Dual mechanism: Epigenetic inhibitor apabetalone reduces SARS-CoV-2 Delta and Omicron variant spike binding and attenuates SARS-CoV-2 RNA induced inflammation.

The SARS-CoV-2 virus initiates infection via interactions between the viral spike protein and the ACE2 receptors on host cells. Variants of concern have mutations in the spike protein that enhance ACE2 binding affinity, leading to increased virulence and transmission. Viral RNAs released after entry into host cells trigger interferon-I (IFN-I) mediated inflammatory responses for viral clearance and resolution of infection. However, overreactive host IFN-I responses and pro-inflammatory signals drive COVID-19 pathophysiology and disease severity during acute infection. These immune abnormalities also lead to the development of post-COVID syndrome if persistent. Novel therapeutics are urgently required to reduce short- and long-term pathologic consequences associated with SARS-CoV-2 infection. Apabetalone, an inhibitor of epigenetic regulators of the BET protein family, is a candidate for COVID-19 treatment via a dual mechanism of action. In vitro, apabetalone downregulates ACE2 gene expression to limit SARS-CoV-2 entry and propagation. In pre-clinical models and patients treated for cardiovascular disease, apabetalone inhibits expression of inflammatory mediators involved in the pathologic cytokine storm (CS) stimulated by various cytokines. Here we show apabetalone treatment of human lung epithelial cells reduces binding of viral spike protein regardless of mutations found in the highly contagious Delta variant and heavily mutated Omicron. Additionally, we demonstrate that apabetalone counters expression of pro-inflammatory factors with roles in CS and IFN-I signaling in lung cells stimulated with SARS-CoV-2 RNA. Our results support clinical evaluation of apabetalone to treat COVID-19 and post-COVID syndrome regardless of the SARS-CoV-2 variant.

Revealing In Silico that Bacteria's Outer Membrane Proteins may Help our Bodies Replicate and Carry Severe Acute Respiratory Syndrome Coronavirus 2.

Some studies in the literature show that viruses can affect bacteria directly or indirectly, and viruses use their own specific ways to do these interactions. Furthermore, it is said that bacteria are prone to attachment mammalian cells during a viral illness using their surface proteins that bind to host extracellular matrix proteins such as fibronectin, fibrinogen, vitronectin, and elastin. A recent study identified the cooperation between bacteria and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in silico, in vitro, and in vivo. Like this study, we hypothesized that more bacteria protein might help SARS-CoV-2 transport and attach to angiotensin-converting enzyme 2 (ACE2). The bacteria's outer membrane proteins (OMPs) we chose were not random; they had to be on the outer surface of the bacteria because these proteins on the outer surface should have a high probability of interacting with both the spike protein and ACE2. We obtained by using bioinformatics tools that there may be binding between both ACE2 and spike protein of these bacteria's OMPs. Protein-protein interaction results also supported our hypothesis. Therefore, based on our predicted results, these bacteria OMPs may help SARS-CoV-2 move in our body, and both find and attach to ACE2. It is expected that these inferences obtained from the bioinformatics results may play a role in the SARS-CoV-2 virus reaching host cells. Thus, it may bring a different perspective to studies on how the virus can infect host cells.

2,4-Dihydroxycinnamic acid as spike ACE2 inhibitor and apigenin as RdRp inhibitor in Nimbamritadi Panchatiktam Kashayam against COVID-19: an in silico and in vitro approach.

Nimbamritadi Panchatiktam Kashayam (NPK) is an ayurvedic formulation composed of ingredients with potent anti-viral activities. We studied the interaction energy of 144 phytoconstituents present in NPK against spike receptor-binding domain (RBD) complexed with ACE2 protein (PDB ID: 6LZG) and RNA-dependent RNA polymerase protein (PDB ID: 7BTF) using Biovia Drug Discovery studio. The result indicated that 2,4-hydroxycinnamic acid exerts more significant binding affinities (28.43 kcal/mol) than Umifenovir (21.24 kcal/mol) against spike ACE2. Apigenin exhibited the highest binding affinities (54.63 kcal/mol) compared with Remdesivir (24.52 kcal/mol) against RdRp. An in vitro analysis showed a reduction in the number of lentiviral particles on transfected HEK293T-hACE2 cells as assessed by pseudovirus inhibition assay. At the same time, the tested compounds showed non-toxic up to 100 µg/ml in normal cells by MTT assay. The study highlights the plausible clinical utility of this traditional medicine against SARS CoV2.

