Lipid Lowering Natural Products Targeting hPCSK9 May Prevent the Severity of COVID-19 Infection

Background: The deadly outbreak of COVID-19 disease caused by novel SARS CoV2 has created an unprecedented global health crisis affecting every sectors of human life and enor-mous damage to world's economy. With >16.1 million infections and >650,000 deaths worldwide as of July 27, 2020, there is no treatment for this disease neither is there any available vaccine. Seri-ous research efforts are ongoing on all fronts including treatment, prevention and diagnosis to combat the spread of this infection. A number of targets that include both viral and host proteins have been identified and became part of intense investigation. In this respect the viral surface spike (S) glycoprotein caught the attention most. It is cleaved by multiple host proteases to allow recognition by host receptor human Angiotensin Converting Enzyme2 (hACE2) leading to fusion and viral re-plication. Natural products, small compounds, antioxidants, peptides, proteins, oligonucleotides, antibodies and other compounds are under investigation for development of antiviral agents against COVID-19. Objective(s): Recently cholesterol lowering phytocompounds Quercetin, Swertiamarin and Berberine which promote human Low Density Lipoprotein Receptor (hLDLR) via inhibition of human Pro-protein Convertase Subtilisin Kexin9 (hPCSK9) have been shown to block viral infections caused by ebola, influenza, Respiratory Syncytial Virus (RSV), Hepatitis C virus (HCV) and other RNA type viruses. Since SARS CoV2 is a RNA virus with similar genetic structure and infection machin-ery, it is hypothesised that these phytocompounds may also exhibit antiviral property against COVID-19. Method(s): Our above concept is based on recently published studies as well as our herein presented in silico modeling and computational data which suggested strong interactions of hPCSK9 with above phytocompounds and most importantly with hACE2 following its complexation with receptor binding domain (RBD) of SARS CoV2 S protein. Result(s): These results and a proposed schematic model showing association of hPCSK9 with SARS CoV2 infection are presented in this manuscript. It is proposed that hPCSK9 plays the role of a co-receptor in binding with hACE2:RBD complex and thereby facilitates viral fusion. Conclusion(s): Our studies suggest that PCSK9 inhibitors may provide beneficial effect against COVID-19 infection by retarding viral fusion through displacement of bound hPCSK9 from its complex with ACE2:RBD of SARS CoV2 S protein.Copyright © 2021 Bentham Science Publishers.

Probable pathways of SARS-CoV-2 to central nervous system

Various reported cases related to the COVID-19 pandemic since 2019 has shown that SARS-CoV-2 directly or indirectly affects the nervous system besides the upper respiratory tract (Whittaker et al. 2020). SARS-CoV-2 is a zoonotic strain of coronavirus with various structural proteins. It is reported that spike protein, which is one of its structural proteins, can bind to or interact with Neuropilin-1, CD147 (Basigin), KREMEN1, ASGR1 (Asialoglycoprotein Receptor), Furin, LRP1 (Lipoprotein Receptor-Related Protein 1) and Ephrin receptors as well as ACE-2 (Angiotensin Converting Enzyme-2) and TMPRSS2 (Transmembrane Serine Protease 2) receptors. The related studies suggest that these possible receptors in the target of SARS-CoV- 2 cooperate with ACE-2, hence make the central nervous system an open target for the virus (Zalpoor et al. 2022). The first possible route of SARS-CoV-2 is seen as the route from the olfactory epithelium to its bulb via the trigeminal nerve (CN V) and olfactory nerve (CN I) pathway. Additionally, the virus entering the bloodstream can reach the Blood Brain Barier, cross the barrier and spread to neurons through the oligodendrocyte in a process called “Trojen horse”. Lastly, another route considered focuses on the vagal nerves of the enteric system associated with the central nervous system (Guadarrama-Ortiz et al. 2020). This literature review focuses on possible entry routes of SARS-CoV-2 into the nervous system. In this context, a route has been established based on the receptors in the nervous system cells, which are reported to be the target of SARS-CoV-2, based on the studies.

