Combination of antiviral drugs inhibits SARS-CoV-2 polymerase and exonuclease and demonstrates COVID-19 therapeutic potential in viral cell culture.

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.

Activity of Galidesivir in a Hamster Model of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) has claimed the lives of millions of people worldwide since it first emerged. The impact of the COVID-19 pandemic on public health and the global economy has highlighted the medical need for the development of broadly acting interventions against emerging viral threats. Galidesivir is a broad-spectrum antiviral compound with demonstrated in vitro and in vivo efficacy against several RNA viruses of public health concern, including those causing yellow fever, Ebola, Marburg, and Rift Valley fever. In vitro studies have shown that the antiviral activity of galidesivir also extends to coronaviruses. Herein, we describe the efficacy of galidesivir in the Syrian golden hamster model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Treatment with galidesivir reduced lung pathology in infected animals compared with untreated controls when treatment was initiated 24 h prior to infection. These results add to the evidence of the applicability of galidesivir as a potential medical intervention for a range of acute viral illnesses, including coronaviruses.

Elevation in sphingolipid upon SARS-CoV-2 infection: possible implications for COVID-19 pathology.

Understanding pathways that might impact coronavirus disease 2019 (COVID-19) manifestations and disease outcomes is necessary for better disease management and for therapeutic development. Here, we analyzed alterations in sphingolipid (SL) levels upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infection induced elevation of SL levels in both cells and sera of infected mice. A significant increase in glycosphingolipid levels was induced early post SARS-CoV-2 infection, which was essential for viral replication. This elevation could be reversed by treatment with glucosylceramide synthase inhibitors. Levels of sphinganine, sphingosine, GA1, and GM3 were significantly increased in both cells and the murine model upon SARS-CoV-2 infection. The potential involvement of SLs in COVID-19 pathology is discussed.

A Novel Phenylpyrrolidine Derivative: Synthesis and Effect on Cognitive Functions in Rats with Experimental Ishemic Stroke.

We performed an in silico, in vitro, and in vivo assessment of a potassium 2-[2-(2-oxo-4-phenylpyrrolidin-1-yl) acetamido]ethanesulfonate (compound 1) as a potential prodrug for cognitive function improvement in ischemic brain injury. Using in silico methods, we predicted the pharmacological efficacy and possible safety in rat models. In addition, in silico data showed neuroprotective features of compound 1, which were further supported by in vitro experiments in a glutamate excitotoxicity-induced model in newborn rat cortical neuron cultures. Next, we checked whether compound 1 is capable of crossing the blood-brain barrier in intact and ischemic animals. Compound 1 improved animal behavior both in intact and ischemic rats and, even though the concentration in intact brains was low, we still observed a significant anxiety reduction and activity escalation. We used molecular docking and molecular dynamics to support our hypothesis that compound 1 could affect the AMPA receptor function. In a rat model of acute focal cerebral ischemia, we studied the effects of compound 1 on the behavior and neurological deficit. An in vivo experiment demonstrated that compound 1 significantly reduced the neurological deficit and improved neurological symptom regression, exploratory behavior, and anxiety. Thus, here, for the first time, we show that compound 1 can be considered as an agent for restoring cognitive functions.

Effect of drug metabolism in the treatment of SARS-CoV-2 from an entirely computational perspective.

Understanding the effects of metabolism on the rational design of novel and more effective drugs is still a considerable challenge. To the best of our knowledge, there are no entirely computational strategies that make it possible to predict these effects. From this perspective, the development of such methodologies could contribute to significantly reduce the side effects of medicines, leading to the emergence of more effective and safer drugs. Thereby, in this study, our strategy is based on simulating the electron ionization mass spectrometry (EI-MS) fragmentation of the drug molecules and combined with molecular docking and ADMET models in two different situations. In the first model, the drug is docked without considering the possible metabolic effects. In the second model, each of the intermediates from the EI-MS results is docked, and metabolism occurs before the drug accesses the biological target. As a proof of concept, in this work, we investigate the main antiviral drugs used in clinical research to treat COVID-19. As a result, our strategy made it possible to assess the biological activity and toxicity of all potential by-products. We believed that our findings provide new chemical insights that can benefit the rational development of novel drugs in the future.