Lonicerae Japonicae Flos Attenuates Neutrophilic Inflammation by Inhibiting Oxidative Stress.

Lonicerae japonicae flos (LJ) is an Asian traditional herb that is used as a dietary supplement, tea, and beverage to clear heat and quench thirst. However, no studies investigated its effect on activated human neutrophils, which played a crucial role in the bad prognosis of coronavirus disease of 2019 (COVID-19) patients by aggravating lung inflammation and respiratory failure. Herein, we evaluated the anti-inflammatory effect of LJ ethanol extract (LJEE) on human neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine (fMLF). Our experimental results indicated that LJEE suppressed fMLF-activated superoxide anion (O2•-) generation, the expression of CD11b, and cell adhesion and migration, as well as the formation of neutrophil extracellular traps in human neutrophils. Further in-depth mechanical investigation revealed that pretreatment with LJEE accelerated the Ca2+ clearance, but did not affect the phosphorylation of mitogen-activated protein kinases (MAPKs) and protein kinase B (Akt) in activated human neutrophils. In addition, LJEE displayed a dose-dependent reactive oxygen species (ROS) scavenger activity, which assisted its anti-inflammatory activity. From the bioassay-coupled chromatographic profile, chlorogenic acids were found to dominate the anti-inflammatory effects of LJEE. Moreover, LJ water extract (LJWE) demonstrated an interrupting effect on the severe acute respiratory syndrome coronavirus-2 spike protein (SARS-CoV-2-Spike)/angiotensin-converting enzyme 2 (ACE2) binding. In conclusion, the obtained results not only supported the traditional use of LJ for heat-clearance, but also suggested its potential application in daily health care during the COVID-19 pandemic.

In silico investigation of ACE2 and the main protease of SARS-CoV-2 with phytochemicals from Myristica fragrans (Houtt.) for the discovery of a novel COVID-19 drug.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), is a new coronavirus strain that was first reported in December 2019 in Wuhan, China. A specific treatment for COVID-19 has yet to be identified. Potential therapeutic targets include SARS-CoV-2 main protease (Mpro) and the SARS-CoV-2 spike-ACE2 interaction. Molecular docking, molecular dynamics (MD), solvent screening for the extraction of the specified compounds, and prediction of the drug properties of certain molecules were the methods used in this study to investigate compounds from the medicinal plant Myristica fragrans, which is one of twelve herbs in Prasachandaeng remedy (PSD). ArgusLab, AutoDock Vina, and AutoDock were used to perform docking tasks. The examined ligands were compared with panduratin A as a standard (Kanjanasirirat et al., 2020), which is a promising medicinal plant molecule for the treatment of COVID-19. Molecular docking revealed that malabaricones B and C and licarins A, B and C bound to SARS-CoV-2/ACE2 and SARS-CoV-2 Mpro with low binding energies compared to that of the standard ligand. Furthermore, appropriate solvent usage is important. Acetone was selected by COSMOquick software for compound extraction in this investigation because it can extract large amounts of all five of the abovementioned M. fragrans compounds. Furthermore, the drug-like properties of these compounds were studied utilizing the Lipinski, Veber, and Ghose criteria. The results revealed that these M. fragrans compounds have potential as effective medicines to combat the COVID-19 pandemic. However, to assess the therapeutic potential of these ligands, additional research is needed, which will use our findings as a foundation.

The effect of various compounds on the COVID mechanisms, from chemical to molecular aspects.

