Current thoughts on cellular functions of numb-associated kinases.

Members of the Numb-associated kinase family of serine/threonine kinases play an essential role in many cellular processes, such as endocytosis, autophagy, dendrite morphogenesis, osteoblast differentiation, and the regulation of the Notch pathway. Numb-associated kinases have been relevant to diverse diseases, including neuropathic pain, Parkinson's disease, and prostate cancer. Therefore, they are considered potential therapeutic targets. In addition, it is reported that Numb-associated kinases have been involved in the life cycle of multiple viruses such as hepatitis C virus (HCV), Ebola virus (EBOV), and dengue virus (DENV). Recently, Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten global health. Studies show that Numb-associated kinases are implicated in the infection of SARS-CoV-2 which can be suppressed by Numb-associated kinases inhibitors. Thus, Numb-associated kinases are proposed as potential host targets for broad-spectrum antiviral strategies. We will focus on the recent advances in Numb-associated kinases-related cellular functions and their potential as host targets for viral infections in this review. Questions that remained unknown on the cellular functions of Numb-associated kinases will also be discussed.

Effects of Drugs Formerly Suggested for COVID-19 Repurposing on Pannexin1 Channels.

Although many efforts have been made to elucidate the pathogenesis of COVID-19, the underlying mechanisms are yet to be fully uncovered. However, it is known that a dysfunctional immune response and the accompanying uncontrollable inflammation lead to troublesome outcomes in COVID-19 patients. Pannexin1 channels are put forward as interesting drug targets for the treatment of COVID-19 due to their key role in inflammation and their link to other viral infections. In the present study, we selected a panel of drugs previously tested in clinical trials as potential candidates for the treatment of COVID-19 early on in the pandemic, including hydroxychloroquine, chloroquine, azithromycin, dexamethasone, ribavirin, remdesivir, favipiravir, lopinavir, and ritonavir. The effect of the drugs on pannexin1 channels was assessed at a functional level by means of measurement of extracellular ATP release. Immunoblot analysis and real-time quantitative reversetranscription polymerase chain reaction analysis were used to study the potential of the drugs to alter pannexin1 protein and mRNA expression levels, respectively. Favipiravir, hydroxychloroquine, lopinavir, and the combination of lopinavir with ritonavir were found to inhibit pannexin1 channel activity without affecting pannexin1 protein or mRNA levels. Thusthree new inhibitors of pannexin1 channels were identified that, though currently not being used anymore for the treatment of COVID-19 patients, could be potential drug candidates for other pannexin1-related diseases.

Possible role of pannexin 1 channels and purinergic receptors in the pathogenesis and mechanism of action of SARS-CoV-2 and therapeutic potential of targeting them in COVID-19.

Identifying signaling pathways and molecules involved in SARS-CoV-2 pathogenesis is pivotal for developing new effective therapeutic or preventive strategies for COVID-19. Pannexins (PANX) are ATP-release channels in the plasma membrane essential in many physiological and immune responses. Activation of pannexin channels and downstream purinergic receptors play dual roles in viral infection, either by facilitating viral replication and infection or inducing host antiviral defense. The current review provides a hypothesis demonstrating the possible contribution of the PANX1 channel and purinergic receptors in SARS-CoV-2 pathogenesis and mechanism of action. Moreover, we discuss whether targeting these signaling pathways may provide promising preventative therapies and treatments for patients with progressive COVID-19 resulting from excessive pro-inflammatory cytokines and chemokines production. Several inhibitors of this pathway have been developed for the treatment of other viral infections and pathological consequences. Specific PANX1 inhibitors could be potentially included as part of the COVID-19 treatment regimen if, in future, studies demonstrate the role of PANX1 in COVID-19 pathogenesis. Of note, any ATP therapeutic modulation for COVID-19 should be carefully designed and monitored because of the complex role of extracellular ATP in cellular physiology.

IDEAS: individual level differential expression analysis for single-cell RNA-seq data.