A fluorescence-based, gain-of-signal, live cell system to evaluate SARS-CoV-2 main protease inhibition.

The likelihood of continued circulation of COVID-19 and its variants, and novel coronaviruses due to future zoonotic transmissions, combined with the current paucity of coronavirus antivirals, emphasize the need for improved screening in developing effective antivirals for the treatment of infection by SARS-CoV-2 (CoV2) and other coronaviruses. Here we report the development of a live-cell based assay for evaluating the intracellular function of the critical, highly-conserved CoV2 target, the Main 3C-like protease (Mpro). This assay is based on expression of native wild-type mature CoV2 Mpro, the function of which is quantitatively evaluated in living cells through cleavage of a biosensor leading to loss of fluorescence. Evaluation does not require cell harvesting, allowing for multiple measurements from the same cells facilitating quantification of Mpro inhibition, as well as recovery of function upon removal of inhibitory drugs. The pan-coronavirus Mpro inhibitor, GC376, was utilized in this assay and effective inhibition of intracellular CoV2 Mpro was found to be consistent with levels required to inhibit CoV2 infection of human lung cells. We demonstrate that GC376 is an effective inhibitor of intracellular CoV2 Mpro at low micromolar levels, while other predicted Mpro inhibitors, bepridil and alverine, are not. Results indicate this system can provide a highly effective high-throughput coronavirus Mpro screening system.

Viral polymerase binding and broad-spectrum antiviral activity of molnupiravir against human seasonal coronaviruses.

Endemic seasonal coronaviruses cause morbidity and mortality in a subset of patients, but no specific treatment is available. Molnupiravir is a promising pipeline antiviral drug for treating SARS-CoV-2 infection potentially by targeting RNA-dependent RNA polymerase (RdRp). This study aims to evaluate the potential of repurposing molnupiravir for treating seasonal human coronavirus (HCoV) infections. Molecular docking revealed that the active form of molnupiravir, ß-D-N4-hydroxycytidine (NHC), has similar binding affinity to RdRp of SARS-CoV-2 and seasonal HCoV-NL63, HCoV-OC43 and HCoV-229E. In cell culture models, treatment of molnupiravir effectively inhibited viral replication and production of infectious viruses of the three seasonal coronaviruses. A time-of-drug-addition experiment indicates the specificity of molnupiravir in inhibiting viral components. Furthermore, combining molnupiravir with the protease inhibitor GC376 resulted in enhanced antiviral activity. Our findings highlight that the great potential of repurposing molnupiravir for treating seasonal coronavirus infected patients.

An update on the progress of galidesivir (BCX4430), a broad-spectrum antiviral.

Galidesivir (BCX4430) is an adenosine nucleoside analog that is broadly active in cell culture against several RNA viruses of various families. This activity has also been shown in animal models of viral disease associated with Ebola, Marburg, yellow fever, Zika, and Rift Valley fever viruses. In many cases, the compound is more efficacious in animal models than cell culture activity would predict. Based on favorable data from in vivo animal studies, galidesivir has recently undergone evaluation in several phase I clinical trials, including against severe acute respiratory syndrome coronavirus 2, and as a medical countermeasure for the treatment of Marburg virus disease.

GC-MS, LC-MS/MS, Docking and Molecular Dynamics Approaches to Identify Potential SARS-CoV-2 3-Chymotrypsin-Like Protease Inhibitors from Zingiber officinale Roscoe.

