localization signal (NLS) at amino acids 354-370 and that K(354), R(355), and K(367) are important for the NLS function, whereas UL30 has no NLS. Coimmunoprecipitation assays verified that UL42 interacts with importins α3 and α4 through its NLS. In vitro nuclear import assays demonstrated that nuclear accumulation of UL42 is a temperature- and energy-dependent process and requires both importins α and β, confirming that UL42 utilizes the importin α/β-mediated pathway for nuclear entry. In an UL42 NLS-null mutant, the UL42/UL30 heterodimer was completely confined to the cytoplasm when UL42 was coexpressed with UL30, indicating that UL30 utilizes the NLS function of UL42 for its translocation into the nucleus. Collectively, these findings suggest that UL42 contains an importin α/β-mediated bipartite NLS that transports the viral DNA polymerase holoenzyme into the nucleus in an in vitro expression system.
- 17Surnar, B., Kamran, M. Z., Shah, A. S., Basu, U., Kolishetti, N., Deo, S., Jayaweera, D. T., Daunert, S., and Dhar, S. (2019) Orally Administrable Therapeutic Synthetic Nanoparticle for Zika Virus. ACS Nano 13, 11034– 11048, DOI: 10.1021/acsnano.9b02807[ACS Full Text
], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvFeqtrvP&md5=b23f2a1279b8ad8fc09ac8b09be9fbe1Orally Administrable Therapeutic Synthetic Nanoparticle for Zika VirusSurnar, Bapurao; Kamran, Mohammad Z.; Shah, Anuj S.; Basu, Uttara; Kolishetti, Nagesh; Deo, Sapna; Jayaweera, Dushyantha T.; Daunert, Sylvia; Dhar, ShantaACS Nano (2019), 13 (10), 11034-11048CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The spread of Zika virus (ZIKV) infection across the USA and various countries in the last three years will not only have a direct impact on the U. S. health care system but has caused international concerns as well. The ultimate impact of ZIKV infection remains to be understood. Currently, there are no therapeutic or vaccine options available to protect those infected by ZIKV. The drug ivermectin (IVM) was found to be a viable agent for the prevention of transmission of ZIKV. Ivermectin is both unstable in the presence of water and does not remain in adequate concn. in the human bloodstream to be effective in treatment for ZIKV. Biodegradable nanoparticles would aid in the delivery of ivermectin, by providing a high enough concn. of drug and ensuring the drug is gradually released to maintain an appropriate level in the body. The overall goal of this study was to develop and optimize an orally administrable nanoformulation of IVM which can circulate in the blood for a long period for efficient delivery. To achieve the goal, we synthesized and optimized a synthetic nanoformulation of IVM for oral use which can cross the intestinal epithelial barrier to enter the bloodstream. Our studies documented that when delivered with the synthetic nanoparticle (NP), IVM can be accumulated in the blood at a higher concn. and preliminary studies highlighted that NP delivered IVM has the ability to target nonstructural 1 protein of ZIKV. For potential clin. relevance, long-term storable formulation of IVM-nanoparticle in dry powder state for inclusion in a capsule form and cryoprotectant contg. frozen forms revealed promising findings. Further, our preliminary in vitro studies documented that ivermectin crosses the placental barrier thus making it unsafe for pregnant ZIKV population whereas ivermectin-loaded nanoparticle did not show any significant placental barrier crossing, thus indicating its potential suitability for such population. We envision that this work will fill a great unmet need by developing safer and more effective therapies for the treatment of viral infections, including ZIKV. - 18Elkassaby, M. H. (1991) Ivermectin uptake and distribution in the plasma and tissue of Sudanese and Mexican patients infected with Onchocerca volvulus. Trop. Med. Parasitol. 42 (2), 79– 81[PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK3Mzns1GmsA%253D%253D&md5=86e32c02975dab98c9ba807500f452a9Ivermectin uptake and distribution in the plasma and tissue of Sudanese and Mexican patients infected with Onchocerca volvulusElkassaby M HTropical medicine and parasitology : official organ of Deutsche Tropenmedizinische Gesellschaft and of Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZ) (1991), 42 (2), 79-81 ISSN:0177-2392.Ten Sudanese patients with Onchocerca volvulus infection were treated with a single oral dose of 150 micrograms/kg of ivermectin. Plasma samples were collected before treatment, 0.5, 1, 3, 4, 6, 12 hours and 1, 2, 3, 7, and 30 days. Four patients were selected for nodulectomies and skin biopsies at 6, 18 and 30 hours and 3 days post treatment. Using these samples O. volvulus worm fragments were dissected free of host nodular tissues for ivermectin extraction. Ivermectin was present in the nodular tissue at 6 hr and persisted for 3 days. It was also detected in an individual worm tissue extract at a concentration similar to the nodule, but in subcutaneous fascial tissue higher concentrations were sometimes found. Ivermectin was detected by radioimmunoassay in the plasma of all patients at 1 hr and peak concentrations were reached in an average of 5.6 hr. The drug persisted at detectable levels for 7 days in 70% of the studied patients. Plasma samples were also collected from 16 treated Mexican onchocerciasis patients before ivermectin treatment and 4 hr treatment and from six individuals who served as controls. The Mexican patients had concentrations of ivermectin in their plasma similar to those in the Sudanese patients.
- 19Dashraath, P., Wong, J. L. J., Lim, M. X. K., Lim, L. M., Li, S., Biswas, A., Choolani, M., Mattar, C., and Su, L. L. (2020) Coronavirus disease 2019 (COVID-19) pandemic and pregnancy. Am. J. Obstet. Gynecol. 222 (6), 521– 531, DOI: 10.1016/j.ajog.2020.03.021[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnsleksLY%253D&md5=daae72f12be4c4583a87e38436524408Coronavirus disease 2019 (COVID-19) pandemic and pregnancyDashraath, Pradip; Wong, Jing Lin Jeslyn; Lim, Mei Xian Karen; Lim, Li Min; Li, Sarah; Biswas, Arijit; Choolani, Mahesh; Mattar, Citra; Su, Lin LinAmerican Journal of Obstetrics and Gynecology (2020), 222 (6), 521-531CODEN: AJOGAH; ISSN:0002-9378. (Elsevier Inc.)A review. The current coronavirus disease 2019 (COVID-19) pneumonia pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading globally at an accelerated rate, with a basic reprodn. no. (R0) of 2-2.5, indicating that 2-3 persons will be infected from an index patient. A serious public health emergency, it is particularly deadly in vulnerable populations and communities in which healthcare providers are insufficiently prepd. to manage the infection. As of March 16, 2020, there are more than 180,000 confirmed cases of COVID-19 worldwide, with more than 7000 related deaths. The SARS-CoV-2 virus has been isolated from asymptomatic individuals, and affected patients continue to be infectious 2 wk after cessation of symptoms. The substantial morbidity and socioeconomic impact have necessitated drastic measures across all continents, including nationwide lockdowns and border closures. Pregnant women and their fetuses represent a high-risk population during infectious disease outbreaks. To date, the outcomes of 55 pregnant women infected with COVID-19 and 46 neonates have been reported in the literature, with no definite evidence of vertical transmission. Physiol. and mech. changes in pregnancy increase susceptibility to infections in general, particularly when the cardiorespiratory system is affected, and encourage rapid progression to respiratory failure in the gravida. Furthermore, the pregnancy bias toward T-helper 2 (Th2) system dominance, which protects the fetus, leaves the mother vulnerable to viral infections, which are more effectively contained by the Th1 system. These unique challenges mandate an integrated approach to pregnancies affected by SARS-CoV-2. Here we present a review of COVID-19 in pregnancy, bringing together the various factors integral to the understanding of pathophysiol. and susceptibility, diagnostic challenges with real-time reverse transcription polymerase chain reaction (RT-PCR) assays, therapeutic controversies, intrauterine transmission, and maternal-fetal complications. We discuss the latest options in antiviral therapy and vaccine development, including the novel use of chloroquine in the management of COVID-19. Fetal surveillance, in view of the predisposition to growth restriction and special considerations during labor and delivery, is addressed. In addn., we focus on keeping frontline obstetric care providers safe while continuing to provide essential services. Our clin. service model is built around the principles of workplace segregation, responsible social distancing, containment of cross-infection to healthcare providers, judicious use of personal protective equipment, and telemedicine. Our aim is to share a framework that can be adopted by tertiary maternity units managing pregnant women in the flux of a pandemic while maintaining the safety of the patient and healthcare provider at its core.
