Biology subjects

Moorman, N. J.

Publications and source records attributed to Moorman, N. J..

At least 19 recordsLinked to original sources

bioRxiv2026

No-known-vector flaviviruses exhibit diverse replication and virulence phenotypes in mice

The Orthoflavivirus genus (flaviviruses) includes globally significant arboviruses, which cycle between arthropod vectors (mosquitoes and ticks) and vertebrate hosts. In contrast, no-known-vector flaviviruses (NKVFVs) have been isolated from rodents and bats, but not arthropods, so are thought to spread by vector-independent routes. However, little is known about the host range and pathogenic mechanisms of these viruses. To evaluate NKVFV pathogenesis, we infected wild-type, Ifnar1-/-, and Ifnar1-/- Ifngr1-/- mice by footpad inoculation with 12 NKVFVs: Entebbe bat virus (ENTV), Sokuluk virus (SOKV), Yokose virus (YOKV), Modoc virus (MODV), Apoi virus (APOIV), Jutiapa virus (JUTV), Sal Vieja virus (SVV), Dakar bat virus (DBV), Rio Bravo virus (RBV), Montana myotis leukoencephalitis virus (MMLV), Phnom Penh bat virus (PPBV), and Tamana bat virus (TABV). We compared these NKVFVs to the mosquito-borne Zika virus and Kedougou virus and to the tick-borne Langat virus and Kadam virus. We monitored disease signs and measured viremia. 7 NKVFVs (ENTV, SOKV, YOKV, MODV, APOIV, DBV, RBV) were virulent in Ifnar1-/- mice, causing 100% lethality within 9 dpi, accompanied by viremia. All viruses tested were virulent in Ifnar1-/- Ifngr1-/- mice and produced greater viremia compared to Ifnar1-/- mice. No viremia or disease signs were detected in wild-type mice. We further evaluated RBV, MMLV, and PPBV replication in mouse primary fibroblasts and bone marrow-derived macrophages, as well as in cell lines from three bat species. Altogether, our results provide new information about the virulence and replication phenotypes of NKVFVs, supporting future studies investigating NKVFV-specific and pan-flavivirus pathogenic mechanisms. IMPORTANCEFlaviviruses that cause human disease are transmitted by mosquitoes and ticks (e.g. West Nile virus, yellow fever virus, tick-borne encephalitis virus). But there are related flaviviruses that are not known to infect arthropods, so are thought to spread by vector-independent routes (no-known-vector flaviviruses, NKVFVs). Not much is known about NKVFVs, but they provide an opportunity to understand flavivirus replication, tropism, and pathogenesis more broadly. We evaluated a panel of 12 NKVFVs for their ability to cause disease in mice with and without antiviral interferon responses as well as their replication in mouse cells. Our findings provide new information about these under-studied viruses and demonstrate which mouse models may be appropriate to use for further studies with NKVFVs.

microbiology↗

bioRxiv2025

Functional Roles for RNA Ribosylation in Mammalian Cells

In mammalian cells, the addition of ADP-ribose to proteins and DNA plays well established roles in regulating cell function. Recently, RNA ribosylation was also found in mammalian cells under conditions of cell stress, though the functional consequences remain unclear. Here we find that infection with chikungunya virus, a positive strand RNA virus that causes frequent widespread epidemics, increases overall levels of RNA ribosylation in human fibroblasts. During infection, viral RNA is ribosylated by the PARP12 ribosyltransferase, which is counteracted by a virally-encoded. Increased viral RNA ribosylation resulted in decreased translation in cell-free systems and infected fibroblasts, and more rapid viral RNA decay. Further, ribosylated RNA potently induced the expression of antiviral host response genes. Together these data show the first functional consequences of RNA ribosylation in mammalian cells by showing that RNA ribosylation inhibits translation, decreases RNA stability, and creates a novel pathogen-associated molecular pattern (PAMP) that activates the host innate immune response. As macrodomains are present in multiple unrelated viruses, our data suggest RNA ribosylation is a novel component of cellular antiviral sensing pathway. These results also provide a starting point for defining functional roles for RNA ribosylation in other mammalian cell stress conditions beyond viral infection.

