GRP78 Inhibitor YUM70 Suppresses SARS-CoV-2 Viral Entry, Spike Protein Production and Ameliorates Lung Damage.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has given rise to many new variants with increased transmissibility and the ability to evade vaccine protection. The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum (ER) chaperone that has been recently implicated as an essential host factor for SARS-CoV-2 entry and infection. In this study, we investigated the efficacy of YUM70, a small molecule inhibitor of GRP78, to block SARS-CoV-2 viral entry and infection in vitro and in vivo. Using human lung epithelial cells and pseudoviral particles carrying spike proteins from different SARS-CoV-2 variants, we found that YUM70 was equally effective at blocking viral entry mediated by original and variant spike proteins. Furthermore, YUM70 reduced SARS-CoV-2 infection without impacting cell viability in vitro and suppressed viral protein production following SARS-CoV-2 infection. Additionally, YUM70 rescued the cell viability of multi-cellular human lung and liver 3D organoids transfected with a SARS-CoV-2 replicon. Importantly, YUM70 treatment ameliorated lung damage in transgenic mice infected with SARS-CoV-2, which correlated with reduced weight loss and longer survival. Thus, GRP78 inhibition may be a promising approach to augment existing therapies to block SARS-CoV-2, its variants, and other viruses that utilize GRP78 for entry and infection.

SARS-CoV-2 Receptors and Their Role in Cell Infection

The new Coronavirus infection (COVI D-19) pandemic caused by the SARS-CoV-2 virus has many times surpassed the epidemic caused by SARS-CoV. The reason for this is the presence of amino acid sequences in the peptide chain of the SARS-CoV-2 S-protein that ensure interaction with a wider range of receptors on the host cell surface. The review considers both already known receptors common to SARS-CoV and SARS-CoV-2 and new receptors specific to SARS-CoV-2. © 2022 Russian Academy of Sciences, Institute of Archaeology. All rights reserved.

In silico targeting of SARS-CoV-2 spike receptor-binding domain from different variants with chaga mushroom terpenoids.

Terpenoids from the chaga mushroom have been identified as potential antiviral agents against SARS-CoV-2. This is because it can firmly bind to the viral spike receptor binding domain (RBD) and the auxiliary host cell receptor glucose-regulated protein 78 (GRP78). The current work examines the association of the chaga mushroom terpenoids with the RBD of various SARS-CoV-2 variants, including alpha, beta, gamma, delta, and omicron. This association was compared to the SARS-CoV-2 wild-type (WT) RBD using molecular docking analysis and molecular dynamics modeling. The outcomes demonstrated that the mutant RBDs, which had marginally greater average binding affinities (better binding) than the WT, were successfully inhibited by the chaga mushroom terpenoids. The results suggest that the chaga mushroom can be effective against various SARS-CoV-2 variants by targeting both the host-cell surface receptor GRP78 and the viral spike RBD.Communicated by Ramaswamy H. Sarma.

Porcine Epidemic Diarrhea Virus and Its nsp14 Suppress ER Stress Induced GRP78.

Porcine epidemic diarrhea virus (PEDV), a member of the α-coronavirus genus, can cause vomiting, diarrhea, and dehydration in piglets. Neonatal piglets infected with PEDV have a mortality rate as high as 100%. PEDV has caused substantial economic losses to the pork industry. Endoplasmic reticulum (ER) stress, which can alleviate the accumulation of unfolded or misfolded proteins in ER, involves in coronavirus infection. Previous studies have indicated that ER stress could inhibit the replication of human coronaviruses, and some human coronaviruses in turn could suppress ER stress-related factors. In this study, we demonstrated that PEDV could interact with ER stress. We determined that ER stress could potently inhibit the replication of GⅠ, GⅡ-a, and GⅡ-b PEDV strains. Moreover, we found that these PEDV strains can dampen the expression of the 78 kDa glucose-regulated protein (GRP78), an ER stress marker, while GRP78 overexpression showed antiviral activity against PEDV. Among different PEDV proteins, PEDV non-structural protein 14 (nsp14) was revealed to play an essential role in the inhibition of GRP78 by PEDV, and its guanine-N7-methyltransferase domain is necessary for this role. Further studies show that both PEDV and its nsp14 negatively regulated host translation, which could account for their inhibitory effects against GRP78. In addition, we found that PEDV nsp14 could inhibit the activity of GRP78 promotor, helping suppress GRP78 transcription. Our results reveal that PEDV possesses the potential to antagonize ER stress, and suggest that ER stress and PEDV nsp14 could be the targets for developing anti-PEDV drugs.