- 20Caly, L., Druce, J. D., Catton, M. G., Jans, D. A., and Wagstaff, K. M. (2020) The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in vitro. Antiviral Res. 179, 104787, DOI: 10.1016/j.antiviral.2020.104787
- 21Rowland, R. R., Chauhan, V., Fang, Y., Pekosz, A., Kerrigan, M., and Burton, M. D. (2005) Intracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: absence of nucleolar accumulation during infection and after expression as a recombinant protein in Vero cells. J. Virol. 79 (17), 11507– 12, DOI: 10.1128/JVI.79.17.11507-11512.2005[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXps1Gltbw%253D&md5=1131e6faf604e2e9916968033354cdaaIntracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: Absence of nucleolar accumulation during infection and after expression as a recombinant protein in Vero cellsRowland, Raymond R. R.; Chauhan, Vinita; Fang, Ying; Pekosz, Andrew; Kerrigan, Maureen; Burton, Miriam D.Journal of Virology (2005), 79 (17), 11507-11512CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)The nucleocapsid (N) protein of several members within the order Nidovirales localizes to the nucleolus during infection and after transfection of cells with N genes. However, confocal microscopy of N protein localization in Vero cells infected with the severe acute respiratory syndrome coronavirus (SARS-CoV) or transfected with the SARS-CoV N gene failed to show the presence of N in the nucleoplasm or nucleolus. Amino acids 369 to 389, which contain putative nuclear localization signal (NLS) and nucleolar localization signal motifs, failed to restore nuclear localization to an NLS-minus mutant Rev protein. These data indicate that nuclear localization is not a conserved property among all nidoviruses.
- 22Timani, K. A., Liao, Q., Ye, L., Zeng, Y., Liu, J., Zheng, Y., Ye, L., Yang, X., Lingbao, K., Gao, J., and Zhu, Y. (2005) Nuclear/nucleolar localization properties of C-terminal nucleocapsid protein of SARS coronavirus. Virus Res. 114 (1–2), 23– 34, DOI: 10.1016/j.virusres.2005.05.007[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1WjtLfI&md5=9e6fff0b59f1df7f6702573df681d9c9Nuclear/nucleolar localization properties of C-terminal nucleocapsid protein of SARS coronavirusTimani, Khalid Amine; Liao, Qingjiao; Ye, Linbai; Zeng, Yingchun; Liu, Jing; Zheng, Yi; Ye, Li; Yang, Xiaojun; Lingbao, Kong; Gao, Jingrong; Zhu, YingVirus Research (2005), 114 (1-2), 23-34CODEN: VIREDF; ISSN:0168-1702. (Elsevier B.V.)A novel coronavirus (CoV) has recently been identified as the etiol. agent of severe acute respiratory syndrome (SARS). Nucleocapsid (N) proteins of the Coronaviridae family have no discernable homol., but they share a common nucleolar-cytoplasmic distribution pattern. There are three putative nuclear localization signal (NLS) motifs present in the N. To det. the role of these putative NLSs in the intracellular localization of the SARS-CoV N, we performed a confocal microscopy anal. using rabbit anti-N antisera. In this report, we show that the wild type N was distributed mainly in the cytoplasm. The N-terminal of the N, which contains the NLS1 (aa38-44), was localized to the nucleus. The C-terminus of the N, which contains both NLS2 (aa257-265) and NLS3 (aa369-390) was localized to the cytoplasm and the nucleolus. Results derived from anal. of various deletion mutations show that the region contg. amino acids 226-289 is able to mediate nucleolar localization. The deletion of two hydrophobic regions that flanked the NLS3 recovered its activity and localized to the nucleus. Furthermore, deletion of leucine rich region (220-LALLLLDRLNRL) resulted in the accumulation of N to the cytoplasm and nucleolus, and when fusing this peptide to EGFP localization was cytoplasmic, suggesting that the N may act as a shuttle protein. Differences in nuclear/nucleolar localization properties of N from other members of coronavirus family suggest a unique function for N, which may play an important role in the pathogenesis of SARS.
- 23Wagstaff, K. M., Rawlinson, S. M., Hearps, A. C., and Jans, D. A. (2011) An AlphaScreen(R)-based assay for high-throughput screening for specific inhibitors of nuclear import. J. Biomol. Screening 16 (2), 192– 200, DOI: 10.1177/1087057110390360[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjsVGlu7w%253D&md5=3a2163c020ec13b2074e444ad0ac4d82An AlphaScreen-based assay for high-throughput screening for specific inhibitors of nuclear importWagstaff, Kylie M.; Rawlinson, Stephen M.; Hearps, Anna C.; Jans, David A.Journal of Biomolecular Screening (2011), 16 (2), 192-200CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)Specific viral proteins enter the nucleus of infected cells to perform essential functions, as part of the viral life cycle. The integrase (IN) mol. of human immunodeficiency virus (HIV)-1 is of particular interest in this context due to its integral role in integrating the HIV genome into that of the infected host cell. Most IN-based antiviral compds. target the IN/DNA interaction, but since IN must first enter the nucleus before it can perform these crit. functions, nuclear transport of IN is also an attractive target for therapeutic intervention. Here the authors describe a novel high-throughput screening assay for identifying inhibitors of nuclear import, particularly IN, based on amplified luminescent proximity homogeneous assay (AlphaScreen) technol., which is high throughput, requires low amts. of material, and is efficient and cost-effective. The authors use the assay to screen for specific inhibitors of the interaction between IN and its nuclear transport receptor importin α/β, successfully identifying several inhibitors of the IN/importin α/β interaction. Importantly, they demonstrate that one of the identified compds., mifepristone, is effective in preventing active nuclear transport of IN in transfected cells and hence may represent a useful anti-HIV therapeutic. The screen also identified broad-spectrum importin α/β inhibitors such as ivermectin, which may represent useful tools for nuclear transport research in the future. The authors validate the activity and specificity of mifepristone and ivermectin in inhibiting nuclear protein import in living cells, underlining the utility of the screening approach.
- 24Wagstaff, K. M., Sivakumaran, H., Heaton, S. M., Harrich, D., and Jans, D. A. (2012) Ivermectin is a specific inhibitor of importin alpha/beta-mediated nuclear import able to inhibit replication of HIV-1 and dengue virus. Biochem. J. 443 (3), 851– 6, DOI: 10.1042/BJ20120150[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xls1Sht70%253D&md5=19879ceff24d84ea0f16f437f8c921fdIvermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of HIV-1 and dengue virusWagstaff, Kylie M.; Sivakumaran, Haran; Heaton, Steven M.; Harrich, David; Jans, David A.Biochemical Journal (2012), 443 (3), 851-856CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)The movement of proteins between the cytoplasm and nucleus mediated by the importin superfamily of proteins is essential to many cellular processes, including differentiation and development, and is crit. to disease states such as viral disease and oncogenesis. We recently developed a high-throughput screen to identify specific and general inhibitors of protein nuclear import, from which ivermectin was identified as a potential inhibitor of importin α/β-mediated transport. In the present study, we characterized in detail the nuclear transport inhibitory properties of ivermectin, demonstrating that it is a broad-spectrum inhibitor of importin α/β nuclear import, with no effect on a range of other nuclear import pathways, including that mediated by importin β1 alone. Importantly, we establish for the first time that ivermectin has potent antiviral activity towards both HIV-1 and dengue virus, both of which are strongly reliant on importin α/β nuclear import, with respect to the HIV-1 integrase and NS5 (non-structural protein 5) polymerase proteins resp. Ivermectin would appear to be an invaluable tool for the study of protein nuclear import, as well as the basis for future development of antiviral agents.
- 25Wulan, W. N., Heydet, D., Walker, E. J., Gahan, M. E., and Ghildyal, R. (2015) Nucleocytoplasmic transport of nucleocapsid proteins of enveloped RNA viruses. Front. Microbiol. 6, 553, DOI: 10.3389/fmicb.2015.00553[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MbksVWlsA%253D%253D&md5=c2bd7c868440b2aba4f3ad1cb267de81Nucleocytoplasmic transport of nucleocapsid proteins of enveloped RNA virusesWulan Wahyu N; Ghildyal Reena; Heydet Deborah; Walker Erin J; Gahan Michelle EFrontiers in microbiology (2015), 6 (), 553 ISSN:1664-302X.Most viruses with non-segmented single stranded RNA genomes complete their life cycle in the cytoplasm of infected cells. However, despite undergoing replication in the cytoplasm, the structural proteins of some of these RNA viruses localize to the nucleus at specific times in the virus life cycle, primarily early in infection. Limited evidence suggests that this enhances successful viral replication by interfering with or inhibiting the host antiviral response. Nucleocapsid proteins of RNA viruses have a well-established, essential cytoplasmic role in virus replication and assembly. Intriguingly, nucleocapsid proteins of some RNA viruses also localize to the nucleus/nucleolus of infected cells. Their nuclear function is less well understood although significant advances have been made in recent years. This review will focus on the nucleocapsid protein of cytoplasmic enveloped RNA viruses, including their localization to the nucleus/nucleolus and function therein. A greater understanding of the nuclear localization of nucleocapsid proteins has the potential to enhance therapeutic strategies as it can be a target for the development of live-attenuated vaccines or antiviral drugs.