cell biology↗

bioRxiv2025

N-Alkyl Sulfamates as a New Class of nsP2 Cysteine Protease Inhibitors with Broad Spectrum Antialphaviral Activity

The emergence of mosquito-borne alphaviruses that cause chronic arthritis or encephalitis underscores the urgent need for broad-spectrum antiviral therapeutics. The viral nsP2 cysteine protease, which is essential for alphavirus replication, is a promising antiviral target. Vinyl sulfone-based inhibitors, such as RA-2034, potently inhibit nsP2 protease but suffer from glutathione reactivity and species-dependent systemic clearance catalyzed by glutathione S-transferase. To address these liabilities, we explored alternative electrophilic warheads and identified reverse amide inhibitors bearing N-alkyl sulfamate warheads with improved biochemical and antiviral profiles. N-methyl sulfamate acetamide 5 emerged as a lead compound with potency against both New and Old World alphaviruses, low GSH reactivity, and high proteome-wide selectivity. Despite its promising antialphaviral activity, 5 exhibited rapid clearance due to hepatic glucuronidation. Structure-activity studies revealed modifications that improve metabolic stability while retaining antiviral activity. These findings introduce sulfamate acetamides as a new class of covalent nsP2 protease inhibitors and advance the discovery of direct acting pan-alphavirus drugs.

pharmacology and toxicology↗

bioRxiv2025

Identification of a Broadly Acting Inhibitor of the Alphavirus Non-Structural Protein 2 Helicase

Alphaviruses are mosquito-borne viruses that have caused significant outbreaks in the 21st century. Despite multiple recent outbreaks, there are no approved antiviral drugs to treat any alphavirus infection. Therefore, developing broadly acting antiviral drugs effective against multiple alphaviruses is necessary and could provide protection from both current and emerging alphavirus threats. A critical component of the alphavirus replication complex is non-structural protein 2 (nsP2), which is a multifunctional enzyme containing a helicase domain connected to a protease domain by a flexible linker. nsP2 functions as an ATP-dependent helicase, is conserved across the alphavirus genus, and is essential for virus replication, making it a promising target for development of alphavirus broad-acting antiviral drugs. Previous studies identified an enantioselective compound RA-0025298 that inhibited nsP2 ATPase activity and chikungunya virus CHIKV replication. Antiviral testing of RA-25298. against a diverse group of alphaviruses found broad activity except for Sindbis-like viruses. Using this information along with mutational profiling of virus passaged with RA-0025298 we identified the site of RA-0025298 action and confirmed the binding site via biophysical analyses. Finally, we found that the active enantiomer of RA-0025298 (SGC-NSP2hel-1) reduced viral loads in vivo and protected mice from tissue damage and disease caused by CHIKV infection. These findings further describe the mechanism of action of a first-in-class nsP2 helicase inhibitor with the potential for development as a broad spectrum drug for treating or preventing disease caused by current and emerging alphaviruses. One Sentence SummaryThis study describes the mechanism of action and in vivo efficacy of a first in class broadly acting inhibitor of alphavirus nsP2 helicase activity.

microbiology↗

bioRxiv2025

An Enantioselective Chemical Probe for Chikungunya nsP2 Helicase with Antialphaviral Activity

