https://doi.org/10.1016/j.ajp.2022.103129

Psychiatric adverse reactions to COVID-19 vaccines: A rapid review of published case reports

http://www.cell.com/article/S0969212622002295/pdf

Binding of the SARS-CoV-2 envelope E protein to human BRD4 is essential for infection

The emergence of fast-spreading SARS-CoV-2 mutants has sparked a new phase of COVID-19 pandemic. There is a dire necessity for antivirals targeting highly conserved genomic domains on SARS-CoV-2 that are less prone to mutation. The nsp12, also known as the RNA-dependent RNA-polymerase (RdRp), the core component of 'SARS-CoV-2 replication-transcription complex', is a potential well-conserved druggable antiviral target. Several FDA-approved RdRp 'nucleotide analog inhibitors (NAIs)' such as remdesivir, have been repurposed to treat COVID-19 infections. The NAIs target RdRp protein translation and competitively block the nucleotide insertion into the RNA chain, resulting in the inhibition of viral replication. However, the replication proofreading function of nsp14-ExoN could provide resistance to SARS-CoV-2 against many NAIs. Conversely, the 'non-nucleoside analog inhibitors (NNAIs)' bind to allosteric sites on viral polymerase surface, change the redox state; thereby, exert antiviral activity by altering interactions between the enzyme substrate and active core catalytic site of the RdRp. NNAIs neither require metabolic activation (unlike NAIs) nor compete with intracellular pool of nucleotide triphosphates (NTPs) for anti-RdRp activity. The NNAIs from phytonutrient origin are potential antiviral candidates compared to their synthetic counterparts. Several in-silico studies reported the antiviral spectrum of natural phytonutrient-NNAIs such as Suramin, Silibinin (flavonolignan), Theaflavin (tea polyphenol), Baicalein (5,6,7-trihydroxyflavone), Corilagin (gallotannin), Hesperidin (citrus bioflavonoid), Lycorine (pyrrolidine alkaloid), with superior redox characteristics (free binding energy, hydrogen-bonds, etc.) than antiviral drugs (i.e. remdesivir, favipiravir). These phytonutrient-NNAIs also exert anti-inflammatory, antioxidant, immunomodulatory and cardioprotective functions, with multifunctional therapeutic benefits in the clinical management of COVID-19.

Plant-Derived Natural Non-Nucleoside Analog Inhibitors (NNAIs) against RNA-Dependent RNA Polymerase Complex (nsp7/nsp8/nsp12) of SARS-CoV-2

