Endocytic trafficking of GAS6-AXL complexes is associated with sustained AKT activation.

AXL, a TAM receptor tyrosine kinase (RTK), and its ligand growth arrest-specific 6 (GAS6) are implicated in cancer metastasis and drug resistance, and cellular entry of viruses. Given this, AXL is an attractive therapeutic target, and its inhibitors are being tested in cancer and COVID-19 clinical trials. Still, astonishingly little is known about intracellular mechanisms that control its function. Here, we characterized endocytosis of AXL, a process known to regulate intracellular functions of RTKs. Consistent with the notion that AXL is a primary receptor for GAS6, its depletion was sufficient to block GAS6 internalization. We discovered that upon receptor ligation, GAS6-AXL complexes were rapidly internalized via several endocytic pathways including both clathrin-mediated and clathrin-independent routes, among the latter the CLIC/GEEC pathway and macropinocytosis. The internalization of AXL was strictly dependent on its kinase activity. In comparison to other RTKs, AXL was endocytosed faster and the majority of the internalized receptor was not degraded but rather recycled via SNX1-positive endosomes. This trafficking pattern coincided with sustained AKT activation upon GAS6 stimulation. Specifically, reduced internalization of GAS6-AXL upon the CLIC/GEEC downregulation intensified, whereas impaired recycling due to depletion of SNX1 and SNX2 attenuated AKT signaling. Altogether, our data uncover the coupling between AXL endocytic trafficking and AKT signaling upon GAS6 stimulation. Moreover, our study provides a rationale for pharmacological inhibition of AXL in antiviral therapy as viruses utilize GAS6-AXL-triggered endocytosis to enter cells.

Hesperetin and the PI3K/AKT pathway: Could their interaction play a role in the entry and replication of the SARS-CoV-2?

Hesperetin, mainly found in citrus honey, has antioxidant, anti-inflammatory, and antiviral properties. Recently, the effect of hesperetin on different aspects of SARS-CoV-2 infection such as viral entry, replication, and inflammatory responses has attracted a lot of attention. However, the exact molecular mechanism for its effects on SARS-CoV-2 infection is not stated. The PI3K/AKT signaling pathway is an intracellular pathway involved in cell proliferation, protein synthesis, and response to environmental changes. Since the role of this pathway in the pathogenesis of SARS-CoV-2 has recently been considered, this letter assumes the probable role of this pathway in the function of hesperetin against SARS-CoV-2 infection. PRACTICAL APPLICATIONS: In this paper, we have discussed the therapeutic effects of hesperetin on SARS-CoV-2 infection. Additionally, we have hypothesized the molecular mechanism of hesperetin in suppression of SARS-CoV-2 entry to the host cells, its replication and inhibition of inflammatory responses. Based on this evidence, the pharmacological properties of hesperetin make this natural compound a potential treatment for suppression of SARS-CoV-2 entry into host cells and the subsequent replication of viral particles.

Bioinformatics and in-silico findings reveal medical features and pharmacological targets of biochanin A against colorectal cancer and COVID-19.

Severe mortality due to the COVID-19 pandemic resulted from the lack of effective treatment. Although COVID-19 vaccines are available, their side effects have become a challenge for clinical use in patients with chronic diseases, especially cancer patients. In the current report, we applied network pharmacology and systematic bioinformatics to explore the use of biochanin A in patients with colorectal cancer (CRC) and COVID-19 infection. Using the network pharmacology approach, we identified two clusters of genes involved in immune response (IL1A, IL2, and IL6R) and cell proliferation (CCND1, PPARG, and EGFR) mediated by biochanin A in CRC/COVID-19 condition. The functional analysis of these two gene clusters further illustrated the effects of biochanin A on interleukin-6 production and cytokine-cytokine receptor interaction in CRC/COVID-19 pathology. In addition, pathway analysis demonstrated the control of PI3K-Akt and JAK-STAT signaling pathways by biochanin A in the treatment of CRC/COVID-19. The findings of this study provide a therapeutic option for combination therapy against COVID-19 infection in CRC patients.

α-Ketoglutarate Inhibits Thrombosis and Inflammation by Prolyl Hydroxylase-2 Mediated Inactivation of Phospho-Akt.