Chikungunya virus (CHIKV) replication relies on the multifunctional nsP2 protein, making it an attractive target for antiviral drug discovery. Here, we report the resolution of oxaspiropiperidine 1, a first-in-class inhibitor of the CHIKV nsP2 RNA helicase (nsP2hel), into its constitutive enantiomers and characterization of their antiviral activity. The enantiomer (R)-1 exhibited potent inhibition of viral replication, nsP2hel ATPase activity, and dsRNA unwinding, while the (S)-1 enantiomer was >100-fold less active. The (R)-1 enantiomer also demonstrated high selectivity for CHIKV over other RNA viruses and for nsP2hel over other RNA helicases. Direct binding of (R)-1 to nsP2hel protein was confirmed by 19F NMR. Biophysical and structural studies revealed conformational polymorphism in the spirocyclic scaffold of (R)-1, suggesting a potential role of thermal mobility of the ligand in allosteric inhibition of nsP2hel. Collectively, these findings designate (R)-1 (RA-NSP2-1) as a high-quality chemical probe and (S)-1 (RA-NSP2-1N) as a negative control for probing the biology of alphavirus RNA helicases.

pharmacology and toxicology↗

bioRxiv2025

Identification of Direct-acting nsP2 Helicase Inhibitors with Anti-alphaviral Activity

Alphaviruses are mosquito-borne RNA viruses that pose a significant public health threat, with no FDA-approved antiviral therapeutics available. The non-structural protein 2 helicase (nsP2hel) is an enzyme involved in unwinding dsRNA essential for alphavirus replication. This study reports the discovery and optimization of first-in-class oxaspiropiperidine inhibitors targeting nsP2hel. Structure-activity relationship (SAR) studies identified potent cyclic sulfonamide analogs with nanomolar antiviral activity against chikungunya virus (CHIKV). Biochemical analyses of nsP2hel ATPase and RNA unwindase activities showed these compounds act by a non-competitive mode suggesting that they are allosteric inhibitors. Viral resistance mutations mapped to nsP2hel and a fluorine-labeled analog exhibited direct binding to the protein by 19F NMR. The lead inhibitor, 2o, demonstrated broad-spectrum antialphaviral activity, reducing titers of CHIKV, Mayaro virus (MAYV), and Venezuelan equine encephalitis virus (VEEV). These findings support nsP2hel as a viable target for development of broad-spectrum direct-acting antialphaviral drugs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/641060v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@856df5org.highwire.dtl.DTLVardef@1f6225borg.highwire.dtl.DTLVardef@4997d5org.highwire.dtl.DTLVardef@18f42f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Species Dependent Metabolism of a Covalent nsP2 Protease Inhibitor with in Vivo Anti-alphaviral Activity

RA-0002034 (1) is a potent covalent inhibitor targeting the alphavirus nsP2 cysteine protease. The species-dependent pharmacokinetics and metabolism of 1 were investigated to evaluate its therapeutic potential. Pharmacokinetic profiling revealed rapid clearance in mice, predominantly mediated by glutathione S-transferase (GST)-catalyzed conjugation. This metabolic liability contrasted with slower clearance observed in human hepatocytes and preclinical species such as rats, dogs, and monkeys. Cross-species studies confirmed the dominance of GST-driven metabolism in mice, whereas oxidative pathways were more pronounced in dogs. Despite rapid systemic clearance, 1 achieved antiviral efficacy in mice, reducing CHIKV viral loads in multiple tissues. Initial estimations of human hepatic clearance and half-life extrapolated from animal data indicate that b.i.d. dosing of 1 will be possible to maintain concentrations sufficient for antiviral activity in humans. These cross-species pharmacokinetic and metabolism studies support the continued evaluation of 1 as a promising anti-alphaviral therapeutic. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/632788v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@8c1558org.highwire.dtl.DTLVardef@cd7e21org.highwire.dtl.DTLVardef@113e88aorg.highwire.dtl.DTLVardef@7c7dc_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2024

The Human Cytomegalovirus vGPCR UL33 is Essential for Efficient Lytic Replication in Epithelial Cells