The novel coronavirus that caused COVID-19 pandemic is SARS-CoV-2. Although various vaccines are currently being used to prevent the disease's severe consequences, there is still a need for medications for those who become infected. The SARS-CoV-2 has a variety of proteins that have been studied extensively since the virus's advent. In this review article, we looked at chemical to molecular aspects of the various structures studied that have pharmaceutical activity and attempted to find a link between drug activity and compound structure. For example, designing of the compounds which bind to the allosteric site and modify hydrogen bonds or the salt bridges can disrupt SARS-CoV2 RBD-ACE2 complex. It seems that quaternary ammonium moiety and quinolin-1-ium structure could act as a negative allosteric modulator to reduce the tendency between spike-ACE2. Pharmaceutical structures with amino heads and hydrophobic tails can block envelope protein to prevent making mature SARS-CoV-2. Also, structures based on naphthalene pharmacophores or isosteres can form a strong bond with the PLpro and form a π-π and the Mpro's active site can be occupied by octapeptide compounds or linear compounds with a similar fitting ability to octapeptide compounds. And for protein RdRp, it is critical to consider pH and pKa so that pKa regulation of compounds to comply with patients is very effective, thus, the presence of tetrazole, phenylpyrazole groups, and analogs of pyrophosphate in the designed drugs increase the likelihood of the RdRp active site inhibition. Finally, it can be deduced that designing hybrid drug molecules along with considering the aforementioned characteristics would be a suitable approach for developing medicines in order to accurate targeting and complete inhibition this virus.

Accelerating PERx reaction enables covalent nanobodies for potent neutralization of SARS-CoV-2 and variants.

The long-lasting COVID-19 pandemic and increasing SARS-CoV-2 variants demand effective drugs for prophylactics and treatment. Protein-based biologics offer high specificity, yet their noncovalent interactions often lead to drug dissociation and incomplete inhibition. Here, we have developed covalent nanobodies capable of binding with SARS-CoV-2 irreversibly via a proximity-enabled reactive therapeutic (PERx) mechanism. A latent bioreactive amino acid (FFY) was designed and genetically encoded into nanobodies to accelerate the PERx reaction rate. Compared with the noncovalent wild-type nanobody, the FFY-incorporated covalent nanobodies neutralized both wild-type SARS-CoV-2 and its Alpha, Delta, Epsilon, Lambda, and Omicron variants with drastically higher potency. This PERx-enabled covalent-nanobody strategy and the related insights into increased potency can be valuable to developing effective therapeutics for various viral infections.

Discovery of a new generation of angiotensin receptor blocking drugs: Receptor mechanisms and in silico binding to enzymes relevant to SARS-CoV-2.

The discovery and facile synthesis of a new class of sartan-like arterial antihypertensive drugs (angiotensin receptor blockers [ARBs]), subsequently referred to as "bisartans" is reported. In vivo results and complementary molecular modelling presented in this communication indicate bisartans may be beneficial for the treatment of not only heart disease, diabetes, renal dysfunction, and related illnesses, but possibly COVID-19. Bisartans are novel bis-alkylated imidazole sartan derivatives bearing dual symmetric anionic biphenyl tetrazole moieties. In silico docking and molecular dynamics studies revealed bisartans exhibited higher binding affinities for the ACE2/spike protein complex (PDB 6LZG) compared to all other known sartans. They also underwent stable docking to the Zn2 + domain of the ACE2 catalytic site as well as the critical interfacial region between ACE2 and the SARS-CoV-2 receptor binding domain. Additionally, semi-stable docking of bisartans at the arginine-rich furin-cleavage site of the SARS-CoV-2 spike protein (residues 681-686) required for virus entry into host cells, suggest bisartans may inhibit furin action thereby retarding viral entry into host cells. Bisartan tetrazole groups surpass nitrile, the pharmacophoric "warhead" of PF-07321332, in its ability to disrupt the cysteine charge relay system of 3CLpro. However, despite the apparent targeting of multifunctional sites, bisartans do not inhibit SARS-CoV-2 infection in bioassays as effectively as PF-07321332 (Paxlovid).

