RESUMO
Despite the successes of immunotherapy in cancer treatment over recent decades, less than <10%-20% cancer cases have demonstrated durable responses from immune checkpoint blockade. To enhance the efficacy of immunotherapies, combination therapies suppressing multiple immune evasion mechanisms are increasingly contemplated. To better understand immune cell surveillance and diverse immune evasion responses in tumor tissues, we comprehensively characterized the immune landscape of more than 1,000 tumors across ten different cancers using CPTAC pan-cancer proteogenomic data. We identified seven distinct immune subtypes based on integrative learning of cell type compositions and pathway activities. We then thoroughly categorized unique genomic, epigenetic, transcriptomic, and proteomic changes associated with each subtype. Further leveraging the deep phosphoproteomic data, we studied kinase activities in different immune subtypes, which revealed potential subtype-specific therapeutic targets. Insights from this work will facilitate the development of future immunotherapy strategies and enhance precision targeting with existing agents.
Assuntos
Neoplasias , Proteogenômica , Humanos , Terapia Combinada , Genômica , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/terapia , Proteômica , Evasão TumoralRESUMO
Post-translational modifications (PTMs) play key roles in regulating cell signaling and physiology in both normal and cancer cells. Advances in mass spectrometry enable high-throughput, accurate, and sensitive measurement of PTM levels to better understand their role, prevalence, and crosstalk. Here, we analyze the largest collection of proteogenomics data from 1,110 patients with PTM profiles across 11 cancer types (10 from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium [CPTAC]). Our study reveals pan-cancer patterns of changes in protein acetylation and phosphorylation involved in hallmark cancer processes. These patterns revealed subsets of tumors, from different cancer types, including those with dysregulated DNA repair driven by phosphorylation, altered metabolic regulation associated with immune response driven by acetylation, affected kinase specificity by crosstalk between acetylation and phosphorylation, and modified histone regulation. Overall, this resource highlights the rich biology governed by PTMs and exposes potential new therapeutic avenues.
Assuntos
Neoplasias , Processamento de Proteína Pós-Traducional , Proteômica , Humanos , Acetilação , Histonas/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação , Proteômica/métodosRESUMO
Cancer driver events refer to key genetic aberrations that drive oncogenesis; however, their exact molecular mechanisms remain insufficiently understood. Here, our multi-omics pan-cancer analysis uncovers insights into the impacts of cancer drivers by identifying their significant cis-effects and distal trans-effects quantified at the RNA, protein, and phosphoprotein levels. Salient observations include the association of point mutations and copy-number alterations with the rewiring of protein interaction networks, and notably, most cancer genes converge toward similar molecular states denoted by sequence-based kinase activity profiles. A correlation between predicted neoantigen burden and measured T cell infiltration suggests potential vulnerabilities for immunotherapies. Patterns of cancer hallmarks vary by polygenic protein abundance ranging from uniform to heterogeneous. Overall, our work demonstrates the value of comprehensive proteogenomics in understanding the functional states of oncogenic drivers and their links to cancer development, surpassing the limitations of studying individual cancer types.
Assuntos
Neoplasias , Proteogenômica , Humanos , Neoplasias/genética , Oncogenes , Transformação Celular Neoplásica/genética , Variações do Número de Cópias de DNARESUMO
Pancreatic ductal adenocarcinoma (PDA) is characterized by aggressive local invasion and metastatic spread, leading to high lethality. Although driver gene mutations during PDA progression are conserved, no specific mutation is correlated with the dissemination of metastases1-3. Here we analysed RNA splicing data of a large cohort of primary and metastatic PDA tumours to identify differentially spliced events that correlate with PDA progression. De novo motif analysis of these events detected enrichment of motifs with high similarity to the RBFOX2 motif. Overexpression of RBFOX2 in a patient-derived xenograft (PDX) metastatic PDA cell line drastically reduced the metastatic potential of these cells in vitro and in vivo, whereas depletion of RBFOX2 in primary pancreatic tumour cell lines increased the metastatic potential of these cells. These findings support the role of RBFOX2 as a potent metastatic suppressor in PDA. RNA-sequencing and splicing analysis of RBFOX2 target genes revealed enrichment of genes in the RHO GTPase pathways, suggesting a role of RBFOX2 splicing activity in cytoskeletal organization and focal adhesion formation. Modulation of RBFOX2-regulated splicing events, such as via myosin phosphatase RHO-interacting protein (MPRIP), is associated with PDA metastases, altered cytoskeletal organization and the induction of focal adhesion formation. Our results implicate the splicing-regulatory function of RBFOX2 as a tumour suppressor in PDA and suggest a therapeutic approach for metastatic PDA.