Human cytomegalovirus (HCMV) is a {beta}-herpesvirus which is ubiquitous in the human population. HCMV has the largest genome of all known human herpesviruses, and thus encodes a large array of proteins that affect pathogenesis in different cell types. Given the large genome and the ability of HCMV to replicate in a range of cells, investigators have begun to identify viral proteins required for cell type-specific replication. There are four proteins encoded in the HCMV genome that are homologous to human G protein-coupled receptors (GPCRs); these viral-encoded GPCRs (vGPCRs) are UL33, UL78, US27, and US28. In the current study, we find that deletion of all four vGPCR genes from a clinical isolate of HCMV severely attenuates lytic replication in both primary human salivary gland epithelial cells, as well as ARPE-19 retinal epithelial cells as evidenced by significant decreases in immediate early gene expression and virus production. Deletion of UL33 from the HCMV genome also results in a failure to efficiently replicate in epithelial cells, and this defect is manifested by decreased levels of immediate early, early, and late gene expression, as well as reduced viral production. We find that similar to US28, UL33 constitutively activates Gq-dependent PLC-{beta} signaling to high levels in these epithelial cells. We also find that UL33 transcription is more complicated than originally believed, and there is the potential for the virus to utilize various 5 UTRs to create novel UL33 proteins that are all capable of constitutive Gq signaling. Taken together, these studies suggest that UL33 driven signaling is important for lytic HCMV replication in cells of epithelial origin.

microbiology↗

bioRxiv2024

Structure Activity of β-Amidomethyl Vinyl Sulfones as Covalent Inhibitors of Chikungunya nsP2 Cysteine Protease with Anti-alphavirus Activity

Despite their widespread impact on human health there are no approved drugs for combating alphavirus infections. The heterocyclic {beta}-aminomethyl vinyl sulfone RA-0002034 (1a) is a potent irreversible covalent inhibitor of the alphavirus nsP2 cysteine protease with broad spectrum antiviral activity. Analogs of 1a that varied each of three regions of the molecule were synthesized to establish structure-activity relationships for inhibition of Chikungunya (CHIKV) nsP2 protease and viral replication. The covalent warhead was highly sensitive to modifications of the sulfone or vinyl substituents. However, numerous alterations to the core 5-membered heterocycle and its aryl substituent were well tolerated and several analogs were identified that enhanced CHIKV nsP2 binding. For example, the 4-cyanopyrazole analog 8d exhibited a kinact/Ki ratio >10,000 M-1s-1. 3-Arylisoxazole was identified an isosteric replacement for the 5-membered heterocycle, which circumvented the intramolecular cyclization that complicated the synthesis of pyrazole-based inhibitors like 1a. The accumulated structure-activity data was used to build a ligand-based model of the enzyme active site, which can be used to guide the design of covalent nsP2 protease inhibitors as potential therapeutics against alphaviruses.

cell biology↗

bioRxiv2024

Discovery of a cell-active chikungunya virus nsP2 protease inhibitor using a covalent fragment-based screening approach

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has been responsible for numerous large-scale outbreaks in the last twenty years. Currently, there are no FDA-approved therapeutics for any alphavirus infection. CHIKV non-structural protein 2 (nsP2), which contains a cysteine protease domain, is essential for viral replication, making it an attractive target for a drug discovery campaign. Here, we optimized a CHIKV nsP2 protease (nsP2pro) biochemical assay for the screening of a 6,120-compound cysteine-directed covalent fragment library. Using a 50% inhibition threshold, we identified 153 hits (2.5% hit rate). In dose-response follow up, RA-0002034, a covalent fragment that contains a vinyl sulfone warhead, inhibited CHIKV nsP2pro with an IC50 of 58 {+/-} 17 nM, and further analysis with time-dependent inhibition studies yielded a kinact/KI of 6.4 x 103 M-1s-1. LC-MS/MS analysis determined that RA-0002034 covalently modified the catalytic cysteine in a site-specific manner. Additionally, RA-0002034 showed no significant off-target reactivity against a panel of cysteine proteases. In addition to the potent biochemical inhibition of CHIKV nsP2pro activity and exceptional selectivity, RA-0002034 was tested in cellular models of alphavirus infection and effectively inhibited viral replication of both CHIKV and related alphaviruses. This study highlights the discovery and characterization of the chemical probe RA-0002034 as a promising hit compound from covalent fragment-based screening for development toward a CHIKV or pan-alphavirus therapeutic. Significance StatementChikungunya virus is one of the most prominent and widespread alphaviruses and has caused explosive outbreaks of arthritic disease. Currently, there are no FDA-approved drugs to treat disease caused by chikungunya virus or any other alphavirus-caused infection. Here, we report the discovery of a covalent small molecule inhibitor of chikungunya virus nsP2 protease activity and viral replication of four diverse alphaviruses. This finding highlights the utility of covalent fragment screening for inhibitor discovery and represents a starting point towards the development of alphavirus therapeutics targeting nsP2 protease.