https://doi.org/10.1016/j.drudis.2022.06.012

Mutation informatics: SARS-CoV-2 receptor-binding domain of the spike protein

We demonstrated that SF2523, a dual small molecule inhibitor of PI3K-α/mTOR/BRD4 pathways, can inhibit the replication of SARS-CoV-2 and its emerging variants of concern (VOCs), including Delta and Omicron. Further, we also found that SF2523 acts synergistically with remdesivir (RDV) and MU-UNMC-2 (a small molecule entry inhibitor of SARS-CoV-2).1 The ongoing COVID-19 pandemic due to the emergence of a novel coronavirus SARS-CoV-2 remains a significant health concern globally. Several vaccine candidates and anti-virals received emergency use authorization. However, these vaccines/anti-virals safety, efficacy and durability remain unknown, especially for the individuals with comorbid conditions, and VOCs, such as Delta, Omicron, BA.2 and Deltacron, which have evolved mutations in the receptor-binding domain of SARS-CoV-2 spike protein may even evade antibodies induced by vaccines or natural infection.2 Similarly, recently FDA-approved Molnupiravir and PAXLOVID remain sensitive towards VOCs.3 The in-depth understanding of the molecular mechanism of the SARS-CoV-2 lifecycle revealed the interaction of several host factors with viral proteins essential for the reproduction of progeny viruses, such as bromodomain, containing extra-terminal domain proteins (BETs), and the mTOR pathway.4 Recent studies identified 67 potential interactions between host and viral proteins essential for the SARS-CoV-2 lifecycle, like BRD2/BRD4 with the E protein of SARS-CoV-2,4 and suggested that BRD2 inhibition downregulates ACE2 expression, blocks the entry of SARS-CoV-2 into host cells and controls hyperactive immune response in COVID-19 patients through downregulation Interferon stimulated genes (ISGs).5 Targeted therapies that exploit host–virus interaction are likely to be least impacted by the VOCs of SARS-CoV-2 and are expected to produce more robust, durable treatment options. Therefore, we tested the anti-viral potential of SF2523 against the wild-type SARS-CoV-2 and VOCs. In UNCN1T (a bronchial epithelial cell line), Vero STAT1 KO and Calu-3 cells, the CC50 value of SF2523 is above 100 μM (Figure 1A,B; Figure S2A). SF2523 showed potent anti-viral activity with an IC50 of 1.52 μM (RDV IC50 of 1.06 μM) in UNCN1T cells (Figure 1C) and 1.02 μM (RDV IC50 of 1.03 μM) in Vero STAT1 KO cells (Figure 1D) at 24 hpi, respectively. At 48 hpi in UNCN1T cells, SF2523 has an IC50 of 1.58 μM (RDV IC50 of 2.75 μM) and in Vero STAT1 KO cells has an IC50 of 3.22 μM (RDV IC50 of .76 μM), respectively (Figure S1A; 1B). Similarly, in Calu-3 cells, SF2523 has an anti-viral activity with an IC50 of 2.08, 4.03, 0.86 and 4.03 μM against the Delta variant (linage: B.1.617.2; Figure 2A), the Omicron variant (linage: B.1.1.529; Figure 2B), the South African variant (linage: B.1.351; Figure 2C) and the Scotland variant (linage: B.1.222; Figure 2D), respectively. Since BRD4 promotes lung tissue fibrosis through an increase in the expression of pro-fibrotic genes, and the majority of severely ill COVID-19 patients suffer from pulmonary fibrosis,6 the inclusion of BRD4 inhibitor SF2523 in COVID-19 therapy will benefit patients to recover from lung fibrosis. This is further supported by a recent study showing that BET protein inhibition blocks cardiac irregularities and SARS-CoV-2 infection7 and may be useful to controlling the long COVID-19 effects recently seen in many recovered patients. Combining multiple drugs with different modes of action has synergistic effects and has been used as anti-virals. Therefore, we evaluated a series of fixed-dose combinations of SF2523 and RDV or MU-UNMC-2.1 The combined doses of SF2523/RDV and SF2523/MU-UNMC-2 have CC50 values above 100 μM (Figure S2B and C). In UNCN1T cells at 24 hpi, when a fixed dose of 0.1 μM SF2523 is combined with different dosages of RDV, it has an IC50 value of 0.62 μM (Figure 3A) that is lower than individual IC50 of both the compounds. The dose–response percent inhibition matrix of single and combination treatment of SF2523/RDV and SF2523/MU-UNMC-2 is described in Figure 3B and E. The 3D interaction landscape of SF2523/RDV and SF2523/MU-UNMC-2 was computed based on Loewe additive model using SynergyFinder v.2 (Figure 3C and F). The synergistic/antagonistic effects of drug combinations are reconfirmed using Chou and Talalay combination index (CI) theorem8 [CI < 1: synergism; = 1: additive effect; > 1: antagonism]. Using CompuSyn, we computed CI values of SF2523/RDV as CI < 1 and CI of SF2523/MU-UNMC-2 as 0.129, confirming their synergistic effect. For SF2523/RDV, we obtained a dose reduction index (DRI) of 25.33 and 3.75 for SF2523 and RDV, and for SF2523/MU-UNMC-2, we obtained the DRI of 23.03 and 11.56 for SF2523 and MU-UNMC-2, respectively. This synergistic effect of SF2523 with RDV or MU-UNMC-2 is expected in a favourable shift in the plasma Cmax/EC90 ratio. Next, using NMR titration experiments by labelling 15N-labelled BRD4 domains, we showed that RDV is not able to bind to either BRD4-BD1, BRD4-BD2 or the ET domain of BRD4, as no significant chemical shift perturbations were observed in 1H,15N heteronuclear single quantum coherence spectra of these domains (Figure 4). This confirms that RDV does not alter the potency of SF2523 and does not interfere with the binding of SF2523 to BRD4. It is documented that SARS-CoV-2 infection in mammalian cells inhibits autophagy through various