- 26Yang, S. N. Y., Atkinson, S. C., Wang, C., Lee, A., Bogoyevitch, M. A., Borg, N. A., and Jans, D. A. (2020) The broad spectrum antiviral ivermectin targets the host nuclear transport importin alpha/beta1 heterodimer. Antiviral Res. 177, 104760, DOI: 10.1016/j.antiviral.2020.104760[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXntVKrsrY%253D&md5=5273353c8738f614375caab849315874The broad spectrum antiviral ivermectin targets the host nuclear transport importin α/β1 heterodimerYang, Sundy N. Y.; Atkinson, Sarah C.; Wang, Chunxiao; Lee, Alexander; Bogoyevitch, Marie A.; Borg, Natalie A.; Jans, David A.Antiviral Research (2020), 177 (), 104760CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)Infection by RNA viruses such as human immunodeficiency virus (HIV)-1, influenza, and dengue virus (DENV) represent a major burden for human health worldwide. Although RNA viruses replicate in the infected host cell cytoplasm, the nucleus is central to key stages of the infectious cycle of HIV-1 and influenza, and an important target of DENV nonstructural protein 5 (NS5) in limiting the host antiviral response. We previously identified the small mol. ivermectin as an inhibitor of HIV-1 integrase nuclear entry, subsequently showing ivermectin could inhibit DENV NS5 nuclear import, as well as limit infection by viruses such as HIV-1 and DENV. We show here that ivermectin's broad spectrum antiviral activity relates to its ability to target the host importin (IMP) α/β1 nuclear transport proteins responsible for nuclear entry of cargoes such as integrase and NS5. We establish for the first time that ivermectin can dissoc. the preformed IMPα/β1 heterodimer, as well as prevent its formation, through binding to the IMPα armadillo (ARM) repeat domain to impact IMPα thermal stability and α-helicity. We show that ivermectin inhibits NS5-IMPα interaction in a cell context using quant. bimol. fluorescence complementation. Finally, we show for the first time that ivermectin can limit infection by the DENV-related West Nile virus at low (μM) concns. Since it is FDA approved for parasitic indications, ivermectin merits closer consideration as a broad spectrum antiviral of interest.
- 27Singh, K. K., Chaubey, G., Chen, J. Y., and Suravajhala, P. (2020) Decoding SARS-CoV-2 hijacking of host mitochondria in COVID-19 pathogenesis. Am. J. Physiol Cell Physiol 319 (2), C258– C267, DOI: 10.1152/ajpcell.00224.2020
Cited By
This article is cited by 4 publications.
- Tean Zaheer, Kaushik Pal, Rao Zahid Abbas, María del Pilar Rodríguez Torres. COVID-19 and Ivermectin: Potential threats associated with human use. Journal of Molecular Structure 2021, 1243 , 130808. https://doi.org/10.1016/j.molstruc.2021.130808
- Shadpour Mallakpour, Elham Azadi, Chaudhery Mustansar Hussain. Protection, disinfection, and immunization for healthcare during the COVID-19 pandemic: Role of natural and synthetic macromolecules. Science of The Total Environment 2021, 776 , 145989. https://doi.org/10.1016/j.scitotenv.2021.145989
- Anna Paula A. Carvalho, Carlos A. Conte‐Junior. Recent Advances on Nanomaterials to COVID‐19 Management: A Systematic Review on Antiviral/Virucidal Agents and Mechanisms of SARS‐CoV‐2 Inhibition/Inactivation. Global Challenges 2021, 36 , 2000115. https://doi.org/10.1002/gch2.202000115
- Wanru Guo, Harini Lakshminarayanan, Alex Rodriguez-Palacios, Robert A Salata, Kaijin Xu, Mohamed S Draz. Glycan Nanostructures of Human Coronaviruses. International Journal of Nanomedicine 2021, Volume 16 , 4813-4830. https://doi.org/10.2147/IJN.S302516
Abstract
Figure 1
Figure 1. Graphical representation showing that the targeted-Fc-IVM-NPs in the acidic gut lumen bind to FcRn receptors, allowing NPs to transcytose across the intestinal barrier and release at the physiological pH of blood. IVM delivered via T-Fc-IVM-NPs shows the ability to (1) decrease ACE2 receptor levels, (2) decrease SARS-CoV-2 spike protein levels, and (3) decrease levels of the nuclear transport proteins importin α and β1, which leads to (4) an increase in the antiviral activity of infected cells.
Figure 2
Figure 2. (A) Western blot showing expression of ACE2 and spike protein in HEK293T cells transfected with plasmid expressing spike protein with treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 2 or 4 h at a concentration of 10 μM with respect to IVM, after which the media was changed and the cells were further incubated up to a total of 24 h. Immunofluorescence staining showing expression of (B) ACE2 and (C) spike protein in HEK293T cells transfected with a plasmid expressing spike protein with treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 4 h at a concentration of 10 μM with respect to IVM, followed by an additional 20 h incubation. (D) Fold change in mRNA expression level of ACE2 in HEK293T cells upon treatment with articles at a concentration of 10 μM with respect to IVM. (E) Fold change in mRNA expression levels of ACE2 and spike protein in spike-protein expressing HEK293T cells. The cells transfected with spike plasmid were treated with articles at a concentration of 10 μM with respect to IVM. For panels D and E, the treatment time was 4 h, followed by an additional incubation for 20 h.
Figure 3
Figure 3. (A) Western blot showing expression of ACE2 in A549 adenocarcinomic alveolar basal epithelial cells transfected with plasmid expressing spike protein with treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 4 h at a concentration of 10 μM with respect to IVM, followed by incubation in normal media for an additional 20 h. (B) Western blot showing a dose-dependent decrease in basal ACE2 expression after treatment with varying concentrations of T-Fc-IVM-NPs with respect to IVM in A549 cells. (C) Western blot showing expression of ACE2 in HeLa malignant epithelial cells transfected with plasmid expressing spike protein with treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 4 h at a concentration of 10 μM with respect to IVM, followed by incubation in normal media for an additional 20 h. (D) Immunofluorescence staining showing expression of ACE2 in A549 cells transfected with plasmid expressing spike protein with treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 4 h at a concentration of 10 μM with respect to IVM, followed by incubation in normal media for an additional 20 h.
Figure 4
Figure 4. (A) Schematic representation of mNeonGreen pseudovirus reporter protein accumulation in HEK293T cells and the efficacy of T-Fc-IVM-NP showing inhibition of both ACE2 and pseudovirus uptake under (B) therapeutic and (C) preventative treatment methods as measured by the microplate reader. Confocal microscopy images revealing the changes in the expression of red-tagged ACE2 receptor on the cell membrane and mNeonGreen pseudovirus accumulation in the nucleus following the treatment of articles under (D) therapeutic and (E) preventative treatment methods. The article concentration was kept at 10 μM with respect to IVM for 4 h followed by an additional 20 h of incubation.
Figure 5
Figure 5. (A) Western blot showing the change in expression of IMP α and β1 in HEK293T cells following treatment with IVM and its nanoformulations. Cells were treated with articles for 2, 4, and 6 h at a concentration of 10 μM with respect to IVM, followed by incubation in normal media up to a total of 24 h. (B) Western blot showing dose-dependent changes in IMP α and β1 after treatment with varying concentrations of importazole (IMZ) in HEK293T cells. (C) Efficacy of IMZ in combination with IVM, NT-IVM-NP, and T-Fc-IVM-NP on both ACE2 expression and pseudovirus uptake under preventative treatment method as measured by the microplate reader. (D) Western blot showing the change in expression of ACE2, spike protein, and IMP α and β1 in HEK293T cells following treatment with remdesivir, IVM, or its nanoformulations. Cells were treated with articles for 4 h at a concentration of 10 μM with respect to IVM or remdesivir, followed by incubation in normal media up to a total of 24 h. (E) Western blot (top) and RT-PCR (bottom) data showing the change in expression of MERS-CoV spike protein in HEK293T cells following treatment with remdesivir, IVM, or its nanoformulations. Cells were treated with articles for 4 h at a concentration of 10 μM with respect to IVM or remdesivir, followed by incubation in normal media up to a total of 24 h. (F) Schematic representation of how IVM delivered through T-Fc-IVM-NP inhibits IMP α and β1, thus increasing host cell antiviral activity.
Figure 6
Figure 6. (A) Cellular toxicity of IVM and IVM-loaded NPs as measured by mitochondrial respiration profiles in spike protein-expressing HEK293T cells using the Seahorse analyzer and MitoStress assay: oligomycin, ATP synthase inhibitor; FCCP-carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, an ionophore; rotenone, an inhibitor of mitochondrial complex I; and antimycin A, an inhibitor of mitochondrial complex III. (B) Basal respiration and ATP production from the MitoStress assay. (C) Expression of cytokines IL-6, IL-1β, and TNFα in the media of spike protein-expressing HEK293T cells treated with IVM, NT-IVM-NP, or T-Fc-IVM-NP.
References
ARTICLE SECTIONSThis article references 27 other publications.