This study aims to identify and isolate the secondary metabolites of Zingiber officinale using GC-MS, preparative TLC, and LC-MS/MS methods, to evaluate the inhibitory potency on SARS-CoV-2 3 chymotrypsin-like protease enzyme, as well as to study the molecular interaction and stability by using docking and molecular dynamics simulations. GC-MS analysis suggested for the isolation of terpenoids compounds as major compounds on methanol extract of pseudostems and rhizomes. Isolation and LC-MS/MS analysis identified 5-hydro-7, 8, 2'-trimethoxyflavanone (9), (E)-hexadecyl-ferulate (1), isocyperol (2), N-isobutyl-(2E,4E)-octadecadienamide (3), and nootkatone (4) from the rhizome extract, as well as from the leaves extract with the absence of 9. Three known steroid compounds, i.e., spinasterone (7), spinasterol (8), and 24-methylcholesta-7-en-3ß-on (6), were further identified from the pseudostem extract. Molecular docking showed that steroids compounds 7, 8, and 6 have lower predictive binding energies (MMGBSA) than other metabolites with binding energy of -87.91, -78.11, and -68.80 kcal/mole, respectively. Further characterization on the single isolated compound by NMR showed that 6 was identified and possessed 75% inhibitory activity on SARS-CoV-2 3CL protease enzyme that was slightly different with the positive control GC376 (77%). MD simulations showed the complex stability with compound 6 during 100 ns simulation time.

Qing-Fei-Pai-Du decoction and wogonoside exert anti-inflammatory action through down-regulating USP14 to promote the degradation of activating transcription factor 2.

COVID-19 is often characterized by dysregulated inflammatory and immune responses. It has been shown that the Traditional Chinese Medicine formulation Qing-Fei-Pai-Du decoction (QFPDD) is effective in the treatment of the disease, especially for patients in the early stage. Our network pharmacology analyses indicated that many inflammation and immune-related molecules were the targets of the active components of QFPDD, which propelled us to examine the effects of the decoction on inflammation. We found in the present study that QFPDD effectively alleviated dextran sulfate sodium-induced intestinal inflammation in mice. It inhibited the production of pro-inflammatory cytokines IL-6 and TNFα, and promoted the expression of anti-inflammatory cytokine IL-10 by macrophagic cells. Further investigations found that QFPDD and one of its active components wogonoside markedly reduced LPS-stimulated phosphorylation of transcription factor ATF2, an important regulator of multiple cytokines expression. Our data revealed that both QFPDD and wogonoside decreased the half-life of ATF2 and promoted its proteasomal degradation. Of note, QFPDD and wogonoside down-regulated deubiquitinating enzyme USP14 along with inducing ATF2 degradation. Inhibition of USP14 with the small molecular inhibitor IU1 also led to the decrease of ATF2 in the cells, indicating that QFPDD and wogonoside may act through regulating USP14 to promote ATF2 degradation. To further assess the importance of ubiquitination in regulating ATF2, we generated mice that were intestinal-specific KLHL5 deficiency, a CUL3-interacting protein participating in substrate recognition of E3s. In these mice, QFPDD mitigated inflammatory reaction in the spleen, but not intestinal inflammation, suggesting CUL3-KLHL5 may function as an E3 for ATF2 degradation.

A molecular modelling approach for identifying antiviral selenium-containing heterocyclic compounds that inhibit the main protease of SARS-CoV-2: an in silico investigation.

Coronavirus disease 2019 (COVID-19), an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been declared a global pandemic by the World Health Organization, and the situation worsens daily, associated with acute increases in case fatality rates. The main protease (Mpro) enzyme produced by SARS-CoV-2 was recently demonstrated to be responsible for not only viral reproduction but also impeding host immune responses. The element selenium (Se) plays a vital role in immune functions, both directly and indirectly. Thus, we hypothesised that Se-containing heterocyclic compounds might curb the activity of SARS-CoV-2 Mpro. We performed a molecular docking analysis and found that several of the selected selenocompounds showed potential binding affinities for SARS-CoV-2 Mpro, especially ethaselen (49), which exhibited a docking score of -6.7 kcal/mol compared with the -6.5 kcal/mol score for GC376 (positive control). Drug-likeness calculations suggested that these compounds are biologically active and possess the characteristics of ideal drug candidates. Based on the binding affinity and drug-likeness results, we selected the 16 most effective selenocompounds as potential anti-COVID-19 drug candidates. We also validated the structural integrity and stability of the drug candidate through molecular dynamics simulation. Using further in vitro and in vivo experiments, we believe that the targeted compound identified in this study (ethaselen) could pave the way for the development of prospective drugs to combat SARS-CoV-2 infections and trigger specific host immune responses.

Redox-Mediated Artificial Non-Enzymatic Antioxidant MXene Nanoplatforms for Acute Kidney Injury Alleviation.

