Serine-arginine protein kinases and their targets in viral infection and their inhibition.

Accumulating evidence has consolidated the interaction between viral infection and host alternative splicing. Serine-arginine (SR) proteins are a class of highly conserved splicing factors critical for the spliceosome maturation, alternative splicing and RNA metabolism. Serine-arginine protein kinases (SRPKs) are important kinases that specifically phosphorylate SR proteins to regulate their distribution and activities in the central pre-mRNA splicing and other cellular processes. In addition to the predominant SR proteins, other cytoplasmic proteins containing a serine-arginine repeat domain, including viral proteins, have been identified as substrates of SRPKs. Viral infection triggers a myriad of cellular events in the host and it is therefore not surprising that viruses explore SRPKs-mediated phosphorylation as an important regulatory node in virus-host interactions. In this review, we briefly summarize the regulation and biological function of SRPKs, highlighting their involvement in the infection process of several viruses, such as viral replication, transcription and capsid assembly. In addition, we review the structure-function relationships of currently available inhibitors of SRPKs and discuss their putative use as antivirals against well-characterized viruses or newly emerging viruses. We also highlight the viral proteins and cellular substrates targeted by SRPKs as potential antiviral therapeutic candidates.

Major adverse cardiovascular events are associated with necroptosis during severe COVID-19.

BACKGROUND: The mechanisms used by SARS-CoV-2 to induce major adverse cardiac events (MACE) are unknown. Thus, we aimed to determine if SARS-CoV-2 can induce necrotic cell death to promote MACE in patients with severe COVID-19. METHODS: This observational prospective cohort study includes experiments with hamsters and human samples from patients with severe COVID-19. Cytokines and serum biomarkers were analysed in human serum. Cardiac transcriptome analyses were performed in hamsters' hearts. RESULTS: From a cohort of 70 patients, MACE was documented in 26% (18/70). Those who developed MACE had higher Log copies/mL of SARS-CoV-2, troponin-I, and pro-BNP in serum. Also, the elevation of IP-10 and a major decrease in levels of IL-17ɑ, IL-6, and IL-1rɑ were observed. No differences were found in the ability of serum antibodies to neutralise viral spike proteins in pseudoviruses from variants of concern. In hamster models, we found a stark increase in viral titters in the hearts 4 days post-infection. The cardiac transcriptome evaluation resulted in the differential expression of ~ 9% of the total transcripts. Analysis of transcriptional changes in the effectors of necroptosis (mixed lineage kinase domain-like, MLKL) and pyroptosis (gasdermin D) showed necroptosis, but not pyroptosis, to be elevated. An active form of MLKL (phosphorylated MLKL, pMLKL) was elevated in hamster hearts and, most importantly, in the serum of MACE patients. CONCLUSION: SARS-CoV-2 identification in the systemic circulation is associated with MACE and necroptosis activity. The increased pMLKL and Troponin-I indicated the occurrence of necroptosis in the heart and suggested necroptosis effectors could serve as biomarkers and/or therapeutic targets. Trial registration Not applicable.

Targeting Human Proteins for Antiviral Drug Discovery and Repurposing Efforts: A Focus on Protein Kinases.

Despite the great technological and medical advances in fighting viral diseases, new therapies for most of them are still lacking, and existing antivirals suffer from major limitations regarding drug resistance and a limited spectrum of activity. In fact, most approved antivirals are directly acting antiviral (DAA) drugs, which interfere with viral proteins and confer great selectivity towards their viral targets but suffer from resistance and limited spectrum. Nowadays, host-targeted antivirals (HTAs) are on the rise, in the drug discovery and development pipelines, in academia and in the pharmaceutical industry. These drugs target host proteins involved in the virus life cycle and are considered promising alternatives to DAAs due to their broader spectrum and lower potential for resistance. Herein, we discuss an important class of HTAs that modulate signal transduction pathways by targeting host kinases. Kinases are considered key enzymes that control virus-host interactions. We also provide a synopsis of the antiviral drug discovery and development pipeline detailing antiviral kinase targets, drug types, therapeutic classes for repurposed drugs, and top developing organizations. Furthermore, we detail the drug design and repurposing considerations, as well as the limitations and challenges, for kinase-targeted antivirals, including the choice of the binding sites, physicochemical properties, and drug combinations.

Apatinib combined with camrelizumab in advanced acral melanoma patients: An open-label, single-arm phase 2 trial.