Assuntos
Processamento Alternativo , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Processamento Alternativo/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Animais , Metástase Neoplásica , Adesões FocaisRESUMO
Protein phosphorylation is one of the most widespread post-translational modifications in biology1,2. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes3,4. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible3. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.
Assuntos
Fosfoproteínas , Proteínas Serina-Treonina Quinases , Proteoma , Serina , Treonina , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Especificidade por Substrato , Treonina/metabolismo , Proteoma/química , Proteoma/metabolismo , Conjuntos de Dados como Assunto , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Linhagem Celular , Fosfosserina/metabolismo , Fosfotreonina/metabolismoRESUMO
CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Modelos Biológicos , Fator de Transcrição TFIIH/metabolismo , Transcrição Gênica/genética , Processamento Alternativo/genética , Sobrevivência Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Ativação Enzimática/genética , Células HL-60 , Humanos , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
There is an urgent need to create novel models using human disease-relevant cells to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biology and to facilitate drug screening. Here, as SARS-CoV-2 primarily infects the respiratory tract, we developed a lung organoid model using human pluripotent stem cells (hPSC-LOs). The hPSC-LOs (particularly alveolar type-II-like cells) are permissive to SARS-CoV-2 infection, and showed robust induction of chemokines following SARS-CoV-2 infection, similar to what is seen in patients with COVID-19. Nearly 25% of these patients also have gastrointestinal manifestations, which are associated with worse COVID-19 outcomes1. We therefore also generated complementary hPSC-derived colonic organoids (hPSC-COs) to explore the response of colonic cells to SARS-CoV-2 infection. We found that multiple colonic cell types, especially enterocytes, express ACE2 and are permissive to SARS-CoV-2 infection. Using hPSC-LOs, we performed a high-throughput screen of drugs approved by the FDA (US Food and Drug Administration) and identified entry inhibitors of SARS-CoV-2, including imatinib, mycophenolic acid and quinacrine dihydrochloride. Treatment at physiologically relevant levels of these drugs significantly inhibited SARS-CoV-2 infection of both hPSC-LOs and hPSC-COs. Together, these data demonstrate that hPSC-LOs and hPSC-COs infected by SARS-CoV-2 can serve as disease models to study SARS-CoV-2 infection and provide a valuable resource for drug screening to identify candidate COVID-19 therapeutics.
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Antivirais/farmacologia , COVID-19/virologia , Colo/citologia , Avaliação Pré-Clínica de Medicamentos/métodos , Pulmão/citologia , Organoides/efeitos dos fármacos , Organoides/virologia , SARS-CoV-2/efeitos dos fármacos , Animais , COVID-19/prevenção & controle , Colo/efeitos dos fármacos , Colo/virologia , Aprovação de Drogas , Feminino , Xenoenxertos/efeitos dos fármacos , Humanos , Técnicas In Vitro , Pulmão/efeitos dos fármacos , Pulmão/virologia , Masculino , Camundongos , Organoides/citologia , Organoides/metabolismo , SARS-CoV-2/genética , Estados Unidos , United States Food and Drug Administration , Tropismo Viral , Internalização do Vírus/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
Creatine kinases (CKs) provide local ATP production in periods of elevated energetic demand, such as during rapid anabolism and growth. Thus, creatine energetics has emerged as a major metabolic liability in many rapidly proliferating cancers. Whether CKs can be targeted therapeutically is unknown because no potent or selective CK inhibitors have been developed. Here we leverage an active site cysteine present in all CK isoforms to develop a selective covalent inhibitor of creatine phosphagen energetics, CKi. Using deep chemoproteomics, we discover that CKi selectively engages the active site cysteine of CKs in cells. A co-crystal structure of CKi with creatine kinase B indicates active site inhibition that prevents bidirectional phosphotransfer. In cells, CKi and its analogs rapidly and selectively deplete creatine phosphate, and drive toxicity selectively in CK-dependent acute myeloid leukemia. Finally, we use CKi to uncover an essential role for CKs in the regulation of proinflammatory cytokine production in macrophages.