Acute kidney injury (AKI), as a common oxidative stress-related renal disease, causes high mortality in clinics annually, and many other clinical diseases, including the pandemic COVID-19, have a high potential to cause AKI, yet only rehydration, renal dialysis, and other supportive therapies are available for AKI in the clinics. Nanotechnology-mediated antioxidant therapy represents a promising therapeutic strategy for AKI treatment. However, current enzyme-mimicking nanoantioxidants show poor biocompatibility and biodegradability, as well as non-specific ROS level regulation, further potentially causing deleterious adverse effects. Herein, the authors report a novel non-enzymatic antioxidant strategy based on ultrathin Ti3 C2 -PVP nanosheets (TPNS) with excellent biocompatibility and great chemical reactivity toward multiple ROS for AKI treatment. These TPNS nanosheets exhibit enzyme/ROS-triggered biodegradability and broad-spectrum ROS scavenging ability through the readily occurring redox reaction between Ti3 C2 and various ROS, as verified by theoretical calculations. Furthermore, both in vivo and in vitro experiments demonstrate that TPNS can serve as efficient antioxidant platforms to scavenge the overexpressed ROS and subsequently suppress oxidative stress-induced inflammatory response through inhibition of NF-κB signal pathway for AKI treatment. This study highlights a new type of therapeutic agent, that is, the redox-mediated non-enzymatic antioxidant MXene nanoplatforms in treatment of AKI and other ROS-associated diseases.

Zinc supplementation augments the suppressive effects of repurposed NF-κB inhibitors on ACE2 expression in human lung cell lines.

AIMS: Angiotensin-converting enzyme 2 (ACE2) is a key negative regulator of the renin-angiotensin system and also a major receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we reveal a role for NF-κB in human lung cell expression of ACE2, and we further explore the potential utility of repurposing NF-κB inhibitors to downregulate ACE2. MAIN METHODS: Expression of ACE2 was assessed by Western blotting and RT-qPCR in multiple human lung cell lines with or without NF-κB inhibitor treatment. Surface ACE2 expression and intracellular reactive oxygen species (ROS) levels were measured with flow cytometry. p50 was knocked down with siRNA. Cytotoxicity was monitored by PARP cleavage and MTS assay. KEY FINDINGS: Pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor, suppressed endogenous ACE2 mRNA and protein expression in H322M and Calu-3 cells. The ROS level in H322M cells was increased after PDTC treatment, and pretreatment with N-acetyl-cysteine (NAC) reversed PDTC-induced ACE2 suppression. Meanwhile, treatment with hydrogen peroxide augmented ACE2 suppression in H322M cells with p50 knockdown. Two repurposed NF-κB inhibitors, the anthelmintic drug triclabendazole and the antiprotozoal drug emetine, also reduced ACE2 mRNA and protein levels. Moreover, zinc supplementation augmented the suppressive effects of triclabendazole and emetine on ACE2 expression in H322M and Calu-3 cells. SIGNIFICANCE: These results suggest that ACE2 expression is modulated by ROS and NF-κB signaling in human lung cells, and the combination of zinc with triclabendazole or emetine shows promise for clinical treatment of ACE2-related disease.

N-Terminal Finger Stabilizes the S1 Pocket for the Reversible Feline Drug GC376 in the SARS-CoV-2 Mpro Dimer.

The main protease (Mpro, also known as 3CL protease) of SARS-CoV-2 is a high priority drug target in the development of antivirals to combat COVID-19 infections. A feline coronavirus antiviral drug, GC376, has been shown to be effective in inhibiting the SARS-CoV-2 main protease and live virus growth. As this drug moves into clinical trials, further characterization of GC376 with the main protease of coronaviruses is required to gain insight into the drug's properties, such as reversibility and broad specificity. Reversibility is an important factor for therapeutic proteolytic inhibitors to prevent toxicity due to off-target effects. Here we demonstrate that GC376 has nanomolar Ki values with the Mpro from both SARS-CoV-2 and SARS-CoV strains. Restoring enzymatic activity after inhibition by GC376 demonstrates reversible binding with both proteases. In addition, the stability and thermodynamic parameters of both proteases were studied to shed light on physical chemical properties of these viral enzymes, revealing higher stability for SARS-CoV-2 Mpro. The comparison of a new X-ray crystal structure of Mpro from SARS-CoV complexed with GC376 reveals similar molecular mechanism of inhibition compared to SARS-CoV-2 Mpro, and gives insight into the broad specificity properties of this drug. In both structures, we observe domain swapping of the N-termini in the dimer of the Mpro, which facilitates coordination of the drug's P1 position. These results validate that GC376 is a drug with an off-rate suitable for clinical trials.