BACKGROUND: At present, immune monotherapy and combination therapy has not shown satisfactory effects on acral melanoma, and still no standard treatment is available for advanced acral melanoma. Here, a phase II trial was performed to explore the safety and efficacy of apatinib combined with camrelizumab in advanced acral melanoma patients as first-line therapy (NCT03955354). METHODS: Patients with pathologically confirmed, locally unresectable or metastatic treatment native acral melanoma received 250 mg apatinib once daily and camrelizumab 200 mg once every two weeks intravenously every 28-day cycle. The primary end-point was objective response rate and the secondary end-points were disease control rate, overall survival, progression-free survival and safety. RESULTS: Thirty patients were recruited between January 2015 and January 2022. Among them, 21 (70.0%) had stage IV, and a median tumour burden was 50 mm (range: 11-187). Objective response rate was 24.1%, and 7 of 29 patients had an anti-tumour response, including partial response (n = 5) and complete response (n = 2). Disease control rate was 82.8%, median progression-free survival was 7.39 months (confidence interval: 3.65-9.92), and median overall survival was 13.4 months (confidence interval: 1.9-25.0). Grade 3-4 treatment-related toxicity (grade 3 50.5%; grade 4 3.3%) included transaminase elevations, proteinuria, leukocytopenia, vomiting, diarrhea and drug-induced liver injury. No treatment-related mortality occurred. The mutations of TTN, MUC16, VPS13D, ALPK2 and SCUBE1 showed significant alterations with survival outcome. CONCLUSIONS: Apatinib combined with camrelizumab showed manageable safety profile and reasonable anti-tumour activity in advanced acral melanoma patients as first-line therapy.

SARS-CoV-2 immune complex triggers human monocyte necroptosis.

We analyzed the ability of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) itself and SARS-CoV-2-IgG immune complexes to trigger human monocyte necroptosis. SARS-CoV-2 was able to induce monocyte necroptosis dependently of MLKL activation. Necroptosis-associated proteins (RIPK1, RIPK3 and MLKL) were involved in SARS-CoV-2N1 gene expression in monocytes. SARS-CoV-2 immune complexes promoted monocyte necroptosis in a RIPK3- and MLKL-dependent manner, and Syk tyrosine kinase was necessary for SARS-CoV-2 immune complex-induced monocyte necroptosis, indicating the involvement of Fcγ receptors on necroptosis. Finally, we provide evidence that elevated LDH levels as a marker of lytic cell death are associated with COVID-19 pathogenesis.

SARS-CoV-2 spike protein enhances MAP4K3/GLK-induced ACE2 stability in COVID-19.

ACE2 on epithelial cells is the SARS-CoV-2 entry receptor. Single-cell RNA-sequencing data derived from two COVID-19 cohorts revealed that MAP4K3/GLK-positive epithelial cells were increased in patients. SARS-CoV-2-induced GLK overexpression in epithelial cells was correlated with COVID-19 severity and vesicle secretion. GLK overexpression induced the epithelial cell-derived exosomes containing ACE2; the GLK-induced exosomes transported ACE2 proteins to recipient cells, facilitating pseudovirus infection. Consistently, ACE2 proteins were increased in the serum exosomes from another COVID-19 cohort. Remarkably, SARS-CoV-2 spike protein-stimulated GLK, and GLK stabilized ACE2 in epithelial cells. Mechanistically, GLK phosphorylated ACE2 at two serine residues (Ser776, Ser783), leading to the dissociation of ACE2 from its E3 ligase UBR4. Reduction in UBR4-induced Lys48-linked ubiquitination at three lysine residues (Lys26, Lys112, Lys114) of ACE2 prevented its degradation. Furthermore, SARS-CoV-2 pseudovirus or live virus infection in humanized ACE2 mice induced GLK and ACE2 protein levels, and ACE2-containing exosomes. Collectively, ACE2 stabilization by SARS-CoV-2-induced MAP4K3/GLK may contribute to the pathogenesis of COVID-19.

In Vitro Kinase-to-Phosphosite Database (iKiP-DB) Predicts Kinase Activity in Phosphoproteomic Datasets.

Phosphoproteomics routinely quantifies changes in the levels of thousands of phosphorylation sites, but functional analysis of such data remains a major challenge. While databases like PhosphoSitePlus contain information about many phosphorylation sites, the vast majority of known sites is not assigned to any protein kinase. Assigning changes in the phosphoproteome to the activity of individual kinases therefore remains a key challenge. A recent large-scale study systematically identified in vitro substrates for most human protein kinases. Here, we reprocessed and filtered these data to generate an in vitro Kinase-to-Phosphosite database (iKiP-DB). We show that iKiP-DB can accurately predict changes in kinase activity in published phosphoproteomic data sets for both well-studied and poorly characterized kinases. We apply iKiP-DB to a newly generated phosphoproteomic analysis of SARS-CoV-2 infected human lung epithelial cells and provide evidence for coronavirus-induced changes in host cell kinase activity. In summary, we show that iKiP-DB is widely applicable to facilitate the functional analysis of phosphoproteomic data sets.