- 1Li, X., Geng, M., Peng, Y., Meng, L., and Lu, S. (2020) Molecular immune pathogenesis and diagnosis of COVID-19. J. Pharm. Anal. 10 (2), 102– 108, DOI: 10.1016/j.jpha.2020.03.001[Crossref], [PubMed], [CAS], Google Scholar1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB38zksVygtg%253D%253D&md5=a0f4031c4fb4a0ddb72c96905fb0a8f2Molecular immune pathogenesis and diagnosis of COVID-19Li Xiaowei; Geng Manman; Peng Yizhao; Meng Liesu; Lu Shemin; Li Xiaowei; Geng Manman; Peng Yizhao; Meng Liesu; Lu SheminJournal of pharmaceutical analysis (2020), 10 (2), 102-108 ISSN:.Coronavirus disease 2019 (COVID-19) is a kind of viral pneumonia which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The emergence of SARS-CoV-2 has been marked as the third introduction of a highly pathogenic coronavirus into the human population after the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV) in the twenty-first century. In this minireview, we provide a brief introduction of the general features of SARS-CoV-2 and discuss current knowledge of molecular immune pathogenesis, diagnosis and treatment of COVID-19 on the base of the present understanding of SARS-CoV and MERS-CoV infections, which may be helpful in offering novel insights and potential therapeutic targets for combating the SARS-CoV-2 infection.
- 2Yuki, K., Fujiogi, M., and Koutsogiannaki, S. (2020) COVID-19 pathophysiology: A review. Clin. Immunol. 215, 108427, DOI: 10.1016/j.clim.2020.108427[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXosFWitr0%253D&md5=8c97d9785e6bfbf2a1885e12400cc55fCOVID-19 pathophysiology: A reviewYuki, Koichi; Fujiogi, Miho; Koutsogiannaki, SophiaClinical Immunology (Amsterdam, Netherlands) (2020), 215 (), 108427CODEN: CLIIFY; ISSN:1521-6616. (Elsevier B.V.)A review. In Dec. 2019, a novel coronavirus, now named as SARS-CoV-2, caused a series of acute atypical respiratory diseases in Wuhan, Hubei Province, China. The disease caused by this virus was termed COVID-19. The virus is transmittable between humans and has caused pandemic worldwide. The no. of death tolls continues to rise and a large no. of countries have been forced to do social distancing and lockdown. Lack of targeted therapy continues to be a problem. Epidemiol. studies showed that elder patients were more susceptible to severe diseases, while children tend to have milder symptoms. Here we reviewed the current knowledge about this disease and considered the potential explanation of the different symptomatol. between children and adults.
- 3Gheblawi, M., Wang, K., Viveiros, A., Nguyen, Q., Zhong, J., Turner, A. J., Raizada, M. K., Grant, M. B., and Oudit, G. Y. (2020) Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin System. Circ. Res. 126 (10), 1456– 1474, DOI: 10.1161/CIRCRESAHA.120.317015[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXptVaksrY%253D&md5=f240902dd9bf0f970ad227a1c5d4f037Angiotensin-Converting Enzyme 2: SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin System: Celebrating the 20th Anniversary of the Discovery of ACE2Gheblawi, Mahmoud; Wang, Kaiming; Viveiros, Anissa; Nguyen, Quynh; Zhong, Jiu-Chang; Turner, Anthony J.; Raizada, Mohan K.; Grant, Maria B.; Oudit, Gavin Y.Circulation Research (2020), 126 (10), 1456-1474CODEN: CIRUAL; ISSN:0009-7330. (Lippincott Williams & Wilkins)A review. ACE2 (angiotensin-converting enzyme 2) has a multiplicity of physiol. roles that revolve around its trivalent function: a neg. regulator of the renin-angiotensin system, facilitator of amino acid transport, and the severe acute respiratory syndrome-coronavirus (SARS-CoV) and SARS-CoV-2 receptor. ACE2 is widely expressed, including, in the lungs, cardiovascular system, gut, kidneys, central nervous system, and adipose tissue. ACE2 has recently been identified as the SARS-CoV-2 receptor, the infective agent responsible for coronavirus disease 2019, providing a crit. link between immunity, inflammation, ACE2, and cardiovascular disease. Although sharing a close evolutionary relationship with SARS-CoV, the receptor-binding domain of SARS-CoV-2 differs in several key amino acid residues, allowing for stronger binding affinity with the human ACE2 receptor, which may account for the greater pathogenicity of SARS-CoV-2. The loss of ACE2 function following binding by SARS-CoV-2 is driven by endocytosis and activation of proteolytic cleavage and processing. The ACE2 system is a crit. protective pathway against heart failure with reduced and preserved ejection fraction including, myocardial infarction and hypertension, and against lung disease and diabetes mellitus. The control of gut dysbiosis and vascular permeability by ACE2 has emerged as an essential mechanism of pulmonary hypertension and diabetic cardiovascular complications. Recombinant ACE2, gene-delivery of Ace2, Ang 1-7 analogs, and Mas receptor agonists enhance ACE2 action and serve as potential therapies for disease conditions assocd. with an activated renin-angiotensin system. RhACE2 (recombinant human ACE2) has completed clin. trials and efficiently lowered or increased plasma angiotensin II and angiotensin 1-7 levels, resp. Our review summarizes the progress over the past 20 years, highlighting the crit. role of ACE2 as the novel SARS-CoV-2 receptor and as the neg. regulator of the renin-angiotensin system, together with implications for the coronavirus disease 2019 pandemic and assocd. cardiovascular diseases.
- 4Li, G., He, X., Zhang, L., Ran, Q., Wang, J., Xiong, A., Wu, D., Chen, F., Sun, J., and Chang, C. (2020) Assessing ACE2 expression patterns in lung tissues in the pathogenesis of COVID-19. J. Autoimmun. 112, 102463, DOI: 10.1016/j.jaut.2020.102463[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnt1WnsLY%253D&md5=9f7532dfbe5c0d07277a7d290b2761cdAssessing ACE2 expression patterns in lung tissues in the pathogenesis of COVID-19Li, Guoping; He, Xiang; Zhang, Lei; Ran, Qin; Wang, Junyi; Xiong, Anying; Wu, Dehong; Chen, Feng; Sun, Jinlyu; Chang, ChristopherJournal of Autoimmunity (2020), 112 (), 102463CODEN: JOAUEP; ISSN:0896-8411. (Elsevier Ltd.)It has been reported that SARS-CoV-2 may use ACE2 as a receptor to gain entry into human cells, in a way similar to that of SARS-CoV. Analyzing the distribution and expression level of ACE2 may therefore help reveal underlying mechanisms of viral susceptibility and post-infection modulation. In this study, we utilized previously uploaded information on ACE2 expression in various conditions including SARS-CoA to evaluate the role of ACE2 in SARS-CoV and extrapolate that to COVID-19. We found that the expression of ACE2 in healthy populations and patients with underlying diseases was not significantly different. However, based on the elevated expression of ACE2 in cigarette smokers, we speculate that long-term smoking may be a risk factor for COVID-19. Anal. of ACE2 in SARS-CoV infected cells suggests that ACE2 is not only a receptor but is also involved in post-infection regulation, including immune response, cytokine secretion, and viral genome replication. Moreover, we constructed Protein-protein interaction (PPI) networks and identified hub genes in viral activity and cytokine secretion. Our findings may help clinicians and researchers gain more insight into the pathogenesis of SARS-CoV-2 and design therapeutic strategies for COVID-19.
- 5McGonagle, D., Sharif, K., O’Regan, A., and Bridgewood, C. (2020) The Role of Cytokines including Interleukin-6 in COVID-19 induced Pneumonia and Macrophage Activation Syndrome-Like Disease. Autoimmun. Rev. 19 (6), 102537, DOI: 10.1016/j.autrev.2020.102537[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXms1ylt78%253D&md5=d05b5c11ac442ff5e1573fc14db7921cThe Role of Cytokines including Interleukin-6 in COVID-19 induced Pneumonia and Macrophage Activation Syndrome-Like DiseaseMcGonagle, Dennis; Sharif, Kassem; O'Regan, Anthony; Bridgewood, CharlieAutoimmunity Reviews (2020), 19 (6), 102537CODEN: ARUEBU; ISSN:1568-9972. (Elsevier B.V.)A review. Severe COVID-19 assocd. pneumonia patients may exhibit features of systemic hyper-inflammation designated under the umbrella term of macrophage activation syndrome (MAS) or cytokine storm, also known as secondary haemophagocytic lymphohistocytosis (sHLH). This is distinct from HLH assocd. with immunodeficiency states termed primary HLH -with radically different therapy strategies in both situations. COVID-19 infection with MAS typically occurs in subjects with adult respiratory distress syndrome (ARDS) and historically, non-survival in ARDS was linked to sustained IL-6 and IL-1 elevation. We provide a model for the classification of MAS to stratify the MAS-like presentation in COVID-19 pneumonia and explore the complexities of discerning ARDS from MAS. We discuss the potential impact of timing of anti-cytokine therapy on viral clearance and the impact of such therapy on intra-pulmonary macrophage activation and emergent pulmonary vascular disease.