Matrine combined with Osthole inhibited the PERK apoptosis of splenic lymphocytes in PCV2-infected mice model.

BACKGROUND: Porcine circovirus type 2 (PCV2) is one of the major pathogens commonly found in pigs, which causes immunosuppression and apoptosis. Vaccination and a single drug cannot totally prevent and treat PCV2 infection. Our previous in vitro study reported that the synergistic anti-PCV2 effect of Matrine and Osthole was better than that of Matrine or Osthole alone, This study was aimed to evaluate the synergistic anti-PCV2 effect as well as the underline molecular mechanism of Matrine and Osthole in Kunming (KM) mice model infected with PCV2. KM mice were randomly divided into 8 groups namely control group, PCV2 infected, Matrine combined with Osthole high dose treatment (40 mg/kg + 12 mg/kg), medium dose treatment (20 mg/kg + 6 mg/kg), low dose treatment (10 mg/kg + 3 mg/kg), Matrine treatment (40 mg/kg), Osthole treatment (12 mg/kg) and Ribavirin positive control (40 mg/kg) groups. PCV2 was intraperitoneally (i.p.) injected in all mice except the control group. 5 days of post-infection (dpi), mice in different treatment groups were injected i.p. with various doses of Matrine, Osthole and Ribavirin once daily for the next 5 consecutive days. RESULTS: The synergistic inhibitory effect of Matrine and Osthole on PCV2 replication in mouse liver was significantly heigher than that of Matrine and Osthole alone. The expression of GRP78, p-PERK, p-eIF2α, ATF4, CHOP, cleaved caspase-3 and Bax proteins were significantly reduced, while that of Bcl-2 was significantly increased in Matrine combined with Osthole groups, which alleviated the pathological changes caused by PCV2, such as interstitial pneumonia, loss of spleen lymphocytes, infiltration of macrophages and eosinophils. CONCLUSIONS: The synergistic anti-apoptotic effect of Matrine and Osthole was better than their alone effect, Both Matrine and Osthole had directly inhibited the expression of PCV2 Cap and the apoptosis of spleen cells induced by PCV2 Cap through the PERK pathway activated by endoplasmic reticulum (ER) GRP78. These results provided a new insight to control PCV2 infection and provide good component prescription candidate for the development of novel anti-PCV2 drugs.

SARS-CoV-2 Receptors and Their Involvement in Cell Infection.

The new coronavirus infection (COVID-19) pandemic caused by SARS-CoV-2 has many times surpassed the epidemics caused by SARS-CoV and MERS-CoV. The reason for this was the presence of sites in the protein sequence of SARS-CoV-2 that provide interaction with a broader range of receptor proteins on the host cell surface. In this review, we consider both already known receptors common to SARS-CoV and SARS-CoV-2 and new receptors specific to SARS-CoV-2.

Leukocyte surface expression of the endoplasmic reticulum chaperone GRP78 is increased in severe COVID-19.

Hyperinflammation present in individuals with severe COVID-19 has been associated with an exacerbated cytokine production and hyperactivated immune cells. Endoplasmic reticulum stress leading to the unfolded protein response has been recently reported as an active player in inducing inflammatory responses. Once unfolded protein response is activated, GRP78, an endoplasmic reticulum-resident chaperone, is translocated to the cell surface (sGRP78), where it is considered a cell stress marker; however, its presence has not been evaluated in immune cells during disease. Here we assessed the presence of sGRP78 on different cell subsets in blood samples from severe or convalescent COVID-19 patients. The frequency of CD45+sGRP78+ cells was higher in patients with the disease compared to convalescent patients. The latter showed similar frequencies to healthy controls. In patients with COVID-19, the lymphoid compartment showed the highest presence of sGRP78+ cells versus the myeloid compartment. CCL2, TNF-α, C-reactive protein, and international normalized ratio measurements showed a positive correlation with the frequency of CD45+sGRP78+ cells. Finally, gene expression microarray data showed that activated T and B cells increased the expression of GRP78, and peripheral blood mononuclear cells from healthy donors acquired sGRP78 upon activation with ionomycin and PMA. Thus, our data highlight the association of sGRP78 on immune cells in patients with severe COVID-19.