mechanisms, including the activation of autophagy inhibitory proteins (AKT1 and SKP2) and inhibition of proteins involved in autophagy initiation (AMPK, TSC2, Unc-51 Like Autophagy Activating Kinase 1 and Beclin 1), autophagosome formation (VPS34) and in membrane tethering and fusion of autophagosomes to endolysosomes (ATG14)9 and SARS-CoV-2, and its transmembrane protein ORF3a inhibits autophagy. Activating autophagy using small molecules that target PI3Kinase/Akt pathway or stabilize Beclin inhibits SARS-CoV-2 replication.9 Our studies suggest that SF2523 may be modulating the autophagy mechanisms (Figure S3) to limit SARS-CoV-2 infection similar to previously reported HIV-infected macrophages restrict infection through autophagy induction,10 although this warrants further in-depth investigation. In conclusion, we demonstrated that SF2523 could inhibit the replication of SARS-CoV-2 and its VOCs. Further, we found a synergistic effect of SF2523 with RDV or MU-UNMC-2 in a wide dose range. Therefore, we conclude that SF2523 (a PI3K-α/mTOR/BRD4 inhibitor) alone or combined with other anti-virials represents a future therapeutic approach to prevent the severe disease associated with SARS-CoV-2 and its VOCs. We acknowledge the UNMC BSL-3 core facility for allowing us to perform all in vitro experiments involving SARS-CoV-2. The University of Nebraska Medical Center BSL-3 Core Facility is administrated through the Office of the Vice-Chancellor for Research and supported by the Nebraska Research Initiative (NRI). The following reagent was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: (a) SARS-Related Coronavirus 2; Isolate USA-WI1/2020 (NR-52384), (b) isolate hCoV-19/USA/MD-HP01542/2021 (NR-55282; B.1.351), (c) isolate hCoV-19/Scotland/CVR2224/2020 (NR-53945; B.1.222), (d) isolate hCoV-19/USA/MD-HP05647/202 (BEI; cat# NR-55672), (e) isolate hCoV-19/USA/HI-CDC-4359259-001/2021 (BEI; cat#NR-56475), (f) Quantitative PCR (qPCR) Control RNA from Heat-Inactivated SARS-Related Coronavirus 2, Isolate USA-WA1/2020, (NR 52347). This work is partially supported by the National Institute of Allergy and Infectious Diseases Grant R01 AI129745, 5P30 CA036727-33, Frances E. Lageschulte and Evelyn B. Weese New Frontiers in Medical Research Fund and independent research and development (IRAD) funding from the National Strategic Research Institute (NSRI) at the University of Nebraska to SNB and NIH grants HL151334 and CA252707 to T.G.K; and CA215651 to DLD. Fig S1. Wild type SARS-CoV-2 replication inhibition dose-response curves of SF2523 in UNCN1T and Vero STAT1 KO cells. (A, B). UNCN1T and Vero STAT1 knockout cells (20,000 cells/well) were seeded in 96-well plates 24 hours before infection. Different concentrations of SF2523 and remdesivir (100 μM, 10 μM, 5 μM, 1 μM, 0.1 μM, 0.01 μM and 0.001 μM) were added to the cells 2 hours before infection. The cells were infected with 0.1 MOI of SARS-CoV-2 isolate USA-WI1/2020. Culture supernatant was collected at 48 hrs post-infection. The SARS-CoV-2 viral load was quantified in the culture supernatant using RT-QPCR with primer probes targeting E gene of SARS-CoV-2 using PrimeDirect Probe RT-qPCR Mix (TaKaRa Bio USA, Inc) and Applied Biosystems QuantStudio3 real-time PCR system (Applied Biosystems, Waltham, MA, USA) per the manufacturer's instructions. The SARS-CoV-2 genome equivalent copies were calculated using quantitative PCR (qPCR) control RNA from heat-inactivated SARS-CoV-2, isolate USA-WA1/2020 (BEI, Catalog# NR-52347). The percentage inhibition of SARS-CoV-2 replication in SF2523 and remdesivir treated wells was calculated with respect to virus concentration in positive control wells treated with DMSO (considered 0% inhibition) and negative control wells (uninfected cells). SF2523 (in blue) and remdesivir (in green) IC50 values were calculated using four-parameter variable slope sigmoidal dose-response models using Graph Pad Prism 8.0 software; [CC50: Drug concentration that required to reduces cell viability by 50%; IC50: Drug concentration that required to reduce the viral replication by 50%]. Fig S2. Cytotoxicity of SF2523 in Calu-3 cells (A) and combination of SF2523/RDV and SF2523/MU-UNMC-2 in UNCN1T cells (B and C) cells was measured by MTT assay. The cells were treated with increasing concentration of SF2523, SF2523/RDV and SF2523/MU-UNMC-2 (0.001 to 100 μM) and incubated at 37° C in a humidified 5% CO2 incubator. After 72-hour post-treatment, 20 μL of MTT substrate (5 mg/mL) was added to each well and incubated for 4 additional hours at 37° C in the dark. Then the culture media was carefully removed, and blue formazan crystals were dissolved in 200 °l of DMSO, and the purple color was read at 595 nm with a reference filter of 620 nm. In respective cells, SF2523, SF2523/RDV and SF2523/MU-UNMC-2 has a CC50 value above 100 °M. Fig S3. SF2523 may induces autophagy in SARS-CoV-2 infected Vero cells. The cells were treated with the indicated concentration of SF2523 and 5 mM 3-MA and infected with SARS-CoV-2. After 24h, cells were harvested, cell lysate prepared, total protein content quantified, and immunoblotting was performed. The upper and lower panels are the representative immunoblots of p62 and LC3B-I//II. Actin was used as a loading control. The upper and lower bar graphs show densitometric analysis of p62 and LC3B-II immunoblots, respectively normalized with actin. Data is expressed as mean ± SEM derived from two experiments. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. 