- 6Merad, M. and Martin, J. C. (2020) Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages. Nat. Rev. Immunol. 20 (6), 355– 362, DOI: 10.1038/s41577-020-0331-4[Crossref], [PubMed], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXoslOqt7s%253D&md5=d38b037fed1fcc469e6c8caa23777fc7Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophagesMerad, Miriam; Martin, Jerome C.Nature Reviews Immunology (2020), 20 (6), 355-362CODEN: NRIABX; ISSN:1474-1733. (Nature Research)A review. Abstr.: The COVID-19 pandemic caused by infection with SARS-CoV-2 has led to more than 200,000 deaths worldwide. Several studies have now established that the hyperinflammatory response induced by SARS-CoV-2 is a major cause of disease severity and death in infected patients. Macrophages are a population of innate immune cells that sense and respond to microbial threats by producing inflammatory mols. that eliminate pathogens and promote tissue repair. However, a dysregulated macrophage response can be damaging to the host, as is seen in the macrophage activation syndrome induced by severe infections, including in infections with the related virus SARS-CoV. Here we describe the potentially pathol. roles of macrophages during SARS-CoV-2 infection and discuss ongoing and prospective therapeutic strategies to modulate macrophage activation in patients with COVID-19.
- 7Ou, X., Liu, Y., Lei, X., Li, P., Mi, D., Ren, L., Guo, L., Guo, R., Chen, T., Hu, J., Xiang, Z., Mu, Z., Chen, X., Chen, J., Hu, K., Jin, Q., Wang, J., and Qian, Z. (2020) Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat. Commun. 11, 1620, DOI: 10.1038/s41467-020-15562-9[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlvFyjt78%253D&md5=6b0b1ef5a68f4a35da4aabecb0f99544Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoVOu, Xiuyuan; Liu, Yan; Lei, Xiaobo; Li, Pei; Mi, Dan; Ren, Lili; Guo, Li; Guo, Ruixuan; Chen, Ting; Hu, Jiaxin; Xiang, Zichun; Mu, Zhixia; Chen, Xing; Chen, Jieyong; Hu, Keping; Jin, Qi; Wang, Jianwei; Qian, ZhaohuiNature Communications (2020), 11 (1), 1620CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biol. of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are crit. for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.
- 8Qin, C., Zhou, L., Hu, Z., Zhang, S., Yang, S., Tao, Y., Xie, C., Ma, K., Shang, K., Wang, W., and Tian, D. S. (2020) Dysregulation of immune response in patients with COVID-19 in Wuhan, China. Clin. Infect. Dis. 71, 762, DOI: 10.1093/cid/ciaa248[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsF2mt7fF&md5=ff15e4dfcc73998aaa84b06b798cf0a5Dysregulation of immune response in patients with coronavirus 2019 (COVID-19) in Wuhan, ChinaQin, Chuan; Zhou, Luoqi; Hu, Ziwei; Zhang, Shuoqi; Yang, Sheng; Tao, Yu; Xie, Cuihong; Ma, Ke; Shang, Ke; Wang, Wei; Tian, Dai-ShiClinical Infectious Diseases (2020), 71 (15), 762-768CODEN: CIDIEL; ISSN:1537-6591. (Oxford University Press)Background. In Dec. 2019, coronavirus 2019 (COVID-19) emerged in Wuhan and rapidly spread throughout China. Methods. Demog. and clin. data of all confirmed cases with COVID-19 on admission at Tongji Hospital from 10 Jan. to 12 Feb. 2020 were collected and analyzed. The data on lab. examns., including peripheral lymphocyte subsets, were analyzed and compared between patients with severe and nonsevere infection. Results. Of the 452 patients with COVID-19 recruited, 286 were diagnosed as having severe infection. The median age was 58 years and 235 were male. The most common symptoms were fever, shortness of breath, expectoration, fatigue, dry cough, and myalgia. Severe cases tend to have lower lymphocyte counts, higher leukocyte counts and neutrophil-lymphocyte ratio (NLR), as well as lower percentages of monocytes, eosinophils, and basophils. Most severe cases demonstrated elevated levels of infection-related biomarkers and inflammatory cytokines. The no. of T cells significantly decreased, and were more impaired in severe cases. Both helper T (Th) cells and suppressor T cells in patients with COVID-19 were below normal levels, with lower levels of Th cells in the severe group. The percentage of naive Th cells increased and memory Th cells decreased in severe cases. Patients with COVID-19 also have lower levels of regulatory T cells, which are more obviously decreased in severe cases. Conclusions. The novel coronavirus might mainly act on lymphocytes, esp. T lymphocytes. Surveillance of NLR and lymphocyte subsets is helpful in the early screening of crit. illness, diagnosis, and treatment of COVID-19.
- 9Sungnak, W., Huang, N., Becavin, C., Berg, M., Queen, R., Litvinukova, M., Talavera-Lopez, C., Maatz, H., Reichart, D., Sampaziotis, F., Worlock, K. B., Yoshida, M., and Barnes, J. L. (2020) SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes. Nat. Med. 26, 681– 687, DOI: 10.1038/s41591-020-0868-6[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXotVCjurY%253D&md5=cc313df9c190333b98af43951a2146faSARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genesSungnak, Waradon; Huang, Ni; Becavin, Christophe; Berg, Marijn; Queen, Rachel; Litvinukova, Monika; Talavera-Lopez, Carlos; Maatz, Henrike; Reichart, Daniel; Sampaziotis, Fotios; Worlock, Kaylee B.; Yoshida, Masahiro; Barnes, Josephine L.; HCA Lung Biological NetworkNature Medicine (New York, NY, United States) (2020), 26 (5), 681-687CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)We investigated SARS-CoV-2 potential tropism by surveying expression of viral entry-assocd. genes in single-cell RNA-sequencing data from multiple tissues from healthy human donors. We co-detected these transcripts in specific respiratory, corneal and intestinal epithelial cells, potentially explaining the high efficiency of SARS-CoV-2 transmission. These genes are co-expressed in nasal epithelial cells with genes involved in innate immunity, highlighting the cells' potential role in initial viral infection, spread and clearance. The study offers a useful resource for further lines of inquiry with valuable clin. samples from COVID-19 patients and we provide our data in a comprehensive, open and user-friendly fashion at www.covid19cellatlas.org.
- 10Yan, R., Zhang, Y., Li, Y., Xia, L., Guo, Y., and Zhou, Q. (2020) Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 367 (6485), 1444– 1448, DOI: 10.1126/science.abb2762[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlslymsLo%253D&md5=ff4dfdfc646ea878cfb325019160e94aStructural basis for the recognition of SARS-CoV-2 by full-length human ACE2Yan, Renhong; Zhang, Yuanyuan; Li, Yaning; Xia, Lu; Guo, Yingying; Zhou, QiangScience (Washington, DC, United States) (2020), 367 (6485), 1444-1448CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for severe acute respiratory syndrome coronavirus (SARS-CoV) and the new coronavirus (SARS-CoV-2) that is causing the serious coronavirus disease 2019 (COVID-19) epidemic. Here, we present cryo-electron microscopy structures of full-length human ACE2 in the presence of the neutral amino acid transporter B0AT1 with or without the receptor binding domain (RBD) of the surface spike glycoprotein (S protein) of SARS-CoV-2, both at an overall resoln. of 2.9 angstroms, with a local resoln. of 3.5 angstroms at the ACE2-RBD interface. The ACE2-B0AT1 complex is assembled as a dimer of heterodimers, with the collectrin-like domain of ACE2 mediating homodimerization. The RBD is recognized by the extracellular peptidase domain of ACE2 mainly through polar residues. These findings provide important insights into the mol. basis for coronavirus recognition and infection.
- 11Zhang, H., Penninger, J. M., Li, Y., Zhong, N., and Slutsky, A. S. (2020) Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target. Intensive Care Med. 46 (4), 586– 590, DOI: 10.1007/s00134-020-05985-9[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkt1erurY%253D&md5=705f72887420f24bdc15084bb6fdf193Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic targetZhang, Haibo; Penninger, Josef M.; Li, Yimin; Zhong, Nanshan; Slutsky, Arthur S.Intensive Care Medicine (2020), 46 (4), 586-590CODEN: ICMED9; ISSN:0342-4642. (Springer)A review. A novel infectious disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was detected in Wuhan, China, in Dec. 2019. The disease (COVID-19) spread rapidly, reaching epidemic proportions in China, and has been found in 27 other countries. As of Feb. 27, 2020, over 82,000 cases of COVID-19 were reported, with > 2800 deaths. No specific therapeutics are available, and current management includes travel restrictions, patient isolation, and supportive medical care. There are a no. of pharmaceuticals already being tested, but a better understanding of the underlying pathobiol. is required. In this context, this article will brief review the rationale for angiotensin-converting enzyme 2 (ACE2) receptor as a specific target.