Host-Cell Surface Binding Targets in SARS-COV-2 for Drug Design.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a major public health threat to all countries worldwide. SARS-CoV-2 interactions with its receptor are the first step in the invasion of the host cell. The coronavirus spike protein (S) is crucial in binding to receptors on host cells. Additionally, targeting the SARS-CoV-2 viral receptors is considered a therapeutic option in this regard. In this review of literature, we summarized five potential host cell receptors, as host-cell surface bindings, including angiotensin-converting enzyme 2 (ACE2), neuropilin 1 (NRP-1), dipeptidyl peptidase 4 (DPP4), glucose regulated protein-78 (GRP78), and cluster of differentiation 147 (CD147) related to the SARS-CoV-2 infection. Among these targets, ACE2 was recognized as the main SARS-CoV-2 receptor, expressed at a low/moderate level in the human respiratory system, which is also involved in SARS-CoV-2 entrance, so the virus may utilize other secondary receptors. Besides ACE2, CD147 was discovered as a novel SARS-CoV-2 receptor, CD147 appears to be an alternate receptor for SARS-CoV-2 infection. NRP-1, as a single- transmembrane glycoprotein, has been recently found to operate as an entrance factor and enhance SARS Coronavirus 2 (SARS-CoV-2) infection under in-vitro. DPP4, which was discovered as the first gene clustered with ACE2, may serve as a potential SARS-CoV-2 spike protein binding target. GRP78 could be recognized as a secondary receptor for SARS-CoV-2 because it is widely expressed at substantially greater levels, rather than ACE2, in bronchial epithelial cells and the respiratory mucosa. This review highlights recent literature on this topic.

Cellular stress modulates severity of the inflammatory response in lungs via cell surface BiP.

Inflammation is a central pathogenic feature of the acute respiratory distress syndrome (ARDS) in COVID-19. Previous pathologies such as diabetes, autoimmune or cardiovascular diseases become risk factors for the severe hyperinflammatory syndrome. A common feature among these risk factors is the subclinical presence of cellular stress, a finding that has gained attention after the discovery that BiP (GRP78), a master regulator of stress, participates in the SARS-CoV-2 recognition. Here, we show that BiP serum levels are higher in COVID-19 patients who present certain risk factors. Moreover, early during the infection, BiP levels predict severe pneumonia, supporting the use of BiP as a prognosis biomarker. Using a mouse model of pulmonary inflammation, we observed increased levels of cell surface BiP (cs-BiP) in leukocytes during inflammation. This corresponds with a higher number of neutrophiles, which show naturally high levels of cs-BiP, whereas alveolar macrophages show a higher than usual exposure of BiP in their cell surface. The modulation of cellular stress with the use of a clinically approved drug, 4-PBA, resulted in the amelioration of the lung hyperinflammatory response, supporting the anti-stress therapy as a valid therapeutic strategy for patients developing ARDS. Finally, we identified stress-modulated proteins that shed light into the mechanism underlying the cellular stress-inflammation network in lungs.

SARS-CoV-2 Receptors and Their Role in Cell Infection

The new coronavirus infection (COVID-19) pandemic caused by the SARS-CoV-2 virus has many times surpassed the epidemic caused by SARS-CoV. The reason for this is the presence of amino acid sequences in the peptide chain of the SARS-CoV-2 S-protein that ensure interaction with a wider range of receptors on the host cell surface. The review considers both already known receptors common to SARS-CoV and SARS-CoV-2 and new receptors specific to SARS-CoV-2.

Potential of green tea EGCG in neutralizing SARS-CoV-2 Omicron variant with greater tropism toward the upper respiratory tract.