We consider an increasingly popular study design where single-cell RNA-seq data are collected from multiple individuals and the question of interest is to find genes that are differentially expressed between two groups of individuals. Towards this end, we propose a statistical method named IDEAS (individual level differential expression analysis for scRNA-seq). For each gene, IDEAS summarizes its expression in each individual by a distribution and then assesses whether these individual-specific distributions are different between two groups of individuals. We apply IDEAS to assess gene expression differences of autism patients versus controls and COVID-19 patients with mild versus severe symptoms.

Rh-CXCL-12 Attenuates Neuronal Pyroptosis after Subarachnoid Hemorrhage in Rats via Regulating the CXCR4/NLRP1 Pathway.

Subarachnoid hemorrhage (SAH) is a cerebrovascular disease associated with high morbidity and mortality. CXCR4 provides neuroprotective effects, which can alleviate brain injury and inflammation induced by stroke. Previous studies have suggested that CXCR4 reduces the pyroptosis of LPS-stimulated BV2 cells. The purpose of this study was to evaluate the antipyroptosis effects and mechanisms of CXCR4 after SAH. SAH animal model was induced via endovascular perforation. A total of 136 male Sprague-Dawley rats were used. Recombinant human cysteine-X-cysteine chemokine ligand 12 (rh-CXCL-12) was administered intranasally at 1 h after SAH induction. To investigate the underlying mechanism, the inhibitor of CXCR4, AMD3100, was administered intraperitoneally at 1 h before SAH. The neurobehavior tests were assessed, followed by performing Western blot and immunofluorescence staining. The Western blot results suggested that the expressions of endogenous CXCL-12, CXCR4, and NLRP1 were increased and peaked at 24 h following SAH. Immunofluorescence staining showed that CXCR4 was expressed on neurons, microglia, and astrocytes. Rh-CXCL-12 treatment improved the neurological deficits and reduced the number of FJC-positive cells, IL-18-positive neurons, and cleaved caspase-1(CC-1)-positive neurons after SAH. Meanwhile, rh-CXCL-12 treatment increased the levels of CXCL-12 and CXCR4, and reduced the levels of NLRP1, IL-18, IL-1ß, and CC-1. Moreover, the administration of AMD3100 abolished antipyroptosis effects of CXCL-12 and its regulation of CXCR4 post-SAH. The CXCR4/NLRP1 signaling pathway may be involved in CXCL-12-mediated neuronal pyroptosis after SAH. Early administration of CXCL-12 may be a preventive and therapeutic strategy against brain injury after SAH.

PDZ-Containing Proteins Targeted by the ACE2 Receptor.

Angiotensin-converting enzyme 2 (ACE2) is a main receptor for SARS-CoV-2 entry to the host cell. Indeed, the first step in viral entry is the binding of the viral trimeric spike (S) protein to ACE2. Abundantly present in human epithelial cells of many organs, ACE2 is also expressed in the human brain. ACE2 is a type I membrane protein with an extracellular N-terminal peptidase domain and a C-terminal collectrin-like domain that ends with a single transmembrane helix and an intracellular 44-residue segment. This C-terminal segment contains a PDZ-binding motif (PBM) targeting protein-interacting domains called PSD-95/Dlg/ZO-1 (PDZ). Here, we identified the human PDZ specificity profile of the ACE2 PBM using the high-throughput holdup assay and measuring the binding intensities of the PBM of ACE2 against the full human PDZome. We discovered 14 human PDZ binders of ACE2 showing significant binding with dissociation constants' values ranging from 3 to 81 µM. NHERF, SHANK, and SNX27 proteins found in this study are involved in protein trafficking. The PDZ/PBM interactions with ACE2 could play a role in ACE2 internalization and recycling that could be of benefit for the virus entry. Interestingly, most of the ACE2 partners we identified are expressed in neuronal cells, such as SHANK and MAST families, and modifications of the interactions between ACE2 and these neuronal proteins may be involved in the neurological symptoms of COVID-19.

Identification of ZDHHC17 as a Potential Drug Target for Swine Acute Diarrhea Syndrome Coronavirus Infection.