biochemistry↗

bioRxiv2024

Synthesis of 5-Benzylamino and 5-Alkylamino-substituted Pyrimidoquinoline Derivatives as CSNK2A Inhibitors with Antiviral Activity

A series of 5-benzylamine-substituted pyrimido[4,5-c]quinoline derivatives of the CSNK2A chemical probe SGC-CK2-2 were synthesized with the goal of improving kinase inhibitor cellular potency and antiviral phenotypic activity while maintaining aqueous solubility. Among the range of analogs, those bearing electron-withdrawing (4c and 4g) or donating (4f) substituents on the benzyl ring as well as introduction of non-aromatic groups such as the cyclohexylmethyl (4t) were shown to maintain CSNK2A activity. The CSNK2A activity was also retained with N-methylation of SGC-CK2-2, but -methyl substitution of the benzyl substituent led to a 10-fold reduction in potency. CSNK2A inhibition potency was restored with indene-based compound 4af, with activity residing in the S-enantiomer (4ag). Analogs with the highest CSNK2A potency showed good activity for inhibition of Mouse Hepatitis Virus (MHV) replication. Conformational analysis indicated that analogs with the best CSNK2A inhibition (4t, 4ac, and 4af) exhibited smaller differences between their ground state conformation and their predicted binding pose. Analogs with reduced activity (4ad, 4ae, and 4ai) required more substantial conformational changes from their ground state within the CSNK2A protein pocket.

cell biology↗

bioRxiv2023

Identification of 2-(4'-carboxyphenyl)-6-indolopyrazines as CSNK2A inhibitors with antiviral activity and improved selectivity over PIM3

We report the synthesis of 2,6-disubstituted pyrazines as potent cell active CSNK2A inhibitors. 4-Carboxyphenyl was found to be the optimal 2-pyrazine substituent for CSNK2A activity, with little tolerance for additional modification. At the 6-position, modifications of the 6-isopropylaminoindazole substituent were explored to improve selectivity over PIM3 while maintaining potent CSNK2A inhibition. The 6-isopropoxyindole analogue 6c was identified as a nanomolar CSNK2A inhibitor with 30-fold selectivity over PIM3 in cells. Replacement of the 6-isopropoxyindole by isosteric ortho-methoxy anilines, such as 7c, generated analogues with selectivity for CSNK2A over PIM3 and improved the kinome-wide selectivity. The optimized 2,6-disubstituted pyrazines showed inhibition of viral replication consistent with their CSNK2A activity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/569845v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1b0a36corg.highwire.dtl.DTLVardef@1b940bdorg.highwire.dtl.DTLVardef@14953d9org.highwire.dtl.DTLVardef@11fd67a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Optimization of 3-Cyano-7-cyclopropylamino-pyrazolopyrimidines Toward the Development of an In Vivo Chemical Probe for CSNK2