Background: COVID-19 due to SARS-CoV-2 infection has had an enormous adverse impact on global public health. As the COVID-19 pandemic evolves, the WHO declared several variants of concern (VOCs), including Alpha, Beta, Gamma, Delta, and Omicron. Compared with earlier variants, Omicron, now a dominant lineage, exhibits characteristics of enhanced transmissibility, tropism shift toward the upper respiratory tract, and attenuated disease severity. The robust transmission of Omicron despite attenuated disease severity still poses a great challenge for pandemic control. Under this circumstance, its tropism shift may be utilized for discovering effective preventive approaches. Scope and approach: This review aims to estimate the potential of green tea epigallocatechin gallate (EGCG), the most potent antiviral catechin, in neutralizing SARS-CoV-2 Omicron variant, based on current knowledge concerning EGCG distribution in tissues and Omicron tropism. Key findings and conclusions: EGCG has a low bioavailability. Plasma EGCG levels are in the range of submicromolar concentrations following green tea drinking, or reach at most low µM concentrations after pharmacological intervention. Nonetheless, its levels in the upper respiratory tract could reach concentrations as high as tens or even hundreds of µM following green tea consumption or pharmacological intervention. An approach for delivering sufficiently high concentrations of EGCG in the pharynx has been developed. Convincing data have demonstrated that EGCG at tens to hundreds of µM can dramatically neutralize SARS-CoV-2 and effectively eliminate SARS-CoV-2-induced cytopathic effects and plaque formation. Thus, EGCG, which exhibits hyperaccumulation in the upper respiratory tract, deserves closer investigation as an antiviral in the current global battle against COVID-19, given Omicron's greater tropism toward the upper respiratory tract.

Mucormycosis in the COVID-19 Environment: A Multifaceted Complication.

The second wave of coronavirus disease 2019 (COVID-19) caused severe infections with high mortality. An increase in the cases of COVID-19-associated mucormycosis (CAM) was reported predominantly in India. Commonly present in immunocompromised individuals, mucormycosis is often a life-threatening condition. Confounding factors and molecular mechanisms associated with CAM are still not well understood, and there is a need for careful research in this direction. In this review, a brief account of the diagnosis, management, and advancement in drug discovery for mucormycosis has been provided. Here, we summarize major factors that dictate the occurrence of mucormycosis in COVID-19 patients through the analysis of published literature and case reports. Major predisposing factors to mucormycosis appear to be uncontrolled diabetes, steroid therapy, and certain cancers. At the molecular level, increased levels of iron in COVID-19 might contribute to mucormycosis. We have also discussed the potential role and regulation of iron metabolism in COVID-19 patients in establishing fungal growth. Other factors including diabetes prevalence and fungal spore burden in India as contributing factors have also been discussed.

Vulnerability of The Male Reproductive System to SARS-CoV-2 Invasion: Potential Role for The Endoplasmic Reticulum Chaperone Grp78/HSPA5/BiP.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) may adversely affect male reproductive tissues and male
fertility. This concern is elicited by the higher susceptibility and mortality rate of men to the SARS-CoV-2 mediated coronavirus disease-19 (COVID-19), compared to the women. SARS-CoV-2 enters host cells after binding to a functional receptor named angiotensin-converting enzyme-2 (ACE2) and then replicates in the host cells and gets released into the plasma. SARS-CoVs use the endoplasmic reticulum (ER) as a site for viral protein synthesis and processing, as well as glucose-regulated protein 78 (Grp78) is a key ER chaperone involved in protein folding by preventing newly synthesized proteins from aggregation.
Therefore, we analyzed Grp78 expression in various human organs, particularly male reproductive organs, using Broad
Institute Cancer Cell Line Encyclopedia (CCLE), the Genotype-Tissue Expression (GTEx), and Human Protein Atlas online
datasets. Grp78 is expressed in male reproductive tissues such as the testis, epididymis, prostate, and seminal vesicle. It can facilitate the coronavirus entry into the male reproductive tract, providing an opportunity for its replication. This link between the SARS-CoV-2 and the Grp78 protein could become a therapeutic target to mitigate its harmful effects on male fertility.