Assuntos
Creatina Quinase , Creatina , Creatina Quinase/química , Creatina Quinase/metabolismo , Creatina/farmacologia , Cisteína , Fosfotransferases , Isoformas de ProteínasRESUMO
Purines represent a class of essential metabolites produced by the cell to maintain cellular homeostasis and facilitate cell proliferation. In times of high purine demand, the de novo purine biosynthetic pathway is activated; however, the mechanisms that facilitate this process are largely unknown. One plausible mechanism is through intracellular signaling, which results in enzymes within the pathway becoming post-translationally modified to enhance their individual enzyme activities and the overall pathway metabolic flux. Here, we employ a proteomic strategy to investigate the extent to which de novo purine biosynthetic pathway enzymes are post-translationally modified in 293T cells. We identified 7 post-translational modifications on 135 residues across the 6 human pathway enzymes. We further asked whether there were differences in the post-translational modification state of each pathway enzyme isolated from cells cultured in the presence or absence of purines. Of the 174 assigned modifications, 67% of them were only detected in one experimental growth condition in which a significant number of serine and threonine phosphorylations were noted. A survey of the most-probable kinases responsible for these phosphorylation events uncovered a likely AKT phosphorylation site at residue Thr397 of PPAT, which was only detected in cells under purine-supplemented growth conditions. These data suggest that this modification might alter enzyme activity or modulate its interaction(s) with downstream pathway enzymes. Together, these findings propose a role for post-translational modifications in pathway regulation and activation to meet intracellular purine demand.
Assuntos
Amidofosforribosiltransferase/metabolismo , Mapeamento de Peptídeos/métodos , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Purinas/metabolismo , Acetilação , Adenilossuccinato Liase/genética , Adenilossuccinato Liase/metabolismo , Amidofosforribosiltransferase/genética , Sequência de Aminoácidos , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Peptídeos/síntese química , Peptídeos/metabolismo , Fosforribosilglicinamido Formiltransferase/genética , Fosforribosilglicinamido Formiltransferase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Transdução de Sinais , Treonina/metabolismo , UbiquitinaçãoRESUMO
Despite recent clinical successes for irreversible drugs, potential toxicities mediated by unpredictable modification of off-target cysteines represents a major hurdle for expansion of covalent drug programs. Understanding the proteome-wide binding profile of covalent inhibitors can significantly accelerate their development; however, current mass spectrometry strategies typically do not provide a direct, amino acid level readout of covalent activity for complex, selective inhibitors. Here we report the development of CITe-Id, a novel chemoproteomic approach that employs covalent pharmacologic inhibitors as enrichment reagents in combination with an optimized proteomic platform to directly quantify dose-dependent binding at cysteine-thiols across the proteome. CITe-Id analysis of our irreversible CDK inhibitor THZ1 identified dose-dependent covalent modification of several unexpected kinases, including a previously unannotated cysteine (C840) on the understudied kinase PKN3. These data streamlined our development of JZ128 as a new selective covalent inhibitor of PKN3. Using JZ128 as a probe compound, we identified novel potential PKN3 substrates, thus offering an initial molecular view of PKN3 cellular activity. CITe-Id provides a powerful complement to current chemoproteomic platforms to characterize the selectivity of covalent inhibitors, identify new, pharmacologically addressable cysteine-thiols, and inform structure-based drug design programs.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteômica , Sequência de Aminoácidos , Domínio Catalítico , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/química , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Modelos Moleculares , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/química , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Subcellular location and activation of Tank Binding Kinase 1 (TBK1) govern precise progression through mitosis. Either loss of activated TBK1 or its sequestration from the centrosomes causes errors in mitosis and growth defects. Yet, what regulates its recruitment and activation on the centrosomes is unknown. We identified that NAK-associated protein 1 (NAP1) is essential for mitosis, binding to and activating TBK1, which both localize to centrosomes. Loss of NAP1 causes several mitotic and cytokinetic defects due to inactivation of TBK1. Our quantitative phosphoproteomics identified numerous TBK1 substrates that are not only confined to the centrosomes but are also associated with microtubules. Substrate motifs analysis indicates that TBK1 acts upstream of other essential cell cycle kinases like Aurora and PAK kinases. We also identified NAP1 as a TBK1 substrate phosphorylating NAP1 at S318 to promote its degradation by the ubiquitin proteasomal system. These data uncover an important distinct function for the NAP1-TBK1 complex during cell division.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Citocinese , Mitose , Proteínas Serina-Treonina Quinases , Humanos , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