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/ctm2.806

PI3K‐α/mTOR/BRD4 inhibitor alone or in combination with other anti‐virals blocks replication of SARS‐CoV‐2 and its variants of concern including Delta and Omicron

Mental Health Issues During and After COVID-19 Vaccine Era.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 19 (COVID-19) pandemic. Despite unprecedented research and developmental efforts, SARS-CoV-2-specific antivirals are still unavailable for the treatment of COVID-19. In most instances, SARS-CoV-2 infection initiates with the binding of Spike glycoprotein to the host cell ACE2 receptor. Utilizing the crystal structure of the ACE2/Spike receptor-binding domain (S-RBD) complex (PDB file 6M0J) in a computer-aided drug design approach, we identified and validated five potential inhibitors of S-RBD and ACE-2 interaction. Two of the five compounds, MU-UNMC-1 and MU-UNMC-2, blocked the entry of pseudovirus particles expressing SARS-CoV-2 Spike glycoprotein. In live SARS-CoV-2 infection assays, both compounds showed antiviral activity with IC50 values in the micromolar range (MU-UNMC-1: IC50 = 0.67 μM and MU-UNMC-2: IC50 = 1.72 μM) in human bronchial epithelial cells. Furthermore, MU-UNMC-1 and MU-UNMC-2 effectively blocked the replication of rapidly transmitting variants of concern: South African variant B.1.351 (IC50 = 9.27 and 3.00 μM) and Scotland variant B.1.222 (IC50 = 2.64 and 1.39 μM), respectively. Following these assays, we conducted "induced-fit (flexible) docking" to understand the binding mode of MU-UNMC-1/MU-UNMC-2 at the S-RBD/ACE2 interface. Our data showed that mutation N501Y (present in B.1.351 variant) alters the binding mode of MU-UNMC-2 such that it is partially exposed to the solvent and has reduced polar contacts. Finally, MU-UNMC-2 displayed high synergy with remdesivir, the only approved drug for treating hospitalized COVID-19 patients. IMPORTANCE The ongoing coronavirus infectious disease 2019 (COVID-19) pandemic is caused by a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). More than 207 million people have been infected globally, and 4.3 million have died due to this viral outbreak. While a few vaccines have been deployed, a SARS-CoV-2-specific antiviral for the treatment of COVID-19 is yet to be approved. As the interaction of SARS-CoV-2 Spike protein with ACE2 is critical for cellular entry, using a combination of a computer-aided drug design (CADD) approach and cell-based in vitro assays, we report the identification of five potential SARS-CoV-2 entry inhibitors. Out of the five, two compounds (MU-UNMC-1 and MU-UNMC-2) have antiviral activity against ancestral SARS-CoV-2 and emerging variants from South Africa and Scotland. Furthermore, MU-UNMC-2 acts synergistically with remdesivir (RDV), suggesting that RDV and MU-UNMC-2 can be developed as a combination therapy to treat COVID-19 patients.