Interferon-γ Preferentially Promotes Necroptosis of Lung Epithelial Cells by Upregulating MLKL.

Necroptosis, a form of programmed lytic cell death, has emerged as a driving factor in the pathogenesis of acute lung injury (ALI). As ALI is often associated with a cytokine storm, we determined whether pro-inflammatory cytokines modulate the susceptibility of lung cells to necroptosis and which mediators dominate to control necroptosis. In this study, we pretreated/primed mouse primary lung epithelial and endothelial cells with various inflammatory mediators and assessed cell type-dependent responses to different necroptosis inducers and their underlying mechanisms. We found that interferon-γ (IFNγ) as low as 1 ng/mL preferentially promoted necroptosis and accelerated the release of damage-associated molecular patterns from primary alveolar and airway epithelial cells but not lung microvascular endothelial cells. Type-I IFNα was about fifty-fold less effective than IFNγ. Conversely, TNFα or agonists of Toll-like receptor-3 (TLR3), TLR4, TLR7 and TLR9 had a minor effect. The enhanced necroptosis in IFNγ-activated lung epithelial cells was dependent on IFNγ signaling and receptor-interacting protein kinase-3. We further showed that necroptosis effector mixed lineage kinase domain-like protein (MLKL) was predominantly induced by IFNγ, contributing to the enhanced necroptosis in lung epithelial cells. Collectively, our findings indicate that IFNγ is a potent enhancer of lung epithelial cell susceptibility to necroptosis.

Mass spectrometry-based proteomic platforms for better understanding of SARS-CoV-2 induced pathogenesis and potential diagnostic approaches.

While protein-protein interaction is the first step of the SARS-CoV-2 infection, recent comparative proteomic profiling enabled the identification of over 11,000 protein dynamics, thus providing a comprehensive reflection of the molecular mechanisms underlying the cellular system in response to viral infection. Here we summarize and rationalize the results obtained by various mass spectrometry (MS)-based proteomic approaches applied to the functional characterization of proteins and pathways associated with SARS-CoV-2-mediated infections in humans. Comparative analysis of cell-lines versus tissue samples indicates that our knowledge in proteome profile alternation in response to SARS-CoV-2 infection is still incomplete and the tissue-specific response to SARS-CoV-2 infection can probably not be recapitulated efficiently by in vitro experiments. However, regardless of the viral infection period, sample types, and experimental strategies, a thorough cross-comparison of the recently published proteome, phosphoproteome, and interactome datasets led to the identification of a common set of proteins and kinases associated with PI3K-Akt, EGFR, MAPK, Rap1, and AMPK signaling pathways. Ephrin receptor A2 (EPHA2) was identified by 11 studies including all proteomic platforms, suggesting it as a potential future target for SARS-CoV-2 infection mechanisms and the development of new therapeutic strategies. We further discuss the potentials of future proteomics strategies for identifying prognostic SARS-CoV-2 responsive age-, gender-dependent, tissue-specific protein targets.

A proposed role for the SARS-CoV-2 nucleocapsid protein in the formation and regulation of biomolecular condensates.

To date, the recently discovered SARS-CoV-2 virus has afflicted >6.9 million people worldwide and disrupted the global economy. Development of effective vaccines or treatments for SARS-CoV-2 infection will be aided by a molecular-level understanding of SARS-CoV-2 proteins and their interactions with host cell proteins. The SARS-CoV-2 nucleocapsid (N) protein is highly homologous to the N protein of SARS-CoV, which is essential for viral RNA replication and packaging into new virions. Emerging models indicate that nucleocapsid proteins of other viruses can form biomolecular condensates to spatiotemporally regulate N protein localization and function. Our bioinformatic analyses, in combination with pre-existing experimental evidence, suggest that the SARS-CoV-2 N protein is capable of forming or regulating biomolecular condensates in vivo by interaction with RNA and key host cell proteins. We discuss multiple models, whereby the N protein of SARS-CoV-2 may harness this activity to regulate viral life cycle and host cell response to viral infection.

Yeast-based high-throughput screens for discovery of kinase inhibitors for neglected diseases.

The discovery and development of a new drug is a complex, time consuming and costly process that typically takes over 10 years and costs around 1 billion dollars from bench to market. This scenario makes the discovery of novel drugs targeting neglected tropical diseases (NTDs), which afflict in particular people in low-income countries, prohibitive. Despite the intensive use of High-Throughput Screening (HTS) in the past decades, the speed with which new drugs come to the market has remained constant, generating doubts about the efficacy of this approach. Here we review a few of the yeast-based high-throughput approaches that can work synergistically with parasite-based, in vitro, or in silico methods to identify and optimize novel antiparasitic compounds. These yeast-based methods range from HTP screens to identify novel hits against promising parasite kinase targets to the identification of potential antiparasitic kinase inhibitors extracted from databases of yeast chemical genetic screens.