ENTERIC NEURONAL VASOACTIVE INTESTINAL PEPTIDE MEDIATES SARS-COV-2 ASSOCIATED DIARRHEA

Background: Diarrhea is present in up to 36.6% of patients with COVID-19. The mechanism of SARS-CoV-2-induced diarrhea remains unclear. We hypothesized that enterocyte-enteric neuron interactions were important in SARS-CoV-2-induced diarrhea. SARS-CoV-2 induces endoplasmic reticulum (ER) stress in enterocytes causing the release of Damage Associated Molecular Patterns (DAMPs). The DAMPs then stimulate the release of enteric neurotransmitters that disrupt gut electrolyte homeostasis. The influence of ER stress and enteric neuronderived vasoactive intestinal peptide (VIP) on the expression of Na+/H+ exchanger 3 (NHE3), an important transporter that mediates intestinal Na+/fluid absorption, was further examined. Methods: SARS-CoV-2 propagated in Vero-E6 cells was used to infect Caco-2, a human colon epithelial cell line that expresses SARS-CoV-2 entry receptor ACE2. The expression of ER stress markers, phospho-PERK, Xbp1s, and DAMP proteins, was examined by Western blotting. Primary mouse enteric neurons were treated with a conditioned medium of Caco- 2 cells that were infected with SARS-CoV-2 or treated with tunicamycin. VIP expression by cultured enteric neurons was assessed by RT-qPCR, Western blotting, and ELISA. Membrane expression of NHE3 was determined by surface biotinylation. Results: SARS-CoV-2 infection of Caco-2 cells led to increased expression of phospho-PERK and Xbp1s indicating increased ER stress. Infected Caco-2 cells secreted DAMP proteins, including HSP70 and calreticulin, as revealed by proteomic and Western analyses. The expression of VIP mRNA in enteric neurons was up-regulated after treatment with a conditioned medium of SARS-CoV-2- infected Caco-2 cells (Mock, 1 ± 0.0885;and SARS-CoV-2, 1.351 ± 0.020, P=.005). CD91, a receptor for HSP70 and calreticulin, is abundantly expressed in cultured mouse and human enteric neurons and was up-regulated by a conditioned medium of SARS-CoV-2-infected Caco-2 cells. Tunicamycin, an inducer of ER stress, also induced the secretion of HSP70 and calreticulin, mimicking SARS-CoV-2 infection. Moreover, co-culture of enteric neurons with tunicamycin-treated Caco-2 cells stimulated VIP production as determined by ELISA. Co-treatment of Caco-2 cells with tunicamycin (apical) and VIP (basolateral) induced a synergistic decrease in the membrane expression of NHE3. Conclusions: Our findings demonstrate that SARS-CoV-2 infection of enterocytes leads to ER stress and the release of DAMPs that up-regulate the expression and release of VIP by enteric neurons. The presence of ER stress together with the secreted VIP, in turn, inhibits fluid absorption through the downregulation of brush-border membrane expression of NHE3 in the enterocytes. These data highlight epithelial-neuronal crosstalk in COVID-19 related diarrhea. (Figure Presented)