SARS-CoV-2 Delta Variant is Recognized Through GRP78 Host-Cell Surface Receptor, In Silico Perspective.

Different SARS-CoV-2 new variants emerged and spread during the past few months, sparking infections and death counts. The new variant B.1.617 (delta variant) sparked in India in the past few months, causing the highest records. The B.1.617 variant of SARS-CoV-2 has the double mutations E484Q and L452R on its spike Receptor Binding Domain (RBD). The first mutation is like the reported South African and the Brazilian variants (501.V2 and B.1.1.248). This mutation lies in the region C480-C488, which we predicted before to be recognized by the host-cell receptor; Glucose Regulated Protein 78 (GRP78). In the current study, we test the binding affinity of the host-cell receptor GRP78 to the delta variant spike RBD using molecular docking and molecular dynamics simulations of up to 100 ns. Additionally, the ACE2-RBD is tested by protein-protein docking. The results reveal equal average binding affinities of the GRP78 against wildtype and delta variant spikes. This supports our previous predictions of the contribution of GRP78 in SARS-CoV-2 spike recognition as an auxiliary route for entry.

Insights from Surgically treated Post Covid Acute Invasive Fungal Rhino-Orbital sinusitis in Chandrapur Study (SPAROS): A Population Based study of Coronavirus Associated Mucormycosis (CAM) characteristics in India.

Introduction: Coronavirus disease 2019 (COVID-19)-associated mucormycosis (CAM) became a public health problem in India in 2021. However, information about the incidence, presentation and prognosis of CAM remains sparse. Methods: This study describes 100 cases from the Surgically treated Post COVID Acute invasive fungal Rhino-Orbital Sinusitis in Chandrapur (SPAROS) study, a prospective observational follow-up study of patients with CAM diagnosed in Chandrapur district, India. Two-step cluster analysis using four input variables - blood glucose on admission, diabetes status, glucocorticoid exposure and severity of COVID-19 - was used to define three distinct CAM clusters. Results: The incidence of CAM in the general population was 7.1 cases/1000 patients hospitalized with COVID-19. Steroid exposure and pre-existing diabetes were present in 76% and 55% of cases, respectively. At median follow-up of 18 days, only two deaths had been recorded, while 93 cases were stable. Glucocorticoids, particularly methylprednisolone, seemed to precipitate CAM. Admission to the intensive care unit appeared to be predictive of less extensive surgery. Discussion: Three subtypes of CAM were identified: COVID-19-associated diabetes and mucormycosis, COVID-19-associated classical mucormycosis, and COVID-19-induced mucormycosis. A CAM hypothesis was proposed based on the dynamics of severe acute respiratory syndrome coronavirus-2 and glucose regulated protein. Conclusion: The clinical characteristics, natural course and pathogenesis of CAM differ from mucormycosis in the pre-COVID era. It is hoped that this classification will be useful in CAM management.

GRP78, a Novel Host Factor for SARS-CoV-2: The Emerging Roles in COVID-19 Related to Metabolic Risk Factors.

The outbreak of coronavirus disease 19 (COVID-19), caused by the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in an unprecedented amount of infection cases and deaths, leading to the global health crisis. Despite many research efforts, our understanding of COVID-19 remains elusive. Recent studies have suggested that cell surface glucose-regulated protein 78 (GRP78) acts as a host co-receptor for SARS-CoV-2 infection and is related to COVID-19 risks, such as older age, obesity, and diabetes. Given its significance in a wide range of biological processes, such as protein homeostasis and cellular signaling, GRP78 might also play an important role in various stages of the viral life cycle and pathology of SARS-CoV-2. In this perspective, we explore the emerging and potential roles of GRP78 in SARS-CoV-2 infection. Additionally, we discuss the association with COVID-19 risks and symptoms. We hope this review article will be helpful to understand COVID-19 pathology and promote attention and study of GRP78 from many clinical and basic research fields.

Soluble RAGE as a Prognostic Marker of Worsening in Patients Admitted to the ICU for COVID-19 Pneumonia: A Prospective Cohort Study.