KEA3: improved kinase enrichment analysis via data integration.

Phosphoproteomics and proteomics experiments capture a global snapshot of the cellular signaling network, but these methods do not directly measure kinase state. Kinase Enrichment Analysis 3 (KEA3) is a webserver application that infers overrepresentation of upstream kinases whose putative substrates are in a user-inputted list of proteins. KEA3 can be applied to analyze data from phosphoproteomics and proteomics studies to predict the upstream kinases responsible for observed differential phosphorylations. The KEA3 background database contains measured and predicted kinase-substrate interactions (KSI), kinase-protein interactions (KPI), and interactions supported by co-expression and co-occurrence data. To benchmark the performance of KEA3, we examined whether KEA3 can predict the perturbed kinase from single-kinase perturbation followed by gene expression experiments, and phosphoproteomics data collected from kinase-targeting small molecules. We show that integrating KSIs and KPIs across data sources to produce a composite ranking improves the recovery of the expected kinase. The KEA3 webserver is available at https://maayanlab.cloud/kea3.

VPTMdb: a viral posttranslational modification database.

In viruses, posttranslational modifications (PTMs) are essential for their life cycle. Recognizing viral PTMs is very important for a better understanding of the mechanism of viral infections and finding potential drug targets. However, few studies have investigated the roles of viral PTMs in virus-human interactions using comprehensive viral PTM datasets. To fill this gap, we developed the first comprehensive viral posttranslational modification database (VPTMdb) for collecting systematic information of PTMs in human viruses and infected host cells. The VPTMdb contains 1240 unique viral PTM sites with 8 modification types from 43 viruses (818 experimentally verified PTM sites manually extracted from 150 publications and 422 PTMs extracted from SwissProt) as well as 13 650 infected cells' PTMs extracted from seven global proteomics experiments in six human viruses. The investigation of viral PTM sequences motifs showed that most viral PTMs have the consensus motifs with human proteins in phosphorylation and five cellular kinase families phosphorylate more than 10 viral species. The analysis of protein disordered regions presented that more than 50% glycosylation sites of double-strand DNA viruses are in the disordered regions, whereas single-strand RNA and retroviruses prefer ordered regions. Domain-domain interaction analysis indicating potential roles of viral PTMs play in infections. The findings should make an important contribution to the field of virus-human interaction. Moreover, we created a novel sequence-based classifier named VPTMpre to help users predict viral protein phosphorylation sites. VPTMdb online web server (http://vptmdb.com:8787/VPTMdb/) was implemented for users to download viral PTM data and predict phosphorylation sites of interest.

Downfalls of Chemical Probes Acting at the Kinase ATP-Site: CK2 as a Case Study.

Protein kinases are a large class of enzymes with numerous biological roles and many have been implicated in a vast array of diseases, including cancer and the novel coronavirus infection COVID-19. Thus, the development of chemical probes to selectively target each kinase is of great interest. Inhibition of protein kinases with ATP-competitive inhibitors has historically been the most widely used method. However, due to the highly conserved structures of ATP-sites, the identification of truly selective chemical probes is challenging. In this review, we use the Ser/Thr kinase CK2 as an example to highlight the historical challenges in effective and selective chemical probe development, alongside recent advances in the field and alternative strategies aiming to overcome these problems. The methods utilised for CK2 can be applied to an array of protein kinases to aid in the discovery of chemical probes to further understand each kinase's biology, with wide-reaching implications for drug development.

Targeting MMP-Regulation of Inflammation to Increase Metabolic Tolerance to COVID-19 Pathologies: A Hypothesis.

Many individuals infected with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) develop no or only mild symptoms, but some can go on onto develop a spectrum of pathologies including pneumonia, acute respiratory distress syndrome, respiratory failure, systemic inflammation, and multiorgan failure. Many pathogens, viral and non-viral, can elicit these pathologies, which justifies reconsidering whether the target of therapeutic approaches to fight pathogen infections should be (a) the pathogen itself, (b) the pathologies elicited by the pathogen interaction with the human host, or (c) a combination of both. While little is known about the immunopathology of SARS-CoV-2, it is well-established that the above-mentioned pathologies are associated with hyper-inflammation, tissue damage, and the perturbation of target organ metabolism. Mounting evidence has shown that these processes are regulated by endoproteinases (particularly, matrix metalloproteinases (MMPs)). Here, we review what is known about the roles played by MMPs in the development of COVID-19 and postulate a mechanism by which MMPs could influence energy metabolism in target organs, such as the lung. Finally, we discuss the suitability of MMPs as therapeutic targets to increase the metabolic tolerance of the host to damage inflicted by the pathogen infection, with a focus on SARS-CoV-2.