- 12Pandey, P., Khan, F., Kumar, A., Srivastava, A., and Jha, N. K. (2021) Screening of Potent Inhibitors Against 2019 Novel Coronavirus (Covid-19) from Alliumsativum and Allium cepa: An In Silico Approach. Biointerface Res. Appl. Chem. 11 (1), 7981– 7993, DOI: 10.33263/BRIAC111.79817993
- 13Pandey, P., Khan, F., Rana, A. K., Srivastava, Y., Jha, S. K., and Jha, N. K. (2021) A Drug Repurposing Approach Towards Elucidating the Potential of Flavonoids as COVID-19 Spike Protein Inhibitors. Biointerface Res. Appl. Chem. 11 (1), 8482– 8501, DOI: 10.33263/BRIAC111.84828501
- 14Mastrangelo, E., Pezzullo, M., De Burghgraeve, T., Kaptein, S., Pastorino, B., Dallmeier, K., de Lamballerie, X., Neyts, J., Hanson, A. M., Frick, D. N., Bolognesi, M., and Milani, M. (2012) Ivermectin Is a Potent Inhibitor of Flavivirus Replication Specifically Targeting NS3 Helicase Activity: New Prospects for an Old Drug. J. Antimicrob. Chemother. 67 (8), 1884– 1894, DOI: 10.1093/jac/dks147[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtVeisbzF&md5=a3bf7d857cd11b9bfed8e6780b501b39Ivermectin is a potent inhibitor of flavivirus replication specifically targeting NS3 helicase activity: new prospects for an old drugMastrangelo, Eloise; Pezzullo, Margherita; De Burghgraeve, Tine; Kaptein, Suzanne; Pastorino, Boris; Dallmeier, Kai; de Lamballerie, Xavier; Neyts, Johan; Hanson, Alicia M.; Frick, David N.; Bolognesi, Martino; Milani, MarioJournal of Antimicrobial Chemotherapy (2012), 67 (8), 1884-1894CODEN: JACHDX; ISSN:0305-7453. (Oxford University Press)Objectives Infection with yellow fever virus (YFV), the prototypic mosquito-borne flavivirus, causes severe febrile disease with hemorrhage, multi-organ failure and a high mortality. Moreover, in recent years the Flavivirus genus has gained further attention due to re-emergence and increasing incidence of West Nile, dengue and Japanese encephalitis viruses. Potent and safe antivirals are urgently needed. Methods Starting from the crystal structure of the NS3 helicase from Kunjin virus (an Australian variant of West Nile virus), we identified a novel, unexploited protein site that might be involved in the helicase catalytic cycle and could thus in principle be targeted for enzyme inhibition. In silico docking of a library of small mols. allowed us to identify a few selected compds. with high predicted affinity for the new site. Their activity against helicases from several flaviviruses was confirmed in in vitro helicase/enzymic assays. The effect on the in vitro replication of flaviviruses was then evaluated. Results Ivermectin, a broadly used anti-helminthic drug, proved to be a highly potent inhibitor of YFV replication (EC50 values in the sub-nanomolar range). Moreover, ivermectin inhibited, although less efficiently, the replication of several other flaviviruses, i.e. dengue fever, Japanese encephalitis and tick-borne encephalitis viruses. Ivermectin exerts its effect at a timepoint that coincides with the onset of intracellular viral RNA synthesis, as expected for a mol. that specifically targets the viral helicase. Conclusions The well-tolerated drug ivermectin may hold great potential for treatment of YFV infections. Furthermore, structure-based optimization may result in analogs exerting potent activity against flaviviruses other than YFV.
- 15Heidary, F. and Gharebaghi, R. (2020) Ivermectin: a systematic review from antiviral effects to COVID-19 complementary regimen. J. Antibiot. 73 (9), 593– 602, DOI: 10.1038/s41429-020-0336-z[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtFGgsLbN&md5=5ce416e511fb3575df954eb294ac05cfIvermectin: a systematic review from antiviral effects to COVID-19 complementary regimenHeidary, Fatemeh; Gharebaghi, RezaJournal of Antibiotics (2020), 73 (9), 593-602CODEN: JANTAJ; ISSN:0021-8820. (Nature Research)A review. A reviews. Ivermectin proposes many potentials effects to treat a range of diseases, with its antimicrobial, antiviral, and anti-cancer properties as a wonder drug. It is highly effective against many microorganisms including some viruses. In this comprehensive systematic review, antiviral effects of ivermectin are summarized including in vitro and in vivo studies over the past 50 years. Several studies reported antiviral effects of ivermectin on RNA viruses such as Zika, dengue, yellow fever, West Nile, Hendra, Newcastle, Venezuelan equine encephalitis, chikungunya, Semliki Forest, Sindbis, Avian influenza A, Porcine Reproductive and Respiratory Syndrome, Human immunodeficiency virus type 1, and severe acute respiratory syndrome coronavirus 2. Furthermore, there are some studies showing antiviral effects of ivermectin against DNA viruses such as Equine herpes type 1, BK polyomavirus, pseudorabies, porcine circovirus 2, and bovine herpesvirus 1. Ivermectin plays a role in several biol. mechanisms, therefore it could serve as a potential candidate in the treatment of a wide range of viruses including COVID-19 as well as other types of pos.-sense single-stranded RNA viruses. In vivo studies of animal models revealed a broad range of antiviral effects of ivermectin, however, clin. trials are necessary to appraise the potential efficacy of ivermectin in clin. setting.
- 16Wang, Y. P., Du, W. J., Huang, L. P., Wei, Y. W., Wu, H. L., Feng, L., and Liu, C. M. (2016) The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the Holoenzyme. Front. Microbiol. 7, 124, DOI: 10.3389/fmicb.2016.00124[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28jksVenuw%253D%253D&md5=a0e9889fdb01480d9b5b50ae01ce34c4The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the HoloenzymeWang Yi-Ping; Du Wen-Juan; Huang Li-Ping; Wei Yan-Wu; Wu Hong-Li; Feng Li; Liu Chang-MingFrontiers in microbiology (2016), 7 (), 124 ISSN:1664-302X.Pseudorabies virus (PRV) DNA replication occurs in the nuclei of infected cells and requires the viral DNA polymerase. The PRV DNA polymerase comprises a catalytic subunit, UL30, and an accessory subunit, UL42, that confers processivity to the enzyme. Its nuclear localization is a prerequisite for its enzymatic function in the initiation of viral DNA replication. However, the mechanisms by which the PRV DNA polymerase holoenzyme enters the nucleus have not been determined. In this study, we characterized the nuclear import pathways of the PRV DNA polymerase catalytic and accessory subunits. Immunofluorescence analysis showed that UL42 localizes independently in the nucleus, whereas UL30 alone predominantly localizes in the cytoplasm. Intriguingly, the localization of UL30 was completely shifted to the nucleus when it was coexpressed with UL42, demonstrating that nuclear transport of UL30 occurs in an UL42-dependent manner. Deletion analysis and site-directed mutagenesis of the two proteins showed that UL42 contains a functional and transferable bipartite nuclear localization signal (NLS) at amino acids 354-370 and that K(354), R(355), and K(367) are important for the NLS function, whereas UL30 has no NLS. Coimmunoprecipitation assays verified that UL42 interacts with importins α3 and α4 through its NLS. In vitro nuclear import assays demonstrated that nuclear accumulation of UL42 is a temperature- and energy-dependent process and requires both importins α and β, confirming that UL42 utilizes the importin α/β-mediated pathway for nuclear entry. In an UL42 NLS-null mutant, the UL42/UL30 heterodimer was completely confined to the cytoplasm when UL42 was coexpressed with UL30, indicating that UL30 utilizes the NLS function of UL42 for its translocation into the nucleus. Collectively, these findings suggest that UL42 contains an importin α/β-mediated bipartite NLS that transports the viral DNA polymerase holoenzyme into the nucleus in an in vitro expression system.
- 17Surnar, B., Kamran, M. Z., Shah, A. S., Basu, U., Kolishetti, N., Deo, S., Jayaweera, D. T., Daunert, S., and Dhar, S. (2019) Orally Administrable Therapeutic Synthetic Nanoparticle for Zika Virus. ACS Nano 13, 11034– 11048, DOI: 10.1021/acsnano.9b02807[ACS Full Text], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvFeqtrvP&md5=b23f2a1279b8ad8fc09ac8b09be9fbe1Orally Administrable Therapeutic Synthetic Nanoparticle for Zika VirusSurnar, Bapurao; Kamran, Mohammad Z.; Shah, Anuj S.; Basu, Uttara; Kolishetti, Nagesh; Deo, Sapna; Jayaweera, Dushyantha T.; Daunert, Sylvia; Dhar, ShantaACS Nano (2019), 13 (10), 11034-11048CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)The spread of Zika virus (ZIKV) infection across the USA and various countries in the last three years will not only have a direct impact on the U. S. health care system but has caused international concerns as well. The ultimate impact of ZIKV infection remains to be understood. Currently, there are no therapeutic or vaccine options available to protect those infected by ZIKV. The drug ivermectin (IVM) was found to be a viable agent for the prevention of transmission of ZIKV. Ivermectin is both unstable in the presence of water and does not remain in adequate concn. in the human bloodstream to be effective in treatment for ZIKV. Biodegradable nanoparticles would aid in the delivery of ivermectin, by providing a high enough concn. of drug and ensuring the drug is gradually released to maintain an appropriate level in the body. The overall goal of this study was to develop and optimize an orally administrable nanoformulation of IVM which can circulate in the blood for a long period for efficient delivery. To achieve the goal, we synthesized and optimized a synthetic nanoformulation of IVM for oral use which can cross the intestinal epithelial barrier to enter the bloodstream. Our studies documented that when delivered with the synthetic nanoparticle (NP), IVM can be accumulated in the blood at a higher concn. and preliminary studies highlighted that NP delivered IVM has the ability to target nonstructural 1 protein of ZIKV. For potential clin. relevance, long-term storable formulation of IVM-nanoparticle in dry powder state for inclusion in a capsule form and cryoprotectant contg. frozen forms revealed promising findings. Further, our preliminary in vitro studies documented that ivermectin crosses the placental barrier thus making it unsafe for pregnant ZIKV population whereas ivermectin-loaded nanoparticle did not show any significant placental barrier crossing, thus indicating its potential suitability for such population. We envision that this work will fill a great unmet need by developing safer and more effective therapies for the treatment of viral infections, including ZIKV.