The recent emergence and spread of zoonotic viruses highlights that animal-sourced viruses are the biggest threat to global public health. Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an HKU2-related bat coronavirus that was spilled over from Rhinolophus bats to swine, causing large-scale outbreaks of severe diarrhea disease in piglets in China. Unlike other porcine coronaviruses, SADS-CoV possesses broad species tissue tropism, including primary human cells, implying a significant risk of cross-species spillover. To explore host dependency factors for SADS-CoV as therapeutic targets, we employed genome-wide CRISPR knockout library screening in HeLa cells. Consistent with two independent screens, we identified the zinc finger DHHC-type palmitoyltransferase 17 (ZDHHC17 or ZD17) as an important host factor for SADS-CoV infection. Through truncation mutagenesis, we demonstrated that the DHHC domain of ZD17 that is involved in palmitoylation is important for SADS-CoV infection. Mechanistic studies revealed that ZD17 is required for SADS-CoV genomic RNA replication. Treatment of infected cells with the palmitoylation inhibitor 2-bromopalmitate (2-BP) significantly suppressed SADS-CoV infection. Our findings provide insight on SADS-CoV-host interactions and a potential therapeutic application. IMPORTANCE The recent emergence of deadly zoonotic viral diseases, including Ebola virus and SARS-CoV-2, emphasizes the importance of pandemic preparedness for the animal-sourced viruses with potential risk of animal-to-human spillover. Over the last 2 decades, three significant coronaviruses of bat origin, SARS-CoV, MERS-CoV, and SARS-CoV-2, have caused millions of deaths with significant economy and public health impacts. Lack of effective therapeutics against these coronaviruses was one of the contributing factors to such losses. Although SADS-CoV, another coronavirus of bat origin, was only known to cause fatal diarrhea disease in piglets, the ability to infect cells derived from multiple species, including human, highlights the potential risk of animal-to-human spillover. As part of our effort in pandemic preparedness, we explore SADS-CoV host dependency factors as targets for host-directed therapeutic development and found zinc finger DHHC-type palmitoyltransferase 17 is a promising drug target against SADS-CoV replication. We also demonstrated that a palmitoylation inhibitor, 2-bromopalmitate (2-BP), can be used as an inhibitor for SADS-CoV treatment.

TMEM106B in humans and Vac7 and Tag1 in yeast are predicted to be lipid transfer proteins.

TMEM106B is an integral membrane protein of late endosomes and lysosomes involved in neuronal function, its overexpression being associated with familial frontotemporal lobar degeneration, and point mutation linked to hypomyelination. It has also been identified in multiple screens for host proteins required for productive SARS-CoV-2 infection. Because standard approaches to understand TMEM106B at the sequence level find no homology to other proteins, it has remained a protein of unknown function. Here, the standard tool PSI-BLAST was used in a nonstandard way to show that the lumenal portion of TMEM106B is a member of the late embryogenesis abundant-2 (LEA-2) domain superfamily. More sensitive tools (HMMER, HHpred, and trRosetta) extended this to predict LEA-2 domains in two yeast proteins. One is Vac7, a regulator of PI(3,5)P2 production in the degradative vacuole, equivalent to the lysosome, which has a LEA-2 domain in its lumenal domain. The other is Tag1, another vacuolar protein, which signals to terminate autophagy and has three LEA-2 domains in its lumenal domain. Further analysis of LEA-2 structures indicated that LEA-2 domains have a long, conserved lipid-binding groove. This implies that TMEM106B, Vac7, and Tag1 may all be lipid transfer proteins in the lumen of late endocytic organelles.

Nsp2 has the potential to be a drug target revealed by global identification of SARS-CoV-2 Nsp2-interacting proteins.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global health threat since December 2019, and there is still no highly effective drug to control the pandemic. To facilitate drug target identification for drug development, studies on molecular mechanisms, such as SARS-CoV-2 protein interactions, are urgently needed. In this study, we focused on Nsp2, a non-structural protein with largely unknown function and mechanism. The interactome of Nsp2 was revealed through the combination of affinity purification mass spectrometry (AP-MS) and stable isotope labeling by amino acids in cell culture (SILAC), and 84 proteins of high-confidence were identified. Gene ontology analysis demonstrated that Nsp2-interacting proteins are involved in several biological processes such as endosome transport and translation. Network analysis generated two clusters, including ribosome assembly and vesicular transport. Bio-layer interferometry (BLI) assay confirmed the bindings between Nsp2- and 4-interacting proteins, i.e. STAU2 (Staufen2), HNRNPLL, ATP6V1B2, and RAP1GDS1 (SmgGDS), which were randomly selected from the list of 84 proteins. Our findings provide insights into the Nsp2-host interplay and indicate that Nsp2 may play important roles in SARS-CoV-2 infection and serve as a potential drug target for anti-SARS-CoV-2 drug development.