BACKGROUND: Phospho-Akt1 (pAkt1) undergoes prolyl hydroxylation at Pro125 and Pro313 by the prolyl hydroxylase-2 (PHD2) in a reaction decarboxylating α-ketoglutarate (αKG). We investigated whether the αKG supplementation could inhibit Akt-mediated activation of platelets and monocytes, in vitro as well as in vivo, by augmenting PHD2 activity. METHODS: We treated platelets or monocytes isolated from healthy individuals with αKG in presence of agonists in vitro and assessed the signalling molecules including pAkt1. We supplemented mice with dietary αKG and estimated the functional responses of platelets and monocytes ex vivo. Further, we investigated the impact of dietary αKG on inflammation and thrombosis in lungs of mice either treated with thrombosis-inducing agent carrageenan or infected with SARS-CoV-2. FINDINGS: Octyl αKG supplementation to platelets promoted PHD2 activity through elevated intracellular αKG to succinate ratio, and reduced aggregation in vitro by suppressing pAkt1(Thr308). Augmented PHD2 activity was confirmed by increased hydroxylated-proline and enhanced binding of PHD2 to pAkt in αKG-treated platelets. Contrastingly, inhibitors of PHD2 significantly increased pAkt1 in platelets. Octyl-αKG followed similar mechanism in monocytes to inhibit cytokine secretion in vitro. Our data also describe a suppressed pAkt1 and reduced activation of platelets and leukocytes ex vivo from mice supplemented with dietary αKG, unaccompanied by alteration in their number. Dietary αKG significantly reduced clot formation and leukocyte accumulation in various organs including lungs of mice treated with thrombosis-inducing agent carrageenan. Importantly, in SARS-CoV-2 infected hamsters, we observed a significant rescue effect of dietary αKG on inflamed lungs with significantly reduced leukocyte accumulation, clot formation and viral load alongside down-modulation of pAkt in the lung of the infected animals. INTERPRETATION: Our study suggests that dietary αKG supplementation prevents Akt-driven maladies such as thrombosis and inflammation and rescues pathology of COVID19-infected lungs. FUNDING: Study was funded by the Department of Biotechnology (DBT), Govt. of India (grants: BT/PR22881 and BT/PR22985); and the Science and Engineering Research Board, Govt. of India (CRG/000092).

Targeting the PI3K/AKT pathway: a potential new weapon in the global fight against SARS-CoV-2?

Commentary on 'Capivasertib restricts SARS-CoV-2 cellular entry: a potential clinical application for COVID-19' by Sun et al.

The Pro-Inflammatory Chemokines CXCL9, CXCL10 and CXCL11 Are Upregulated Following SARS-CoV-2 Infection in an AKT-Dependent Manner.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a 'cytokine storm.' In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.

Lessons from Bacillus Calmette-Guérin: Harnessing Trained Immunity for Vaccine Development.

Vaccine design traditionally focuses on inducing adaptive immune responses against a sole target pathogen. Considering that many microbes evade innate immune mechanisms to initiate infection, and in light of the discovery of epigenetically mediated innate immune training, the paradigm of vaccine design has the potential to change. The Bacillus Calmette-Guérin (BCG) vaccine induces some level of protection against Mycobacterium tuberculosis (Mtb) while stimulating trained immunity that correlates with lower mortality and increased protection against unrelated pathogens. This review will explore BCG-induced trained immunity, including the required pathways to establish this phenotype. Additionally, potential methods to improve or expand BCG trained immunity effects through alternative vaccine delivery and formulation methods will be discussed. Finally, advances in new anti-Mtb vaccines, other antimicrobial uses for BCG, and "innate memory-based vaccines" will be examined.

Akt-independent effects of triciribine on ACE2 expression in human lung epithelial cells: Potential benefits in restricting SARS-CoV2 infection.

The severe acute respiratory syndrome coronavirus 2 that causes coronavirus disease 2019 (COVID-19) binds to the angiotensin-converting enzyme 2 (ACE2) to gain cellular entry. Akt inhibitor triciribine (TCBN) has demonstrated promising results in promoting recovery from advanced-stage acute lung injury in preclinical studies. In the current study, we tested the direct effect of TCBN on ACE2 expression in human bronchial (H441) and lung alveolar (A549) epithelial cells. Treatment with TCBN resulted in the downregulation of both messenger RNA and protein levels of ACE2 in A549 cells. Since HMGB1 plays a vital role in the inflammatory response in COVID-19, and because hyperglycemia has been linked to increased COVID-19 infections, we determined if HMGB1 and hyperglycemia have any effect on ACE2 expression in lung epithelial cells and whether TCBN has any effect on reversing HMGB1- and hyperglycemia-induced ACE2 expression. We observed increased ACE2 expression with both HMGB1 and hyperglycemia treatment in A549 as well as H441 cells, which were blunted by TCBN treatment. Our findings from this study, combined with our previous reports on the potential benefits of TCBN in the treatment of acute lung injury, generate reasonable optimism on the potential utility of TCBN in the therapeutic management of patients with COVID-19.

Exosomes from adipose tissue-derived mesenchymal stem cells ameliorate histone-induced acute lung injury by activating the PI3K/Akt pathway in endothelial cells.