Discovery and evaluation of entry inhibitors for SARS-CoV-2 and its emerging variants.

The emergence and rapid spread of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused over 180 million confirmed cases resulting in over 4 million deaths worldwide with no clear end in sight for the coronavirus disease 19 (COVID-19) pandemic. Most SARS-CoV-2 exposed individuals experience mild to moderate symptoms, including fever, cough, fatigue, and loss of smell and taste. However, many individuals develop pneumonia, acute respiratory distress syndrome, septic shock, and multiorgan dysfunction. In addition to these primarily respiratory symptoms, SARS-CoV-2 can also infiltrate the central nervous system, which may damage the blood-brain barrier and the neuron's synapses. Resultant inflammation and neurodegeneration in the brain stem can further prevent efferent signaling to cranial nerves, leading to the loss of anti-inflammatory signaling and normal respiratory and gastrointestinal functions. Additionally, SARS-CoV-2 can infect enterocytes resulting in gut damage followed by microbial dysbiosis and translocation of bacteria and their byproducts across the damaged epithelial barrier. As a result, this exacerbates pro-inflammatory responses both locally and systemically, resulting in impaired clinical outcomes. Recent evidence has highlighted the complex interactions that mutually modulate respiratory, neurological, and gastrointestinal function. In this review, we discuss the ways SARS-CoV-2 potentially disrupts the gut-brain-lung axis. We further highlight targeting specific responses to SARS-CoV-2 for the development of novel, urgently needed therapeutic interventions. Finally, we propose a prospective related to the individuals from Low- and Middle-Income countries. Here, the underlying propensity for heightened gut damage/microbial translocation is likely to result in worse clinical outcomes during this COVID-19 pandemic.

Therapeutic implications of SARS-CoV-2 dysregulation of the gut-brain-lung axis.

SARS-CoV-2 infection initiates with the attachment of spike protein to the ACE2 receptor. While vaccines have been developed, no SARS-CoV-2 specific small molecule inhibitors have been approved. Herein, utilizing the crystal structure of the ACE2/Spike receptor binding domain (S-RBD) complex in computer-aided drug design (CADD) approach, we docked ~8 million compounds within the pockets residing at S-RBD/ACE2 interface. Five best hits depending on the docking score, were selected and tested for their in vitro efficacy to block SARS-CoV-2 replication. Of these, two compounds (MU-UNMC-1 and MU-UNMC-2) blocked SARS-CoV-2 replication at sub-micromolar IC 50 in human bronchial epithelial cells (UNCN1T) and Vero cells. Furthermore, MU-UNMC-2 was highly potent in blocking the virus entry by using pseudoviral particles expressing SARS-CoV-2 spike. Finally, we found that MU-UNMC-2 is highly synergistic with remdesivir (RDV), suggesting that minimal amounts are needed when used in combination with RDV, and has the potential to develop as a potential entry inhibitor for COVID-19.

https://www.biorxiv.org/content/biorxiv/early/2021/04/02/2021.04.02.438204.full.pdf

Elizabeth A. Klug

Papers

Mental Health Issues During and After COVID-19 Vaccine Era.

Discovery and evaluation of entry inhibitors for SARS-CoV-2 and its emerging variants.

Therapeutic implications of SARS-CoV-2 dysregulation of the gut-brain-lung axis.

Discovery and in-vitro evaluation of potent SARS-CoV-2 entry inhibitors