- 18Elkassaby, M. H. (1991) Ivermectin uptake and distribution in the plasma and tissue of Sudanese and Mexican patients infected with Onchocerca volvulus. Trop. Med. Parasitol. 42 (2), 79– 81[PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaK3Mzns1GmsA%253D%253D&md5=86e32c02975dab98c9ba807500f452a9Ivermectin uptake and distribution in the plasma and tissue of Sudanese and Mexican patients infected with Onchocerca volvulusElkassaby M HTropical medicine and parasitology : official organ of Deutsche Tropenmedizinische Gesellschaft and of Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZ) (1991), 42 (2), 79-81 ISSN:0177-2392.Ten Sudanese patients with Onchocerca volvulus infection were treated with a single oral dose of 150 micrograms/kg of ivermectin. Plasma samples were collected before treatment, 0.5, 1, 3, 4, 6, 12 hours and 1, 2, 3, 7, and 30 days. Four patients were selected for nodulectomies and skin biopsies at 6, 18 and 30 hours and 3 days post treatment. Using these samples O. volvulus worm fragments were dissected free of host nodular tissues for ivermectin extraction. Ivermectin was present in the nodular tissue at 6 hr and persisted for 3 days. It was also detected in an individual worm tissue extract at a concentration similar to the nodule, but in subcutaneous fascial tissue higher concentrations were sometimes found. Ivermectin was detected by radioimmunoassay in the plasma of all patients at 1 hr and peak concentrations were reached in an average of 5.6 hr. The drug persisted at detectable levels for 7 days in 70% of the studied patients. Plasma samples were also collected from 16 treated Mexican onchocerciasis patients before ivermectin treatment and 4 hr treatment and from six individuals who served as controls. The Mexican patients had concentrations of ivermectin in their plasma similar to those in the Sudanese patients.
- 19Dashraath, P., Wong, J. L. J., Lim, M. X. K., Lim, L. M., Li, S., Biswas, A., Choolani, M., Mattar, C., and Su, L. L. (2020) Coronavirus disease 2019 (COVID-19) pandemic and pregnancy. Am. J. Obstet. Gynecol. 222 (6), 521– 531, DOI: 10.1016/j.ajog.2020.03.021[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnsleksLY%253D&md5=daae72f12be4c4583a87e38436524408Coronavirus disease 2019 (COVID-19) pandemic and pregnancyDashraath, Pradip; Wong, Jing Lin Jeslyn; Lim, Mei Xian Karen; Lim, Li Min; Li, Sarah; Biswas, Arijit; Choolani, Mahesh; Mattar, Citra; Su, Lin LinAmerican Journal of Obstetrics and Gynecology (2020), 222 (6), 521-531CODEN: AJOGAH; ISSN:0002-9378. (Elsevier Inc.)A review. The current coronavirus disease 2019 (COVID-19) pneumonia pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading globally at an accelerated rate, with a basic reprodn. no. (R0) of 2-2.5, indicating that 2-3 persons will be infected from an index patient. A serious public health emergency, it is particularly deadly in vulnerable populations and communities in which healthcare providers are insufficiently prepd. to manage the infection. As of March 16, 2020, there are more than 180,000 confirmed cases of COVID-19 worldwide, with more than 7000 related deaths. The SARS-CoV-2 virus has been isolated from asymptomatic individuals, and affected patients continue to be infectious 2 wk after cessation of symptoms. The substantial morbidity and socioeconomic impact have necessitated drastic measures across all continents, including nationwide lockdowns and border closures. Pregnant women and their fetuses represent a high-risk population during infectious disease outbreaks. To date, the outcomes of 55 pregnant women infected with COVID-19 and 46 neonates have been reported in the literature, with no definite evidence of vertical transmission. Physiol. and mech. changes in pregnancy increase susceptibility to infections in general, particularly when the cardiorespiratory system is affected, and encourage rapid progression to respiratory failure in the gravida. Furthermore, the pregnancy bias toward T-helper 2 (Th2) system dominance, which protects the fetus, leaves the mother vulnerable to viral infections, which are more effectively contained by the Th1 system. These unique challenges mandate an integrated approach to pregnancies affected by SARS-CoV-2. Here we present a review of COVID-19 in pregnancy, bringing together the various factors integral to the understanding of pathophysiol. and susceptibility, diagnostic challenges with real-time reverse transcription polymerase chain reaction (RT-PCR) assays, therapeutic controversies, intrauterine transmission, and maternal-fetal complications. We discuss the latest options in antiviral therapy and vaccine development, including the novel use of chloroquine in the management of COVID-19. Fetal surveillance, in view of the predisposition to growth restriction and special considerations during labor and delivery, is addressed. In addn., we focus on keeping frontline obstetric care providers safe while continuing to provide essential services. Our clin. service model is built around the principles of workplace segregation, responsible social distancing, containment of cross-infection to healthcare providers, judicious use of personal protective equipment, and telemedicine. Our aim is to share a framework that can be adopted by tertiary maternity units managing pregnant women in the flux of a pandemic while maintaining the safety of the patient and healthcare provider at its core.
- 20Caly, L., Druce, J. D., Catton, M. G., Jans, D. A., and Wagstaff, K. M. (2020) The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in vitro. Antiviral Res. 179, 104787, DOI: 10.1016/j.antiviral.2020.104787
- 21Rowland, R. R., Chauhan, V., Fang, Y., Pekosz, A., Kerrigan, M., and Burton, M. D. (2005) Intracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: absence of nucleolar accumulation during infection and after expression as a recombinant protein in Vero cells. J. Virol. 79 (17), 11507– 12, DOI: 10.1128/JVI.79.17.11507-11512.2005[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXps1Gltbw%253D&md5=1131e6faf604e2e9916968033354cdaaIntracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: Absence of nucleolar accumulation during infection and after expression as a recombinant protein in Vero cellsRowland, Raymond R. R.; Chauhan, Vinita; Fang, Ying; Pekosz, Andrew; Kerrigan, Maureen; Burton, Miriam D.Journal of Virology (2005), 79 (17), 11507-11512CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)The nucleocapsid (N) protein of several members within the order Nidovirales localizes to the nucleolus during infection and after transfection of cells with N genes. However, confocal microscopy of N protein localization in Vero cells infected with the severe acute respiratory syndrome coronavirus (SARS-CoV) or transfected with the SARS-CoV N gene failed to show the presence of N in the nucleoplasm or nucleolus. Amino acids 369 to 389, which contain putative nuclear localization signal (NLS) and nucleolar localization signal motifs, failed to restore nuclear localization to an NLS-minus mutant Rev protein. These data indicate that nuclear localization is not a conserved property among all nidoviruses.
- 22Timani, K. A., Liao, Q., Ye, L., Zeng, Y., Liu, J., Zheng, Y., Ye, L., Yang, X., Lingbao, K., Gao, J., and Zhu, Y. (2005) Nuclear/nucleolar localization properties of C-terminal nucleocapsid protein of SARS coronavirus. Virus Res. 114 (1–2), 23– 34, DOI: 10.1016/j.virusres.2005.05.007[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1WjtLfI&md5=9e6fff0b59f1df7f6702573df681d9c9Nuclear/nucleolar localization properties of C-terminal nucleocapsid protein of SARS coronavirusTimani, Khalid Amine; Liao, Qingjiao; Ye, Linbai; Zeng, Yingchun; Liu, Jing; Zheng, Yi; Ye, Li; Yang, Xiaojun; Lingbao, Kong; Gao, Jingrong; Zhu, YingVirus Research (2005), 114 (1-2), 23-34CODEN: VIREDF; ISSN:0168-1702. (Elsevier B.V.)A novel coronavirus (CoV) has recently been identified as the etiol. agent of severe acute respiratory syndrome (SARS). Nucleocapsid (N) proteins of the Coronaviridae family have no discernable homol., but they share a common nucleolar-cytoplasmic distribution pattern. There are three putative nuclear localization signal (NLS) motifs present in the N. To det. the role of these putative NLSs in the intracellular localization of the SARS-CoV N, we performed a confocal microscopy anal. using rabbit anti-N antisera. In this report, we show that the wild type N was distributed mainly in the cytoplasm. The N-terminal of the N, which contains the NLS1 (aa38-44), was localized to the nucleus. The C-terminus of the N, which contains both NLS2 (aa257-265) and NLS3 (aa369-390) was localized to the cytoplasm and the nucleolus. Results derived from anal. of various deletion mutations show that the region contg. amino acids 226-289 is able to mediate nucleolar localization. The deletion of two hydrophobic regions that flanked the NLS3 recovered its activity and localized to the nucleus. Furthermore, deletion of leucine rich region (220-LALLLLDRLNRL) resulted in the accumulation of N to the cytoplasm and nucleolus, and when fusing this peptide to EGFP localization was cytoplasmic, suggesting that the N may act as a shuttle protein. Differences in nuclear/nucleolar localization properties of N from other members of coronavirus family suggest a unique function for N, which may play an important role in the pathogenesis of SARS.