3-cyano-7-cyclopropylamino-pyrazolo[1,5-a]pyrimidines, including the chemical probe SGC-CK2-1, are potent and selective inhibitors of CSNK2A in cells but have limited utility in animal models due to their poor pharmacokinetic properties. While developing analogs with reduced intrinsic clearance and the potential for sustained exposure in mice, we discovered that Phase II conjugation by GST enzymes was a major metabolic transformation in hepatocytes. A protocol for co-dosing with ethacrynic acid, a covalent reversible GST inhibitor, was developed to improve the exposure of analog 2h in mice. A double co-dosing protocol, using a combination of ethacrynic acid and irreversible P450 inhibitor 1-aminobenzotriazole increased the blood level of 2h by 40-fold at a 5 h time point. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/540828v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1ae672borg.highwire.dtl.DTLVardef@f7777corg.highwire.dtl.DTLVardef@c5aae8org.highwire.dtl.DTLVardef@1579f2f_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2023

Human cytomegalovirus attenuates AKT activity by destabilizing insulin receptor substrate proteins

The phosphoinositide 3-kinase (PI3K)/AKT pathway plays crucial roles in cell viability and protein synthesis and is frequently co-opted by viruses to support their replication. Although many viruses maintain high levels of AKT activity during infection, other viruses, such as vesicular stomatitis virus and human cytomegalovirus (HCMV), cause AKT to accumulate in an inactive state. To efficiently replicate, HCMV requires FoxO transcription factors to localize to the infected cell nucleus (Zhang et. al. mBio 2022), a process directly antagonized by AKT. Therefore, we sought to investigate how HCMV inactivates AKT to achieve this. Subcellular fractionation and live cell imaging studies indicated that AKT failed to recruit to membranes upon serum-stimulation of infected cells. However, UV-inactivated virions were unable to render AKT non-responsive to serum, indicating a requirement for de novo viral gene expression. Interestingly, we were able to identify that UL38 (pUL38), a viral activator of mTORC1, is required to diminish AKT responsiveness to serum. mTORC1 contributes to insulin resistance by causing proteasomal degradation of insulin receptor substrate (IRS) proteins, such as IRS1, which are necessary for the recruitment of PI3K to growth factor receptors. In cells infected with a recombinant HCMV disrupted for UL38, AKT responsiveness to serum is retained and IRS1 is not degraded. Furthermore, ectopic expression of UL38 in uninfected cells induces IRS1 degradation, inactivating AKT. These effects of UL38 were reversed by the mTORC1 inhibitor, rapamycin. Collectively, our results demonstrate that HCMV relies upon a cell-intrinsic negative feedback loop to render AKT inactive during productive infection.

microbiology↗

bioRxiv2022

Targeted Down Regulation Of Core Mitochondrial Genes During SARS-CoV-2 Infection

Defects in mitochondrial oxidative phosphorylation (OXPHOS) have been reported in COVID-19 patients, but the timing and organs affected vary among reports. Here, we reveal the dynamics of COVID-19 through transcription profiles in nasopharyngeal and autopsy samples from patients and infected rodent models. While mitochondrial bioenergetics is repressed in the viral nasopharyngeal portal of entry, it is up regulated in autopsy lung tissues from deceased patients. In most disease stages and organs, discrete OXPHOS functions are blocked by the virus, and this is countered by the host broadly up regulating unblocked OXPHOS functions. No such rebound is seen in autopsy heart, results in severe repression of genes across all OXPHOS modules. Hence, targeted enhancement of mitochondrial gene expression may mitigate the pathogenesis of COVID-19. One-Sentence SummaryCovid-19 is associated with targeted inhibition of mitochondrial gene transcription.

molecular biology↗

bioRxiv2022

Host kinase CSNK2 is a target for inhibition of pathogenic β-coronaviruses including SARS-CoV-2