SARS-CoV-2 Infection in the Central and Peripheral Nervous System-Associated Morbidities and Their Potential Mechanism.

The recent outbreak of SARS-CoV-2 infections that causes coronavirus-induced disease of 2019 (COVID-19) is the defining and unprecedented global health crisis of our time in both the scale and magnitude. Although the respiratory tract is the primary target of SARS-CoV-2, accumulating evidence suggests that the virus may also invade both the central nervous system (CNS) and the peripheral nervous system (PNS) leading to numerous neurological issues including some serious complications such as seizures, encephalitis, and loss of consciousness. Here, we present a comprehensive review of the currently known role of SARS-CoV-2 and identify all the neurological problems reported among the COVID-19 case reports throughout the world. The virus might gain entry into the CNS either through the trans-synaptic route via the olfactory neurons or through the damaged endothelium in the brain microvasculature using the ACE2 receptor potentiated by neuropilin-1 (NRP-1). The most critical of all symptoms appear to be the spontaneous loss of breathing in some COVID-19 patients. This might be indicative of a dysfunction within the cardiopulmonary regulatory centers in the brainstem. These pioneering studies, thus, lay a strong foundation for more in-depth basic and clinical research required to confirm the role of SARS-CoV-2 infection in neurodegeneration of critical brain regulatory centers.

Neurological manifestations of COVID-19: available evidences and a new paradigm.

The recent pandemic outbreak of coronavirus is pathogenic and a highly transmittable viral infection caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2). In this time of ongoing pandemic, many emerging reports suggested that the SARS-CoV-2 has inimical effects on neurological functions, and even causes serious neurological damage. The neurological symptoms associated with COVID-19 include headache, dizziness, depression, anosmia, encephalitis, stroke, epileptic seizures, and Guillain-Barre syndrome along with many others. The involvement of the CNS may be related with poor prognosis and disease worsening. Here, we review the evidence of nervous system involvement and currently known neurological manifestations in COVID-19 infections caused by SARS-CoV-2. We prioritize the 332 human targets of SARS-CoV-2 according to their association with brain-related disease and identified 73 candidate genes. We prioritize these 73 genes according to their spatio-temporal expression in the different regions of brain and also through evolutionary intolerance analysis. The prioritized genes could be considered potential indicators of COVID-19-associated neurological symptoms and thus act as a possible therapeutic target for the prevention and treatment of CNS manifestations associated with COVID-19 patients.

Can COVID-19 induce glioma tumorogenesis through binding cell receptors?

The outbreak of Novel Coronavirus 2019 (COVID-19) represents a global threat to the public healthcare. The viral spike (S) glycoprotein is the key molecule for viral entry through interaction with angiotensin converting enzyme 2 (ACE2) receptor molecules present on the cell membranes. Moreover, it has been established that COVID-19 interacts and infects brain cells in humans via ACE2. Therefore in the light of these known facts we hypothesized that viral S protein molecule may bind to the other overexpressed receptor molecules in glioma cells and may play some role in glioma tumorogenesis. Thus we leverage docking analysis (HEX and Z-DOCK) between viral S protein and epidermal growth factor receptors (EGFR), vascular endothelial growth factor receptors (VEGFR) and hepatocyte growth factor receptors (HGFR/c-MET) to investigate the oncogenic potential of COVID-19. Our findings suggested higher affinity of Viral S protein towards EGFR and VEGFR. Although, the data presented is preliminary and need to be validated further via molecular dynamics studies, however it paves platform to instigate further investigations on this aspect considering the aftermath of COVID-19 pandemic in oncogenic perspective.