Evidence for a mouse origin of the SARS-CoV-2 Omicron variant.

The rapid accumulation of mutations in the SARS-CoV-2 Omicron variant that enabled its outbreak raises questions as to whether its proximal origin occurred in humans or another mammalian host. Here, we identified 45 point mutations that Omicron acquired since divergence from the B.1.1 lineage. We found that the Omicron spike protein sequence was subjected to stronger positive selection than that of any reported SARS-CoV-2 variants known to evolve persistently in human hosts, suggesting a possibility of host-jumping. The molecular spectrum of mutations (i.e., the relative frequency of the 12 types of base substitutions) acquired by the progenitor of Omicron was significantly different from the spectrum for viruses that evolved in human patients but resembled the spectra associated with virus evolution in a mouse cellular environment. Furthermore, mutations in the Omicron spike protein significantly overlapped with SARS-CoV-2 mutations known to promote adaptation to mouse hosts, particularly through enhanced spike protein binding affinity for the mouse cell entry receptor. Collectively, our results suggest that the progenitor of Omicron jumped from humans to mice, rapidly accumulated mutations conducive to infecting that host, then jumped back into humans, indicating an inter-species evolutionary trajectory for the Omicron outbreak.

Screening of Clinically Approved and Investigation Drugs as Potential Inhibitors of SARS-CoV-2: A Combined in silico and in†vitro Study.

In the current study, we used 7922 FDA approved small molecule drugs as well as compounds in clinical investigation from NIH's NPC database in our drug repurposing study. SARS-CoV-2 main protease as well as Spike protein/ACE2 targets were used in virtual screening and top-100 compounds from each docking simulations were considered initially in short molecular dynamics (MD) simulations and their average binding energies were calculated by MM/GBSA method. Promising hit compounds selected based on average MM/GBSA scores were then used in long MD simulations. Based on these numerical calculations following compounds were found as hit inhibitors for the SARS-CoV-2 main protease: Pinokalant, terlakiren, ritonavir, cefotiam, telinavir, rotigaptide, and cefpiramide. In addition, following 3 compounds were identified as inhibitors for Spike/ACE2: Denopamine, bometolol, and rotigaptide. In order to verify the predictions of in silico analyses, 4 compounds (ritonavir, rotigaptide, cefotiam, and cefpiramide) for the main protease and 2 compounds (rotigaptide and denopamine) for the Spike/ACE2 interactions were tested by in vitro experiments. While the concentration-dependent inhibition of the ritonavir, rotigaptide, and cefotiam was observed for the main protease; denopamine was effective at the inhibition of Spike/ACE2 binding.

Computational studies reveal Fluorine based quinolines to be potent inhibitors for proteins involved in SARS-CoV-2 assembly.

World is witnessing one of the worst pandemics of this century caused by SARS-CoV-2 virus which has affected millions of individuals. Despite rapid efforts to develop vaccines and drugs for COVID-19, the disease is still not under control. Chloroquine (CQ) and Hydroxychloroquine (HCQ) are two very promising inhibitors which have shown positive effect in combating the disease in preliminary experimental studies, but their use was reduced due to severe side-effects. Here, we performed a theoretical investigation of the same by studying the binding of the molecules with SARS-COV-2 Spike protein, the complex formed by Spike and ACE2 human receptor and a human serine protease TMPRSS2 which aids in cleavage of the Spike protein to initiate the viral activation in the body. Both the molecules had shown very good docking energies in the range of -6kcal/mol. Subsequently, we did a high throughput screening for other potential quinoline candidates which could be used as inhibitors. From the large pool of ligand candidates, we shortlisted the top three ligands (binding energy -8kcal/mol). We tested the stability of the docked complexes by running Molecular Dynamics (MD) simulations where we observed the stability of the quinoline analogues with the Spike-ACE2 and TMPRSS2 nevertheless the quinolines were not stable with the Spike protein alone. Thus, although the inhibitors bond very well with the protein molecules their intrinsic binding affinity depends on the protein dynamics. Moreover, the quinolines were stable when bound to electronegative pockets of Spike-ACE2 or TMPRSS2 but not with Viral Spike protein. We also observed that a Fluoride based compound: 3-[3-(Trifluoromethyl)phenyl]quinoline helps the inhibitor to bind with both Spike-ACE2 and TMPRSS2 with equal probability. The molecular details presented in this study would be very useful for developing quinoline based drugs for COVID-19 treatment.