Inhibition of the protein kinase CSNK2 with any of 30 specific and selective inhibitors representing different chemotypes, blocked replication of pathogenic human and murine {beta}-coronaviruses. The potency of in-cell CSNK2A target engagement across the set of inhibitors correlated with antiviral activity and genetic knockdown confirmed the essential role of the CSNK2 holoenzyme in {beta}-coronavirus replication. Spike protein uptake was blocked by CSNK2A inhibition, indicating that antiviral activity was due in part to a suppression of viral entry. CSNK2A inhibition may be a viable target for development of new broad spectrum anti-{beta}-coronavirus drugs. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/474779v3_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@5d2799org.highwire.dtl.DTLVardef@1d2de35org.highwire.dtl.DTLVardef@fa852eorg.highwire.dtl.DTLVardef@13da300_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Vandetanib Reduces Inflammatory Cytokines and Ameliorates COVID-19 in Infected Mice

The portfolio of SARS-CoV-2 small molecule drugs is currently limited to a handful that are either approved (remdesivir), emergency approved (dexamethasone, baricitinib) or in advanced clinical trials. We have tested 45 FDA-approved kinase inhibitors in vitro against murine hepatitis virus (MHV) as a model of SARS-CoV-2 replication and identified 12 showing inhibition in the delayed brain tumor (DBT) cell line. Vandetanib, which targets the vascular endothelial growth factor receptor (VEGFR), the epidermal growth factor receptor (EGFR), and the RET-tyrosine kinase showed the most promising results on inhibition versus toxic effect on SARS-CoV-2-infected Caco-2 and A549-hACE2 cells (IC50 0.79 M) while also showing a reduction of > 3 log TCID50/mL for HCoV-229E. The in vivo efficacy of vandetanib was assessed in a mouse model of SARS-CoV-2 infection and statistically significantly reduced the levels of IL-6, IL-10, TNF-, and mitigated inflammatory cell infiltrates in the lungs of infected animals but did not reduce viral load. Vandetanib rescued the decreased IFN-1{beta} caused by SARS-CoV-2 infection in mice to levels similar to that in uninfected animals. Our results indicate that the FDA-approved vandetanib is a potential therapeutic candidate for COVID-19 positioned for follow up in clinical trials either alone or in combination with other drugs to address the cytokine storm associated with this viral infection.

immunology↗

bioRxiv2020

Repurposing the Ebola and Marburg Virus Inhibitors Tilorone, Quinacrine and Pyronaridine: In vitro Activity Against SARS-CoV-2 and Potential Mechanisms

SARS-CoV-2 is a newly identified virus that has resulted in over 1.3 M deaths globally and over 59 M cases globally to date. Small molecule inhibitors that reverse disease severity have proven difficult to discover. One of the key approaches that has been widely applied in an effort to speed up the translation of drugs is drug repurposing. A few drugs have shown in vitro activity against Ebola virus and demonstrated activity against SARS-CoV-2 in vivo. Most notably the RNA polymerase targeting remdesivir demonstrated activity in vitro and efficacy in the early stage of the disease in humans. Testing other small molecule drugs that are active against Ebola virus would seem a reasonable strategy to evaluate their potential for SARS-CoV-2. We have previously repurposed pyronaridine, tilorone and quinacrine (from malaria, influenza, and antiprotozoal uses, respectively) as inhibitors of Ebola and Marburg virus in vitro in HeLa cells and of mouse adapted Ebola virus in mouse in vivo. We have now tested these three drugs in various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7 and monocytes) infected with SARS-CoV-2 as well as other viruses (including MHV and HCoV 229E). The compilation of these results indicated considerable variability in antiviral activity observed across cell lines. We found that tilorone and pyronaridine inhibited the virus replication in A549-ACE2 cells with IC50 values of 180 nM and IC50 198 nM, respectively. We have also tested them in a pseudovirus assay and used microscale thermophoresis to test the binding of these molecules to the spike protein. They bind to spike RBD protein with Kd values of 339 nM and 647 nM, respectively. Human Cmax for pyronaridine and quinacrine is greater than the IC50 hence justifying in vivo evaluation. We also provide novel insights into their mechanism which is likely lysosomotropic.

pharmacology and toxicology↗