BACKGROUND: Mesenchymal stem cells (MSCs), including adipose-derived mesenchymal stem cells (ADSCs), have been shown to attenuate organ damage in acute respiratory distress syndrome (ARDS) and sepsis; however, the underlying mechanisms are not fully understood. In this study, we aimed to explore the potential roles and molecular mechanisms of action of ADSCs in histone-induced endothelial damage. METHODS: Male C57BL/6 N mice were intravenously injected with ADSCs, followed by histones or a vehicle. The mice in each group were assessed for survival, pulmonary vascular permeability, and histological changes. A co-culture model with primary human umbilical vein endothelial cells (HUVECs) exposed to histones was used to clarify the paracrine effect of ADSCs. Overexpression and inhibition of miR-126 ADSCs were also examined as causative factors for endothelial protection. RESULTS: The administration of ADSCs markedly improved survival, inhibited histone-mediated lung hemorrhage and edema, and attenuated vascular hyper-permeability in mice. ADSCs were engrafted in the injured lung and attenuated histone-induced endothelial cell apoptosis. ADSCs showed endothelial protection (via a paracrine effect) and Akt phosphorylation in the histone-exposed HUVECs. Notably, increased Akt phosphorylation by ADSCs was mostly mediated by exosomes in histone-induced cytotoxicity and lung damage. Moreover, the expression of miR-126 was increased in exosomes from histone-exposed ADSCs. Remarkably, the inhibition of miR-126 in ADSCs failed to increase Akt phosphorylation in histone-exposed HUVECs. CONCLUSION: ADSC-derived exosomes may exert protective effects on endothelial cells via activation of the PI3K/Akt pathway.

System biological investigations of hydroxychloroquine and azithromycin targets and their implications in QT interval prolongation.

COVID-2019 pandemic is affecting people worldwide in the absence of an effective treatment strategy. Several suggestive therapeutic options through drug repurposing are recommended, but a complete consensus is not reached. A combination of Hydroxychloroquine (HCQ) and Azithromycin (AZM) has been widely tried and discussed but its administration has also led to potential adversities in patients. Studies are suggesting that most prominent adverse event with HCQ and AZM combination is QT interval prolongation. We studied interaction of HCQ with AZM and subsequent effect of this drug combination on QT interval prolongation. We performed system biological investigation of HCQ and AZM targets and screened important targets and pathways possibly involved in QT interval prolongation. The best core hub protein drug targets involved in QT interval prolongation were identified as HSP90AA1 exclusively associated with HCQ, while AKT1 exclusively associated with AZM on the basis of node degree value. It was found that PI3K/Akt, VEGF, ERBB2 pathways must be given consideration for understanding the role of HCQ and AZM in QT interval prolongation. Conclusion: Computational methods have certain limitations based on source database coverage and prediction algorithms and therefore this data needs experimental correlation to draw final conclusion, but current findings screen targets for QT interval prolongation associated with HCQ and AZM. These proteins and pathways may provide ways to reduce this major risk associated with this combination.

Decreased NHE3 activity and trafficking in TGEV-infected IPEC-J2 cells via the SGLT1-mediated P38 MAPK/AKt2 pathway.

Transmissible gastroenteritis virus (TGEV) primarily replicates in intestinal epithelial cells and causes severe damage to host cells, resulting in diarrhea. Surface NHE3 serves as the key regulatory site controlling electroneutral Na+ absorption. In this study, our results showed that the surface NHE3 content was significantly reduced following TGEV infection, whereas the total level of protein expression was not significantly changed, and NHE3 activity gradually decreased with prolonged infection time. We then inhibited SGLT1 expression by lentiviral interference and drug inhibition, respectively. Inhibition studies showed that the level of phosphorylation of the downstream key proteins, MAPKAPK-2 and EZRIN, in the SGLT1-mediated p38MAPK/AKt2 signaling pathway was significantly increased. The surface NHE3 expression was also significantly increased, and NHE3 activity was also significantly enhanced. These results demonstrate that a TGEV infection can inhibit NHE3 translocation and attenuates sodium-hydrogen exchange activity via the SGLT1-mediated p38MAPK/AKt2 signaling pathway, affecting cellular electrolyte absorption leading to diarrhea.

Dysregulation in Akt/mTOR/HIF-1 signaling identified by proteo-transcriptomics of SARS-CoV-2 infected cells.

How severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections engage cellular host pathways and innate immunity in infected cells remains largely elusive. We performed an integrative proteo-transcriptomics analysis in SARS-CoV-2 infected Huh7 cells to map the cellular response to the invading virus over time. We identified four pathways, ErbB, HIF-1, mTOR and TNF signaling, among others that were markedly modulated during the course of the SARS-CoV-2 infection in vitro. Western blot validation of the downstream effector molecules of these pathways revealed a dose-dependent activation of Akt, mTOR, S6K1 and 4E-BP1 at 24 hours post infection (hpi). However, we found a significant inhibition of HIF-1α through 24hpi and 48hpi of the infection, suggesting a crosstalk between the SARS-CoV-2 and the Akt/mTOR/HIF-1 signaling pathways. Inhibition of the mTOR signaling pathway using Akt inhibitor MK-2206 showed a significant reduction in virus production. Further investigations are required to better understand the molecular sequelae in order to guide potential therapy in the management of severe coronavirus disease 2019 (COVID-19) patients.