To delineate the mechanisms by which the ERK1 and ERK2 mitogen-activated protein kinases support mutant KRAS-driven cancer growth, we determined the ERK-dependent phosphoproteome in KRAS-mutant pancreatic cancer. We determined that ERK1 and ERK2 share near-identical signaling and transforming outputs and that the KRAS-regulated phosphoproteome is driven nearly completely by ERK. We identified 4666 ERK-dependent phosphosites on 2123 proteins, of which 79 and 66%, respectively, were not previously associated with ERK, substantially expanding the depth and breadth of ERK-dependent phosphorylation events and revealing a considerably more complex function for ERK in cancer. We established that ERK controls a highly dynamic and complex phosphoproteome that converges on cyclin-dependent kinase regulation and RAS homolog guanosine triphosphatase function (RHO GTPase). Our findings establish the most comprehensive molecular portrait and mechanisms by which ERK drives KRAS-dependent pancreatic cancer growth.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Neoplasias Pancreáticas , Fosfoproteínas , Proteoma , Proteínas Proto-Oncogênicas p21(ras) , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/genética , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Fosforilação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Células HEK293RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, drastically modifies infected cells to optimize virus replication. One such modification is the activation of the host p38 mitogen-activated protein kinase (MAPK) pathway, which plays a major role in inflammatory cytokine production, a hallmark of severe COVID-19. We previously demonstrated that inhibition of p38/MAPK activity in SARS-CoV-2-infected cells reduced both cytokine production and viral replication. Here, we combined quantitative genetic screening, genomics, proteomics, and phosphoproteomics to better understand mechanisms underlying the dependence of SARS-CoV-2 on the p38 pathway. We found that p38ß is a critical host factor for SARS-CoV-2 replication in multiple relevant cell lines and that it functions at a step after viral mRNA expression. We identified putative host and viral p38ß substrates in the context of SARS-CoV-2 infection and found that most host substrates have intrinsic antiviral activities. Taken together, this study reveals a unique proviral function for p38ß and supports exploring p38ß inhibitor development as a strategy toward creating a new class of COVID-19 therapies. IMPORTANCE SARS-CoV-2 is the causative agent of the COVID-19 pandemic that has claimed millions of lives since its emergence in 2019. SARS-CoV-2 infection of human cells requires the activity of several cellular pathways for successful replication. One such pathway, the p38 MAPK pathway, is required for virus replication and disease pathogenesis. Here, we applied systems biology approaches to understand how MAPK pathways benefit SARS-CoV-2 replication to inform the development of novel COVID-19 drug therapies.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Citocinas , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Pandemias , SARS-CoV-2/metabolismo , Replicação Viral , Proteína Quinase 11 Ativada por Mitógeno/metabolismoRESUMO
The phosphoinositide 3-kinase p110α is an essential mediator of insulin signaling and glucose homeostasis. We interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate p110α at T1061, which inhibits its activity. This inhibitory state corresponds to a conformational change of a membrane-binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases MST1/2 using forskolin or epinephrine is associated with phosphorylation of T1061 and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules or if the T1061 is mutated to alanine. Our study defines an inhibitory pathway of PI3K signaling and a link between epinephrine and insulin signaling.
Assuntos
Proteínas Serina-Treonina Quinases , Humanos , Animais , Camundongos , Linhagem Celular , Camundongos Endogâmicos C57BL , Masculino , Feminino , Epinefrina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Deleção de Genes , Colforsina/farmacologia , Insulina/metabolismo , Fosforilação/efeitos dos fármacos , Via de Sinalização Hippo/efeitos dos fármacos , Via de Sinalização Hippo/genéticaRESUMO
Previously, we showed that coagulation factors directly cleave SARS-CoV-2 spike and promote viral entry (Kastenhuber et al., 2022). Here, we show that substitutions in the S1/S2 cleavage site observed in SARS-CoV-2 variants of concern (VOCs) exhibit divergent interactions with host proteases, including factor Xa and furin. Nafamostat remains effective to block coagulation factor-mediated cleavage of variant spike sequences. Furthermore, host protease usage has likely been a selection pressure throughout coronavirus evolution, and we observe convergence of distantly related coronaviruses to attain common host protease interactions, including coagulation factors. Interpretation of genomic surveillance of emerging SARS-CoV-2 variants and future zoonotic spillover is supported by functional characterization of recurrent emerging features.