BACKGROUND: The different waves of SARS-CoV-2 infection have strained hospital resources and, notably, intensive care units (ICUs). Identifying patients at risk of developing a critical condition is essential to correctly refer patients to the appropriate structure and to spare limited resources. The soluble form of RAGE (sRAGE), the endoplasmic stress response and its surrogates, GRP78 and VEGF-A, may be interesting markers. METHODS: This was a prospective monocenter cohort study of adult patients admitted to the ICU for severe COVID-19 pneumonia. The plasma levels of sRAGE, GRP78 and VEGF-A were measured within the first 24 h. Patients were classified as critical if they further needed vasopressor therapy, renal replacement therapy, or invasive mechanical ventilation, or died during their ICU stay, and were otherwise classified as not critical. RESULTS: A total of 98 patients were included and 39 developed a critical condition. Critical patients presented higher sRAGE (626 [450-1043] vs. 227 [137-404] pg/mL, p < 0.0001), interleukin-6 (43 [15-112] vs. 11 [5-20] pg/mL, p < 0.0001), troponin T (17 [9-39] vs. 10 [6-18] pg/mL, p = 0.003) and NT-pro-BNP (321 [118-446] vs. 169 [63-366] pg/mL, p = 0.009) plasma levels. No difference was observed for VEGF-A and GRP78. The variables independently associated with worsening in the ICU were sRAGE (1.03 [1.01-1.05] per 10 pg/mL) and age (1.7 [1.2-2.4] per 5 years). An sRAGE value of 449.5 pg/mL predicted worsening with a sensitivity of 77% and a specificity of 80%. CONCLUSION: sRAGE may allow the identification of patients at risk of developing a critical form of COVID-19 pneumonia, and thus may be useful to correctly refer patients to the appropriate structure of care.

Endoplasmic Reticulum Chaperones in Viral Infection: Therapeutic Perspectives.

Viruses are intracellular parasites that subvert the functions of their host cells to accomplish their infection cycle. The endoplasmic reticulum (ER)-residing chaperone proteins are central for the achievement of different steps of the viral cycle, from entry and replication to assembly and exit. The most abundant ER chaperones are GRP78 (78-kDa glucose-regulated protein), GRP94 (94-kDa glucose-regulated protein), the carbohydrate or lectin-like chaperones calnexin (CNX) and calreticulin (CRT), the protein disulfide isomerases (PDIs), and the DNAJ chaperones. This review will focus on the pleiotropic roles of ER chaperones during viral infection. We will cover their essential role in the folding and quality control of viral proteins, notably viral glycoproteins which play a major role in host cell infection. We will also describe how viruses co-opt ER chaperones at various steps of their infectious cycle but also in order to evade immune responses and avoid apoptosis. Finally, we will discuss the different molecules targeting these chaperones and the perspectives in the development of broad-spectrum antiviral drugs.

Chitosan, from crayfish wastes, as a Possible Therapeutic Option against COVID-19;an In-Silico Perspective

Human coronavirus SARS-CoV-2 is the causative agent for the COVID-19 pandemic we are encountering nowadays. With the rapid spread of the contagious virus, it is urgent to find possible therapeutics. Chitosan is a naturally occurring polymer found in animals and plants. Antiviral and antibacterial activities of chitosan were documented. In the current study, the association of chitosan with the viral spike protein was assessed utilizing computational techniques. Molecular docking, combined with molecular dynamics, was used to test the binding affinity of chitosan to SARS-CoV-2 spike protein. The results suggest excellent binding potency of the chitosan to SARS-CoV-2 spike receptor-binding domain (RBD). These results are based on the docking and the interaction dynamics. Chitosan was able to form eight contacts (H-bonds and few salt bridges) with the spike receptor-binding domain (RBD) near the angiotensin-converting enzyme 2 (ACE2) and glucose regulating protein 78 (GRP78) recognition surfaces. Thus, chitosan can be a successful candidate against COVID-19;however, the in-silico results are yet to be validated experimentally. The current data were submitted to the Egyptian Academy of Scientific Research and Technology (ASRT) patent office with the registration number of 1602/2020. © 2022, Egyptian Society for the Development of Fisheries and Human Health. All rights reserved.