In Silico, In Vitro and In Cellulo Models for Monitoring SARS-CoV-2 Spike/Human ACE2 Complex, Viral Entry and Cell Fusion.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent responsible for the recent coronavirus disease 2019 (COVID-19) pandemic. Productive SARS-CoV-2 infection relies on viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). Indeed, viral entry into cells is mostly mediated by the early interaction between the viral spike protein S and its ACE2 receptor. The S/ACE2 complex is, thus, the first contact point between the incoming virus and its cellular target; consequently, it has been considered an attractive therapeutic target. To further characterize this interaction and the cellular processes engaged in the entry step of the virus, we set up various in silico, in vitro and in cellulo approaches that allowed us to specifically monitor the S/ACE2 association. We report here a computational model of the SARS-CoV-2 S/ACE2 complex, as well as its biochemical and biophysical monitoring using pulldown, AlphaLISA and biolayer interferometry (BLI) binding assays. This led us to determine the kinetic parameters of the S/ACE2 association and dissociation steps. In parallel to these in vitro approaches, we developed in cellulo transduction assays using SARS-CoV-2 pseudotyped lentiviral vectors and HEK293T-ACE2 cell lines generated in-house. This allowed us to recapitulate the early replication stage of the infection mediated by the S/ACE2 interaction and to detect cell fusion induced by the interaction. Finally, a cell imaging system was set up to directly monitor the S/ACE2 interaction in a cellular context and a flow cytometry assay was developed to quantify this association at the cell surface. Together, these different approaches are available for both basic and clinical research, aiming to characterize the entry step of the original SARS-CoV-2 strain and its variants as well as to investigate the possible chemical modulation of this interaction. All these models will help in identifying new antiviral agents and new chemical tools for dissecting the virus entry step.

Quinolines-Based SARS-CoV-2 3CLpro and RdRp Inhibitors and Spike-RBD-ACE2 Inhibitor for Drug-Repurposing Against COVID-19: An in silico Analysis.

The novel coronavirus SARS-CoV-2 disease "COVID-19" emerged in China and rapidly spread to other countries; due to its rapid worldwide spread, the WHO has declared this as a global emergency. As there is no specific treatment prescribed to treat COVID-19, the seeking of suitable therapeutics among existing drugs seems valuable. The structure availability of coronavirus macromolecules has encouraged the finding of conceivable anti-SARS-CoV-2 therapeutics through in silico analysis. The results reveal that quinoline,1,2,3,4-tetrahydro-1-[(2-phenylcyclopropyl)sulfonyl]-trans-(8CI) and saquinavir strongly interact with the active site (Cys-His catalytic dyad), thereby are predicted to hinder the activity of SARS-CoV-2 3CLpro. Out of 113 quinoline-drugs, elvitegravir and oxolinic acid are able to interact with the NTP entry-channel and thus interfere with the RNA-directed 5'-3' polymerase activity of SARS-CoV-2 RdRp. The bioactivity-prediction results also validate the outcome of the docking study. Moreover, as SARS-CoV-2 Spike-glycoprotein uses human ACE2-receptor for viral entry, targeting the Spike-RBD-ACE2 has been viewed as a promising strategy to control the infection. The result shows rilapladib is the only quinoline that can interrupt the Spike-RBD-ACE2 complex. In conclusion, owing to their ability to target functional macromolecules of SARS-CoV-2, along with positive ADMET properties, quinoline,1,2,3,4-tetrahydro-1-[(2-phenylcyclopropyl)sulfonyl]-trans-(8CI), saquinavir, elvitegravir, oxolinic acid, and rilapladib are suggested for the treatment of COVID-19.