- 23Wagstaff, K. M., Rawlinson, S. M., Hearps, A. C., and Jans, D. A. (2011) An AlphaScreen(R)-based assay for high-throughput screening for specific inhibitors of nuclear import. J. Biomol. Screening 16 (2), 192– 200, DOI: 10.1177/1087057110390360[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjsVGlu7w%253D&md5=3a2163c020ec13b2074e444ad0ac4d82An AlphaScreen-based assay for high-throughput screening for specific inhibitors of nuclear importWagstaff, Kylie M.; Rawlinson, Stephen M.; Hearps, Anna C.; Jans, David A.Journal of Biomolecular Screening (2011), 16 (2), 192-200CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)Specific viral proteins enter the nucleus of infected cells to perform essential functions, as part of the viral life cycle. The integrase (IN) mol. of human immunodeficiency virus (HIV)-1 is of particular interest in this context due to its integral role in integrating the HIV genome into that of the infected host cell. Most IN-based antiviral compds. target the IN/DNA interaction, but since IN must first enter the nucleus before it can perform these crit. functions, nuclear transport of IN is also an attractive target for therapeutic intervention. Here the authors describe a novel high-throughput screening assay for identifying inhibitors of nuclear import, particularly IN, based on amplified luminescent proximity homogeneous assay (AlphaScreen) technol., which is high throughput, requires low amts. of material, and is efficient and cost-effective. The authors use the assay to screen for specific inhibitors of the interaction between IN and its nuclear transport receptor importin α/β, successfully identifying several inhibitors of the IN/importin α/β interaction. Importantly, they demonstrate that one of the identified compds., mifepristone, is effective in preventing active nuclear transport of IN in transfected cells and hence may represent a useful anti-HIV therapeutic. The screen also identified broad-spectrum importin α/β inhibitors such as ivermectin, which may represent useful tools for nuclear transport research in the future. The authors validate the activity and specificity of mifepristone and ivermectin in inhibiting nuclear protein import in living cells, underlining the utility of the screening approach.
- 24Wagstaff, K. M., Sivakumaran, H., Heaton, S. M., Harrich, D., and Jans, D. A. (2012) Ivermectin is a specific inhibitor of importin alpha/beta-mediated nuclear import able to inhibit replication of HIV-1 and dengue virus. Biochem. J. 443 (3), 851– 6, DOI: 10.1042/BJ20120150[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xls1Sht70%253D&md5=19879ceff24d84ea0f16f437f8c921fdIvermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of HIV-1 and dengue virusWagstaff, Kylie M.; Sivakumaran, Haran; Heaton, Steven M.; Harrich, David; Jans, David A.Biochemical Journal (2012), 443 (3), 851-856CODEN: BIJOAK; ISSN:0264-6021. (Portland Press Ltd.)The movement of proteins between the cytoplasm and nucleus mediated by the importin superfamily of proteins is essential to many cellular processes, including differentiation and development, and is crit. to disease states such as viral disease and oncogenesis. We recently developed a high-throughput screen to identify specific and general inhibitors of protein nuclear import, from which ivermectin was identified as a potential inhibitor of importin α/β-mediated transport. In the present study, we characterized in detail the nuclear transport inhibitory properties of ivermectin, demonstrating that it is a broad-spectrum inhibitor of importin α/β nuclear import, with no effect on a range of other nuclear import pathways, including that mediated by importin β1 alone. Importantly, we establish for the first time that ivermectin has potent antiviral activity towards both HIV-1 and dengue virus, both of which are strongly reliant on importin α/β nuclear import, with respect to the HIV-1 integrase and NS5 (non-structural protein 5) polymerase proteins resp. Ivermectin would appear to be an invaluable tool for the study of protein nuclear import, as well as the basis for future development of antiviral agents.
- 25Wulan, W. N., Heydet, D., Walker, E. J., Gahan, M. E., and Ghildyal, R. (2015) Nucleocytoplasmic transport of nucleocapsid proteins of enveloped RNA viruses. Front. Microbiol. 6, 553, DOI: 10.3389/fmicb.2015.00553[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MbksVWlsA%253D%253D&md5=c2bd7c868440b2aba4f3ad1cb267de81Nucleocytoplasmic transport of nucleocapsid proteins of enveloped RNA virusesWulan Wahyu N; Ghildyal Reena; Heydet Deborah; Walker Erin J; Gahan Michelle EFrontiers in microbiology (2015), 6 (), 553 ISSN:1664-302X.Most viruses with non-segmented single stranded RNA genomes complete their life cycle in the cytoplasm of infected cells. However, despite undergoing replication in the cytoplasm, the structural proteins of some of these RNA viruses localize to the nucleus at specific times in the virus life cycle, primarily early in infection. Limited evidence suggests that this enhances successful viral replication by interfering with or inhibiting the host antiviral response. Nucleocapsid proteins of RNA viruses have a well-established, essential cytoplasmic role in virus replication and assembly. Intriguingly, nucleocapsid proteins of some RNA viruses also localize to the nucleus/nucleolus of infected cells. Their nuclear function is less well understood although significant advances have been made in recent years. This review will focus on the nucleocapsid protein of cytoplasmic enveloped RNA viruses, including their localization to the nucleus/nucleolus and function therein. A greater understanding of the nuclear localization of nucleocapsid proteins has the potential to enhance therapeutic strategies as it can be a target for the development of live-attenuated vaccines or antiviral drugs.
- 26Yang, S. N. Y., Atkinson, S. C., Wang, C., Lee, A., Bogoyevitch, M. A., Borg, N. A., and Jans, D. A. (2020) The broad spectrum antiviral ivermectin targets the host nuclear transport importin alpha/beta1 heterodimer. Antiviral Res. 177, 104760, DOI: 10.1016/j.antiviral.2020.104760[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXntVKrsrY%253D&md5=5273353c8738f614375caab849315874The broad spectrum antiviral ivermectin targets the host nuclear transport importin α/β1 heterodimerYang, Sundy N. Y.; Atkinson, Sarah C.; Wang, Chunxiao; Lee, Alexander; Bogoyevitch, Marie A.; Borg, Natalie A.; Jans, David A.Antiviral Research (2020), 177 (), 104760CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)Infection by RNA viruses such as human immunodeficiency virus (HIV)-1, influenza, and dengue virus (DENV) represent a major burden for human health worldwide. Although RNA viruses replicate in the infected host cell cytoplasm, the nucleus is central to key stages of the infectious cycle of HIV-1 and influenza, and an important target of DENV nonstructural protein 5 (NS5) in limiting the host antiviral response. We previously identified the small mol. ivermectin as an inhibitor of HIV-1 integrase nuclear entry, subsequently showing ivermectin could inhibit DENV NS5 nuclear import, as well as limit infection by viruses such as HIV-1 and DENV. We show here that ivermectin's broad spectrum antiviral activity relates to its ability to target the host importin (IMP) α/β1 nuclear transport proteins responsible for nuclear entry of cargoes such as integrase and NS5. We establish for the first time that ivermectin can dissoc. the preformed IMPα/β1 heterodimer, as well as prevent its formation, through binding to the IMPα armadillo (ARM) repeat domain to impact IMPα thermal stability and α-helicity. We show that ivermectin inhibits NS5-IMPα interaction in a cell context using quant. bimol. fluorescence complementation. Finally, we show for the first time that ivermectin can limit infection by the DENV-related West Nile virus at low (μM) concns. Since it is FDA approved for parasitic indications, ivermectin merits closer consideration as a broad spectrum antiviral of interest.
- 27Singh, K. K., Chaubey, G., Chen, J. Y., and Suravajhala, P. (2020) Decoding SARS-CoV-2 hijacking of host mitochondria in COVID-19 pathogenesis. Am. J. Physiol Cell Physiol 319 (2), C258– C267, DOI: 10.1152/ajpcell.00224.2020
Supporting Information
Supporting Information
ARTICLE SECTIONSThe Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.0c00179.
Description of materials and methods, cell lines, chemicals, biochemicals, tables showing completed and ongoing clinical trial for different therapeutic options of COVID-19, and additional data (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.