Profiling viral receptors in nasal and amniotic samples at birth

Introduction/Aim: Children with wheeze and asthma present with airway epithelial vulnerabilities, such as impaired responses to viral infection. It is postulated that the in utero environment may contribute to the development of such airway epithelial vulnerabilities, that may predispose children to wheeze and asthma outcomes. To explore developmental mechanisms, further research is required using epithelial samples at birth. Our study asked whether amniotic epithelial samples from placentas show similar viral receptor expression to nasal epithelial cells at birth. We aimed to investigate expression of respiratory viral receptors for human rhinovirus (HRV), respiratory syncytial virus (RSV) and COVID-19-causing coronavirus (SARS-CoV-2) in nasal and amniotic epithelial samples. Methods: Unmatched nasal (n = 20 births) and amniotic (n = 33 newborns) epithelial samples were collected from ORIGINS cohort participants recruited into the AERIAL study. Using purified RNA, receptor expression for HRV (ICAM-1, LDLR, CDHR3), RSV (NCL, TLR4) and SARSCoV- 2 (ACE2, TMPRSS2) was assessed by qPCR. In addition, receptor protein expression was quantified through western blot and localized using immunohistochemistry in amniotic samples only. Results: Nasal epithelial and amniotic samples expressed various receptors for HRV, RSV and SARS-CoV-2 at the gene level in nasal (median(IQR) arbitrary units (AU);ICAM-1: 11.44(63.18);LDLR: 4.00(7.32);CDHR3: 0.40 (1.14);NCL: 2.32(2.18);CX3CR1: 2.17(2.33);TLR4: 2.20 (6.20);TMPRSS2: 1.99(4.85);ACE2: 0.36(0.52) AU) and amnion (ICAM-1: 0.69(2.21);LDLR: 0.39(1.38);CDHR3: 1.0 x 10-4(3.0x10-4);NCL: 1.03(0.55);CX3CR1: 0.12(0.24);TLR4: 0.10(0.13);TMPRSS2: 3.0 x 10-4 (16.0x10-4);ACE2: 0.01(0.02) AU). Amniotic samples also expressed these receptors at the protein level (ICAM-1: 0.03(0.05);LDLR: 0.06(0.03);CDHR3: 0.28(0.15);NCL: 0.96(1.19);CX3CR1: 0.08(0.08);TMPRSS2: 0.09(0.06);ACE2: 0.34(0.92) AU) and expression within the amniotic epithelium was confirmed by immunohistochemistry. Conclusion: Newborn nasal and amniotic epithelial samples expressed receptors for respiratory viruses, HRV, RSV, SARS-CoV-2. These findings warrant further investigation of the clinical significance of receptor expression in relation to prenatal and postnatal exposures, as well as childhood asthma development.

Nano-triciribine reduces sars-cov-2 infection by sequestering ace2 and the novel host factor LDLR

Introduction: People with previous CVD hospitalized for COVID-19 have elevated death rate. We reported that patients with diabetes and HF higher protein levels of the low density lipoprotein receptor (LDLR). We hypothesized that LDLR is a novel host factor for the SARS-CoV-2-Spike (S2S) protein that may be regulated by the Akt inhibitor Triciribine (TCN), a drug being tested in Phase III studies for breast cancer. We also hypothesized that nano-formulation of Triciribine (NanoTriciribine;NTCN) would enhance its efficacy and allow for intranasal delivery. Methods: Interactions between the recombinant proteins Spike-RBD (receptor binding domain), ACE2, LDLR and its ectodomains (EGFA-EFFB, C2-C5 and C2) were analyzed by binding assays and co-IP in HepG2, HK2, and 293T cells. Viral entry assays were performed with 2 S2S pseudoviruses using 293T cells + hACE2 and TMPRSS2 or Furin protease. The effect of NTCN or the LXR agonist GW-3965 on viral uptake (pseudotyped VSVΔG-GFP∗S2S or chimera VSV-S2SeGFP virus) was assessed. Akt, pAkt, ACE2, and LDLR levels were determined in 293T+hACE2 by flow cytometry. Assays were done in triplicates and 1-way-ANOVA with Tukey's correction was used for statistics. Results: RBD protein binds modestly to the human LDLR (EC50:10μM) and its C2-C5 ectodomain (EC50:13.8μM). Co-IP revealed a novel and strong LDLR-ACE2 interaction in several human cell lines. LDLR overexpression in human cells increased the uptake of VSVΔG-GFP∗S2S (FC=2.32;p<0.001) and chimera virus (FC=.33;p<.0001). NTCN and TCN each reduced pAkt/Akt ratio. 1μM TCN or NTCN reduced LDLR (7.2%;p<.01 &15.6%;p<0.0001) and ACE2 (32%;p<0.05 &44.7%;p<.01) cell surface expression, respectively. 1μM NTCN or GW-3965 reduced S2S viral entry by 64.2% (p<.0001) and 40.7% (p<.01), respectively, confirming a role for LDLR in S2S infection. In hACE2tg mice, chimera VSV-S2S caused significant lung infection as measured by qPCR, GFP expression in proximal and distal lung airway epithelial cells, and electron microscopy. Intranasal delivery of NTCN was well tolerated. Conclusions: LDLR enhanced S2S viral entry supporting the elevated COVID-19 susceptibility seen in patients with heart disease. NTCN is a promising candidate for prophylactic treatment against COVID-19.