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Multiple coronaviruses have emerged independently in the past 20 years that cause lethal human diseases. Although vaccine development targeting these viruses has been accelerated substantially, there remain patients requiring treatment who cannot be vaccinated or who experience breakthrough infections. Understanding the common host factors necessary for the life cycles of coronaviruses may reveal conserved therapeutic targets. Here, we used the known substrate specificities of mammalian protein kinases to deconvolute the sequence of phosphorylation events mediated by three host protein kinase families (SRPK, GSK-3, and CK1) that coordinately phosphorylate a cluster of serine and threonine residues in the viral N protein, which is required for viral replication. We also showed that loss or inhibition of SRPK1/2, which we propose initiates the N protein phosphorylation cascade, compromised the viral replication cycle. Because these phosphorylation sites are highly conserved across coronaviruses, inhibitors of these protein kinases not only may have therapeutic potential against COVID-19 but also may be broadly useful against coronavirus-mediated diseases.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Humanos , SARS-CoV-2/genética , Fosforilação , Quinase 3 da Glicogênio Sintase/metabolismo , Replicação Viral , Proteínas do Nucleocapsídeo/metabolismo , Nucleocapsídeo/metabolismo , Serina/metabolismo , Treonina/metabolismo , Mamíferos/metabolismo , Proteínas Serina-Treonina QuinasesRESUMO
Cyclin-dependent kinases (CDKs) constitute a family of 20 serine/threonine protein kinases that play pivotal roles in the regulation of numerous important molecular and cellular processes. CDKs have long been considered promising therapeutic targets in a variety of pathologies, and the recent therapeutic success of CDK4/6 inhibitors in breast cancers has renewed interest in their therapeutic potential. Small-molecule inhibitors have been identified for every human CDK, except for CDK10. The only recent discovery of an activating cyclin (CycM) for CDK10 enabled us to identify its first phosphorylation substrates and gain insights into its biological functions. Yet, our knowledge of this kinase remains incomplete, despite it being the only member of its family that causes severe human developmental syndromes, when mutated either on the cyclin or the CDK moiety. CDK10 small-molecule inhibitors would be useful in exploring the functions of this kinase and gauging its potential as a therapeutic target for some cancers. Here, we report the identification of an optimized peptide phosphorylation substrate of CDK10/CycM and the development of the first homogeneous, miniaturized CDK10/CycM in vitro kinase assay. We reveal the ability of known CDK inhibitors, among which clinically tested SNS-032, riviciclib, flavopiridol, dinaciclib, AZD4573 and AT7519, to potently inhibit CDK10/CycM. We also show that NVP-2, a strong, remarkably selective CDK9 inhibitor is an equally potent CDK10/CycM inhibitor. Finally, we validate this kinase assay for applications in high-throughput screening campaigns to discover new, original CDK10 inhibitors.
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The SARS-CoV-2 virus has caused already over 3.5 million COVID-19 cases and 250,000 deaths globally. There is an urgent need to create novel models to study SARS-CoV-2 using human disease-relevant cells to understand key features of virus biology and facilitate drug screening. As primary SARS-CoV-2 infection is respiratory-based, we developed a lung organoid model using human pluripotent stem cells (hPSCs) that could be adapted for drug screens. The lung organoids, particularly aveolar type II cells, express ACE2 and are permissive to SARS-CoV-2 infection. Transcriptomic analysis following SARS-CoV-2 infection revealed a robust induction of chemokines and cytokines with little type I/III interferon signaling, similar to that observed amongst human COVID-19 pulmonary infections. We performed a high throughput screen using hPSC-derived lung organoids and identified FDA-approved drug candidates, including imatinib and mycophenolic acid, as inhibitors of SARS-CoV-2 entry. Pre- or post-treatment with these drugs at physiologically relevant levels decreased SARS-CoV-2 infection of hPSC-derived lung organoids. Together, these data demonstrate that hPSC-derived lung cells infected by SARS-CoV-2 can model human COVID-19 disease and provide a valuable resource to screen for FDA-approved drugs that might be repurposed and should be considered for COVID-19 clinical trials.
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While vaccines are vital for preventing COVID-19 infections, it is critical to develop new therapies to treat patients who become infected. Pharmacological targeting of a host factor required for viral replication can suppress viral spread with a low probability of viral mutation leading to resistance. In particular, host kinases are highly druggable targets and a number of conserved coronavirus proteins, notably the nucleoprotein (N), require phosphorylation for full functionality. In order to understand how targeting kinases could be used to compromise viral replication, we used a combination of phosphoproteomics and bioinformatics as well as genetic and pharmacological kinase inhibition to define the enzymes important for SARS-CoV-2 N protein phosphorylation and viral replication. From these data, we propose a model whereby SRPK1/2 initiates phosphorylation of the N protein, which primes for further phosphorylation by GSK-3a/b and CK1 to achieve extensive phosphorylation of the N protein SR-rich domain. Importantly, we were able to leverage our data to identify an FDA-approved kinase inhibitor, Alectinib, that suppresses N phosphorylation by SRPK1/2 and limits SARS-CoV-2 replication. Together, these data suggest that repurposing or developing novel host-kinase directed therapies may be an efficacious strategy to prevent or treat COVID-19 and other coronavirus-mediated diseases.