Improved SARS-CoV-2 Mpro inhibitors based on feline antiviral drug GC376: Structural enhancements, increased solubility, and micellar studies.

Replication of SARS-CoV-2, the coronavirus causing COVID-19, requires a main protease (Mpro) to cleave viral proteins. Consequently, Mpro is a target for antiviral agents. We and others previously demonstrated that GC376, a bisulfite prodrug with efficacy as an anti-coronaviral agent in animals, is an effective inhibitor of Mpro in SARS-CoV-2. Here, we report structure-activity studies of improved GC376 derivatives with nanomolar affinities and therapeutic indices >200. Crystallographic structures of inhibitor-Mpro complexes reveal that an alternative binding pocket in Mpro, S4, accommodates the P3 position. Alternative binding is induced by polar P3 groups or a nearby methyl. NMR and solubility studies with GC376 show that it exists as a mixture of stereoisomers and forms colloids in aqueous media at higher concentrations, a property not previously reported. Replacement of its Na+ counter ion with choline greatly increases solubility. The physical, biochemical, crystallographic, and cellular data reveal new avenues for Mpro inhibitor design.

Expedited Approach toward the Rational Design of Noncovalent SARS-CoV-2 Main Protease Inhibitors.

The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II, and XII, with each containing a reactive warhead that covalently modifies the catalytic Cys145. Coupling structure-based drug design with the one-pot Ugi four-component reaction, we discovered one of the most potent noncovalent inhibitors, 23R (Jun8-76-3A) that is structurally distinct from the canonical Mpro inhibitor GC376. Significantly, 23R is highly selective compared with covalent inhibitors such as GC376, especially toward host proteases. The cocrystal structure of SARS-CoV-2 Mpro with 23R revealed a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study discovered 23R, one of the most potent and selective noncovalent SARS-CoV-2 Mpro inhibitors reported to date, and a novel binding pocket in Mpro that can be explored for inhibitor design.

Lead compounds for the development of SARS-CoV-2 3CL protease inhibitors.

We report the identification of three structurally diverse compounds - compound 4, GC376, and MAC-5576 - as inhibitors of the SARS-CoV-2 3CL protease. Structures of each of these compounds in complex with the protease revealed strategies for further development, as well as general principles for designing SARS-CoV-2 3CL protease inhibitors. These compounds may therefore serve as leads for the basis of building effective SARS-CoV-2 3CL protease inhibitors.

Glucosylceramide synthase inhibitors prevent replication of SARS-CoV-2 and influenza virus.

The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health. Vaccines are ideal solutions to prevent infection, but treatments are also needed for those who have contracted the virus to limit negative outcomes, when vaccines are not applicable. Viruses must cross host cell membranes during their life cycle, creating a dependency on processes involving membrane dynamics. Thus, in this study, we examined whether the synthetic machinery for glycosphingolipids, biologically active components of cell membranes, can serve as a therapeutic target to combat SARS-CoV-2. We examined the antiviral effect of two specific inhibitors of glucosylceramide synthase (GCS): (i) Genz-123346, an analogue of the United States Food and Drug Administration-approved drug Cerdelga and (ii) GENZ-667161, an analogue of venglustat, which is currently under phase III clinical trials. We found that both GCS inhibitors inhibit replication of SARS-CoV-2. Moreover, these inhibitors also disrupt replication of influenza virus A/PR/8/34 (H1N1). Our data imply that synthesis of glycosphingolipids is necessary to support viral life cycles and suggest that GCS inhibitors should be further explored as antiviral therapies.

Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2.

SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme's active site. Our findings provide new insights for the development of uracil scaffold-based drugs.